Peptide-conjugates of sn38 useful for the treatment of cancer

Abstract

Peptide conjugates of SN38, methods of making the peptide conjugates, pharmaceutical compositions comprising the peptide conjugates, and therapeutic indications for the peptide conjugates as anticancer drugs.

Description

[0001] This application claims the benefit of European patent application EP20382854.6, filed on September 28, 2020. Technical Field

[0002] This invention relates to the field of delivery systems for the anticancer drug SN38, methods for its preparation, and its therapeutic indications. Background Technology

[0003] Cancer is a heterogeneous disease characterized by the aggregation of tumor cells that can cause death in animals and humans. It is also one of the leading causes of death from disease in children and adolescents. While substantial progress has been made in treating several types of cancer, particularly childhood cancers, over the past fifty years, progress in treating other types has been limited. The annual incidence of childhood cancers is between 100 and 160 cases per million people. One in 500 children under the age of 15 is at risk each year. This incidence is slightly lower in industrialized countries. Brain and spinal cord tumors account for 25% of all lesions (and 40-50% of all pediatric solid tumors). Despite progress, survival rates for these patients remain low, at approximately 55%. This contrasts sharply with the significant improvements in survival rates seen in recent years in other types of patients, such as those affected by leukemia or extracranial tumors.

[0004] Conventional methods of treating cancer include surgery, chemotherapy, and more recently, immune-responsive therapies involving the administration of antibodies or antibody fragments that can bind to the treatment. However, to date, such treatments have achieved limited success.

[0005] Camptothecin is one of the four major structural classes of plant-derived anticancer compounds. It is a cytotoxic alkaloid composed of a pentacyclic structure containing a pyrrole (3,4β)quinoline moiety, an S-configuration lactone form, and a carboxylate form. Irinotecan is made from naturally occurring camptothecin found in the Chinese ornamental tree *Camptotheca acuminata*. Irinotecan has been used to treat several types of cancer, including glioblastoma multiforme (GBM) in adults and diffuse entropional pontine glioma (DIPG), pediatric glioblastoma (pGBM), neuroblastoma, rhabdomyosarcoma, Ewing sarcoma, and retinoblastoma in children.

[0006] Gliomas are a group of tumors that originate in the glial cells of the brain or spinal cord, accounting for approximately 30% of all brain and central nervous system tumors and 80% of all malignant brain tumors. Treatment for gliomas typically involves a combination of surgery, radiation therapy, and chemotherapy. DIPG primarily affects children, usually those aged 5 to 7 years. Unfortunately, this is the majority of brainstem tumors, accounting for 60-70%, and has the worst prognosis of all tumors. No treatment has been proven effective, and the average survival is 9 months. Radiation and steroid administration are the only methods that have shown remission and academically improved survival rates. To date, there are no effective chemotherapy therapies, and numerous clinical trials have been conducted without favorable results. Pediatric glioblastoma is another group of tumors, accounting for one-third of all hemispheric tumors. The incidence of pediatric glioblastoma peaks between 8 and 12 years of age. Glioblastomas also affect adult patients, occurring in approximately 2-3 cases per 100,000 people.

[0007] Ewing sarcoma is the second leading cause of malignant bone tumors in children and adolescents. The annual incidence is 0.6 cases per million inhabitants. It is rare before the age of 5, with a peak incidence between 10 and 15 years of age, affecting more boys than girls, although this gender relationship varies across age ranges. The most common origin of Ewing sarcoma is in the pelvic (hip) bones, chest wall (such as the ribs or scapula), or mid-leg bones. In the absence of bone lesions, Ewing sarcoma can also present as an extraskeletal lesion. In this variant, the risk of lymphatic spread is high, and treatment is generally similar to that for rhabdomyosarcoma.

[0008] Soft tissue sarcomas are classified into rhabdomyosarcomas and non-rhabdomyosarcomas. Rhabdomyosarcomas account for 50% of all soft tissue sarcomas in children. Rhabdomyosarcoma is the third most common extracranial solid tumor after neuroblastoma and Wilms' tumor. The age peak is bimodal, with the first peak between 2 and 5 years of age, and the second peak during puberty, between 15 and 19 years of age. While sarcomas in adults primarily occur in the extremities, in children they can originate from anywhere in the body, including skeletal muscles and soft tissues. The most severely affected areas in children are the head and neck and the genitourinary tract. Limb involvement occurs in 20% of patients. Overall survival is low unless the tumor is located in a location where it can be completely removed, sometimes requiring amputation or drainage. Survival rates range from 7% to 70%, depending on the location.

[0009] Neuroblastoma is the most common extracranial solid tumor in childhood. Due to its embryonic origin, neuroblastoma can actually exist in any part of the sympathetic nervous system, but the most common location for localized disease is on the adrenal glands (44%). Children aged 1.5–6 years at diagnosis may still be curable with conventional treatment, but their survival chances decrease when they are diagnosed with metastatic disease (stage 4 neuroblastoma). Approximately 50% of newly diagnosed patients already have bone (60%), bone marrow (50%), lymph node (42%), and / or liver (15%) metastases and require intensive chemotherapy, surgery, and radiation therapy, but their survival remains poor and has seen little progress in recent decades.

[0010] Finally, retinoblastoma is the most common cause of eye tumors in children, with a global incidence of 1 in 20,000 live births. Retinoblastomas typically occur within the first two years of life. 30-40% are bilateral, and in these cases, there is always a positive family history. In unilateral cases, 10% have a germline mutation in the Rb gene located on chromosome 13. If detected early, their survival rate is 95%. In some parts of the world, when diagnosed later, the survival rate drops sharply to below 20%. Treatment depends on achieving tumor control. When chemotherapy and brachytherapy fail to control tumor regression, surgical removal is recommended. Even after removal, in some cases the tumor may invade the optic nerve, necessitating additional chemotherapy.

[0011] The efficacy of irinotecan has been shown to be limited by its hepatic activation, which leads to low conversion rates, high inter-patient variability, and dose-limiting gastrointestinal toxicity.

[0012] SN38 is the active metabolite of irinotecan and is formed by the hydrolysis of irinotecan by hepatic carboxylesterases and metabolized via glucuronidation by UGT1A1. SN38 has the following formula:

[0013]

[0014] Since its discovery, SN38 has attracted the attention of scientists worldwide due to its potent anti-tumor activity. SN38 is 1000 times more active than irinotecan and can be used to treat the same types of cancer as irinotecan. In vitro cytotoxicity assays show that SN38 is 2 to 2000 times more potent than irinotecan. Even so, SN38 has significant limitations at the chemical, pharmacological, and toxicological levels. At the chemical level, unlike the readily soluble irinotecan, SN38 is almost insoluble in water and insoluble in most solvents and oils, making administration to patients impractical. Many solvents have been tested, and only dimethyl sulfoxide, formic acid, and... HP and SN38 can be dissolved in 0.5% NaOH at 0.1M, but the alkaline pH of this 0.1M NaOH aqueous solution inactivates SN38. The drug's high lipophilicity and instability in aqueous solution necessitate administration using polar aprotic solvents, causing all the inconvenience. Furthermore, the presence of a terminal lactone ring makes the drug unstable in aqueous solution, leading to non-enzymatic and pH-dependent hydrolysis to form a hydroxycarboxylic acid ring, which has weak inhibitory activity against topoisomerases. Therefore, at neutral or alkaline pH, the equilibrium shifts towards the less active species, while at higher acidic pH, lactone formation is favorable, resulting in greater inhibitory activity. The drug also contains an asymmetric carbon at position 20; the S form is the pharmacologically active configuration.

[0015] Given the unfavorable characteristics of this drug, some studies have led to the formulation of SN38 as a prodrug to address the insolubility issue and thus leverage its potent mechanism of action. Several conjugation strategies have been applied to SN38 to release the drug instead of using a prodrug.

[0016] Some of these conjugation strategies are based on the slow release of SN38 from soluble conjugates. For example, US8299089B2 proposes conjugating poly(ethylene glycol) with multi-arm PEG to improve solubility and release SN38 due to ester hydrolysis. However, these conjugates must be applied at high levels to be effective.

[0017] WO2015 / 051307A1 discloses several conjugates that release SN38 from polyethylene glycol at a slow rate via a β-elimination mechanism to enable low-dose and long-term exposure protocols.

[0018] F. Koizumi et al. disclosed in “Novel SN38-incorporating polymeric micelles, NK012, eradicate vascular endothelial growth factor-secreting bulky tumors”, Cancer Res 2006, Vol. 66(20), pp. 10048-56, that a poly(ethylene glycol)-poly(glutamate) block copolymer chemically conjugated with SN38 from self-assembled nanoparticles (NPs) showed superior performance to irinotecan in preclinical studies.

[0019] Other conjugation strategies include, for example, using peptide conjugations to bypass liver activation, thus reducing gastrointestinal toxicity and inter-patient variability compared to irinotecan.

[0020] Therefore, F. Meyer-Losic et al. proposed in “DTS-108, A novel Peptidic prodrug of SN38: Invivo Efficacy and Toxicokinetic Studies”, Clinical Cancer Research 2008, Vol. 14, No. 7, pp. 2145-2153, that SN38 could be conjugated to a cationic peptide (Vectocell) via an esterase-cleavable linker to deliver significantly higher levels of SN38 than irinotecan without the associated toxicity of irinotecan, resulting in an increased therapeutic window of DTS-108 in preclinical models.

[0021] WO2007 / 113687A2 discloses the preparation of camptothecin-cell-penetrating peptide conjugates, such as the conjugate DPV1047-MIC-SN38 (DPV-1047 is peptide VKRGLKLRHVRPRVTRMDV; MIC is 6-maleimide hexanoic acid residue; SN38 is 7-ethyl-10-hydroxycamptothecin), via linkers and thioether bonds, which have shown significant antitumor activity against the human HCT-116 cell line.

[0022] Finally, WO2018 / 064683A1 discloses the synthesis of the antitumor agent IF7-SN38, which targets tumor angiogenesis. The antitumor agent comprises an annexin-1 binding peptide having the sequence IFLLWQR(IF7) and the anticancer drug (SN38) via a linker of 4-{4-[(N-maleimidemethyl)cyclohexanecarbamate]methyl}cyclohexane-1-carboxylic acid. This compound targets brain tumors and overcomes the blood-brain barrier through passive transcytosis.

[0023] Despite efforts to provide conjugates of SN38 to allow for adequate administration and bioavailability of SN38 for cancer treatment, there remains an unmet medical need to find improved systems for the effective administration of SN38. Summary of the Invention

[0024] The inventors have developed a conjugate of SN38 with a specific set of peptides linked to SN38 via a specific type of linker, maintaining antitumor activity against several cell lines from brain and extracranial cancers, similar to SN38. The novel conjugates of SN38 exhibit high solubility in water, unlike the nearly insoluble SN38, which allows them to be administered to patients. In vitro, the activity of the novel conjugates of SN38 is significantly higher than that of irinotecan. Advantageously, they exhibit good stability in human serum in vitro.

[0025] Peptides that form part of the conjugate are known and previously described in WO2015 / 001015A1. This document discloses that these peptides have the ability to cross the blood-brain barrier (BBB) ​​and to transport objects that they themselves cannot cross the BBB (particularly large objects such as proteins and antibodies). They cross the BBB using an active transport mechanism. B. Oller et al. highlighted the peptide DapKAPETALD in “MiniAp-4: A Venom-Inspired Peptidomimetic for Brain Delivery”, AngewChem Int Ed 2016, Vol. 55, pp. 572-575, and disclosed that it is resistant to proteases and capable of efficiently delivering objects across the blood-brain barrier in human cell-based models and in vivo.

[0026] The inventors have discovered that, in the case of SN38, favorable antitumor activity was obtained by conjugating it with certain peptides disclosed in WO2015 / 001015A1 and with a specific size of linker larger than that disclosed in the aforementioned documents. These conjugates are advantageous compared to other conjugates because they allow for the maintenance of antitumor activity similar to SN38 and are 100 times more potent than irinotecan in vitro.

[0027] The comparative data provided in the experimental section show that when SN38 is conjugated to another peptide (THRre) through the same linker, there is a partial / complete loss of antitumor activity (Example 7 of the present invention and Comparative Example 1), and the results are even worse if a different linker than that of the present invention is used (Comparative Example 2).

