Prodrug Kit for Multi-Stage Chemotherapy

Abstract

A prodrug kit for multi-factor dynamic chemotherapy comprises S i =Fc i -L i -Ct i where i = 1, 2, 3, …, 360 and contains N different small molecule drug conjugates selected from the group comprising where 2 ≦ N ≦ 360, Fc i is a part cleavable by fibroblast activation protein, L i is a self-sacrificing linker, and Ct i is a known chemotherapeutic agent.

Description

Technical Field

[0001] The present invention relates to S i =Fc i -L i -Ct i where i = 1, 2, 3, …, 360 a prodrug kit for multi - factor dynamic chemotherapy, comprising N different small - molecule drug conjugates selected from the group consisting of where 2 ≦ N ≦ 360, Fc i is a moiety cleavable by fibroblast activation protein, L i is a self - sacrificial linker, and Ct i is a known chemotherapeutic agent, and relates to the prodrug kit.

Background Art

[0002] Cancer remains a major global health problem. Despite significant research efforts to understand cancer biology and devise new treatments, only limited success has been achieved in treating leukemia and non - solid or soft - tissue tumors. According to recent statistics from the International Agency for Research on Cancer (IARC), which is supported by the World Health Organization (WHO) or the American Cancer Society (ACS), the incidence, mortality, and economic burden of cancer are increasing at an alarming rate worldwide. In 2014, IARC reported that the fight against global cancer cannot be won with treatment alone and promoted the implementation of preventive strategies to mitigate the imminent cancer crisis.

[0003] Most cancer tumors are diagnosed at an early stage and can be removed by surgical resection. Treatments for advanced or inoperable malignancies include - radiotherapy, - systemic or targeted chemotherapy using cytotoxic agents, kinase inhibitors, immune checkpoint inhibitors, antibody - drug conjugates (ADCs) or small - molecule drug conjugates (SMDCs), and - immunotherapy, particularly chimeric antigen receptor therapy (CAR - T) can be mentioned.

[0004] Unfortunately, despite a good initial response, progressive resistance to these treatments remains a major cause of cancer recurrence and death. To identify novel treatment strategies and improve clinical outcomes, a better understanding of the molecular mechanisms underlying cancer progression and acquired drug resistance is urgently needed. In recent years, numerous biological mechanisms leading to treatment resistance have been identified, including activation of growth factor receptors and their downstream signaling pathways, DNA repair mechanisms, metabolic rewiring, miRNA expression and trafficking, drug extrusion mediated by ATP-binding cassette transporters, and enrichment of cancer stem cell populations. Another important mechanism involved in drug and radiation resistance is the bidirectional communication between cancer cells and their microenvironment (stromal cells, vascular endothelial cells, immune cells), which is thought to be mediated by extracellular vesicles and their molecular cargo. More recently, the important role of the gut microbiota in tumor progression and patient response to various anticancer agents has been recognized. In this context, naturally occurring phytochemical compounds have also regained attention.

[0005] Increasing efforts are being made to identify new biomarkers that can predict treatment response and prevent cancer recurrence. Circulating cancer cells, as well as circulating tumor DNA, cancer cell secretome, and tumor-derived extracellular vesicles, and miRNAs can be easily isolated from patient body fluids. Therefore, liquid biopsy is currently considered a promising tool for cancer detection and determination of appropriate treatment regimens.

[0006] Most targeted, accurate, or personalized cancer therapies, as well as recent immunotherapies, use drugs that address and regulate specific overexpressed or underexpressed cancer-related molecules such as hormones, enzymes, epitopes, growth factors, kinases, cytokines, chemokines, cell receptors, or adapter proteins (e.g., Kras, P-gp, BCR, PI3K, CD11, CD22, CD44, Myc, BRCA2, ALK, IL-10, IL-12, p53, p27, p70, MAPK, TKI, VEGF, EGF). These molecular targets are derived from altered or mutated genes (e.g., DNA damage, hypomethylated or hypermethylated genes, and expression products). Targeted molecular entities are part of the highly heterogeneous biochemical landscape of cancer. However, despite promising results in in vitro studies and xenograft tumors in mice, most targeted molecular entities are, by themselves, limited in their use for translation to the clinic. This dichotomy is borne out by clinical trials, where approximately 97% of new drug candidates do not result in a quantitative improvement.

[0007] Furthermore, cancer patients with stage III or IV cancer treated according to established chemotherapy regimens often develop drug resistance and progress to metastatic stages involving lymph nodes, liver, lung, bone, and brain, ultimately leading to multiple organ failure, vascular damage, induction of proteolytic cascades, and disseminated intravascular coagulation, which is most difficult to cure.

[0008] In contrast to the extensive research on the molecular mechanisms of resistance, the understanding of how resistance develops remains limited. Recent studies suggest that resistance may arise from heterogeneous and weak resistant cell subpopulations with different sensitivities to chemotherapeutic agents. Instead of the commonly assumed stochastic single-hit (epi)mutational transition or drug-induced reprogramming, experimental studies have pointed to a hybrid scenario involving stepwise multi-factor adaptation by various genetic and epigenetic synergistic changes.

[0009] Nevertheless, most of the first- and second-line treatment regimens rely on cytotoxic agents, partially complemented by one or two chemotherapeutic agents, mainly adjuvants that improve side effects. Considering the transient multi-factorial adaptation ability of cancer cells, clinically established treatment regimens based on one or two chemotherapeutic agents administered repeatedly over a long period of time seem insufficient.

[0010] Since 1980, VAMP and RCHOP have achieved cure rates exceeding 90% and 60% in pediatric ALL (acute lymphoblastic leukemia) and DLBCL (diffuse large B-cell lymphoma), respectively, by combining powerful drugs with different mechanisms of action. In the case of solid tumors, the cure rate is much lower because cancer cells are protected from the immune system and large foreign biomolecules (such as antibodies). CAF / FAP-targeted prodrugs with reduced systemic toxicity can overcome the stromal barrier.

[0011] The treatment regimen and FAP-prodrug of the present invention are inspired by and utilize the following (i) A large list of vintage and more recent cancer parent drugs with clinically proven efficacy, (ii) The increasing popularity of combination therapies, as well as (iii) Recent advances in cancer research, such as those cited below. -A.E. Pomeroy, E.V. Schmidt, P.K. Sorger, A.C. Palmer, Drug independence and the curability of cancer by combination chemotherapy, Trends in Cancer, November 2022, Vol. 8, No. 11, https: / / doi.org / 10.1016 / j.trecan.2022.06.009: Conclusion: In this paper, we outlined three historical principles that explain how combinations of independently active therapies can address the problem of tumor heterogeneity and kill more cancer cells in more patients. None of these principles require synergistic drug interactions (activity exceeding additivity) to improve treatment outcomes, although their substantial clinical benefits are often colloquially referred to as synergistic (meaning beneficial for the patient). Thus, the common perception that "synergistic combinations of drugs are required to overcome drug resistance" is false in a quantitative sense. The multiple meanings of "synergy" have been a longstanding cause of confusion regarding the mechanisms of combination therapies

[38] , and have led to the oversight of the importance of tumor heterogeneity and drug cross-resistance as critical factors in the effectiveness of combination therapies. -A.O. Pisco, A. Brock, J. Zhou, A. Moor, M. Mojtahedi, D. Jackson, S. Huang, Non-Darwinian dynamics in therapy-induced cancer drug resistance, Nat Commun 4, 2467 (2013), https: / / doi.org / 10.1038 / ncomms3467: The emergence of drug resistance, a major cause of cancer treatment failure, is generally explained by the selection of resistant mutant cancer cells. However, the dynamic non-genetic heterogeneity of clonal cell populations continuously generates metastable phenotypic variants (persisters), some of which represent stem-like states conferring resistance.... We show by quantitative measurement and modeling that the appearance of MDR1-positive cells 1-2 days after treatment with vincristine (VINC) is mediated mainly by cell-autonomous induction of MDR1 expression rather than by selection of MDR1-expressing cells. -https: / / www.sciencedirect.com / science / article / pii / S2589004220308531