[0028] Therefore, the first aspect of the present invention relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0029] (Z)-(L)-P-(W) s -(Y)(I)

[0030] in:

[0031] Z is a radical of the pharmaceutical active ingredient SN38 or a pharmaceutically acceptable salt thereof, wherein the pharmaceutical active ingredient SN38 has formula (II), and wherein Z is independently linked to the connector L via only one of the two hydroxyl groups (a) or (b) of the pharmaceutical active ingredient;

[0032]

[0033] L is a connector, which is a diradical composed of 2 to 8 diradicals L' and has the following formula: -L' a -(L'b ) n -L' c ;

[0034] L a The diradical is selected from the group consisting of: -C(=O)-(CH2) r -C(=O)-;-C(=O)-(CH2) r -NH-;-C(=O)-(CH2) r -S-;-C(=O)-(CH2) r -O-;-C(=O)-NH-(CH2) r -C(=O)-;-C(=O)-NH-(CH2) r -NH-;-C(=O)-NH-(CH2) r -S-;-C(=O)-NH-(CH2) r -O-;-(CH2) r -C(=O)-;-(CH2) r -NH-;-(CH2) r -S-;-(CH2) r -O-;-Si(R1)(R2)-(CH2) r -NH-;-Si(R1)(R2)-(CH2) r -C(=O)-;-Si(R1)(R2)-(CH2) r O-;-Si(R1)(R2)-(CH2) r -S-;-SO2-(CH2) r -NH-;-SO2-(CH2) r -C(=O)-;-SO2-(CH2) r -O-;-SO2-(CH2) r -S-;-P(=O)(OR1)-O-(CH2) r -NH-; -P(=O)(OR1)-O-(CH2) r -C(=O)-;-P(=O)(OR1)-O-(CH2) r -O-;-P(=O)(OR1)-O-(CH2) r -S-;-CH(OH)-(CH2) r -NH-;-CH(OH)-(CH2) r -C(=O)-;-CH(OH)-(CH2) r -O-;-CH(OH)-(CH2) r -S-;

[0035]

[0036]

[0037] L8-L 11 The substitution of any one of them is located at any position in the ring;

[0038]

[0039] L b 'A diradical that can be independently selected from the group consisting of: -NH-(CH2)' r -C(=O)-;-C(=O)-(CH2) r -C(=O)-;-S-(CH2) r -C(=O)-;-O-(CH2) r -C(=O)-;-NH-(CH2) r -;-C(=O)-(CH2) r -;-S-(CH2) r -;-O-(CH2) r -;-NH-CH-((CH2) r NH2)-C(=O)-;-S-CH2-CH(NH2)-C(=O)-;-(CH2) r -C(=O)-;-(CH2) r -O-;-(CH2) r -NH-;-(CH2) r -S-;-C(=O)-(CH2) r -NH-;-C(=O)-(CH2) r -O-; -C(=O)-(CH2) r -S-;-NH-(CH2) r -O-;-NH-(CH2) r -NH-; -NH-(CH2) r -S-; L1; L2; L3; L4; and their combinations;

[0040] L c The diradical is selected from the group consisting of: -NH-(CH2) r -C(=O)-;-NH-CH-((CH2) r -NH2)-C(=O)-;-C(=O)-(CH2) r -C(=O)-;-S-(CH2) r -C(=O)-;-S-CH2-CH(NH2)-C(=O)-;-O-(CH2) r-C(=O)-;-(CH2) r -C(=O)-;L1;L2;L3;L4;

[0041]

[0042]

[0043] P is a diradical of a peptide selected from the group consisting of: (a) a peptide containing the amino acid sequence X1KAPETALX2, with an intrapeptide bond between X1 and X2, wherein the intrapeptide bond is an amide bond; wherein X1 is selected from the group consisting of Dap (2,3-diaminopropionic acid) and Dab (2,4-diaminobutyric acid); and X2 is selected from the group consisting of D (aspartic acid) and E (glutamic acid); i.e.

[0044]

[0045] For the amino acid Dap, this article uses the codes Dap and Dpr equivalently;

[0046] (b) A peptide of 12-20 amino acid residues having at least one intrapeptide bond, said intrapeptide bond being a disulfide bond or a diselenole bond, and comprising the amino acid sequence X3KAPETALX4AAA; having at least one intrapeptide disulfide bond or diselenole bond between X3 and X4, wherein X3 and X4 are equivalent and selected from the group consisting of C (cysteine), Sec (selenocysteine), and Pen (penicillamine); i.e.

[0047]

[0048] (c) A peptide of 9-11 amino acid residues having at least one intrapeptide bond, said intrapeptide bond being a disulfide bond or a diselenole bond, and consisting of an amino acid sequence selected from the group consisting of X5KAPETALX6, X5KAPETALX6A, and X5KAPETALX6AA, which have at least one intrapeptide disulfide bond or diselenole bond between X5 and X6; wherein X5 and X6 are equivalent to and selected from the group consisting of C (cysteine), Sec (selenocysteine), and Pen (penicillamine), i.e.

[0049]

[0050] (d) Contains 16 amino acid residues and is contained between X7 and X9 and between X8 and X9. 10 Amino acid sequences containing intrapeptide disulfide bonds or diselenobonds: X7NX8KAPETALX9AAAX 10 H peptide; in which X7-X 10Independently select the group consisting of C (cysteine), Sec (selenocysteine), and Pen (penicillamine); provided that X7 and X9 are equivalent and X8-X 10 Equivalent; that is

[0051]

[0052] And (e) a peptide containing the amino acid sequence X1KAPETALX2, wherein X1 is selected from the group consisting of Dap and Dab; ​​and X2 is selected from the group consisting of D (aspartic acid) and E (glutamic acid) (SEQ ID NO:7), i.e., a linear peptide;

[0053] W is selected freely –NH-(CH2) r -C(=O)- and –NH-CH((CH2) r A diradical selected from the group consisting of -NH2)-C(=O)-; Y is a radical selected from the group consisting of -NH2, -OH, -OR3 and -NHR3;

[0054] s is an integer independently selected from 0 to 1; n is an integer from 0 to 6; r is an integer independently selected from 1 to 5; k is an integer from 5 to 8; R1 and R2 are independently selected from (C1-C6)-alkyl; and R3 is a free radical selected from the group consisting of (C1-C6)-alkyl groups;

[0055] L a 'By selecting bonds from the group consisting of ester, ether, carbamate, silyl ether, sulfonate, phosphate, ketal, hemiketal, carbonate, and carbamate bonds, the bonds are connected to the free radical Z, and the bonds are drawn in L...' a The C=O, SO2, Si, P, CH or CH2 group on the left side of the formula forms with one of the hydroxyl groups in the SN38;

[0056] When n = 0, L a 'By selecting chemically feasible bonds consisting of groups of amine, amide, ether, thioether, disulfide, ester, and thioester bonds, they are linked to the free radical L. c ', the key in the drawn L a The functional group on the right side of the equation and L c Formed between the functional groups on the left side of the formula;

[0057] When n=1, L a 'Linked to the free radical L via a chemically feasible bond selected from the group consisting of amine, amide, ether, thioether, disulfide, ester, and thioester bonds.' b ', the key in the drawn L a The functional group on the right side of the equation and L bFormed between the functional groups on the left side of the equation; and L b 'By selecting chemically feasible bonds consisting of groups of amine, amide, ether, thioether, disulfide, ester, and thioester bonds, they are linked to the free radical L. c The bond is formed between the functional group on the right side of the drawn Lb' formula and the functional group on the left side of the drawn Lc' formula;

[0058] When n is greater than 1, L b 'Equivalent or different and connected between them by chemically feasible bonds selected from the group consisting of amine, amide, ether, thioether, disulfide, ester, and thioester bonds; an L b 'By selecting chemically feasible bonds from the group consisting of amine, amide, ether, thioether, disulfide, ester, and thioester bonds, the terminal bond is attached to L.' a ', the key in the drawn L a The functional group on the right side of the equation and the drawn L b Formed between the functional groups on the left side of the equation; and another L b 'By selecting chemically feasible bonds from the group consisting of amine, amide, ether, thioether, disulfide, ester, and thioester bonds, the terminal bond is attached to L.' c ', the key in the drawn L b The functional group on the right side of the equation and the drawn L c Formed between the functional groups on the left side of the formula;

[0059] L c 'By using the drawn L c The carbonyl group on the right side of the formula and the amino group of the first amino acid of peptide sequence P form an amide bond that is attached to the diradical P.

[0060] When s = 0, P is directly linked to Y via an amide bond, carboxylic acid bond, or ester bond. This bond is formed between the C=O terminal of the last amino acid in sequence P and the free radical Y, which is -NH2, -OH, -OR3, or -NHR3.

[0061] When s = 1, P is linked to the free radical W via an amide bond formed by the C=O terminal of the last amino acid of sequence P, the bond being formed between the functional group on the left side of the drawn W formula and the C=O terminal functional group of the last amino acid of sequence P on the right side of the drawn sequence; and W is linked to Y as follows: -C(=O)-NH-(CH2). r- C(=O)-Y or -C(=O)-NH-CH((CH2) r NH2)-C(=O)-Y.

[0062] The line between two amino acids in the preceding or following sequence represents an intrapeptide bond between the side chains of the two amino acids. In a particular embodiment, the line between two amino acids in the preceding or following sequence represents an intrapeptide bond between the side chains of the two amino acids.

[0063] A second aspect of the invention relates to a pharmaceutical composition comprising a therapeutically effective amount of a compound as defined above and an appropriate amount of a pharmaceutically acceptable carrier or excipient.

[0064] A third aspect of the invention relates to compounds as defined above, used for use as pharmaceuticals.

[0065] The fourth aspect of the invention relates to compounds as described above, used for the treatment of cancers in mammals, including humans.

[0066] The fifth aspect of the invention relates to a compound of formula (I) for use in treating cancer, wherein the compound of formula (I) is used in combination therapy with a chemotherapeutic agent and / or with a carboxylesterase inhibitor. Attached Figure Description

[0067] Compound (Ia) is also named G2B-001 or SN38-connector A-MiniAp4. These names are used equivalently herein. Compound (Ib) is also named G2B-001 (linear). Compound (Ic) is also named G2B-003. Compound (Id) is also named G2B-004. Compound (Ie) is also named G2B-005.

[0068] Figure 1 This example shows the comparative antiproliferative activity of compound (Ia) (also referred to herein as G2B-001 or SN38-connector A-MiniAp4) (Example 7) and related compounds SN38 and irinotecan against the adult glioma U87 cancer cell line. Values ​​are expressed as the percentage of the MTS signal considered to be 100% in untreated control cells. Values ​​in point form represent the mean and SD of three to six replicates at the compound concentration. MiniAp4 is DapKAPETALD with an amide intrapeptide bond between D and D.

[0069] Figure 2 This chart shows the comparative antiproliferative activity of the SN38-connector A-MiniAp4(Ia) and related compounds SN38 and irinotecan against the adult glioma U373 cancer cell line. Values ​​are expressed as the percentage of MTS signal considered to be 100% in untreated control cells. Values ​​in point form represent the mean and SD of three to six replicates at the compound concentration.

[0070] Figure 3This chart shows the comparative antiproliferative activity of SN38-linker A-MiniAp4(Ia) and related compounds SN38 and irinotecan against the DIPG cell model HSJD-DIPG-007. Values ​​are expressed as the percentage of MTS signal considered to be 100% in untreated control cells. Values ​​in point form represent the mean and SD of three to six replicates at the compound concentration.

[0071] Figure 4 This chart shows the comparative antiproliferative activity of the SN38-linker A-MiniAp4(Ia) and related compounds SN38 and irinotecan against the DIPG model HSJD-DIPG-011. Values ​​are expressed as the percentage of MTS signal considered to be 100% in untreated control cells. Values ​​in point form represent the mean and SD of three to six replicates at the compound concentration.

[0072] Figure 5 This chart compares the antiproliferative activity of SN38-connector A-MiniAp4(Ia) and related compounds SN38 and irinotecan against the pediatric advanced glioma model HSJD-GBM-001. Values ​​are expressed as the percentage of MTS signal considered to be 100% in untreated control cells. Values ​​in point form represent the mean and SD of three to six replicates at the compound concentration.

[0073] Figure 6 This chart shows the comparative antiproliferative activity of the SN38-connector A-MiniAp4(Ia) and related compounds SN38 and irinotecan against the retinoblastoma cell model HSJD-RBT-5. Values ​​are expressed as the percentage of MTS signal considered to be 100% in untreated control cells. Values ​​in point form represent the mean and SD of three to six replicates at the compound concentration.

[0074] Figure 7 This chart shows the comparative antiproliferative activity of the SN38-connector A-MiniAp4(Ia) and related compounds SN38 and irinotecan against the retinoblastoma cell model HSJD-RBT-7. Values ​​are expressed as the percentage of MTS signal considered to be 100% in untreated control cells. Values ​​in point form represent the mean and SD of three to six replicates at the compound concentration.

[0075] Figure 8 This chart shows the comparative antiproliferative activity of the SN38-linker A-MiniAp4(Ia) and related compounds SN38 and irinotecan against the retinoblastoma cell model HSJD-RBT-14. Values ​​are expressed as the percentage of MTS signal considered to be 100% in untreated control cells. Values ​​in point form represent the mean and SD of three to six replicates at the compound concentration.

[0076] Figure 9 This chart shows the comparative antiproliferative activity of the SN38-connector A-MiniAp4(Ia) and related compounds SN38 and irinotecan against the Ewing sarcoma cell line A673. Values ​​are expressed as the percentage of the MTS signal considered to be 100% in untreated control cells. Values ​​in point form represent the mean and SD of three to six replicates at the compound concentration.

[0077] Figure 10 This chart shows the comparative antiproliferative activity of the SN38-connector A-MiniAp4(Ia) and related compounds SN38 and irinotecan against the rhabdomyosarcoma cell line Rd. Values ​​are expressed as the percentage of MTS signal considered to be 100% in untreated control cells. Values ​​in point form represent the mean and SD of three to six replicates at the compound concentration.

[0078] Figure 11 This chart shows the comparative antiproliferative activity of the SN38-connector A-MiniAp4(Ia) and related compounds SN38 and irinotecan against the rhabdomyosarcoma cell line RH4. Values ​​are expressed as the percentage of MTS signal considered to be 100% in untreated control cells. Values ​​in point form represent the mean and SD of three to six replicates at the compound concentration.

[0079] Figure 12 This chart shows the comparative antiproliferative activity of the SN38-connector A-MiniAp4(Ia) and related compounds SN38 and irinotecan against the neuroblastoma cell line LAN-1. Values ​​are expressed as the percentage of the MTS signal considered to be 100% in untreated control cells. Values ​​in point form represent the mean and SD of three to six replicates at the compound concentration.

[0080] Figure 13 This chart shows the comparative antiproliferative activity of the SN38-connector A-MiniAp4(Ia) and related compounds SN38 and irinotecan against the neuroblastoma cell line Sk-N-JD. Values ​​are expressed as the percentage of MTS signal considered to be 100% in untreated control cells. Values ​​in point form represent the mean and SD of three to six replicates at the compound concentration.