[0012] Professor Kornelia Polyak: "Nevertheless, most cancer treatment research seems to be based on finding new drug targets, ignoring the fact that all cancer drugs invented select for resistance. We need to face this fact head-on and find ways to prevent or control treatment resistance." - J.West, L.You, J.Zhang, R.A.Gatenby, J.S.Brown, P.K.Newton, A.R.A.Anderson, Towards Multidrug Adaptive Therapy, Cancer Res 2020, 80:1578 - 89, doi:10.1158 / 0008 - 5472.CAN - 19 - 2669 - A.H.Briggs et al., An Attribution of Value Framework for Combination Therapies, https: / / assets - dam.takeda.com / raw / upload / v1675187100 / legacy - dotcom / siteassets / en - gb / home / what - we - do / combination - treatments / a - value - attribution - framework - for - combination - therapies - takeda - whitepaper.pdf - https: / / www.fiercepharma.com / pharma / after - ira - victory - senate - doubles - down - more - initiatives - cut - drug - pices - Clinical results of AVA6000: https: / / avacta.wistia.com / medias / tc76pkecuy, https: / / avacta.com / first - patient - dosed - in - fifth - cohort - of - ava6000 - phase - ia - dose - escalation - study / -A. Zana, A. Galbiati, E. Gilardoni, M. Bocci, J. Millul, T. Sturm, R. Stucchi, A. Elsayed, L. Nadal, M. Cirillo, W. Roll, L. Stegger, I. Asmus, P. Backhaus, M. Schaefers, D. Neri, S. Cazzamalli, Fibroblast Activation Protein triggers release of drug payload from non-internalizing small molecule-drug conjugates in solid tumors, Clin Cancer Res CCR-22-1788, October 10, 2022, https: / / doi.org / 10.1158 / 1078-0432.CCR-22-1788 -M. Qi, S. Fan, M. Huang, J. Pan, Y. Li, Q. Miao, W. Lyu, X. Li, L. Deng, S. Qiu, T. Liu, W.-Deng, X. Chu, C. Jiang, W. He, L. Xia, Y. Yang, J. Hong, Q. Qi, W. Yin, X. Liu, C. Shi, M. Chen, W. Ye, D. Zhang, Targeting FAPα-expressing hepatic stellate cells overcomes resistance to antiangiogenics in colorectal cancer liver metastasis models, J Clin Invest, 2022;132(19):e157399, doi:10.1172 / JCI157399, https: / / jci.me / 157399 / pdf -G.Ye, M.Huang, Y.Li, J.Ouyang, M.Chen, Q.Wen, X.Li, H.Zeng, P.Long, Z.Fan, J.Yin, W.Ye, D.Zhang, The FAPα-activated prodrug Z-GP-DAVLBH inhibits the growth and pulmonary metastasis of osteosarcoma cells by suppressing the AXL pathway, Acta Pharmaceutica Sinica B 2022, 12(3):1288e1304, https: / / doi.org / 10.1016 / j.apsb.2021.08.015 -X.Xu, R.Kumari, J.Zhou, J.Chen, B.Mao, J.Wang, M.Zheng, X.Tu, X.An, X.Chen, L.Zhang, X.Tian, H.Wang, X.Dong, Z.Bao, S.Guo, X.Ouyang, L.Shang, F.Wang, X.Yan, R.Zhang, R.G.J.Vries, H.Clevers, Q.-X.Li, A living biobank of matched pairs of patient-derived xenografts and organoids for cancer pharmacology, PLoS ONE 18(1):e0279821, https: / / doi.org / 10.1371 / journal.pone.0279821 - Y. Kieffer, H. R. Hocine, G. Gentric, F. Pelon, C. Bernard, B. Bourachot, S. Lameiras, L. Albergante, C. Bonneau, A. Guyard, K. Tarte, A. Zinovyev, S. Baulande, G. Zalcman, A. Vincent-Salomon, F. Mechta-Grigoriou, Single-Cell Analysis Reveals Fibroblast Clusters Linked to Immunotherapy Resistance in Cancer, Cancer Discov 2020, 10:1330 - 51, doi:10.1158 / 2159-8290.CD-19-1384 - N. Ortiz-Otero, J. R. Marshall, B. Lash, M. R. King, Chemotherapy-induced release of circulating-tumor cells into the bloodstream in collective migration units with cancer-associated fibroblasts in metastatic cancer patients, BMC Cancer(2020)20:873, https: / / doi.org / 10.1186 / s12885-020-07376-1 - J.-W. Seo, K. Fu, S. Correa, M. Eisenstein, E. A. Appel, H. T. Soh, Real-time monitoring of drug pharmacokinetics within tumor tissue in live animals, Sci.Adv. 8, eabk2901(2022), https: / / www.science.org / doi / epdf / 10.1126 / sciadv.abk2901

[0013] Cancer treatment and dosing often follow the "magic bullet" and fractionated, i.e., long-term "maximum tolerated dose" paradigm. However, cancer consists of heterogeneous cell populations that metabolically, transcriptionally, epigenetically, and evolutionarily adapt within hours, days, weeks, and months to immunological, chemical, or radiological stress. To counteract cancer mutability, the present invention - about 360 different small molecule drug conjugates (SMDCs), each comprising one or more FAP-activatable initiator Fc, a self-sacrificing linker L, and a known proven chemotherapeutic agent Ct, - extracellular prodrug cleavage by fibroblast activation protein (FAP), which is overexpressed in solid tumors, metastases, and aggregates with circulating tumor cells (CTCs), - treatment of all tumors, preferably personalized treatment, - simultaneous administration of multiple prodrugs ("combination therapy"), preferably without cross-resistance, - high variability, e.g., selecting 2 prodrugs from 360 results in about 129,000 different combinations, - rapidly changing over time, non-redundant, non-mutagenic, preferably rational combinations of prodrugs, - elimination of heterogeneous cancer cell populations, - avoidance of neoplastic adaptation and resistance, - secondary reduction of cancer-associated fibroblasts (CAF) and tumor microenvironment (TME), enhancing access of the immune system to cancer cells, - reduction to less than 1 / 10 of systemic toxicity, - facilitation of low-risk / moderate clinical trials and approvals due to low toxicity and use of known chemotherapeutic agent Ct, - economical and accessible prodrug kits, - improved PK / PD compared to prior art FAP-prodrugs are included.

[0014] Generally, a small prodrug kit of about 4 of the present invention's CAF / FAP-targeted prodrugs is for combination therapy (i.e., 10 12should be sufficient to achieve the death of more than a few cancer cells and concomitantly restore the immune response.

[0015] Using a novel sensor, Seo et al. measured the pharmacokinetic parameters of the cancer drug doxorubicin in an in vivo tumor model. [Table 1]

[0016] Pisco et al., Seo et al. and a vast number of scientific literature have shown that - cancer cells, like most cells, are resilient and utilize various evolutionary defense mechanisms that allow for a rapid and flexible adaptation to treatment attacks, - intratumoral drug exposure is anisotropic and promotes cancer cell resistance in low-dose regions, - systemic and tumor drug clearance is rapid, requiring rapid and efficient intratumoral drug delivery . [Summary of the Invention]

[0017] Therefore, to overcome refractory cancers, the present invention provides a multi-stage treatment regimen that includes two, three, four, or five or more stages, which - each stage lasts for a period of 48 hours to several weeks, - each stage involves administering, simultaneously once or repeatedly, a set of two, three, four, or five or more different tumor-targeted prodrugs, - the set of prodrugs administered in one stage is different from each set administered in the previous or subsequent stage, .

[0018] The proposed treatment regimen - enables highly tumor-targeted dosing of chemotherapeutic agents with minimal side effects, - exposes cancer cells to a number of different chemotherapeutic agents in a rapidly changing order, - Addressing heterogeneous cancer cell populations, - Countering cancer cell adaptation and resistance, and - Enhancing the potential for complete eradication of cancer stem cells is advantageous in these respects.

[0019] The treatment regimen outlined above is S i =Fc i -L i -Ct i where i = 1, 2, 3, …, 360 and is implemented by the use of a chemotherapeutic drug kit comprising N different off-the-shelf small molecule drug conjugates selected from the group comprising where -2 ≤ N ≤ 360, - each Ct i is a residue of a known chemotherapeutic compound, - when i ≠ j, Ct i ≠ Ct j and - each Fc i is a residue of a fibroblast activation protein (FAP) cleavable moiety, - each L i is a residue of a self-immolative linker, - Fc i and Ct i are covalently bonded to Li.

[0020] In a preferred embodiment of the chemotherapeutic drug kit, each S i is provided in a separate container (e.g., a medical vial or ampoule).

[0021] According to the present invention, each Ct i is a residue of one of the known chemotherapeutic agents shown in Tables 1 and 2 below. Many of the chemotherapeutic agents listed in Tables 1 and 2 have been used in clinical practice for years, and in some cases, decades.

[0022] In the chemical structures shown in Tables 1 and 2, the self-immolative linker L iGroups suitable for covalent bonding are indicated by circles surrounded by dashed lines. Generally, hydroxy (OH-), primary amine (NH2-), or secondary amine (R-NH-R’) groups are suitable for conjugation by replacing hydrogen (H) with a self-sacrificing linker L i is suitable.

[0023]

Table 2-1

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Table 2-87

[0024]

Table 3-1

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Table 3-37

[0025] Most of the chemotherapeutic compounds listed in Tables 1 and 2 can be readily obtained from commercial suppliers or prepared from commercially available compounds through facile derivatization. The same is true for the self-immolative linker L of the present invention i (e.g., https: / / bezwadabiomedical.com / ) and the FAP-cleavable moiety Fc i、 and can be appropriately functionalized and protected for sequential coupling with the hydroxy or amine groups of the chemotherapeutic compounds in Tables 1 and 2. Strategies and schemes for chemical synthesis and coupling via amide or ether linkages are presented in Examples 1-4.

[0026] The present invention offers the following advantages: - Each Ct i is known and has well-characterized pharmaceutical activity, - Each S i is linked to fibroblast activation protein (FAP) mainly expressed by cancer-associated fibroblasts (CAF) in tumor tissue and metastatic lesions and is pharmacologically inactive unless cleaved, - Each S i is pharmacologically compatible with good solubility and stability in serum and long-term systemic retention, - Each S i is suitable for large-scale synthesis and economic production, - The chemotherapeutic drug kit can be manufactured in an efficient and economical manner, - The chemotherapeutic drug kit is versatile and facilitates clinical use.

[0027] The small molecule drug conjugate (or prodrug) S of the present invention iIt contains a portion that is enzymatically cleaved by fibroblast activation protein (FAP). FAP is expressed almost exclusively in somatic healing wounds and in the microenvironment (or stroma) of cancer tumors. Many cancer tumors contain a tumor microenvironment (stroma) that surrounds cancer cells (neoplastic cells). The tumor stroma contains various non-malignant cell types and accounts for up to 90% of the total tumor mass. The tumor stroma plays important roles in the supply of cancer cells and in tumor progression and metastasis. Important components of the tumor stroma are the extracellular matrix (ECM), endothelial cells, pericytes, macrophages, immunomodulatory cells, and activated fibroblasts (commonly called cancer-associated fibroblasts (CAFs)). During tumor progression, CAFs change in morphology and biological function. These changes are induced by cell-cell communication between cancer cells and CAFs. CAFs create an environment that promotes the growth of cancer cells. Treatment methods that simply target cancer cells have been shown to be insufficient. Effective therapies must also address the tumor microenvironment, particularly CAFs.

[0028] In over 90% of human epithelial tumors, CAFs overexpress fibroblast activation protein (FAP). Therefore, FAP is a promising target for cancer drug delivery. The role of FAP in vivo is not fully understood, but it is known to be a serine protease with unique enzymatic activity. FAP exhibits the activities of both dipeptidyl peptidase (DPP) and prolyl oligopeptidase (PREP). Therefore, substrates and inhibitors of DPP, PREP, and FAP are considered as homing ligands for CAF targeting. Suitable FAP ligands must have higher selectivity than related enzymes, such as DPPII, DPPIV, DPP8, DPP9, which are dipeptidyl peptidases ubiquitous in healthy tissues, and the homologous prolyl oligopeptidase.

[0029] Small molecule ligands with high affinity and selectivity for FAP have been known since 2014 and 2019, respectively (see K. Jansen, L. Heirbaut, R. Verkerk, J. D. Cheng, J. Joossens, P. Cos, L. Maes, A.-M. Lambeir, I. De Meester, K. Augustyns, P. Van der Veken, Extended Structure-Activity Relationship and Pharmacokinetic Investigation of (4-Quinolinoyl)glycyl-2-cyanopyrrolidine Inhibitors of Fibroblast Activation Protein (FAP), J. Med. Chem., April 10, 2014, 57(7):3053-74, DOI 10.1021 / jm500031w, A. De Decker, G. Vliegen, D. Van Rompaey, A. Peeraer, A. Bracke, L. Verckist, K. Jansen, R. Geiss-Friedlander, K. Augustyns, H. De Winter, I. De Meester, A.-M. Lambeir, P. Van der Veken, Novel Small Molecule-Derived, Highly Selective Substrates for Fibroblast Activation Protein (FAP), ACS Med. Chem. Lett., 2019, 10, 8, 1173-1179). These ligands contain a modified glycine-proline unit and a quinoline group attached thereto.

[0030] Regarding circulating tumor cells (CTCs), Raskov et al. stated that, "For example, CTCs have a higher survival rate in the bloodstream when accompanied by stromal cells, which also provides advantages regarding the initial survival and growth of tumor cells at the metastatic site (31). When moving within clusters with macrophages, immune cells, and platelets, CAFs support, protect, and increase the survival of CTCs." (H. Raskov, A. Orhan, S. Gaggar, I. Gogenur, Cancer-Associated Fibroblasts and Tumor-Associated Macrophages in Cancer and Cancer Immunotherapy, Frontiers in Oncology, May 2021, Vol. 11, Article 668731, page 5, left column, lines 24 - 29). Therefore, the prodrug of the present invention may also be activated by circulating CAFs and, as a result, affect CTCs.

[0031] Regarding drugs that specifically target CAFs, Raskov et al. (page 12, left column, first paragraph) stated that, "α-SMA" + or FAP + They further stated that "the regulation / eradication of CAFs has various consequences, and currently, targeting CAFs or TAMs individually does not seem to be an appropriate approach."

[0032] However, the present invention utilizes FAP merely as a means for activating chemotherapeutic drugs and does not intend to regulate or eradicate CAFs. Therefore, the prodrug of the present invention contains a chemotherapeutic compound targeting cancer cells. At the same time, CAFs constitute bystanders that can be secondarily affected, particularly by cytotoxic agents. In many cases, the secondary damage to CAFs can promote the antitumor effect of the prodrug of the present invention.

[0033] The chemotherapeutic kit of the present invention easily provides a very large number of possibilities for the selection and co - administration of two or more prodrugs. In particular, in the case of cancer recurrence, clearly different treatment regimens can be pursued flexibly and adaptively.

[0034] In a preferred adaptation mode, the treatment of the present invention involves frequent quantitative diagnostics, such as liquid biopsies and ultrasound-based evaluations of tumor size, vascular system, and perfusion. If the selected combination of prodrugs of the present invention does not result in a quantitative improvement within 2 to 3 weeks, a clearly different combination of prodrugs can be used.

[0035] As embodied above, the present invention - enables the treatment of solid cancer tumors that are easy and cost-effective by exposing a number of different chemotherapeutic agents in a rapidly changing time series, - inhibits cancer growth, - has negligible harmful effects at high tumor target doses, and has the objective of providing a chemotherapeutic drug kit.

[0036] This objective is achieved by S i =Fc i -L i -Ct i where i = 1, 2, 3,..., 360 A chemotherapeutic drug kit comprising N different small molecule drug conjugates selected from the group comprising Here, - 2 ≤ N ≤ 360, - each Fc i independently of one another has the structure

Chemical formula

Chemical formula

[0037] Suitable embodiments of the prodrug compounds of the present invention are characterized by one of the following features, or a combination of features, provided that the features are not mutually exclusive or contradictory, of two or more of the following features: - each Ct iare 1,2,3,4-tetrahydrohydrogen staurosporine, 17-Dmag, 2-aminopropanenitrile, 4SC202, ABBV-CLS-484, abemaciclib, abexinostat, acalabrutinib, acetylbufalin, adelbafib, afatinib, afuresertib, alectinib, alisertib, alpelisib, albosideb, AMD3465, anlotinib, apalutamide, AR-42, asimicinib, atubesiclib, avapritinib, axitinib, AZD7762, BAY1125976, belinostat, β-hydroxy-isovaleric acid, BF211, bicalutamide, binimetinib, bortezomib, bosutinib, brigatinib, bufalin, buparlisib, buthionine sulfoximine, cabozantinib, capivasertib, capmatinib, carfilzomib, CEP-9722, ceralasertib, ceritinib, chidamide, CHR-3996, citrinostat, cobimetinib, CompK, copanlisib, clenolanib, crizotinib, CUDC-101, dabrafenib, daclatrasvir, dacomitinib, darolutamide, dasatinib, dasatinib D1, dasatinib D2, dasatinib D3, dasatinib D4, decitabine, defactinib, degarelix, diethylstilbestrol, dinaciclib, Dp44mT, DpC, DUPA, duvelisib, E7016, ebvaciclib, eganelisib, elimusertib, emavusertib, enasidenib, encorafenib, enitociclib, entinostat, enzastaurin, enzaltamide, epacadostat, epigallocatechin gallate, epoxomicin, erdafitinib, erythromycin dibromide, erlotinib, everolimus, fasudil, fedratinib, filgotinib, foslinanib, fostamatinib, fruquintinib, ganetespib, gedatolisib, gefitinib, GFH018, gilteritinib, gibinostat, glasdegib, goserelin, GSK2256098, GSK269962A, GSK690693, GUL, halofuginone, himechromone, ibrutinib, icotinib, idelalisib, imatinib, imiquimod, infliximab, iniparib,Ipatasertib, Itacitinib, Ivaltinostat, Ivosidenib, Ixazomib, Kebetulin, Lapatinib, Larotrectinib, Lenalidomide, Renolisib, Lenvatinib, Leuprolide, Linifanib, Lonafarnib, Lorlatinib, Losartan, Lucitanib, Luminespib, M1096, Marizomib, ME-344, Melesitnib, Metformin, MG132, Midostaurin, Miransertib, Mivobotinib, MK2206, MMP-9 Inhibitor I, Mobocertinib, Mocetinostat, Motesanib, MRX1133, Navitoclax, Nazartinib, Nedisertib, Neratinib, Nilotinib, Nilutamide, Nintedanib, Niraparib, NMS-P118, NMS-P515, NSC668394, NSC95397, Numidalidostat, NVP-2, Olaparib, Olmutinib, Omipalisib, Oprozomib, Osimertinib, OTS-964, Palbociclib, Pamiparib, Panobinostat, Paricalcitol, Parsaclisib, Pazopanib, Pemetrexed, Pemigatinib, Pevonedistat, Pecidartinib, Pifusertib, Prelixafol, PMPA, Ponatinib, Practinostat, Pralsetinib, Prednisone, Prexasertib, Prinomastat, Propranolol, Xenostatin, Xidazitinib, Larimetinib, Laboxertinib, Regorafenib, Relugolix, Resminostat, Resveratrol, Retaspimycin, Retinoic Acid, Ribociclib, Ricolinostat, Rigosatib, Ripretinib, RO-3306, Rocilinostat, Rogaratinib, Romidepsin, Rubraca, Luxolitinib, S2, S5, Salinomycin, SBI-0654454, SCH772984, Seribasertib, Seritrectinib, Selpercatinib, Selmetinib, SGN-2FF, SGX393, Siconin, Silibinin, Citravatinib, Sonidegib, Sorafenib, Sotorasib, Staurosporine, SU11274, Sunitinib, Sulfatinib, Tacedinaline, Tadalafil, Talazoparib, Talotreclinib, Tarloxotinib, Tacesilib, Tazemetostat, Tefinostat,Selected from the group consisting of deprotonated residues of temsirolimus, tetrazole, cibozanib, tofacitinib, tozasertib, trametinib, tranilast, tretinoin, trichostatin, tsukatinib, tsubesertib, ubenimex, umbralisib, uprosertib, USL311, bactesertib, valproic acid, valsartan, vandetanib, veriparib, bemrafenib, venetoclax, verteporfin, besimodegib, vorinostat, WRG-28, WZ811, xebinaropant, zanubrutinib, zanubrutinib, ZM447439, - each Ct iis selected from the group consisting of abiraterone, aclarubicin, adozelesin, alrestatin, amanitin, amrubicin, anthramycin, arenastatin, bizelesin, bleomycin, camptothecin, capecitabine, carzelesin, CC-1065, chalconein, chlorambucil, cryptophycin-24, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, DAVLBH, dextecan, dexamethasone, dichloroacetic acid, dimethyl-SGD-1882, docetaxel, dirstatin-10, doxorubicin, duocarmycin A, duocarmycin B1, duocarmycin B2, duocarmycin C1, duocarmycin C2, duocarmycin D, duocarmycin GA, duocarmycin SA, emetine, epirubicin, eribulin, etoposide, floxuridine, fludarabine, fluorouracil, flutamide, fulvestrant, gemcitabine, idarubicin, ifosfamide, irinotecan, L-asparaginase, romustatin, melphalan, mertansine, methotrexate, milataxel, mitoxantrone, monomethyl auristatin E, maytansine, maytansinoid, ozogamicin, paclitaxel, pirarubicin, pixantrone, podophyllotoxin, procarbazine, rapamycin, rachelmycin, salinomycin, SB-T-1214, selinexor, SN-38, solamargine, solanine, talirine, temozolomide, tesetaxel, SG3199 (tesirine), thapsigargin, tomatine, topotecan, tubulysin B, valrubicin, vinblastine, vincristine, vinorelbine, VIP126, and the deprotonated residue of zolbicin, -Z and R 1 is [Chemical formula] forms a moiety having a structure selected from the group consisting of -each Fci comprises, independently of one another, a moiety