[0081] Figure 14 This chart shows the comparative antiproliferative activity of the SN38-connector A-MiniAp4(Ia) and related compounds SN38 and irinotecan against the neuroblastoma cell model HSJD-NB-004. Values ​​are expressed as the percentage of MTS signal considered to be 100% in untreated control cells. Values ​​in point form represent the mean and SD of three to six replicates at the compound concentration.

[0082] Figure 15This chart shows the comparative antiproliferative activity of the SN38-connector A-MiniAp4(Ia) and related compounds SN38 and irinotecan against the neuroblastoma cell model HSJD-NB-005. Values ​​are expressed as the percentage of MTS signal considered to be 100% in untreated control cells. Values ​​in point form represent the mean and SD of three to six replicates at the compound concentration.

[0083] Figure 16 This chart shows the comparative antiproliferative activity of the SN38-connector A-MiniAp4(Ia) and related compounds SN38 and irinotecan against the neuroblastoma cell model HSJD-NB-016. Values ​​are expressed as the percentage of MTS signal considered to be 100% in untreated control cells. Values ​​in point form represent the mean and SD of three to six replicates at the compound concentration.

[0084] Figure 17 This shows a comparison of the concentrations (IC50 values) of SN38-linker A-MiniAp4(Ia) drug that inhibited 50% tumor cell proliferation between cultures. Single data points are presented. Statistics: Repeated measures (paired) two-way ANOVA, using Tukey's multiple comparison test.

[0085] Figure 18 The individual body weight curves are shown for three mice (treatment group) treated with a single intravenous injection of SN38-linker A-MiniAp4 compound (Ia) with an MTD of 200 mg / kg on day 0, and two control mice (control group) treated with intravenous injection of saline on day 0.

[0086] Figure 19 The mean weight change of mice treated with irinotecan, SN38-connector A-MiniAp4 (Ia), or saline (control) is shown as a percentage loss of weight from the individual maximum weight, as detailed in Example 10. Mean data (points) and SD (bars) are presented.

[0087] Figure 20 The comparative antiproliferative activity of SN38-adaptor A-MiniAp4 (Ia) and Comparative Example 2 (SN38-adaptor B-THRre) against the HSJD-DIPG-007 cell line is shown. Values ​​are expressed as the percentage of the MTS signal considered to be 100% in untreated control cells. Values ​​in point form represent the mean and SD of six replicates at the compound concentration.

[0088] Figure 21The comparative antiproliferative activity of SN38-adaptor A-MiniAp4 (Ia) and Comparative Example 2 (SN38-adaptor B-THRre) against the HSJD-GBM-001 cell line is shown. Values ​​are expressed as the percentage of the MTS signal considered to be 100% in untreated control cells. Values ​​in point form represent the mean and SD of six replicates at the compound concentration.

[0089] Figure 22 The comparative antiproliferative activities of SN38-linker A-MiniAp4 (Ia), Comparative Example 1 (SN38-linker A-THRre), and Comparative Example 2 (SN38-linker B-THRre) against the HSJD-DIPG-007 cell line are shown. Values ​​are expressed as the percentage of the MTS signal considered to be 100% in untreated control cells. Values ​​in point form represent the mean and SD of six replicates at the compound concentration.

[0090] Figure 23 The comparative antiproliferative activity of SN38-linker A-MiniAp4 (Ia), Comparative Example 1 (SN38-linker A-THRre), and Comparative Example 2 (SN38-linker B-THRre) against the HSJD-GBM-001 cell line is shown. Values ​​are expressed as the percentage of the MTS signal considered to be 100% in untreated control cells. Values ​​in point form represent the mean and SD of six replicates at the compound concentration.

[0091] Figure 24 The stability of SN38-linker A-MiniAp4(Ia) in human serum at 37°C in vitro with respect to time was demonstrated.

[0092] Figure 25 The stability of SN38-linker A-MiniAp4(Ia) in rat, mouse, dog and human plasma with respect to time was demonstrated in vitro at 37°C.

[0093] Figure 26 The stability of G2B-001 linear (Ib) in human serum at 37°C in vitro relative to time was demonstrated.

[0094] Figure 27 This chart shows the comparative antiproliferative activity of G2B-001 linear (Ib), G2B-001 (Ia), and the associated compound SN38 against the DIPG cell model HSJD-DIPG-007. Values ​​are expressed as the percentage of the MTS signal considered to be 100% in untreated control cells. Values ​​in point form represent the mean and SD of three to six replicates at the compound concentration.

[0095] Figure 28The figures show the subcutaneous neuroblastoma tumor volumes (mean and SD of the two tumors) in three mice treated with irinotecan (black dot), G2B-003 (Ic) (solid line), or saline control (dashed line). The tumor model was a patient-derived xenograft named HSJD-NB-013. Detailed Implementation

[0096] Unless otherwise stated, all amino acids referred to herein are L-amino acids. One-letter and three-letter codes are used in a vague manner. The following abbreviations are used for the following amino acids: diaminopropionic acid (Dap), diaminobutyric acid (Dab), selenocysteine ​​(Sec), and penicillamine (Pen). In the context of this invention, penicillamine refers only to D-penicillamine.

[0097] The compounds of the present invention described above may be in the form of pharmaceutically acceptable salts. As used herein, the term "pharmaceutically acceptable salt" encompasses any salt formed from a pharmaceutically acceptable, non-toxic acid or base (including inorganic or organic acids or bases). There are no limitations on the salts, except that they must be pharmaceutically acceptable if used for therapeutic purposes.

[0098] Because some compounds of formula (I) are basic, their salts can be prepared from pharmaceutically acceptable, non-toxic acids (including inorganic and organic acids). Such acids include, for example, hydrochloric acid, acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, fumaric acid, gluconic acid, glutamic acid, hydrobromic acid, hydrochloric acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, phosphoric acid, succinic acid, sulfuric acid, tartaric acid, p-toluenesulfonic acid, etc.

[0099] In one specific implementation, the compounds of formula (I) are those compounds in which Z is attached to the connector L via the hydroxyl group (b) of the active pharmaceutical ingredient.

[0100] In another specific embodiment, the compounds of formula (I) are those compounds in which Z is attached to the connector L via the hydroxyl group (a) of the active pharmaceutical ingredient.

[0101] In another specific embodiment, the compound of formula (I) is a biradical of a peptide selected from the group consisting of: (a) a peptide comprising the amino acid sequence DapKAPETALD, wherein there is an intrapeptide bond between Dap and D, said intrapeptide bond being an amide bond, i.e.

[0102]

[0103] (b) A peptide of 9-20 amino acid residues having at least one intrapeptide bond, said intrapeptide bond being a disulfide bond, and comprising the following amino acid sequence: CKAPETALCAAA; having at least one intrapeptide disulfide bond between cysteine ​​residues 1 and 9, i.e.

[0104]

[0105] (c) A peptide of length 9-11 amino acid residues, having at least one intrapeptide bond, said intrapeptide bond being a disulfide bond, and consisting of an amino acid sequence selected from the group consisting of CKAPETALC, CKAPETALCA, and CKAPETALCAA, having at least one intrapeptide disulfide bond between cysteine ​​residues 1 and 9.

[0106] as well as

[0107] (d) A peptide having 16 amino acid residues and containing the amino acid sequence CNCKAPETALCAAACH, wherein an intrapeptide disulfide bond is present between the first and third cysteine ​​residues (cysteines 1 and 11) and between the second and fourth cysteine ​​residues (cysteines 3 and 15), i.e.

[0108]

[0109] Having already selected X1-X 10 The specific amino acid sequences SEQ ID NO:8 to SEQ ID NO:13 show specific intrapeptide bonds in the sequences.

[0110] In another specific embodiment, at least one intrapeptide bond in the peptide of the present invention is a single intrapeptide bond. In another specific embodiment, at least one intrapeptide bond in the peptide of the present invention refers to two intrapeptide bonds.

[0111] In another specific embodiment, the compound of formula (I) is one of those compounds in which P is a diradical of the peptide DapKAPETALD (SEQ ID NO:14), i.e., a linear peptide.

[0112] In one specific embodiment, in conjunction with any specific embodiment above or below, the compound of formula (I) is one of those compounds in which P is a diradical of a peptide selected from the group consisting of: (a) a peptide comprising the amino acid sequence DapKAPETALD having an intrapeptide bond between Dap and D, said intrapeptide bond being an amide bond (also named MiniAp4) (SEQ ID NO:8); and (b) CKAPETALC having at least one intrapeptide disulfide bond between cysteine ​​residues at positions 1 and 9 (SEQ ID NO:10).

[0113] In another specific embodiment, in conjunction with any specific embodiment above or below, the compound of formula (I) is one of the following compounds, wherein P is a diradical of the peptide DapKAPETALD, which has an intrapeptide bond between Dap and D, said intrapeptide bond being an amide bond (SEQ ID NO:8).

[0114] In another specific embodiment, in conjunction with any specific embodiment above or below, the compound of formula (I) is wherein L a 'For compounds that are diradicals selected from the group consisting of: -C(=O)-(CH2)' r -C(=O)-、-C(=O)-(CH2) r -NH-, -C(=O)-(CH2) r -S-、-C(=O)-(CH2) r -O-, -C(=O)-NH-(CH2) r -C(=O)-, L1, L2, L3, L4, L5, L6, L7 and L 12 .

[0115] In another specific embodiment, in conjunction with any specific embodiment above or below, the compound of formula (I) is wherein L a 'For compounds that are diradicals selected from the group consisting of: -C(=O)-(CH2)' r -C(=O)-、-C(=O)-(CH2) r -NH-, -C(=O)-(CH2) r -S-、-C(=O)-(CH2) r -O-, L1, L2, L3, L4, L5, L6 and L7.

[0116] In another specific embodiment, in conjunction with any of the embodiments above or below, the compound of formula (I) is one of the following compounds, wherein L is a linker, the linker being a diradical consisting of 3 to 8 diradicals, and n is an integer from 1 to 6. In another specific embodiment, in conjunction with any of the embodiments above or below, the compound of formula (I) is one of the following compounds, wherein L is a linker, the linker being a diradical consisting of 5 to 8 diradicals. In another specific embodiment, in conjunction with any of the embodiments above or below, the compound of formula (I) is one of the following compounds, wherein L is a linker, the linker being a diradical consisting of 6 to 8 diradicals. In another specific embodiment, in conjunction with any of the embodiments above or below, the compound of formula (I) is one of the following compounds, wherein L is a linker, the linker being a diradical consisting of 6 to 7 diradicals.

[0117] In another specific embodiment, in conjunction with any specific embodiment above or below, the compound of formula (I) is one of the following compounds, wherein L is a linker, the linker being a diradical consisting of 6 diradicals.

[0118] In another specific embodiment, in conjunction with any specific embodiment above or below, the compound of formula (I) is one of the following compounds, wherein L a 'For L3 and L c 'For -C(=O)-(CH2)' r -C(=O)-. In another specific embodiment, the compound of formula (I) is one of the following compounds, wherein L a 'For L3; L b 'Choose freely -NH-(CH2)' r -O-、-(CH2) r -O- and -(CH2) r The group consisting of -NH- and its combinations; L c 'For -C(=O)-(CH2)' r -C(=O)-.

[0119] In another specific embodiment, in conjunction with any specific embodiment above or below, the compound of formula (I) is one of the following compounds, wherein L is a connector, the connector being a diradical consisting of two diradicals, and n = 0.

[0120] In another specific embodiment, in conjunction with any specific embodiment above or below, the compound of formula (I) is one of the following compounds, wherein L a 'For L 12 And L c 'For L 13 .

[0121] In another specific embodiment, in conjunction with any specific embodiment above or below, the compound of formula (I) is one of the following compounds, wherein L a 'For -C(=O)-NH-(CH2) r -C(=O)- and L c 'For L 15 .

[0122] In another specific embodiment, in conjunction with any specific embodiment above or below, the compound of formula (I) is one of the following compounds, wherein L a 'By using as a reference to the drawn L a The ester bond formed by the C=O group on the left side of the formula is attached to the free radical Z, and is attached to the free radical L through a chemically feasible bond with the functional group on the right side of the drawn formula. bThe chemically feasible bond selection consists of the group consisting of amine bonds, amide bonds, ether bonds, thioether bonds, disulfide bonds, ester bonds, and thioester bonds.

[0123] In another specific embodiment, in conjunction with any specific embodiment above or below, the compound of formula (I) is one of the following compounds, wherein L a 'By drawing L' a The ester bond formed by the C=O group on the left side of the formula is attached to the free radical Z, and is attached to the free radical L through a chemically feasible bond with the functional group on the right side of the drawn formula. b The chemically feasible bond is an amide bond.

[0124] In another specific embodiment, in conjunction with any specific embodiment above or below, the compound of formula (I) is one of the following compounds, wherein L a 'By using as a reference to the drawn L a The C=O group on the left side of the formula forms a carbonate or carbamate bond to the free radical Z, and is further bonded to the free radical L by a chemically feasible bond with the functional group on the right side of the formula. b The chemically feasible bond is an amide bond (NH-CO or CO-NH).

[0125] In another specific embodiment, in conjunction with any of the specific embodiments above or below, the compound of formula (I) is one of the following compounds, wherein L b 'With the drawn L b The functional group on the left side of the equation and the free radical L a 'Form chemically feasible bonds; and L b 'By drawing L b The functional group on the right side of the equation forms a chemically feasible bond that connects to the free radical L. c The chemically feasible bond selection is free from the group consisting of amine bonds, amide bonds, ether bonds, thioether bonds, disulfide bonds, ester bonds, and thioester bonds; wherein when n is greater than 1, L b 'Equivalent or different and connected between them by chemically feasible bonds selected from the group consisting of amine, amide, ether, thioether, disulfide, ester, and thioester bonds; an L b 'Connect to L at the end a 'And another L b 'Connect to L at the end c '.