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[0038] P 1 contains an amine group covalently bonded to Fc, and P i is 1 selected from the group consisting of

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Chemical formula

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[0039] P 1 is

Chemical formula

[0040] Fc i and P 1 are,

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[0041] The second embodiment of the present invention has the structure S=Fc-L-Ct and is a prodrug having wherein -Fc has the structure

Chemical formula

Chemical formula

[0042] Suitable embodiments of the prodrug S=Fc-L-Ct of the present invention are characterized by one of the following features, or a combination of features as long as the combined features are not mutually exclusive or contradictory, according to which, -Ct is a radical of dinaciclib, -Ct is a radical of NVP-2, -Ct is a radical of erlotinib, -Ct is a radical of imatinib, -Ct is a radical of sorafenib, -Ct is a radical of bufalin, -Ct is a radical of acetylbufalin, -Z and R 1 are

Chemical formula

Chemical formula

Chemical formula

Chemical formula

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Chemical formula

[0043] P 1 contains an amine group covalently bonded to Fc, and P 1 is

Chemical formula

Chemical formula

Chemical formula

[0044] P 1 is

Chemical formula

[0045] Fc and P 1 are [Chemical formula] form a moiety having a structure selected from the group consisting of moiety P j (where 2 ≦ j ≦ h and 2 ≦ h ≦ 10) are, independently of one another, [Chemical formula] selected from the group consisting of P where h < j ≦ 10 j does not exist, - linker L has the structure [Chemical formula] having, wherein the amine group is covalently bonded to Fc, r = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and moiety Q j (where 1 ≦ j ≦ h and 1 ≦ h ≦ 10) are, independently of one another, [Chemical formula] selected from the group consisting of Q where h < j ≦ 10 j does not exist, - linker L has the structure [Chemical formula] having, where p = 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 - linker L has the structure [Chemical formula] having, where p = 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 - linker L has the structure [Chemical formula] has, where p = 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 - Linker L has the structure

Chemical formula

Chemical formula

Chemical formula

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Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0046] The present invention further proposes a multivalent prodrug having two or more FAP-activatable initiators. The multivalent prodrug shows increased uptake of each parent drug by the tumor and increased release of the parent drug. Compared to a monovalent prodrug having one FAP-activatable initiator, the multivalent prodrug has a higher docking probability and activation probability, or in physical terms, a larger effective cross-sectional area. The improved tumor uptake and parent drug release result in a decrease in the administered dose and further alleviation of harmful side effects.

[0047] In the prodrug or SMDC of the present invention, each of one or more FAP-activatable initiator moieties or trigger moieties is covalently bonded to a linear or branched self-sacrificing linker, and the self-sacrificing linker is also covalently bonded to a radical or residue of a chemotherapeutic compound (parent drug). The prodrug or SMDC of the present invention is configured for extracellular activation by FAP that is overexpressed in various solid tumors. By FAP-catalyzed cleavage of any one of the initiator or trigger moieties from the linear or branched self-sacrificing linker, the self-sacrificing linker dissociates from the chemotherapeutic compound radical, followed by protonation of the chemotherapeutic compound radical. Thus, the chemotherapeutic compound (parent drug) is released into the extracellular compartment of the tumor.

[0048] Accordingly, the present invention also provides a small molecule drug conjugate (SMDC) comprising a chemotherapeutic compound radical Ct, a linear or branched self-sacrificing linker L, and one, two, three, or four or more initiators (F1, F2, F3, F4), - L is covalently bonded to a nitrogen, amine, or oxygen radical of Ct, - L contains one, two, three, or four or more amine radicals, - Each of the initiators (F1, F2, F3, F4) is covalently bonded to an amine radical of L, - Each of the initiators (F1, F2, F3, F4) is configured for enzymatic cleavage from L by fibroblast activation protein (FAP), -L is configured to release Ct upon cleavage of any one of the initiators (F1, F2, F3, F4), -The initiators (F1, F2, F3, F4) are, independently of one another,

Chemical formula

Chemical formula

Chemical formula

[0049] Suitable embodiments of the small molecule drug conjugate (SMDC) or prodrug of the present invention are characterized by one of the following features, or a combination of features as long as the combined features are not mutually exclusive or contradictory, and according to which, - Ct is a radical of dinaciclib, - Ct is a radical of NVP-2, - Ct is a radical of erlotinib, - Ct is a radical of imatinib, - Ct is a radical of sorafenib, - Ct is a radical of bufalin, - Ct is a radical of acetylbufalin, - The initiators (F1, F2, F3, F4) are, independently of each other,

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[0050] In a preferred embodiment, the SMDC or prodrug of the present invention comprises at least a part of one or more FAP activatable initiators and at least a part of a self-sacrificing linker, and has a type [Chemical formula] [Chemical formula] having one or more parts of the structure of, wherein X = -H or -CH3, Y = -H or -F, and R 1 is H or a pharmacokinetic modulating moiety, and the dotted line indicates the enzyme cleavage site.

[0051] A preferred embodiment of the SMDC or prodrug of the present invention, independently of each other, has a general structure [Chemical formula] having one or more parts of, wherein Fc represents one of the FAP cleavable moieties described throughout the preceding text, and G 1 = -O- or -NH-, G 2 = -CH- or -C(CH3)-, preferably G 2 = -CH-, and G 3 is a structure selected from the group of structures including the moiety [Chemical formula] ​ Preferably, G 3 =-CH2-, or -CH(CH3)-, or -O-, or -CH=CH-, and in particular, G 3 =-CH2- or -CH(CH3)-.

[0052] Preferred embodiments of the SMDC or prodrug of the present invention, independently of each other,

Chemical formula

[0053] Moieties of the type shown above are cleaved very effectively by FAP (k cat / K M >10 6 s -1 ·M -1 ) and have excellent selectivity for FAP as opposed to prolyl oligopeptidase (PREP). PREP, unlike FAP, is expressed throughout healthy tissue. Higher selectivity for FAP over PREP limits off-target prodrug activation and attendant systemic toxicity in healthy tissue.

[0054] To enhance the stereochemical probability and efficiency of intratumoral FAP activation and prodrug delivery, the present invention further proposes a prodrug comprising two, three, or four or more FAP-activatable initiators.

[0055] A variety of heterobifunctional linkers are commercially available as either off-the-shelf compounds, crosslinking kits, or services (e.g., from https: / / www.carbolution.de / , https: / / bezwadabiomedical.com / , https: / / broadpharm.com, https: / / p3bio.com / amino-acids / fmoc-amino-acids / , https: / / www.thermofisher.com, https: / / www.profacgen.com). Some vendors offer comprehensive libraries of Fmoc-protected amino acids and tBu-protected amino acids. The Crosslinking Technical Handbook, ThermoFisher Scientific (2022; https: / / assets.thermo-fisher.com / TFS-Assets / BID / Handbooks / bioconjugation-technical-handbook.pdf) describes numerous linker chemistries and bioconjugation strategies. Commercially available linker compounds offer virtually unlimited possibilities for minor modifications of self-immolative linkers without affecting the important pharmacological properties of the prodrugs of the present invention to a clinically significant extent. Therefore, it should be noted that a partially general description of the self-immolative linker structure in the present invention does not reduce the technical feasibility and efficacy of the prodrugs of the present invention.

[0056] Paragraphs 401-431 (Examples 1 and 2) of US Patent Application Publication No. 2017 / 0119901 (A1) describe the synthesis of FAP-activatable prodrugs. The synthesis schematic of US Patent Application Publication No. 2017 / 0119901 (A1), which is incorporated herein by reference, enables those skilled in the art to prepare a variety of FAP-activatable prodrugs of the Fc-L-Ct type in a similar manner.

[0057] The papers cited below further disclose various self-immolative linkers and methods for their preparation: -A. Alouane, R. Labruere, T. Le Saux, F. Schmidt, L. Jullien, Self-Immolative Spacers: Kinetic Aspects, Structure-Property Relationships, and Applications, Angew. Chem. Int. Ed. 2015, 54, 7492-7509, doi:10.1002 / anie.201500088 -A.G. Gavriel, M.R. Sambrook, A.T. Russell, W. Hayes, Recent advances in self-immolative linkers and their applications in polymeric reporting systems, Polym. Chem., 2022, 13, 3188, doi:10.1039 / d2py00414c; -D. Xiao, L. Zhao, F. Xie, S. Fan, L. Liu, W. Li, R. Cao, S. Li, W. Zhong, X. Zhou, A bifunctional molecule-based strategy for the development of theranostic antibody-drug conjugate, Theranostics 2021, 11(6):2550-2563, doi:10.7150 / thno.51232。

[0058] The technical disclosures of the above papers by Alouane et al., Gavriel et al. and Xiao et al. are incorporated herein by reference into this patent application.

[0059] In the present invention, the terms "small molecule drug conjugate", "SMDC" and "prodrug" are synonymous and refer to a chemical compound comprising one or more initiator or trigger moieties activatable by fibroblast activation protein (FAP), a linear or branched self-immolative linker moiety, and a radical or residue of a chemotherapeutic compound, wherein the self-immolative linker is disposed between the radical or residue of the chemotherapeutic compound and each of the one or more initiator or trigger moieties.

[0060] The term "activatable by fibroblast activation protein (FAP)" is a paraphrase of the catalytic (i.e., rapid and highly efficient) cleavage of the initiator or trigger moiety from the self-immolative linker. The cleavage efficiency is typically represented as the ratio k cat to the Michaelis-Menten constant K M as k cat / K M in units of [s -1 ·M -1 (see https: / / en.wikipedia.org / wiki / Michaelis-Menten_kinetics). K cat and K M are readily determined by generally known enzyme assay techniques.

[0061] In the present invention, the term "residue of..." or "radical of..." refers to a chemical compound having at least one unpaired valence electron (see https: / / en.wikipedia.org / wiki / Radical_(chemistry)).

[0062] The term "residue of a chemotherapeutic compound" or "radical of a chemotherapeutic compound" refers to a chemotherapeutic compound having few positively charged hydrogen ions. The term "protonation" refers to the addition of a positively charged hydrogen ion to the radical or residue of a chemotherapeutic compound.

[0063] The entire contents of all prior art documents cited in this patent application are incorporated by reference. In particular, the chemical synthesis methods described in the cited prior art documents are used, directly or with appropriate adaptation of similar methods, to prepare the prodrugs of the present invention.

[0064] Example Example 1: Synthesis of a conjugate of a FAP-cleavable moiety and a self-immolative linker The general synthetic route outlined below in Scheme 1 is A. De Decker, G. Vliegen, D. Van Rompaey, A. Peeraer, A. Bracke, L. Verckist, K. Jansen, R. Geiss-Friedlander, K. Augustyns, H. De Winter, I. De Meester, A.-M. Lambeir, P. Van der Veken, Novel Small Molecule-Derived, Highly Selective Substrates for Fibroblast Activation Protein (FAP), ACS Med. Chem. Lett. 2019, 10, 8, 1173-1179) based on.

Chemical formula

[0065] Scheme 1: Synthesis of a conjugate of a FAP-cleavable moiety and a self-immolative linker

[0066] The commercially available compound 1 (i.e., Boc-L-proline or Boc-4,4-difluoro-L-proline) is coupled with NH2-LP, which is a protected self-immolative linker precursor (see also Example 2). The resulting compound 2 is deprotected to give intermediate 3. Intermediate 3 is coupled with Boc-protected D-alanine or glycine, thereby yielding the protected conjugate 4. Acidolytic deprotection of compound 4 gives intermediate 5, from which conjugate 6 is synthesized by acylating the free amine group with quinoline-4-carboxylic acid.

[0067] Reagents and conditions: (a) 1-Chloro-N,N,2-trimethyl-1-propenylamine, TEA, DCM:THF (1:1), rt, (b) HCl or TFA, DCM, rt, (c) Boc-Xaa, T3P, DIPEA, DCM, rt, (d) TFA, DCM, rt, (e) Quinoline-4-carboxylic acid, T3P, DIPEA, DCM.

[0068] As shown in Scheme 1, the self-sacrificing linker precursor NH2-LP can be readily prepared from commercially available 6-amino-2-oxochromene-3-carboxylic acid (CAS No. 91587-88-1) by reduction with LiAlH4.

[0069] Example 2: Synthesis of a coumarin-based self-sacrificing linker and conjugation with an FAP-cleavable moiety The synthesis outlined below in Scheme 2 is based on R. Weinstain, E. Segal, R. Satchi-Fainarob, D. Shabat, Real-time monitoring of drug release, Chem. Commun., 2010, 46, 553-555, and N.C. Lim, J.V. Schuster, M.C. Porto, M.A. Tanudra, L. Yao, H.C. Freake, C. Bruckner, Coumarin-Based Chemosensors for Zinc(II): Toward the Determination of the Design Algorithm for CHEF-Type and Ratiometric Probes, Inorganic Chemistry, 2005, Vol. 44, No. 6, 2018-2030. and is based on

Chemical formula

[0070] Scheme 2: Synthesis of Coumarin-based Self-Sacrificing Linker and Conjugation with FAP-Cleavable Moiety

[0071] (i) Dissolve 2,4-dihydroxybenzaldehyde 7 (0.74 g, 5.36 mmol) in EtOH (15 mL). After adding diethyl glutaconate (1.0 mL, 5.65 mmol), add 3 drops of piperidine (dried over KOH pellets). Reflux the resulting solution for 24 h. Slowly cool the reaction mixture to room temperature and then to -20 °C. Filter off the formed yellow crystals and dry to obtain ethyl 3-(7-hydroxy-2-oxo-2H-chromen-3-yl)acrylate 8 (1.24 g, 89% yield).

[0072] (ii) Dissolve ethyl acrylate 8 (0.200 g, 0.77 mmol) in dry pyridine (4 mL) and add acetic anhydride (4 mL). Stir the reaction mixture at ambient temperature for 0.5 h, then pour it onto ice and stir for an additional 10 min. Filter the resulting white precipitate and dry to obtain ethyl 3-(7-acetoxy-2-oxo-2H-chromen-3-yl)acrylate 9 (0.210 g, 90%).

[0073] (iii) Dissolve ethyl acrylate 9 (2.20 g, 7.28 mmol) in THF (200 mL). Add OsO4 (2 mL of 4% w / w aqueous solution) to the mixture and stir for 0.5 h. Add NaIO4 (3.42 g, 16 mmol) and stir the suspension at ambient temperature. When the starting material is consumed (after about 5 d), dry the solution by rotary evaporation. Partition the resulting solid between water and CH2Cl2. Collect the organic layer and dry it by rotary evaporation. Isolate the intermediate 3-formyl-2-oxo-2H-chromen-7-yl acetate 10 as a white solid (1.40 g, 83%) using column chromatography (silica, solvent gradient from CH2Cl2 to CH2Cl2 / 5% CH3CN).

[0074] (iv) Dissolve ester 10 (620 mg, 2.66 mmol) in 32% aqueous NH4OH, and slowly add acetonitrile (MeCN) until the reaction mixture becomes homogeneous. Monitor the reaction to completion by TLC (EtOAc:Hex 1:1) (20 min). Add EtOAc and wash the solution twice with HCl [1 M]. Dry the organic phase over MgSO4, then filter and remove the solvent under reduced pressure to obtain compound 11 (456 mg, 90%).

[0075] (v) Conjugate compound 11 with the FAP-cleavable moiety Fc i by means of a general amide (peptide) bond formation to obtain compound 12.

[0076] (vi) Dissolve conjugate 12 (100 mg, 0.53 mmol, 1 equiv) in MeOH (4 mL), and add sodium borohydride (30 mg, 0.79 mmol, 1.5 equiv). Monitor the reaction to completion by TLC (EtOAc:Hex 1:1) (10 min). Dilute the reaction mixture with EtOAc, wash once with saturated NH4Cl solution, dry over MgSO4, then filter and remove the solvent under reduced pressure. Purify the crude product by column chromatography on silica gel (EtOAc:hexane 1:1) to obtain compound 13 (yield 70 - 86%).

[0077] Example 3: Synthesis of 7-amino-3-(1-hydroxyethyl)-2H-chromen-2-one The synthetic route illustrated below in Scheme 3 is based on D. Xiao, L. Zhao, F. Xie, S. Fan, L. Liu, W. Li, R. Cao, S. Li, W. Zhong, X. Zhou, A bifunctional molecule-based strategy for the development of theranostic antibody-drug conjugate, Theranostics 2021, 11(6):2550 - 2563, doi:10.7150 / thno.51232 as described.

Chemical Structure

[0078] Scheme 3: Synthesis of 7-amino-3-(1-hydroxyethyl)-2H-chromen-2-one

[0079] Synthesis of 3-acetyl-7-nitro-2H-chromen-2-one (1): To a stirred mixture of 2-hydroxy-4-nitrobenzaldehyde (5.00 g, 30 mmol) and ethyl acetoacetate (4.6 mL, 36 mmol) is added 349 μL of piperidine. After refluxing for 1.5 h, the yellowish solid precipitate is filtered off and subsequently washed with ethanol to give Intermediate 1 (4.00 g, yield 57.1%).

[0080] Synthesis of 3-(1-hydroxyethyl)-7-nitro-2H-chromen-2-one (2): To a solution of Intermediate 1 (2.40 g, 10.30 mmol) in methanol and tetrahydrofuran (1:1, total 200 mL) are added sodium borohydride (390 mg, 10.30 mmol) and cerium chloride (2.54 g, 10.3 mmol) at 0 °C. After completion of the reaction within 1.5 h, the solvent is concentrated in vacuo and the crude product is purified by column chromatography (1:1.5 EtOAc / hexane) to give Intermediate 2 as a yellow solid (1.80 g, yield 74.3%).