[0126] In another specific embodiment, in conjunction with any specific embodiment above or below, the compound of formula (I) is one of the following compounds, wherein L c 'By drawing L cThe carbonyl group on the right side of the formula and the amino group of the first amino acid of the peptide sequence P form an amide bond to the diradical P, and are connected to the radical L via a chemically feasible bond with the functional group on the left side of the drawn formula. b The chemically feasible bond selection consists of the group consisting of amine bonds, amide bonds, ether bonds, thioether bonds, disulfide bonds, ester bonds, and thioester bonds.

[0127] In another specific embodiment, the compound of formula (I) is the compound of formula (Ia) or a pharmaceutically acceptable salt thereof, wherein in formula (I), P is MiniAp4=DapKAPETALD having an intrapeptide bond between Dap and D, Y=CONH2, W=O, the linker is the linker A, and SN38 is connected to the linker via a hydroxyl group (b).

[0128]

[0129] (Ia).

[0130] This compound was also named SN38-connector A-MiniAp4.

[0131] In another specific embodiment, the compound of formula (I) is the compound of formula (Ib) or a pharmaceutically acceptable salt thereof, wherein in formula (I), P is a MiniAp4=DapKAPETALD linear compound that does not have an intrapeptide bond between Dap and D, Y=CONH2, W=0, the linker is the linker A, and SN38 is connected to the linker via a hydroxyl group (b).

[0132]

[0133] This compound is also named G2B-001 linear in this paper.

[0134] In another specific embodiment, the compound of formula (I) is the compound of formula (Ic) or a pharmaceutically acceptable salt thereof, wherein in formula (I), P is MiniAp4=DapKAPETALD having an intrapeptide bond between Dap and D, Y=CONH2, W=O, the linker is the linker A, and SN38 is linked to the linker via a hydroxyl group (a).

[0135]

[0136] (Ic).

[0137] This compound is also named G2B-003 in this paper.

[0138] Connector A is formed by the following:

[0139] La':L3, where r = 4 (right) and 3 (left):

[0140]

[0141] Lb': 1 unit of diradical -NH-(CH2)3-O-, 2 units of diradical -(CH2)2-O-, and 1 unit of diradical -(CH2)3-NH-; and Lc': -C(=O)-(CH2)2-C(=O)-; and the diradicals are connected as shown in the following diagram corresponding to connector A:

[0142]

[0143] In another specific embodiment, the compound of formula (I) is the compound of formula (Id) or a pharmaceutically acceptable salt thereof, wherein in formula (I), P is MiniAp4=DapKAPETALD having an intrapeptide bond between Dap and D, Y=CONH2, W=O, the linker is the linker C, and SN38 is linked to the linker via a hydroxyl group (a).

[0144]

[0145] This compound is also named G2B-004 in this paper. The connector C is formed via the following: La':L 12 and Lc':L 13 And n = 0.

[0146] In another specific embodiment, the compound of formula (I) is the compound of formula (Ie) or a pharmaceutically acceptable salt thereof, wherein in formula (I), P is MiniAp4=DapKAPETALD having an intrapeptide bond between Dap and D, Y=CONH2, W=O, the linker is the linker D, and SN38 is linked to the linker via a hydroxyl group (a).

[0147]

[0148] (Ie).

[0149] This compound is also named G2B-005 in this paper. Linker D is formed via the following: La':-C(=O)-NH-(CH2) r -C(=O)-, where r=1; Lb':L 15 And n = 0.

[0150] Compounds of formula (I) can be generated wholly or partially by chemical synthesis. The amino acids required for the preparation of compounds of formula (I) are commercially available. Compounds of formula (I) can be readily prepared, for example by liquid-phase synthesis, or preferably by solid-phase peptide synthesis, for which numerous methods have been disclosed (see M. Amblard et al., “Methods and protocols of modern solid-phase peptide synthesis”. Molecular Biotechnology 2006, Vol. 33, pp. 239-254). Compounds of formula (I) can also be prepared by any combination of liquid-phase synthesis and / or solid-phase synthesis. For example, the body of peptide P can be synthesized by solid-phase synthesis, followed by removal of the protecting group from solution. The binding of SN-38 to linker L and peptide P can be carried out in the solid phase or in solution. The construction of linker L can also be prepared by any combination of liquid-phase synthesis and / or solid-phase synthesis.

[0151] The peptides of the present invention can also be obtained by generating a DNA template and subcloning it into an expression vector (see JHLee et al., Eur. J. 30 Biochem. 2001. Vol. 268, pp. 2004-2012).

[0152] Compound (Ia) SN38-connector A-MiniAp4 can be prepared by a method comprising reacting compound (III) with compound (IV) as described above to produce compound (Ia).

[0153]

[0154] Pharmaceutically acceptable salts of compounds of formula (I) can be prepared by methods known in the art. For example, they can be prepared by conventional chemical methods from parent compounds containing a basic or acidic moiety. Typically, such salts are prepared, for example, by reacting the free acidic or basic form of these compounds with a stoichiometric amount of an appropriate pharmaceutically acceptable base or acid in water, in an organic solvent, or in a mixture thereof.

[0155] This invention also relates to a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I) as defined above, and an appropriate amount of a pharmaceutically acceptable carrier or excipient. As used herein, the term "therapeuticly effective amount" means an amount of medicine, when administered, sufficient to prevent or to some extent alleviate the development of one or more symptoms of the treated disease. The specific dosage of the compound administered according to the invention will, of course, be determined by the specific circumstances surrounding the case, including the compound administered, the route of administration, the specific symptom being treated, and similar considerations.

[0156] The term "pharmaceutical composition" refers to a mixture of the compound described herein with other chemical components, such as diluents or carriers. A pharmaceutical composition facilitates the administration of the compound to a living organism. The term "pharmaceuticalally acceptable excipient or carrier" refers to a pharmaceutically acceptable material, composition, or medium. Each component must be pharmaceutically acceptable in the sense of compatibility with the other components of the pharmaceutical composition. Each component must also be suitable for contact with human or animal tissues or organs without excessive toxicity, irritation, allergic reactions, immunogenicity, or other problems or complications commensurate with the efficacy / risk ratio.

[0157] The compositions of the present invention are suitable for administration in parenteral forms, such as injection, infusion, or implantation.

[0158] A key feature of the compounds of this invention is their bioactivity in inhibiting the cell growth of the tested tumor cell lines. As shown in the examples, the compounds of this invention exhibit antitumor properties in several cancer cell lines.

[0159] As already explained, the comparative data provided in the experimental section indicate that when SN38 is conjugated with another peptide (THRre) of the following formula via the same linker (linker A), some / all of the antitumor activity is lost, and the results are even worse if a different linker (linker B) is used. Linker B is -C(=O)-(CH2)2-C(=O)-. THRre has the following formula: HD-Pro-D-Trp-D-Val-D-Pro-D-Ser-D-Trp-D-Met-D-Pro-D-Pro-D-Arg-D-His-D-Thr-CONH2. However, the exemplary compounds of the present invention unexpectedly maintain antitumor activity against several cell lines from brain and extracranial cancers, similar to SN38, which is much higher than irinotecan, and exhibit good stability in human serum in vitro.

[0160] Therefore, the present invention relates to compounds of formula (I) as defined above or pharmaceutically acceptable salts thereof, which are used as medicines.

[0161] The present invention also relates to compounds of formula (I) as defined above, or pharmaceutically acceptable salts thereof, for the treatment of cancers in mammals (including humans), as they are active in all types of cancer tested. This aspect can also be described as the use of compounds of formula (I) as defined above, or pharmaceutically acceptable salts thereof, in the preparation of medicaments for the treatment and / or prevention of cancers in mammals (including humans). The present invention also relates to a method of treating a mammal (including humans) suffering from or susceptible to cancer, the method comprising administering to the patient a therapeutically effective amount of a compound of formula (I) as defined above, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient or carrier.

[0162] In one specific embodiment, the compound of formula (I) is used for the purpose as defined above, wherein the cancer treatment includes treating tumors selected from the group consisting of extracranial solid tumors, ocular tumors, and CNS tumors. In another specific embodiment, the compound is used for the purpose as defined above, wherein the cancer is selected from the group consisting of: adult gliomas, pediatric gliomas, retinoblastomas, Ewing sarcomas, DIPG, neuroblastomas, and rhabdomyosarcomas. In another specific embodiment, the compound is used for the purpose as defined above, wherein the pediatric glioma is selected from the group consisting of diffuse entropional pontine gliomas (DIPG) and pediatric high-grade gliomas. In another specific embodiment, the compound is used for the purpose as defined above, wherein the cancer is a diffuse entropional pontine glioma. In another specific embodiment, the compound is used for the purpose as defined above, wherein the cancer is a retinoblastoma. In another specific embodiment, the compound is used for the purpose as defined above, wherein the cancer is Ewing sarcoma. In another specific embodiment, the compound is used for the purpose as defined above, wherein the cancer is a neuroblastoma. In another specific embodiment, the compound is used for the purpose as defined above, wherein the cancer is rhabdomyosarcoma. In another specific embodiment, the compound is used for the purpose as defined above, wherein the cancer is adult glioma.

[0163] In another specific embodiment, the compound is used for the purpose as defined above, wherein the compound is active against cancer cell lines selected from the group consisting of: A673, HSJD-DIPG-007, HSJD-DIPG-011, HSJD-GBM-001, Rd, RH4, HSJD-RBT-5, HSJD-RBT-7, HSJD-RBT-14, U373, U87, LAN-1, SK-N-JD, HSJD-NB-004, HSJD-NB-005, HSJD-NB-013, and HSJD-NB-016.

[0164] In another specific embodiment, the compound is used for the purpose as defined above, wherein the compound of formula (I) is administered by intravenous bolus and / or intravenous infusion.

[0165] The compounds of the present invention can be used in the same manner as other known chemotherapeutic agents, i.e., in combination with other treatments simultaneously or sequentially, depending on the condition to be treated. The compounds of the present invention can be used alone or in combination with other suitable bioactive compounds. Therefore, the compounds of formula (I) of the present invention are used to treat cancers in mammals (including humans) in combination therapy with chemotherapeutic agents. Additionally, the compounds of formula (I) of the present invention are used in combination with ester stabilizers to treat cancers in mammals (including humans). Suitable ester stabilizers are carboxylesterase inhibitors. Examples of carboxylesterase inhibitors can be found in MJ Hatfield and PM Potter, Exp. Opin. Ther. Pat. 2011, Vol. 21(8), pp. 1159-1171.

[0166] In one specific embodiment, the compound of formula (I) is administered in combination with a carboxylesterase inhibitor. In another specific embodiment, the compound of formula (I) is administered simultaneously with a carboxylesterase inhibitor. In yet another specific embodiment, the compound of formula (I) is administered separately in any order within an effective treatment interval.

[0167] In one specific embodiment, the compound of formula (I) is administered in combination with another chemotherapeutic agent. In another specific embodiment, the compound of formula (I) is administered simultaneously with another chemotherapeutic agent. In yet another specific embodiment, the compound of formula (I) is administered separately in any order within an effective treatment interval.

[0168] In another specific embodiment, the compound of formula (I) is administered in combination with another chemotherapeutic agent and a carboxylesterase inhibitor. In another specific embodiment, the compound of formula (I) is administered simultaneously with another chemotherapeutic agent and a carboxylesterase inhibitor. In another specific embodiment, the compound of formula (I) is administered separately from another chemotherapeutic agent and a carboxylesterase inhibitor in any order within an effective treatment interval.

[0169] Throughout the specification and claims, the word "comprising" and its variations are not intended to exclude other technical features, additives, components, or steps. Furthermore, the word "comprising" covers situations where it means "composed of." Other objects, advantages, and features of the invention will become apparent to those skilled in the art upon examination of the specification, or may be learned through practice of the invention. The following embodiments and drawings are provided illustratively and are not intended to limit the invention. Reference numerals in relation to the drawings and placed in brackets in the claims are intended only to increase the comprehensibility of the claims and should not be construed as limiting the scope of the claims. Moreover, the invention covers all possible combinations of the specific and preferred embodiments described herein.

[0170] Example

[0171] The protected amino acids, stems, and resins were supplied by: Luxembourg Industries (Tel Aviv, Israel), Neosystem (Strasbourg, France), CalbiochemNovabiochem AG (Laufelfingen, Switzerland), Bachem AG (Bubbendorf, Switzerland), or Iris Biotech (Marktredwitz, Germany). Other reagents and solvents used are summarized in […]. Table 1 middle. Table 1 Commercial suppliers and reagents used. DCM is passed through an Al₂O₃ column. DMF is stored in... On the molecular sieve, nitrogen gas is introduced to eliminate the volatiles.

[0172]

[0173]

[0174]

[0175] General considerations for manual synthesis: Solid-phase peptide elongation and other solid-phase operations were performed manually in a polypropylene syringe equipped with a polyethylene porous disc. Solvents and soluble reagents were removed by aspiration. Washes between different synthetic steps were performed using dimethylformamide (DMF) (5 x 30 s) and dichloromethane (DCM) (5 x 30 s), each using 10 mL of solvent / g of resin.

[0176] General considerations regarding microwave-assisted synthesis: Microwave-assisted solid-phase peptide synthesis was performed on a Liberty Blue automated microwave peptide synthesizer using H-Rink amide Protide resin (load: 0.56 mmol / g). Linear peptides were synthesized at a scale of 0.5 mmol using Fmoc-amino acids (0.2 M) in a 5-fold excess relative to the resin.

[0177] Identification Test: The following tests were used to identify and control the synthesis: A) Kaiser colorimetric assay for detecting primary amines bound to solids (E. Kaiser et al., Anal. Biochem. 1970, Vol. 34, pp. 595-598); B) p-nitrobenzene ester assay for secondary amines bound to solids (A. Madder et al., Eur. J. Org. Chem. 1999, pp. 2787-2791).

[0178] The scheme used during the manual synthesis of the compoundThe following methods and protocols were used to synthesize the compound on a scale of 100 μmol: The resin for manual synthesis was selected based on the free radical Y: if Y is OH, the terminal will be COOH, and 2-chlorotrytil chloride resin was selected from other available resins. If Y is NH2, the terminal will be CONH2, and Rink amide MBHA resin was selected from other available resins.

[0179] Resin initial treatment: The resin was treated with MeOH (5 x 30 s), DMF (5 x 30 s), DCM (5 x 30 s), DCM containing 1% TFA (1 x 30 s and 2 x 10 min), DCM (5 x 30 s), DMF (5 x 30 s), DCM (5 x 30 s), DCM containing 5% DIEA (1 x 30 s, 2 x 10 min), DCM (5 x 30 s), and DMF (5 x 30 s).

[0180] Fmoc group removal: To remove the 9-fluorenylmethoxycarbonyl (Fmoc) protecting group, a treatment with DMF containing 20% ​​(v / v) piperidine was performed for 30 seconds, followed by two treatments of 10 minutes each. Two additional treatments with DBU, toluene, piperidine, and DMF (5%, 5%, 20%, 70%) (2 × 5 min) were performed to ensure removal of the Fmoc group from the secondary amine (proline).

[0181] Coupling methods described for a 100 μmol scale:

[0182] Coupling Method 1 The protected amino acid (4 equivalents, 400 μmol), TBTU dissolved in DMF (1-3 mL / g resin) (4 equivalents, 400 μmol, 128 mg), and DIEA (8 equivalents, 800 μmol, 136 μl) were added sequentially to the resin, followed by DIEA (8 equivalents, 800 μmol, 136 μl). The mixture was allowed to react for 1 h with intermittent manual stirring. The solvent was removed by suction and the resin was washed with DMF (5 x 30 s) and DCM (5 x 30 s). The degree of coupling was checked by Kaiser colorimetry. The Fmoc group was removed with a 20% piperidine DMF solution (v / v) using a 30 s treatment and two 10 min treatments. If the amino acid to be deprotected was proline, an additional treatment (2 x 5 min) was performed using DBU, toluene, piperidine, and DMF (5%, 5%, 20%, 70%) to ensure removal of the Fmoc group.

[0183] Coupling Method 2:Protected amino acids (4 equivalents, 400 μmol), PyBOP (4 equivalents, 400 μmol, 208 mg), and HOAt (12 equivalents, 1.2 mmol, 163 mg) dissolved in DMF (1–3 mL / g resin) were added sequentially to the resin, followed by DIEA (12 equivalents, 1.2 mmol, 204 μL). The mixture was allowed to react for 1 h with intermittent manual stirring. The solvent was removed by suction and the resin was washed with DMF (5 x 30 s) and DCM (5 x 30 s). The coupling reaction was performed twice under the same conditions. The degree of coupling was checked by Kaiser colorimetry. The Fmoc group was removed with a 20% piperidine DMF solution (v / v) using a 30 s treatment and two 10 min treatments. If the amino acid to be deprotected was proline, an additional treatment (2 × 5 min) was performed using DBU, toluene, piperidine, and DMF (5%, 5%, 20%, 70%) to ensure removal of the Fmoc group.

[0184] Coupling Method 3: Protected amino acids (4 equivalents, 400 μmol), PyBOP (4 equivalents, 400 μmol, 208 mg), and HOBt dissolved in DMF (1–3 mL / g resin) (12 equivalents, 1.2 mmol, 162 mg) were added sequentially to the resin, followed by DIEA (12 equivalents, 1.2 mmol, 204 μL). The mixture was allowed to react for 1 h with intermittent manual stirring. The solvent was removed by suction and the resin was washed with DMF (5 x 30 s) and DCM (5 x 30 s). The coupling reaction was performed twice under the same conditions. The degree of coupling was examined by Kaiser colorimetry. Fmoc groups were removed with a 20% piperidine DMF solution (v / v) using a 30 s treatment and two 10-min treatments. If the amino acid to be deprotected is proline, additional treatment (2 × 5 min) with DBU, toluene, piperidine, or DMF (5%, 5%, 20%, 70%) is performed to ensure removal of the Fmoc group.

[0185] Coupling Method 4Scale 100 μmol: Protected amino acid (3 equivalents, 300 μmol), DIC (3 equivalents, 300 μmol, 46 μL), and Oxyma (3 equivalents, 300 μmol, 43 mg) in DCM / DMF (1:1). The mixture was reacted for 45 min with intermittent manual stirring. The solvent was removed by suction and the resin was washed with DMF (5 x 30 s) and DCM (5 x 30 s). The degree of coupling was checked by Kaiser colorimetry. Fmoc groups were removed with a 20% piperidine DMF solution (v / v) using a 30 s treatment and two 10 min treatments. If the amino acid to be deprotected was proline, an additional treatment (2 x 5 min) was performed using DBU, toluene, piperidine, and DMF (5%, 5%, 20%, 70%) to ensure removal of Fmoc groups.

[0186] Coupling Method 5 Scale 100 μmol: Protected amino acid (3 equivalents, 300 μmol), DIC (3 equivalents, 300 μmol, 46 μL), and HOBt (3 equivalents, 300 μmol, 41 mg) in DCM / DMF (1:1). The mixture was reacted for 45 min with intermittent manual stirring. The solvent was removed by suction and the resin was washed with DMF (5 x 30 s) and DCM (5 x 30 s). The degree of coupling was checked by Kaiser colorimetry. Fmoc groups were removed with a 20% piperidine DMF solution (v / v) using a 30 s treatment and two 10 min treatments. If the amino acid to be deprotected was proline, an additional treatment (2 x 5 min) was performed using DBU, toluene, piperidine, and DMF (5%, 5%, 20%, 70%) to ensure removal of Fmoc groups.

[0187] The scheme used during microwave-assisted automatic synthesis: The compound was synthesized at a scale of 500 μmol using the following method and scheme: The resin for microwave-assisted automated synthesis was selected based on the free radical Y: if Y is OH, the terminal will be COOH, and Cl-TCP(Cl)ProTide resin will be selected from other available resins. If Y is NH2, the terminal will be CONH2, and Rink amide ProTide resin will be selected from other available resins.

[0188] Resin initial treatment: The resin was treated with MeOH (5 x 30 s), DMF (5 x 30 s), DCM (5 x 30 s), DCM containing 1% TFA (1 x 30 s and 2 x 10 min), DCM (5 x 30 s), DMF (5 x 30 s), DCM (5 x 30 s), DCM containing 5% DIEA (1 x 30 s, 2 x 10 min), DCM (5 x 30 s), and DMF (5 x 30 s).

[0189] Coupling and deprotection conditions for microwave-assisted automated peptide synthesis:

[0190] Coupling conditions:

[0191]

[0192]

[0193]

[0194] Conditions for deprotection:

[0195] CEM preference Deprotected mixture Microwave method 1 10% (w / v) piperazine, 10:90 (EtOH / NMP) standard 2 DMF or NMP containing 20% ​​piperidine (v / v) standard

[0196] method ramp time Total Time highest temperature Standard deprotection 20-30s 1:05 90℃

[0197] Methods for cyclizing peptide sequence P:

[0198] Cyclization method 1: Disulfide bond or diselenylene bond: Cycloning was performed in solution after cleavage from the resin or after selective deprotection of Cys, Sec, or Pen residues on the resin. The peptide was dissolved at a concentration of 100 μM in 10 mM ammonium bicarbonate aqueous buffer at pH 8.0. The solution was vigorously stirred at room temperature for 24 h. Subsequently, the product was acidified to pH 2–3 with TFA, frozen, and lyophilized.

[0199] Cycloning method 2: Amide bond: Cyclization was performed on the resin. The Fmoc group was removed with a 20% piperidine DMF solution (v / v) using a 30s treatment followed by two 10min treatments. The N-terminal amine was protected with a Boc protecting group using Boc2O (3 equivalents, 1000 μmol, 56 mg) and DIEA (30 equivalents, 3000 μmol, 240 μL). The OAl and Alloc groups were first deprotected by adding tetrakis(triphenylphosphine)palladium(0) (0.1 equivalents, 10 μM, 12 mg) and phenylsilane (10 equivalents, 1000 μmol, 123 mg) to DCM (3 x 15 min). The resin was washed with DCM containing 0.02 M sodium diethylcarbamate (3 x 5 min). The amino group of Dap and the carboxyl ester group of aspartic acid were then coupled by adding PyBOP (4 equivalents, 400 μmol, 208 mg), HOAt (12 equivalents, 1.2 mmol, 163 mg), DMF (1–3 mL / g resin), and DIEA (12 equivalents, 1.2 mmol, 204 μL). The coupling was carried out for 1.5 h and repeated overnight.

[0200] Cyclization method 3: Amide bond:Cyclization was performed on the resin. The Fmoc group was removed with a 20% piperidine DMF solution (v / v) using a 30s treatment followed by two 10min treatments. The N-terminal amine was protected with a Boc protecting group using Boc2O (3 equivalents, 1000 μmol, 56 mg) and DIEA (30 equivalents, 3000 μmol, 240 μL). The OAl and Alloc groups were first deprotected by adding tetrakis(triphenylphosphine)palladium(0) (0.1 equivalents, 10 μM, 12 mg) and phenylsilane (10 equivalents, 1000 μmol, 123 mg) to DCM (3 x 15 min). The resin was washed with DCM containing 0.02 M sodium diethyldithiocarbamate (3 x 5 min). The coupling of the amino group of Dap and the carboxyl ester group of aspartic acid was then achieved by two 30-min cycles of 4 equivalents of Oxyma (400 μmol, 57 mg) and 4 equivalents of N,N'-diisopropylcarbodiimide (DIC) (400 μmol, 61 μL).

[0201] Cycloning method 4: Amide bond: Cyclization was performed on the resin. The Fmoc group was removed with a 20% piperidine DMF solution (v / v) using a 30s treatment followed by two 10min treatments. The N-terminal amine was protected with a Boc protecting group using Boc2O (3 equivalents, 1000 μmol, 56 mg) and DIEA (30 equivalents, 3000 μmol, 240 μL). The OAl and Alloc groups were first deprotected by adding tetrakis(triphenylphosphine)palladium(0) (0.1 equivalents, 10 μM, 12 mg) and phenylsilane (10 equivalents, 1000 μmol, 123 mg) to DCM (3 x 15 min). The resin was washed with DCM containing 0.02 M sodium diethyldithiocarbamate (3 x 5 min). The coupling of the amino group of Dap and the carboxyl ester group of aspartic acid was then achieved by two 1-hour cycles of 4 equivalents of DIC (400 μmol, 61 μL) and 4 equivalents of HOBt (400 μmol, 54 mg).

[0202] General method for constructing connector L:

[0203] General methods for forming disulfide bonds: Disulfide bonds can be formed by the reaction of two thiols. The thiols were dissolved at a concentration of 100 μM in a 10 mM ammonium bicarbonate aqueous buffer at pH 8.0, and the solution was vigorously stirred at room temperature for 24 h. The solution was then acidified to pH 2–3 with TFA, frozen, and lyophilized.

[0204] General methods for forming sulfides:The thioether bond is formed by the reaction of the N-terminal bromoacetyl group with cysteine ​​thiol, as described in P.L. Barker et al., J. Med. Chem. 1992, Vol. 35, pp. 2040-2048.

[0205] General methods for forming ethers: Ether formation can be accomplished by the reaction of hydroxyl groups with haloalkyl compounds, preferably under basic conditions, as described in Greene's Protective Groups in Organic Synthesis, 5th Edition. Peter GMWuts. 2014 John Wiley & Sons, Inc., pp. 26-29.

[0206] General methods for forming esters: Ester formation can be accomplished by the reaction of hydroxyl groups and carboxylic acids, using typical esterification conditions such as Fischer esterification in the presence of acid catalysis, or by the reaction of alternative hydroxyl groups with the corresponding acyl chlorides, as described in Greene's Protective Groups in Organic Synthesis, 5th ed. Peter G.M. Wuts. 2014 John Wiley & Sons, Inc., pp. 271-279.

[0207] General methods for forming thioesters: Thioester bonds are achieved through the reaction of thiols with carboxylic acids, as described by M. Kazemi et al., Journal of Sulfur Chemistry, 2015, Vol. 36:6, pp. 613-623.

[0208] General methods for forming carbamates: The reaction of hydroxyl groups with isocyanates can produce the corresponding carbamates, as described here in MTNguyen et al., J.Org.Chem. 1998, 63, Vol. 20, pp. 6878-6885.

[0209] General methods for forming methyl silyl ethers: The reaction of hydroxyl groups with halotrialkylsilyl groups produces the corresponding methylsilyl ethers. An acid scavenger is usually required, as described in Greene's Protective Groups in Organic Synthesis, 5th Edition, Peter GMWuts, 2014, John Wiley & Sons, Inc., pp. 456-463.

[0210] General methods for forming sulfonates: The reaction of hydroxyl groups with alkyl or aryl sulfonyl halides produces the corresponding sulfonates, as described in F. David et al., Org. Process Res. Dev. 2010, 14, 4, 999-1007.

[0211] General methods for forming phosphate esters: The reaction of a hydroxyl group with a dialkyl or diaryl phosphate having one hydroxyl group under dehydration conditions or using Mitsunobu reaction conditions can form the corresponding phosphate ester, wherein one of the substituents is a chain connected to the hydroxyl group.

[0212] General methods for forming ketals: Ketals can be formed by the reaction of a hydroxyl group with a halomethyleneoxyalkyl compound, or by the addition of a hydroxyl group to a substituted dihydropyran or dihydrofuran under acidic conditions, as described in Greene's Protective Groups in Organic Synthesis, 5th ed. Peter GMWuts. 2014 John Wiley & Sons, Inc., pp. 69-77.