[0081] Synthesis of 7-amino-3-(1-hydroxyethyl)-2H-chromen-2-one (3): Intermediate 2 (500 mg, 2.13 mmol), iron(III) chloride hexahydrate (115 mg, 0.45 mmol), hydrazine hydrate (1.50 g, 25.6 mmol) and activated carbon (305 mg, 25.6 mmol) are mixed in anhydrous ethanol (30 mL) and refluxed for 2 h. The solution is filtered and the filtrate is concentrated in vacuo. The crude product is purified by column chromatography (1:1 EtOAc / hexane) to give 3 as a white solid (300 mg, yield 68.8%).

[0082] Example 4: Ether Bond Formation between Different Alcohols The synthetic strategy outlined below in Scheme 4 follows P.K. Sahoo, S.S. Gawali, C. Gunanathan, Iron-Catalyzed Selective Etherification and Trans-etherification Reactions Using Alcohols, ACS Omega 2018, 3, 124-136 that of

Chem.

[0083] Scheme 4: Iron(III)-Catalyzed Etherification of Two Different Alcohols

[0084] A secondary alcohol (0.5 mmol), a primary alcohol (0.5 mmol), Fe(OTf)3 (0.025 mmol, 5 mol %), and NH4Cl (0.025 mmol, 5 mol %) are heated in DCM (2 mL) at 45 °C for 1 - 24 h. Fe(NO3)3·9H2O (0.025 mmol, 5 mol %) is used as the catalyst. The reaction is carried out at 70 °C. The product is isolated by column chromatography purification, and the typical yield is 40 - 93%.

[0085] Example 5: Conjugation of Alcohols and Amines by N-O Bond Formation The reaction strategy outlined below in Schemes 5a - 5e is based on J. Hill, A.A. Hettikankanamalage, D. Crich, Diversity-Oriented Synthesis of N,N,O-Trisubstituted Hydroxylamines from Alcohols and Amines by N-O Bond Formation, J. Am. Chem. Soc., 2020, 142, 14820-14825 that of

Chem.

[0086] Scheme 5a: Synthesis of 2-Hydroperoxytetrahydro-2H-pyran

[0087] H2SO4 (18.4 M, 0.05 mL, 0.92 mmol, 0.01 eq) is added to a stirred solution of H2O2 (50% v / v) (3.8 mL, 58.8 mmol, 2 eq) at 0 °C. The solution is stirred for 10 minutes, then 3,4-Dihydro-pyran (2.68 mL, 29.4 mmol, 1 eq) is added dropwise at 0 °C and the solution is stirred for 1 hour. Then, the reaction mixture is diluted with Et2O (15 mL) and quenched by the addition of saturated NH4Cl (30 mL) solution. The resulting biphasic mixture is transferred to a separatory funnel and the layers are separated. The aqueous layer is extracted with ethyl acetate (5 × 40 mL), the organic layers are combined, dried over Na2SO4, filtered, and concentrated in vacuo. The resulting residue is purified by silica flash column chromatography (eluent: 5:95 EtOAc:hexane) to afford the compound 2-Hydroperoxytetrahydro-2H-pyran as a colorless oil (2.04 g, 17.3 mmol, 59%).

Chemical Structure

[0088] Scheme 5b: Synthesis of 2-Hydroperoxy-2-methyltetrahydro-2H-pyran (MTHP)

[0089] A 3.0 M solution of CH3MgCl in THF (20 mmol, 6.67 mL, 1.0 eq) is added dropwise over 10 minutes under an argon atmosphere to a solution of δ-Valerolactone (20 mmol, 1.86 mL, 1.0 eq) in 40 mL of anhydrous THF at -40 °C. The reaction is stirred at -40 °C for 1 hour. After consumption of the starting material as indicated by TLC and MS, the reaction mixture is brought to -20 °C and quenched with saturated solution of NH4Cl (40 mL), followed by dilution with DI water (20 mL) at room temperature. The resulting biphasic mixture is separated and the aqueous layer is extracted with EtOAc (5 × 40 mL). The organic layers are combined, dried over Na2SO4, filtered, and concentrated in vacuo. Thereafter, the crude reaction mixture is used without further purification.

[0090] Sulfuric acid (18.4 M, 109 μL, 2.0 mmol, 0.10 equiv) is added to a stirred solution of 2-methyl-tetrahydro-2H-pyran-2-ol (2.3 g, 20 mmol, 1 equiv) in 100 mL of DCM at 0 °C, obtained from the previous step. Aqueous hydrogen peroxide solution (50% w / w) (6.8 mL, 100 mmol, 5.00 equiv) is added dropwise over 5 minutes and the mixture is stirred at 0 °C for an additional 10 minutes. The reaction is warmed to room temperature and stirred for 2 hours. The reaction is quenched with saturated NH4Cl solution (40 mL), the resulting biphasic mixture is separated, and the aqueous layer is extracted with EtOAc (5 × 40 mL). The combined organic layers are dried over Na2SO4, filtered, and concentrated in vacuo. The resulting residue is purified by silica flash column chromatography (eluent: 0:100 DCM ~ 8:92 Et2O:DCM) to afford 2-hydroperoxy-2-methyltetrahydro-2H-pyran as a clear colorless oil (1.72 g, 13.0 mmol, total yield 65%).

Chemical formula

[0091] Scheme 5c: Synthesis of THP and MTHP Monoperoxyacetals of Simple Alcohols

[0092] Anhydrous DCM (0.17 - 0.60 M), alcohol (1.0 equiv), and base (1.5 equiv) are added to an oven-dried flask at 0 °C under an argon atmosphere. The solution is stirred for 10 minutes, then Tf2O (1.2 - 1.5 equiv) is added dropwise. The solution is stirred at 0 °C for 30 - 60 minutes. Then, HCl (10%, 10 mL) is added and the layers are separated. The organic layer is washed with saturated NaHCO3 (1 × 10 mL). The aqueous layer is extracted with EtOAc (3 × 5 mL), the organic layers are combined, washed with saturated NaCl solution (1 × 10 mL), dried over MgSO4, filtered, and concentrated in vacuo. The triflate is extracted by flash silica column chromatography (eluent: EtOAc:hexane) and used in the following step.

[0093] Lithium tert-butoxide or potassium tert-butoxide (1.2 - 1.5 equivalents) is added in one portion to a stirred solution of THP or MTHP (1.0 - 2.0 equivalents) in anhydrous THF (0.2 - 0.5 M) under an argon atmosphere (balloon). The solution is stirred at 0 °C for 10 minutes, and then a portion of the triflate obtained in the previous step (1.0 - 2.0 equivalents) is added dropwise via syringe. The solution is stirred at 0 °C for 1 hour, and then the mixture is allowed to reach room temperature and stirred for an additional 1 - 24 hours. The reaction mixture is quenched with NaHCO3 (20 mL) and diluted with EtOAc (10 mL). The layers are separated, and the aqueous layer is extracted with EtOAc (3 × 5 mL). The combined organic layers are dried over MgSO4 / Na2SO4, filtered, and concentrated in vacuo. The monoperoxyacetal is obtained by silica flash column chromatography (eluent: EtOAc:hexane).

Chemical formula

[0094] Scheme 5d: Synthesis of MTHP Monoperoxyacetal of Complex Alcohol

[0095] Anhydrous DCM (0.13 - 0.50 M), alcohol (1.0 equivalent), and pyridine (2.0 equivalents) are added to an oven-dried flask at 0 °C under an argon atmosphere. The solution is stirred for 10 minutes, and then Tf2O (1.2 - 1.5 equivalents) is added dropwise. The solution is stirred at 0 °C for 30 - 60 minutes, and then diluted with a few drops of MeOH and 10% HCl (1 × 10 mL). The layers are separated, and the organic layer is washed with saturated NaHCO3 (1 × 10 mL). The aqueous layer is extracted with EtOAc (3 × 5 mL), the organic layers are combined, washed with saturated NaCl solution (1 × 10 mL), dried over MgSO4, filtered, and concentrated in vacuo. The triflate is extracted by flash silica column chromatography (eluent: EtOAc:hexane) and used in the following step.

[0096] NaH (60% dispersion in mineral oil, 1.2 - 1.5 equivalents) is added all at once to a stirred solution of MTHP (1.0 equivalent) in anhydrous DMF under an argon atmosphere. The solution is stirred at 0 °C for 10 minutes, and then the triflate (1.3 equivalents) obtained in the previous step is added dropwise via syringe. The solution is stirred for 1 hour. The mixture is allowed to stand at room temperature and stirred for an additional 1 - 16 hours. The reaction mixture is then diluted with EtOAc (10 mL) and quenched with saturated NaHCO3 (10 mL). The layers are separated and the aqueous layer is extracted with EtOAc (3 × 5 mL). The organic layer is dried over Na2SO4 / MgSO4, filtered, and concentrated in vacuo. The monoperoxyacetal is extracted by silica flash column chromatography (eluent: EtOAc:hexane).

Chemical formula

[0097] Scheme 5e: Synthesis of N,N,O-trisubstituted hydroxylamine using an N - O bond

[0098] Amine (0.25 - 11.0 mmol, 2.5 equivalents) and 0.25 - 11.0 mL of anhydrous THF are added to an oven / flame-dried flask at 0 °C under an argon atmosphere. To this solution, EtMgBr (3 M in diethyl ether) (0.2 - 8.7 mmol, 2.0 equivalents) is added dropwise, and the reaction mixture is stirred at 0 °C for 10 - 30 minutes (formation of magnesium amide generates a significant amount of gas, requiring more extensive precautions). Subsequently, the magnesium amide is transferred via syringe to a stirred solution of THP or MTHP monoperoxyacetal (0.10 - 4.36 mmol, 1.0 equivalent) and stirred at 0 °C in an additional 0.25 - 11.0 mL of anhydrous THF (total 0.2 M) under an argon atmosphere. The solution is stirred until the starting materials are consumed as indicated by TLC and MS, after which the mixture is quenched by the addition of ice water and the layers are separated. The aqueous layer is extracted with EtOAc, the combined organics are dried over MgSO4 / Na2SO4, filtered, and concentrated in vacuo. N,N,O-trisubstituted hydroxylamine is obtained by silica or neutral alumina flash column chromatography (eluent: EtOAc:hexane or DCM:EtOAc).