[0213] General methods for forming hemiketals: The contact between a hydroxyl group and an aldehyde can form the corresponding hemiketal, as described here: https: / / www.cliffsnotes.com / study-guides / chemistry / organic-chemistry-ii / aldehydes-and-ketones / reactions-of-aldehydes-and-ketones.

[0214] General methods for forming carbamates: Carbamates can be formed by the reaction of hydroxyl groups with halocarbamates or isocyanates, as described in Greene's Protective Groups in Organic Synthesis, 5th Edition. Peter G.M. Wuts. 2014 John Wiley & Sons, Inc., pp. 371-374.

[0215] General methods for forming carbonates: Carbonates can be formed by the reaction of SN38 with PNPC (as described in Eur J Pharm Biopharm, 2017, Vol. 115, pp. 149-158) or triphosgene (as described in J. Med. Chem. 2008, Vol. 51, pp. 6916-6926).

[0216] Coupling of Fmoc-TTDS-OH:The coupling of Fmoc-TTDS-OH (2 equivalents) was achieved by two 30-min cycles of 4 equivalents of oxyma and 4 equivalents of N,N'-diisopropylcarbodiimide (DIC) in DMF or 4 equivalents of DIC and 4 equivalents of HOBt in DCM for 2 h. The 9-fluorenylmethoxycarbonyl (Fmoc) protecting group was then removed by treatment with DMF containing 20% ​​(v / v) piperidine for 30 s, followed by two 10-min treatments.

[0217] Coupling of 5-hexyneic acid: The coupling of 5-hexynoic acid (2 equivalents, 200 μmol, 23 mg) was achieved by two 30-min cycles of 4 equivalents of Oxyma (400 μmol, 57 mg) and 4 equivalents of N,N'-diisopropylcarbodiimide (DIC) (400 μmol, 61 μL) in DMF:DCM (1:1), or by 4 equivalents of DIC (400 μmol, 61 μL) and 4 equivalents of HOBt (400 μmol, 54 mg) in DMF:DCM 1:1 for 4 h, or by 2 equivalents of PyBOP (400 μmol, 208 mg) in DMF:DCM 1:1, 6 equivalents of HOAt (600 μmol, 81.5 mg) and 6 equivalents of DIEA (600 μmol, 102 μL) in DMF for 1.5 h. Solvent was removed by aspiration, and the resin was washed with DMF (5 x 30 s) and DCM (5 x 30 s). Coupling was repeated under the same conditions. The degree of coupling was monitored using Kaiser colorimetry.

[0218] Coupling with diethylene glycol anhydride: Coupling with diethylene glycol anhydride (10 equivalents, 1000 μmol, 116 mg) was achieved through two 60-min cycles of 10 equivalents of DIEA (1000 μmol, 174 μL) in DMF. The solvent was removed by aspiration, and the resin was washed with DMF (5 x 30 s) and DCM (5 x 30 s). Coupling was repeated under the same conditions. The degree of coupling was monitored using a Kaiser colorimetric assay.

[0219] General methods for cutting from resin: Final cleavage and side-chain deprotection of the resin: This was carried out by treating the resin with TFA (95%), H2O (2.5%), and TIS (2.5%) for 2 h. Tert-butyl methyl ether was added to the obtained product, and the mixture was centrifuged (3 x 8 min). The supernatant was discarded, and the precipitate was resuspended in a mixture of H2O, MeCN, and TFA (1000:1000:1). The product was filtered off and frozen.

[0220] General methods for characterizing compounds:These compounds were analyzed by UPLC (Acquity high-class system with PDA detector, sample manager FNT and quaternary solvent manager, Acquity BEH C18 (50 x 2 mm x 1.7 μm) column, 0.61 mL / min, with MeCN (0.036% TFA) and H2O (0.045% TFA) used as solvents. In all cases, a 2-min linear gradient was used. UPLC-MS spectroscopy was performed using a Waters high-class system (PDA detector, sample manager FNT and quaternary solvent manager), coupled to an ESI-MS Micromass ZQ electrospray ionization source and MassLynx 4.1 software (Waters, Milford, MA). A BEH C18 column (50 x 2.1 mm x 1.7 μm) was used. 1.7 μm, Waters). The flow rate was 0.6 mL / min, and MeCN (0.07% formic acid) and H2O (0.1% formic acid) were used as solvents. Samples were analyzed using positive ionization: the ion ejection voltage was 30 V, and the capillary temperature was 1 kV. Accurate mass was obtained by mass spectrometry: LTQ-FT Ultra (ThermoScientific), with samples introduced via direct injection (automated nanoelectrospray). NanoMate (AdvionBioSciences, Ithaca, NY, USA) used disposable conductive tips to aspirate samples from 384-well plates (protein lobind) and injected them into the mass spectrometer through a nanoESI chip (consisting of 400 nozzles in a 20x20 array). The ejection voltage was 1.70 kV, and the delivery pressure was 0.50 psi; ionization was NanoESI, positive ionization.

[0221] NMR experiments were performed on a Bruker Avance III 600 MHz spectrometer equipped with a TCI cryopreservation probe. Compounds were prepared by dissolving them at 3–4 mM in 90% H₂O / 10% D₂O and adjusting the pH to 2–3. Chemical shifts were referenced to internal sodium 3-(trimethylsilyl)propanesulfonate (DSS). Suppression of the water signal was achieved through excitation molding. Residue-specific allocations were obtained from 2D total correlation spectroscopy (TOCSY) and correlation spectroscopy (COSY) experiments, while 2D nuclear overhauser effect spectroscopy (NOESY) allowed for sequence-specific allocations. 13C resonances were allocated from 2D 1H13C HSQC spectra. All experiments were performed at 298 K except for the NOESY spectra obtained at 278 K. The amide proton temperature coefficient was determined based on a series of one-dimensional spectra obtained between 278 and 308 K. The TOCSY and NOESY mixing times were 70 and 250 ms, respectively.

[0222] Example 1. (S)-(4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano) Preparation of [3',4':6,7]indolazin[1,2-b]quinolin-9-yl)tert-butyl carbonate

[0223] To a 250 mL flask, 1.5 g of 7-ethyl-10-hydroxycamptothecin (SN38), 1.3 equivalents of di-tert-butyl dicarbonate (1.15 mL), and excess anhydrous pyridine (9 mL) were added to 150 mL of anhydrous DCM. The mixture was stirred overnight at room temperature. The reaction mixture was then washed with HCl (0.5N) x 3, saturated NaHCO3 x 1, and brine. The organic layer was dried over MgSO4 and the solvent was removed under vacuum. No further purification was performed. Reversed-phase UPLC-PDA: linear gradient from 0 to 100% MeCN in H2O solution over 2 min, using an Acquity BEH C18 (50 x 2 mm x 1.7 μm) column at 0.61 mL / min, with MeCN (0.036% TFA) and H2O (0.045% TFA) as solvents; retention time: 1.89 min. Yield: 96%, [M+H] exp + :493.5Da. 1H NMR (400MHz, chloroform-d) δ7.81(d,J=9.2Hz,1H),7.45(d,J=2.5Hz,1H),7.25-7.20(m,1H),6.91-6.85(m,1H),5.32(d,J=16.3,1H),4.86(d,J= 1Hz, 6H), 3.85 (s, 1H), 2.73 (q, J = 7.7Hz, 2H), 1.60 (s, 1H), 1.57-1.41 (m, 3H), 1, 21 (s, 10H), 0.99 (t, J = 7.7Hz, 3H), 0.61 (t, J = 7.3Hz, 3H).

[0224] Example 2. (S)-9-((tert-butoxycarbonyl)oxy)-4,11-diethyl-3,14-dioxo-3,4,12,14- Preparation of tetrahydro-1H-pyrano[3',4':6,7]indolazino[1,2-b]quinoline-4-yl 5-azidovalerate

[0225] In a round-bottom flask, 400 mg of (S)-(4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolazino[1,2-b]quinoline-9-yl) tert-butyl carbonate (Example 1) and 1.6 equivalents of 5-azido-valeric acid (193 mg) were purged with N2 and then dissolved in anhydrous DCM. The mixture was cooled to 0°C. Then, 1.4 equivalents of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (218 mg) were added, and the mixture was stirred at 0°C for 1 hour, then stirred overnight at room temperature. The mixture was washed with 3 x saturated NaHCO3, 2 x HCl (0,1N), and brine. The organic layer was then dried with MgSO4 and removed under vacuum. The compound was used without further purification. [M+H] exp + : 618.36Da.

[0226] Example 3. (S)-4,11-Diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano Preparation of [3',4':6,7]indolazino[1,2-b]quinolin-4-yl 5-azidopentanoate (SN38 modified with azide, as...) (Results of Examples 1-3)

[0227] In a round-bottom flask, 1.5 g of (S)-9-((tert-butoxycarbonyl)oxy)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolazino[1,2-b]quinoline-4-yl 5-azidopentanoate (Example 2) was stirred in 50 mL of HCl (4N, in dioxane) at room temperature for 2 hours. The solvent was then removed under vacuum, and the crude mixture was purified with silica using (DCM / MeOH (10%)) to a purity greater than 95%. Overall yield of Example 1: 8%, [M+H] + 518.58 Da. Reversed-phase UPLC-PDA: Linear gradient from 0% to 100% MeCN in H2O solution over 2 min, using an Acquity BEH C18 (50 x 2 mm x 1.7 μm) column at 0.61 mL / min, with MeCN (0.036% TFA) and H2O (0.045% TFA) as solvents; retention time: 1.819 min. [M+H] exp + : 518.58Da. 1H-NMR (400MHz, chloroform-d) δ1.00(t,3H),1.38(t,3H),1.65(m,2H),1.69(m,2H),2.20(m,2H),2.56(m,2H),3.13( q,3H),3.27(td,2H),5.20(s,2H),5.39-5.72(dd,2H),7.42(d,1H),7.48(s,1H),7.62(dd,1H),8.45(d,1H).

[0228] Example 4. (S)-9-((tert-butoxycarbonyl)oxy)-4,11-diethyl-3,14-dioxo-3,4,12,14- Preparation of tetrahydro-1H-pyrano[3',4':6,7]indolazino[1,2-b]quinoline-4-yl tert-butylsuccinate

[0229] 1 mmol of (S)-tert-butyl (4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolazino[1,2-b]quinoline-9-yl) from Example 1 and 1.5 equivalents of monotert-butyl succinate were added to a round-bottom flask and purged with N2. The reagents were dissolved in 20 mL of anhydrous DCM and cooled at 0 °C. Then N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC·HCl) was added, and the mixture was stirred at 0 °C for 1 hour and then stirred overnight at room temperature. The reaction mixture was washed with 3 x saturated NaHCO3, 2 x HCl (0,1N), and brine. It was then dried over MgSO4, the evaporation was evaporated, and the mixture was dried. The compound was used without further purification. Yield: 72%. 1 H-NMR (400MHz, chloroform-d) δ0.92(t,3H),1.30(s,9H),1.33(m,3H),1.38(q,2H),1.42(s,2H),1.55(s,9H),2.1 4(ddq,2H),2.49(td,2H),2.69(m,2H),2.86(dd,2H),3.09(m,2H),5.20(s,2H),5.32(d,2H),5.61(d,2H).

[0230] Example 5: (S)-4-((4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyran) Preparation of [3',4':6,7]indolazin[1,2-b]quinolin-4-yl)oxy)-4-oxobutyric acid (modified with succinic acid) SN38 (as a result of Examples 4 and 5)

[0231] In a round-bottom flask, (S)-9-((tert-butoxycarbonyl)oxy)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolazino[1,2-b]quinoline-4-yltert-butylsuccinate (Example 4) was stirred in 20 mL of HCl (4N, in dioxane) at room temperature for 2 hours. The solvent was then evaporated, and the crude mixture was purified using Teledyne-ISCO in a gradient of 0-50. The desired compound was obtained in 32% yield and with a purity greater than 95%. Yield: 32%. Reversed-phase UPLC-PDA: Linear gradient from 0% to 100% MeCN in H₂O solution over 2 min, using an Acquity BEHC18 (50 x 2 mm x 1.7 μm) column at 0.61 mL / min, with MeCN (0.036% TFA) and H₂O (0.045% TFA) as solvents; retention time: 1.498 min. [M+H] exp + : 491.27Da. 1 ¹H-NMR (400MHz, chloroform-d) δ 1.02 (t, 3H), 1.39 (t, 3H), 2.22 (m, 2H), 2.61 (t, 2H), 2.84 (m, 2H), 3.16 (q, 2H), 5.27 (s, 2H), 5.45 (d, 2H), 5.60 (d, 2H), 7.33 (s, 1H), 7.42 (dq, 1H), 8.03 (d, 1H).

[0232] Methods for cycloaddition of alkynes-azides: The coupling of alkyne-azide cycloaddition (Click reaction) in solution was carried out using the scheme described in SFM van Dongen et al.; Bioconjugate Chem. 2009, Vol. 20, pp. 20-23.

[0233] This reaction was carried out following the procedure described in lumiprobe (https: / / www.lumiprobe.com / protocols / click-chemistry-dna-labeling) without microwaves. However, this reaction takes approximately two days to complete. Cu was used with the ligand THTPA. Since SN38-N3 is insoluble in H2O, only DMF was used instead of buffer solution and DMSO for the reaction, as it is insoluble in water and even in mixtures (water / DMSO).

[0234] Alternatively, the cycloaddition (Click reaction) of acetylene-TTDS-DapKAPETALD with SN38-N3 to an acetylene-azide was carried out using a microwave: 10 mL of SN38-N3 (1.5 equivalents), acetylene-TTDS-DapKAPETALD (1 equivalent), CuTHTPA (0.15 equivalents), and sodium ascorbate (0.3 equivalents) were added to a microwave-safe vial and dissolved in 3 mL of DMF. Throughout the reaction, the mixture was stirred at 30 °C for 2 to 4 hours with MW (Discover SP MW) assistance. The crude mixture was purified by semi-preparative HPLC (C18).