[0099] Example 6: Amide Bond Formation A general example of an amide coupling reaction is shown in Scheme 6.

Chemical Structure

[0100] Scheme 6: Amide Coupling

[0101] Since the set of readily available carboxylic acids and amine derivatives is virtually unlimited, the amide coupling strategy opens up a simple route for the synthesis of novel compounds. Those skilled in the art are aware of numerous reagents and protocols for amide coupling. The most commonly used amide coupling strategy is based on the condensation of carboxylic acids and amines. For this purpose, carboxylic acids are generally activated. Prior to activation, the remaining functional groups are protected. The reaction is carried out in either of two processes, namely, either using the direct conversion of the activated carboxylic acid in one reaction medium (single pot) or using a two-step process involving the isolation of the activated and "trapped" carboxylic acid and its reaction with the amine.

[0102] Here, the carboxylic acid can be reacted with a coupling agent to form a reactive intermediate, which can be reacted with an amine either in isolated form or directly. For carboxylic acid activation, a number of reagents are available, such as acid halides (chloride, fluoride), azides, anhydrides or carbodiimides. In addition, the reactive intermediate formed may be an ester such as a pentafluorophenyl ester or a hydroxysuccinimide ester. Intermediates formed from acyl chlorides or azides are very reactive. However, the harsh reaction conditions and high reactivity are often a barrier for use with sensitive substrates or amino acids. In contrast, amide coupling strategies utilizing carbodiimides such as DCC (dicyclohexylcarbodiimide) or DIC (diisopropylcarbodiimide) open up a wide range of applications. Often, especially in solid-phase synthesis, additives are used to improve reaction efficiency. Ammonium salts are very efficient peptide coupling reagents with short reaction times and minimal racemization. For some additives, such as HOBt, it is not possible to completely prevent racemization. Ammonium reagents are used in equimolar amounts with the carboxylic acid to prevent the overreaction of the free amine of the peptide. Phosphonium salts react with carboxylates, which generally requires 2 equivalents of a base, such as DIEA. An important advantage of phosphonium salts over iminium reagents is that phosphonium does not react with the free amino group of the amine component. This allows coupling in a 1:1 molar ratio of acid to amine and helps prevent intramolecular cyclization of linear peptides and the overuse of expensive amine components.

[0103] A broad summary of reaction strategies and reagents for amide coupling is provided in the following review article -Analysis of Past and Present Synthetic Methodologies on Medicinal Chemistry: Where Have All the New Reactions Gone?, D.G. Brown, J. Bostrom, J. Med. Chem., 2016, 59, 4443 - 4458, -Peptide Coupling Reagents, More than a Letter Soup, A. El-Faham, F. Albericio, Chem. Rev., 2011, 111, 6557 - 6602, -Rethinking amide bond synthesis, V. R. Pattabiraman, J. W. Bode, Nature, Vol. 480, (2011), 22 / 29, -Amide bond formation: beyond the myth of coupling reagents, E. Valeur, M. Bradley, Chem. Soc. Rev., 2009, 38, 606 - 631 can be found in.

[0104] Example 7: Prodrug of the present invention having a single FAP-activatable initiator Schemes 7a - 7m show exemplary prodrugs having one FAP-activatable initiator. The dotted lines indicate enzymatic cleavage by FAP and drug release from the self-sacrificing linker.

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0105] Example 8: Prodrug of the present invention having two FAP-activatable initiators Schemes 8a - 8c show exemplary prodrugs having two FAP-activatable initiators. The dotted lines indicate enzymatic cleavage by FAP and drug release from the self-sacrificing linker.

Chemical formula

Chem.

Claims

1. A prodrug comprising a chemotherapeutic compound radical Ct, a linear or branched self-sacrificing linker L, and one, two, three, or four or more initiators (F1, F2, F3, F4), -L is covalently bonded to the nitrogen, amine, or oxygen radical of Ct. -L contains one, two, three, or four or more amine radicals, - Each of the initiators (F1, F2, F3, F4) is covalently bonded to the amine radical of L. - Each of the initiators (F1, F2, F3, F4) is configured for enzymatic cleavage from L by fibroblast-activating protein (FAP), -L is configured to release Ct when any one of the initiators (F1, F2, F3, F4) is cleaved. - The initiators (F1, F2, F3, F4) act independently of each other. 【Chemistry 1】 It includes or has a structure selected from the group of structures that include, In the formula, the terminal carbonyl is covalently bonded to the amine radical of the self-sacrificing linker L, and X = -H or -CH ３ And Y = -H or -F, and -R １ However, Z is a radical of the first pharmacokinetic modulatory moiety, and Z is a moiety having a structure selected from the group including structures (1), (2), (3), (4), (5), (6), (7), (8), (9), (10), (11), (12), (13), (14), (15), (16), (17), (18), (19), (20), (21), (22), (23), (24), and (25). 【Chemistry 2-1】 【Chemistry 2-2】 -Ct is 1,2,3,4-tetrahydrogen staurosporine, 17-Dmag, 2-aminopropanenitrile, 4SC202, ABBV-CLS-484, abemaciclib, avexinostat, acalabrutinib, acetylbufarin, adelbacib, afatinib, afrecertib, alectinib, alicertib, alpelicib, albocidib, AMD3465, anlotinib, apalutamide, AR-42, asciminib, atsubeciclib, avapritinib, axitinib, AZD7762, BAY1125976, bellinostat, β-H Droxyisovalerate, BF211, bicalutamide, binimetinib, bortezomib, bosutinib, brigatinib, bufarin, buparulisib, butionine sulfoximine, cabozantinib, capivacertib, capmatinib, carfilzomib, CEP-9722, ceraracertib, ceritinib, tucidinostat (Chidamide), CHR-3996, sitalinostat, cobimetinib, CompK, copanlisib, clenolanib, crizotinib, CUDC-101, dabrafenib, daclatasvir, dacomitinib, darolutamide, dasatinib Dasatinib D1, Dasatinib D2, Dasatinib D3, Dasatinib D4, Decitabine, Defactinib, Degarelix, Diethylstilbestrol, Dinacyclib, Dp44mT, DpC, DUPA, Dubellisib, E7016, Ebvaciclib, Eganericib, Elimusertib, Emavasertib, Enasidenib, Encorafenib, Enitociclib, Entinostat, Entrectinib, Enzalutamide, Epacadostat, Epigallocatechin gallate, Epoxo Micin, erdafitinib, erythmodegib, erlotinib, everolimus, fasudil, phedratinib, filgotinib, foslinanib, hostamatinib, fluquintinib, garnicerutib, ganetespib, gedatricib, gefitinib, GFH018, gilteritinib, zibinostat, gladegib, goserelin, GSK2256098, GSK269962A, GSK690693, GUL, halofudinon, himechromon, ibrutinib, icotinib, idelalisib, imatinib, imiquimod, infiglatinib,Iniparib, Ipatasertib, Itacitinib, Ivaltinostat, Ivosidenib, Ixazomib, Kevetrin, Lapatinib, Larotrectinib, Lenalidomide, Reniolisib, Lenvatinib, Leuprolide, Lincitinib, Ronafarnib, Lorlatinib, Losartan, Lusitanib, Luminespib, M1096, Marizomib, ME-344, Merestinib, Metformin, MG132, Midostaurin, Miransertib, Mibabotinib, MK2206, MMP-9 Inhibitor I, Mobosertinib, Mosetinostat, Motesanib MRTX1133, Navitoclax, Nazartinib, Nedisertib, Neratinib, Nilotinib, Niltamide, Nintedanib, Niraparib, NMS-P118, NMS-P515, NSC668394, NSC95397, Numidalzistat, NVP-2, Olaparib, Olmutinib, Omiparisib, Oprozomib, Osimertinib, OTS-964, Palbociclib, Pamiparib, Panobinostat, Palicalcitol, Palsacricib, Pazopanib, Pemetrexed, Pemigatinib, Pebonezistat, Pexidartinib Pifusertib, prelixafor, PMPA, ponatinib, practinostat, pralcetinib, prednisone, prexasertib, prinomast, propranolol, xinostat, quizartinib, larimetinib, laboxertinib, regorafenib, relugolix, resminostat, resveratrol, letaspimycin, retinoic acid, ribociclib, licorinostat, rigosatib, lipretinib, RO-3306, rosilinostat, rogalatinib Bu, Romidepsin, Lucaparib, Ruxolitinib, S2, S5, Salidegib, SBI-0654454, SCH772984, Sericiclib, Seritrectinib, Serpercatinib, Selmethinib, SGN-2FF, SGX393, Shikonin, Silibinin, Citrabatinib, Sonidegib, Sorafenib, Sotrasib, Staurosporine, SU11274, Sunitinib, Sulfatinib, Tasejinarin, Tadalafil, Talazoparib, Tarretrectinib, Tarloxotinib, Taselicib, Tazemettostat, Tefinostat,Temsirolimus, tetrazole, tivozanib, tofacitinib, tozacertib, trametinib, tranilast, tretinoin, trichostatin, tucatinib, tucidinostat, tubucertib, ubenimex, umbralicib, uprosertib, USL311, bactocertib, valproic acid, valsartan, vandetanib, veliparib, vemurafenib, venetoclax, verteporfin, bismodegib, vorinostat, WRG-28, WZ811, xebinapant, zandelicib, zanubrutinib, ZM447439, abiraterone, Akral Bizerecin, Adozelesin, Alrestatin, Amanitin, Amrubicin, Anthramycin, Arenastatin, Bizerecin, Bleomycin, Camptothecin, Capecitabine, Carzerecin, CC-1065, Chaconine, Chlorambucil, Cryptophycin-24, Cyclophosphamide, Cytarabine, Dacarbazine, Dactinomycin, Daunorubicin, DAVLBH, Deruxtecan, Dexamethasone, Dichloroacetic Acid, Dimethyl-SG D-1882, docetaxel, dorastatin-10, doxorubicin, duocalmycin A, duocalmycin B1, duocalmycin B2, duocalmycin C1, duocalmycin C2, duocalmycin D, duocalmycin GA, duocalmycin SA, emetine, epirubicin, eribulin, etoposide, phloxuridine, fludarabine, fluorouracil, flutamide, fulvestrant, gemcitabine, idarubicin, ifosfamide, irinotecan, L-asparaginase, lomustine, melphalan, meltansine Methotrexate, Mirataxel, Mitoxantrone, Monomethyl Auristatin E, Maytansine, Maytansinoid, Ozogamicin, Paclitaxel, Pirarubicin, Pixantrone, Podophyllotoxin, Procarbazine, Rapamycin, Rachelmycin, Salinomycin, SB-T-1214, Selinexol, SN-38, Soramardine, Solanine, Talirine, Temozolomide, Tesetaxel, SG3199 (Tesirin), Tapsigargin, Tomatine, Topotecan,The radical is a chemotherapeutic compound selected from the group including tubulysin B, barrubicin, vinblastine, vincristine, vinorelbine, VIP126, and zolubicin. Prodrug.

2. The prodrug according to claim 1, characterized in that Ct is a radical of acetylbufarin, bufarin, dinaciclib, erlotinib, ibrutinib, imatinib, lenalidomide, NVP-2, osimertinib, palbociclib, or sorafenib.

3. The initiators (F1, F2, F3, F4) act independently of each other. 【Transformation 3】 In the formula, the terminal carbonyl is covalently bonded to the amine radical of the self-sacrificing linker L, Y = -H or -F, and X = -H or -CH ３ That is The prodrug according to claim 1, characterized in that

4. The initiators (F1, F2, F3, F4) act independently of each other. 【Chemistry 4】 In the formula, the terminal carbonyl is covalently bonded to the amine radical of the self-sacrificing linker L. The prodrug according to claim 1, characterized in that

5. The prodrug according to claim 1, characterized in that two, three, or four or more initiators (F1, F2, F3, F4) are different from each other.

6. The prodrug according to claim 1, characterized in that two, three, or four or more initiators (F1, F2, F3, F4) are equal.

7. The prodrug according to claim 1, characterized by comprising one initiator F1.

8. The prodrug according to claim 1, characterized by comprising two initiators (F1, F2).

9. The prodrug according to claim 1, characterized by comprising four initiators (F1, F2, F3, F4).

10. L includes a coupling portion for Ct, and the coupling portion is 【Transformation 5】 The prodrug according to claim 1, characterized by having a structure selected from the group of structures including the above.

11. L includes a coupling portion for Ct, and the coupling portion is structure 【Transformation 6】 It has, In the formula, the terminal carbonyl is covalently bonded to the nitrogen radical of Ct. The prodrug according to claim 1, characterized in that

12. L includes a coupling portion for Ct, and the coupling portion is structure 【Transformation 7】 It has, In the formula, the terminal carbonyl is covalently bonded to the amine radical of Ct. The prodrug according to claim 1, characterized in that

13. L includes a coupling portion for Ct, and the coupling portion is structure 【Transformation 8】 It has, In the formula, the terminal carbonyl is covalently bonded to the oxygen radical of Ct. The prodrug according to claim 1, characterized in that

14. L includes a coupling portion for Ct, and the coupling portion is structure 【Chemistry 9】 It has, In the formula, the terminal carbonyl is covalently bonded to the nitrogen radical of Ct. The prodrug according to claim 1, characterized in that

15. L includes a coupling portion for Ct, and the coupling portion is structure 【Chemistry 10】 It has, In the formula, the terminal carbonyl is covalently bonded to the amine radical of Ct. The prodrug according to claim 1, characterized in that

16. L includes a coupling portion for Ct, and the coupling portion is structure 【Chemistry 11】 It has, In the formula, the terminal carbonyl is covalently bonded to the oxygen radical of Ct. The prodrug according to claim 1, characterized in that

17. L includes a coupling portion for Ct, and the coupling portion is structure 【Chemistry 12】 It has, In the formula, the terminal carbonyl is covalently bonded to the nitrogen radical of Ct. The prodrug according to claim 1, characterized in that

18. L includes a coupling portion for Ct, and the coupling portion is structure 【Chemistry 13】 It has, In the formula, the terminal carbonyl is covalently bonded to the amine radical of Ct. The prodrug according to claim 1, characterized in that

19. L includes a coupling portion for Ct, and the coupling portion is structure 【Chemistry 14】 It has, In the formula, the terminal carbonyl is covalently bonded to the oxygen radical of Ct. The prodrug according to claim 1, characterized in that

20. L includes a coupling portion for Ct, and the coupling portion is structure 【Chemistry 15】 It has, In the formula, the terminal carbonyl is covalently bonded to the nitrogen radical of Ct. The prodrug according to claim 1, characterized in that

21. L includes a coupling portion for Ct, and the coupling portion is structure 【Chemistry 16】 It has, In the formula, the terminal carbonyl is covalently bonded to the amine radical of Ct. The prodrug according to claim 1, characterized in that

22. L includes a coupling portion for Ct, and the coupling portion is structure 【Chemistry 17】 It has, In the formula, the terminal carbonyl is covalently bonded to the oxygen radical of Ct. The prodrug according to claim 1, characterized in that

23. L is structure [Chemistry 18] Includes a portion having, In the formula, P １０ It is covalently bonded to Ct, and partial P ｊ (where 2 ≤ j ≤ h and 2 ≤ h ≤ 10) are independent of each other. 【Chemistry 19】 Having a structure selected from the group of structures including, P such that h < j ≤ 10 ｊ does not exist The prodrug according to claim 1, characterized in that

24. L is structure 【Chemistry 20】 It includes one or more branched parts having, In the formula, the terminal carbonyl is either oriented toward Ct or covalently bonded to Ct. The prodrug according to claim 1, characterized in that

25. L is structure 【Chemistry 21】 It includes one or more branched portions having In the formula, the terminal carbonyl is either oriented toward Ct or covalently bonded to Ct. The prodrug according to claim 1, characterized in that

26. L comprises one, two, three, or four or more coupling portions for initiators (F1, F2, F3, F4), and the one, two, three, or four or more coupling portions are independent of each other and structure 【Chemistry 22】 In the formula, the terminal amine is covalently bonded to the initiator (F1, F2, F3, F4). The prodrug according to claim 1, characterized in that

27. L comprises one, two, three, or four or more coupling portions for initiators (F1, F2, F3, F4), and the one, two, three, or four or more coupling portions are independent of each other and structure 【Chemistry 23】 In the formula, the terminal amines are covalently bonded to the initiators (F1, F2, F3, F4), and B1 is independent of each other. 【Chemistry 24】 Selected from the group of parts that include The prodrug according to claim 26, characterized in that

28. L comprises one, two, three, or four or more coupling portions for initiators (F1, F2, F3, F4), and the one, two, three, or four or more coupling portions are independent of each other. 【Chemistry 25】 Having a structure selected from the group including, In the formula, the terminal amine is covalently bonded to the initiator (F1, F2, F3, F4). The prodrug according to claim 26, characterized in that

29. The prodrug according to claim 26, characterized by comprising one initiator F1.

30. The prodrug according to claim 29, characterized in that L has the structure described in claim 26.

31. L comprises one, two, three, or four or more coupling portions for initiators (F1, F2, F3, F4), and the one, two, three, or four or more coupling portions are independent of each other and structure 【Chemistry 26】 It has, In the formula, the terminal amine is covalently bonded to the initiator (F1, F2, F3, F4). The prodrug according to claim 1, characterized in that

32. L comprises one, two, three, or four or more coupling portions for initiators (F1, F2, F3, F4), and the one, two, three, or four or more coupling portions are independent of each other and structure 【Chemistry 27】 It has, In the formula, the terminal amine is covalently bonded to the initiator (F1, F2, F3, F4). The prodrug according to claim 31, characterized in that

33. L comprises one, two, three, or four or more coupling portions for initiators (F1, F2, F3, F4), and the one, two, three, or four or more coupling portions are independent of each other and structure 【Chemistry 28】 Having a structure selected from the group including, In the formula, the terminal amine is covalently bonded to the initiator (F1, F2, F3, F4), R ２ However, it is the radical of the second pharmacokinetic regulatory part. The prodrug according to claim 1, characterized in that

34. L comprises one, two, three, or four or more coupling portions for initiators (F1, F2, F3, F4), and the one, two, three, or four or more coupling portions are independent of each other and structure 【Chemistry 29】 Having a structure selected from the group including, In the formula, the terminal amine is covalently bonded to the initiator (F1, F2, F3, F4), R ２ However, it is the radical of the second pharmacokinetic regulatory part. The prodrug according to claim 33, characterized in that

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