[0235] Preparation of reagents for cycloaddition of alkynes and azides (Click Chemistry): 100 mM ketone(II)-THPTA stock solution in 55% DMSO: Dissolve 50 mg of copper(II) sulfate pentahydrate in 1 mL of distilled water and dissolve 116 mg of tris(3-hydroxypropyltriazolylmethyl)amine (THPTA) ligand in 1.1 mL of DMSO. Then, mix the two solutions. 5 mM ascorbic acid stock solution: Dissolve 18 mg of ascorbic acid in 20 mL of distilled water.

[0236] General methods for product purification and characterization:The crude product was purified by semi-preparative-scale RP-HPLC and further purified by UPLC (Acquity high-class system (PDA detector, sample manager FNT and quaternary solvent manager, Acquity BEHC18 (50 x 2 mm x 1.7 μm) column, 0.61 mL / min with MeCN (0.036% TFA) and H2O (0.045% TFA) as solvents. A linear gradient of 2 min was used in all cases) and UPLC-MS (Waters high-class system (PDA detector, sample manager FNT and quaternary solvent manager), which was coupled to an ESI-MS Micromass ZQ electrospray ionization source and MassLynx 4.1 software was used). A BEH C18 column (50 x 2.1 mm x 1.7 μm) was used. 1.7 μm (Waters). Flow rate was 0.6 mL / min, and MeCN (0.07% formic acid) and H₂O (0.1% formic acid) were used as solvents. Samples were analyzed by positive ionization: ion ejection voltage was 30 V, and capillary temperature was 1 kV. Accurate mass was obtained by mass spectrometry: LTQ-FT Ultra (Thermo Scientific), with samples introduced via direct injection (automated nanoelectrospray). NanoMate (Advion BioSciences, Ithaca, NY, USA) used disposable conductive tips to aspirate samples from 384-well plates (protein lobind) and injected them into the mass spectrometer through a nanoESI chip (consisting of 400 nozzles in a 20x20 array). Ejection voltage was 1.70 kV, and delivery pressure was 0.50 psi; ionization was NanoESI, positive ionization. All peptides were obtained with a purity greater than 95%.

[0237] Example 6. Hexynoic acid-TTDS-Dap- with an amide bond between the amino group on the Dap side chain and the carboxylic acid group on the Asp side chain. Preparation of Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH2 (hexynodic acid-TTDS-SEQ ID NO:7).

[0238] For the manual coupling of the first protected amino acid with the resin, coupling method 4 was applied using Fmoc-Asp(OAl)-OH (118.5 mg). Subsequent amino acids were coupled using coupling method 4 in the following order:

[0239]

[0240] 46 μL of DIC and 43 mg of Oxyma were used in a DMF / DCM (1:1) mixture. The mixture was allowed to react for 45 min with intermittent manual stirring. After each coupling, a treatment of 30 s with DMF containing 20% ​​(v / v) piperidine was performed, followed by two treatments of 10 min each to remove the 9-fluorenylmethoxycarbonyl (Fmoc) protecting group. Two additional treatments were performed with DBU, toluene, piperidine, and DMF (5%, 5%, 20%, 70%) (2 × 5 min) to ensure removal of the Fmoc group from the secondary amine (proline). Cyclization on the resin was performed following cyclization method 2: a 30 s treatment and two 10 min treatments were performed to remove the Fmoc group with a 20% piperidine DMF solution (v / v). The N-terminal amine was protected with Boc protecting groups using Boc2O (3 equivalents, 1000 μmol, 56 mg) and DIEA (30 equivalents, 3000 μmol, 240 μL). The OAl and Alloc groups were first deprotected by adding tetrakis(triphenylphosphine)palladium(0) (0.1 equivalents, 10 μM, 12 mg) and phenylsilane (10 equivalents, 1000 μmol, 123 mg) to DCM (3 x 15 min). The resin was washed with DCM containing 0.02 M sodium diethylcarbamate (3 x 5 min). Coupling of the amino group of Dap to the carboxylic acid ester group of aspartic acid was then achieved by adding PyBOP (4 equivalents, 400 μmol, 208 mg), HOAt (12 equivalents, 1.2 mmol, 163 mg), DMF (1-3 mL / g resin), and DIEA (12 equivalents, 1.2 mmol, 204 μL). The coupling was performed for 1.5 hours and repeated overnight.

[0241] Coupling of Fmoc-TTDS-OH: Coupling of Fmoc-TTDS-OH (2 equivalents, 200 μmol, 108.53 mg) was achieved over 2 h with 4 equivalents of DIC (400 μmol, 61 μL) and 4 equivalents of HOBt (400 μmol, 54 mg) in DCM. The 9-fluorenylmethoxycarbonyl (Fmoc) protecting group was then removed by treatment with DMF containing 20% ​​(v / v) piperidine for 30 s, followed by two treatments of 10 min each.

[0242] Coupling of 5-hexynoic acid: To couple 5-hexynoic acid to peptides immobilized on resin, the following protocol was used: hexynoic acid (4 equivalents, 400 μmol, 45 mg), PyBOP (4 equivalents, 400 μmol, 208 mg), and HOAt (12 equivalents, 1.2 mmol, 163 mg) in DMF (1–3 mL / g resin) were added sequentially to the resin, followed by 12 equivalents of DIEA (1.2 mmol, 204 μL). The mixture was allowed to react for 1.5 h with intermittent manual stirring. The solvent was removed by aspiration, and the resin was washed with DMF (5 x 30 s) and DCM (5 x 30 s). Coupling was repeated under the same conditions. The degree of coupling was monitored using a Kaiser assay.

[0243] The peptide was then cleaved and lyophilized. Product characterization. Reversed-phase UPLC: linear gradient from 20% MeCN to 60% MeCN in H2O solution over 2 min, using an Acquity BEH C18 (50 x 2 mm x 1.7 μm) column at 0.61 mL / min, with MeCN (0.036% TFA) and H2O (0.045% TFA) as solvents; retention time: 0.939 min. UPLC-MS [M+H] exp + : 1308.15 Da; Yield (synthesis and purification): 7.5%.

[0244] Example 7: Preparation of compound (Ia) SN38-connector A-MiniAp4

[0245] hexyne prepared as in Example 6 acid Starting with -TTDS-MiniAp4, and using SN38-N3 prepared as in Example 3, the following scheme is followed to obtain compound of formula (Ia), which is also named SN38-connector A-MiniAp4:

[0246] Add 10 mL of 1.5 equivalents of (S)-4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolazino[1,2-b]quinoline-4-yl-5-azidovallotate, 1 equivalent of acetylacetonate-TTDS-DapKAPETALD, 0.15 equivalents of CuTHPTA, and 0.3 equivalents of sodium ascorbate to a microwave-safe vial, and dissolve it in 3 mL of DMF. Throughout the reaction, stir the mixture at 30 °C for 2 to 4 hours with MW (CEM discover SP MW) assistance. Purify the crude product by semi-preparative-scale RP-HPLC. Compounds with purities greater than 95% were obtained. Product characterization: Reversed-phase UPLC-PDA: linear gradient from 0% to 100% MeCN in H2O solution over 2 min, using an Acquity BEHC18 (50 x 2 mm x 1.7 μm) column at 0.61 mL / min, with MeCN (0.036% TFA) and H2O (0.045% TFA) as solvents; retention time: 1.462 min. UPLC-MS [M+H] exp + : 1824.76 Da; Yield (synthesis and purification): 30%.

[0247] Comparative Example 1: SN38-Connector A-THRre

[0248] In this comparative example, all amino acids used were D-amino acids. For the manual coupling of the first protected amino acid to the resin, coupling method 4 was applied using Fmoc-D-Thr(tBu)-OH (159 mg). Subsequent amino acids were coupled using coupling method 4 in the following order:

[0249]

[0250] 46 μL of DIC and 43 mg of Oxyma were used in a DMF / DCM (1:1) mixture. The mixture was allowed to react for 45 min with intermittent manual stirring. After each coupling, the mixture was treated with DMF containing 20% ​​(v / v) piperidine for 30 s, followed by two treatments of 10 min each to remove the 9-fluorenylmethoxycarbonyl (Fmoc) protecting group. Two additional treatments were performed with DBU, toluene, piperidine, and DMF (5%, 5%, 20%, 70%) (2 × 5 min) to ensure removal of the Fmoc group from the secondary amine (proline).

[0251] Coupling of Fmoc-TTDS-OH: Coupling of Fmoc-TTDS-OH (2 equivalents, 200 μmol, 108.53 mg) was achieved over 2 h with 4 equivalents of DIC (400 μmol, 61 μL) and 4 equivalents of HOBt (400 μmol, 54 mg) in DCM. The 9-fluorenylmethoxycarbonyl (Fmoc) protecting group was then removed by treatment with DMF containing 20% ​​(v / v) piperidine for 30 s, followed by two treatments of 10 min each.

[0252] Coupling of 5-hexynoic acid: To couple 5-hexynoic acid to peptides immobilized on resin, the following protocol was used: hexynoic acid (4 equivalents, 400 μmol, 45 mg), PyBOP (4 equivalents, 400 μmol, 208 mg), and HOAt (12 equivalents, 1.2 mmol, 163 mg) in DMF (1–3 mL / g resin) were added sequentially to the resin, followed by 12 equivalents of DIEA (1.2 mmol, 204 μL). The mixture was allowed to react for 1.5 h with intermittent manual stirring. The solvent was removed by aspiration, and the resin was washed with DMF (5 x 30 s) and DCM (5 x 30 s). Coupling was repeated under the same conditions. The degree of coupling was monitored using a Kaiser assay. The peptide was then cleaved and lyophilized.

[0253] Use this hexyne acid -TTDS-THRre, and using SN38-N3 as prepared in Example 3, the following protocol was followed to obtain SN38-connector A-THRre: 10 ml of 1.5 equivalents of (S)-4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolazino[1,2-b]quinoline-4-yl 5-azidovallotate, 1 equivalent of acetylene-TTDS-THRre, 0.15 equivalents of CuTHPTA, and 0.3 equivalents of sodium ascorbate were added to a microwave-safe vial and dissolved in 3 ml of DMF. The mixture was stirred at 30°C for 2 to 4 hours with MW (CEM discover SP MW) assistance throughout the reaction. The crude product was purified by semi-preparative-scale RP-HPLC. Compounds with a purity greater than 95% were obtained. Product characterization: Reversed-phase UPLC-PDA: linear gradient from 0% to 100% MeCN in H2O solution over 2 min, using an Acquity BEH C18 (50 x 2 mm x 1.7 μm) column at 0.61 mL / min, with MeCN (0.036% TFA) and H2O (0.045% TFA) as solvents; retention time: 1.597 min. UPLC-MS [M+H] exp +: 2403.86Da.

[0254] Comparative Example 2: SN38-Connector B–THRre

[0255] In this comparative example, all amino acids used were D-amino acids. For the manual coupling of the first protected amino acid to the resin, coupling method 4 was applied using Fmoc-D-Thr(tBu)-OH (159 mg). Subsequent amino acids were coupled using coupling method 4 in the following order:

[0256]

[0257] 46 μL of DIC and 43 mg of Oxyma were used in a DMF / DCM (1:1) mixture. The mixture was allowed to react for 45 min with intermittent manual stirring. After each coupling, the mixture was treated with DMF containing 20% ​​(v / v) piperidine for 30 s, followed by two treatments of 10 min each to remove the 9-fluorenylmethoxycarbonyl (Fmoc) protecting group. Two additional treatments were performed with DBU, toluene, piperidine, and DMF (5%, 5%, 20%, 70%) (2 × 5 min) to ensure removal of the Fmoc group from the secondary amine (proline).

[0258] The peptide was then cleaved and lyophilized. Using this THRre and SN38-succinic acid prepared as in Example 5, the following protocol was followed to obtain SN38-linker B-THRre: In a round-bottom flask, under N2 atmosphere and at 0°C, 1.5 equivalents of (S)-4-((4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolazino[1,2-b]quinolin-4-yl)oxy)-4-oxobutyric acid, 1.2 equivalents of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC·HCl), and 1.2 equivalents of N-hydroxybenzotriazole (HOBt) were added, followed by anhydrous DMF, and the mixture was stirred for 10 min. THRre was then added, and the reaction was allowed to proceed overnight at room temperature. The crude product was purified by semi-preparative-scale RP-HPLC. Compounds with a purity higher than 95% were obtained. Product characterization: Reversed-phase UPLC-PDA: linear gradient from 0 to 100% MeCN in H2O solution over 2 min, using an Acquity BEH C18 (50 x 2 mm x 1.7 μm) column at 0.61 mL / min, with MeCN (0.036% TFA) and H2O (0.045% TFA) as solvents; retention time: 1.543 min. UPLC-MS [M+2H] exp +2 : 982.32Da.

[0259] Example 8a: Evaluation of solubility in water.

[0260] Dissolving 25 mg of SN38-connector A-MiniAp4 in H2O resulted in a completely clear solution. This means that the solubility of SN38-connector A-MiniAp4 in water is greater than 5 mg / mL, which is more than 400 times greater than the water solubility described for SN38 (Mol. Pharmaceutics, 2016, 13, 379-390).

[0261] G2B-001 linear (Ib), G2B-003 (Ic), G2B-004 (Id), and G2B-005 (Ie) showed good water solubility at 1 mg / mL.

[0262] Example 8b: Evaluation of the turbidity solubility of SN38-connector A-MiniAp4.

[0263] The SN38-connector A-MiniAp4 compound (10 mM in DMSO) was serially diluted to provide 0.1, 0.3, 1, and 3 mM solutions in DMSO. Each test compound concentration was then further diluted 1:100 in buffer (0.01 M phosphate-buffered saline, pH 7.4) to a final DMSO concentration of 1%, and final test compound concentrations of 1, 3, 10, 30, and 100 μM. Experiments were performed at 37 °C, with each concentration incubated in seven replicate wells. The plates were incubated at 37 °C for 2 h, after which absorbance was measured at 620 nm. Nicardipine and pyrene were included as control compounds. The solubility of nicardipine was pH-dependent, while the solubility of pyrene was pH-independent. Solubility was estimated based on the concentration of the test compound that increased absorbance above that of the carrier control (1% DMSO in buffer). See Table 2.

[0264] Table 2 Turbidimetric solubility data for water-based products.

[0265]

[0266] As expected, no solubility issues were found in G2B-001 up to 100 micromoles.

[0267] Example 9. Antitumor activity of compound (Ia): SN38-linker A-MiniAp4 (also known as G2B-001) targets Anti-cancer cell lines: diffuse engenerative pontine glioma, adult glioma and pediatric solid tumor retinoblastoma, Ewing Sarcoma, rhabdomyosarcoma, and neuroblastoma.

[0268] The inventors compared the activity of the novel compound (Ia) SN38-linker A-MIniAp4 (also known as G2B-001) with that of one of the closely related drugs, SN38 and irinotecan.

[0269] Cancer cell lines were obtained from a resource repository held at Hospital Sant Joan de Deu (Barcelona, ​​Spain). The cancer cell lines used in these experiments included the following cancer types: adult gliomas (U373 and U87 cell lines), pediatric gliomas including diffuse entropional pontine gliomas (HSJD-DIPG-007, HSJD-DIPG-011) and pediatric high-grade gliomas (HSJD-GBM-001), retinoblastomas (HSJD-RBT-5, HSJD-RBT-7, and HSJD-RBT-14), Ewing sarcoma (A673), rhabdomyosarcoma (Rd and RH4), and neuroblastomas (LAN-1 and SK-N-JD cell lines and patient-derived cell models HSJD-NB-004, HSJD-NB-005, and HSJD-NB-016). In summary, 3,000 to 30,000 cancer cells were cultured in 96-well plates and exposed to compounds SN38-connector A-MiniAp4(Ia) (concentration range 1–0.00000001 μM), SN38 (concentration range 1–0.00000001 μM), or irinotecan (concentration range 100–0.000001 μM) after 24 hours. To add the drugs to the cultured cells, SN38-connector A-MiniAp4(Ia) and irinotecan were prepared in culture medium from stock solutions at a concentration of 1 mg / mL in water. SN38 was prepared in culture medium from a stock solution at a concentration of 1 mg / mL in DMSO. Cell viability was determined using a tetrazolium compound [3-(4,5-dimethylthiazolyl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazole] (MTS assay; Promega, Fitchburg, WI) after 72 h of incubation with the drug. The drug concentration required to induce a 50% reduction in cell proliferation (IC50 and 95% confidence interval) was calculated using Graphpad Prism 8 software (La Jolla, CA). The following activity curves were constructed for the adult glioma cell line U87 (…). Figure 1 ) and U373 ( Figure 2 Pediatric gliomas, including the DIPG model HSJD-DIPG-007 ( Figure 3 ) and HSJD-DIPG-011 ( Figure 4 ); and the pediatric advanced glioma model HSJD-GBM-001 ( Figure 5 ); retinoblastoma model HSJD-RBT-5 ( Figure 6 ), HSJD-RBT-7 ( Figure 7 ) and HSJD-RBT-14 ( Figure 8 ); Ewing sarcoma cell line A673 ( Figure 9 ); Rhabdomyosarcoma cell line Rd( Figure 10 ) and RH4 ( Figure 11 ); Neuroblastoma cell line LAN-1 ( Figure 12 ) and SK-N-JD ( Figure 13 ); and the cell model HSJD-NB-004 derived from neuroblastoma patients ( Figure 14 ), HSJD-NB-005 Figure 15 ) and HSJD-NB-016 ( Figure 16 ).

[0270] The determined IC50 values ​​are shown in Table 3.

[0271] Table 3. IC50 values ​​of compounds SN38-linker A-MiniAp4(Ia), SN38, and irinotecan against cancer cell lines.

[0272]

[0273]

[0274] The IC50 value obtained for the SN38-connector A-MiniAp4(Ia) was significantly lower than that for the drug irinotecan (P<0.0001; paired ANOVA test). Figure 17 As shown.

[0275] Example 10. In vivo tolerability of SN38-connector A-MiniAp4(Ia) in single and repeated intravenous injections.

[0276] To evaluate the toxicity of irinotecan and SN38-connector A-MiniAp4(Ia) (also known as G2B-001) in athymic nude mice (10 weeks old; 8 mice per group), the maximum tolerated dose (MTD) of SN38-connector A-MiniAp4(Ia) was first assessed by intravenous injection. Three mice were injected at dose levels of 200, 500, and 1000 mg / kg, and symptoms, including death or weight loss, were observed over the next 14 days. The MTD was determined to be 200 mg / kg. At this dose level, mice survived and did not exhibit immediate signs of distress or significant weight loss over the next 14 days compared to untreated control animals. Figure 18 ).

[0277] After determining the MTD of SN38-connector A-MiniAp4(Ia) with a single intravenous injection, we compared the mean body weight of mice treated with irinotecan at a clinically relevant multiple-dose regimen with that treated with SN38-connector A-MiniAp4(Ia) at an equimolar dose. Briefly, mice were treated with irinotecan 10 mg / kg (15 doses, on days 1–5, 8–12, and 29–33) via intraperitoneal (ip) or with SN38-connector A-MiniAp4(Ia) 30 mg / kg (9 doses, on days 1, 3, 5, 8, 10, 12, 29, 31, and 33) via intravenous (iv). This regimen was considered the maximum feasible number of iv injections in mice. Control mice were treated with saline via ip on days 1–5, 8–12, and 29–33.

[0278] Mice were weighed until day 40, and... Figure 19 The values ​​in the table represent the mean and SD for each group. Compared to the control, mice treated with SN38-connector A-MiniAp4(Ia) did not experience significant weight loss. The body weight of mice treated with SN38-connector A-MiniAp4(Ia) was similar to that of mice treated with clinically relevant doses of irinotecan.

[0279] Example 11: SN38, SN38 and MiniAp4 (SN38-connector A-MiniAp4, Example 7) and SN38-connector B- Comparative activity of the conjugate of THRre (Comparative Example 2).

[0280] The inventors compared the activities of the conjugate of the novel compound SN38 with MiniAp4 (SN38-connector A-MiniAp4, Example 7, also known as G2B-001) and SN38-connector B-THRre (Comparative Example 2). The original compound SN38 was used as a reference. The cancer cell lines used in these experiments were pediatric gliomas, including diffuse endophytic pontine glioma HSJD-DIPG-007 and pediatric high-grade glioma HSJD-GBM-001. Briefly, 3000 cancer cells were cultured in 96-well plates and exposed to compound (Ia) (concentration range 1-0.00000001 μM), SN38-connector B-THRre (concentration range 1-0.00000001 μM), and SN38 (concentration range 1-0.00000001 μM) after 24 h. To add the drug to cultured cells, SN38-linker A-MiniAp4(Ia) and SN38-linker B-THRre were prepared in culture medium from a stock solution at a concentration of 1 mg / mL in water. SN38 was prepared in culture medium from a stock solution at a concentration of 1 mg / mL in DMSO. Cell viability after 72 h of incubation with the drug was determined using MTS assay. The drug concentration required to induce a 50% reduction in cell proliferation (IC50 and 95% confidence interval) was calculated using Graphpad Prism 8 software (La Jolla, CA). HSJD-DIPG-007 cells were established. Figure 20 ) and HSJD-GBM-001 ( Figure 21 The activity curves of SN38 and THRre are shown. The determined IC50 values ​​are in Table 4. As can be observed, the conjugation of SN38 with THRre results in lower activity than the conjugation with MiniAp4.

[0281] Table 4. IC50 value of the compound

[0282]

[0283] Example 12. Compound SN38-connector A-MiniAp4 (Ia), Comparative Example 1 (SN38-connector A-THRre) and Comparative Example 2. Example 2 (SN38-linker B-THRre) Comparative activity (peptide and the effect of both peptide and linker).

[0284] Since compound (Ia) and Comparative Example 2 (SN38-connector B-THRre) in the preceding Example 11 were composed of connectors of different sizes, the inventors compared the activity of these two compounds with that of Comparative Example 1 (SN38-connector A-THRre), which had the same connectors as compound (Ia) and Comparative Example 1.

[0285] The cancer cell lines used in these experiments were pediatric gliomas, including diffuse endophytic pontine glioma HSJD-DIPG-007 and pediatric high-grade glioma HSJD-GBM-001. In short, 3000 cancer cells were cultured in 96-well plates and exposed to compounds (Ia), Comparative Example 1, and Comparative Example 2 (each with a concentration range of 1–0.001 μM) after 24 h. All compounds were prepared in culture medium from stock solutions at a concentration of 1 mg / mL in water. Cell viability after 72 h of incubation with the drugs was determined using MTS assays. The drug concentrations required to reduce cell proliferation by 50% (IC50 and 95% confidence interval) were calculated using Graphpad Prism 8 software (La Jolla, CA). The HSJD-DIPG-007 cell line was established... Figure 22 ) and HSJD-GBM-001 ( Figure 23 The activity curves of the peptide MiniAp4 were obtained. Comparative Example 1 (SN38-linker A-THRre) and Comparative Example 2 (SN38-linker B-THRre) showed lower activity than compound (Ia). Therefore, the surprising activity of compound (Ia) compared to Comparative Examples 1 and 2 is attributed to the peptide MiniAp4, but also to the steric linker. The determined IC50 values ​​are shown in Table 5.

[0286] Table 5. IC50 values ​​(μM) of compound (Ia), comparative example 3 and comparative example 4 against cancer cell lines.

[0287]

[0288] Example 13. Stability of SN38-connector A-MiniAp4 in human serum at 37°C in vitro, relative to time.

[0289] Regarding the stability of SN38-connector A-MiniAp4(Ia) (Example 7) in human serum, it was incubated at 37°C in the presence of 90% human serum at a concentration of 200 μM in buffer HBSS. Within a certain time range, 100 μL aliquots were collected, and 400 μL of cold methanol was added to precipitate serum proteins. The samples were centrifuged at 3000 rpm for 30 min at 4°C, filtered, and analyzed by UPLC-PDA and UPLC-MS to determine the degree of degradation of SN38-connector A-MiniAp4(Ia). No significant degradation was observed within 24 h, implying a longer half-life than this time range suggests. Figure 24 ).

[0290] Example 14: SN38-connector A-MiniAp4(G2B-001)(Ia) in vitro at 37°C in rats, mice, dogs, and... Stability of human blood plasma over time.

[0291] To evaluate the stability of SN38-connector A-MiniAp4(Ia) (Example 7) in human, mouse, dog, and rat plasma at 37°C, the compound was prepared in triplicate at 200 μM in plasma samples at each time point (0h, 1h, 4h, 8h, and 24h) for each species. Once incubation was complete, plasma proteins were immediately precipitated by adding cold methanol (ratio 4:1, methanol:plasma) and vortexing the tubes for a few seconds. The samples were centrifuged at 3,000 rpm for 30 minutes at 4°C, the supernatant was collected, transferred to a sample plate, and injected into an LC-MS system.

[0292] Compound recoveries were determined by comparing the analyte response in a analyte-spiked and treated biological sample with the response in a analyte-spiked and treated methanol sample.

[0293] Plasma stability was determined using blank plasma from untreated volunteers / animals. K2-EDTA was used as an anticoagulant. Human blank plasma was obtained from Hospital Sant Pau, mouse blank plasma from Janvier, dog blank plasma from Isoquimen, and rat blank plasma from Dronis Pharma.

[0294] The half-life of 200 μM G2B-001 in human, mouse, dog, and rat plasma is greater than 24 hours, but the half-life of compound G2B-001 in mouse and dog plasma is 8–24 hours. Figure 25 ).

[0295] Table 6. Percentage recovery of G2B-001 in human, mouse, dog and rat plasma after 24 h.

[0296]

[0297]

[0298] Example 15. Hexyneic acid without an amide bond between the Dap side chain amino group and the Asp side chain carboxylic acid group. - TTDS-Dap- Preparation of Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH2 (hexynodic acid-TTDS-SEQ ID NO:14) linear form. DapKAPETALD (SEQ ID NO:14) is a linear peptide.

[0299] For the manual coupling of the first protected amino acid with the resin, coupling method 4 was applied using Fmoc-Asp(OAl)-OH (118.5 mg). Subsequent amino acids were coupled using coupling method 4 in the following order:

[0300]

[0301] 46 μL of DIC and 43 mg of Oxyma were used in a DMF / DCM (1:1) mixture. The mixture was allowed to react for 45 min with intermittent manual stirring. After each coupling, the mixture was treated with DMF containing 20% ​​(v / v) piperidine for 30 s, followed by two treatments of 10 min each to remove the 9-fluorenylmethoxycarbonyl (Fmoc) protecting group. Two additional treatments were performed with DBU, toluene, piperidine, and DMF (5%, 5%, 20%, 70%) (2 × 5 min) to ensure removal of the Fmoc group from the secondary amine (proline).

[0302] Coupling of Fmoc-TTDS-OH: Coupling of Fmoc-TTDS-OH (2 equivalents, 200 μmol, 108.53 mg) was achieved over 2 h with 4 equivalents of DIC (400 μmol, 61 μL) and 4 equivalents of HOBt (400 μmol, 54 mg) in DCM. The 9-fluorenylmethoxycarbonyl (Fmoc) protecting group was then removed by treatment with DMF containing 20% ​​(v / v) piperidine for 30 s, followed by two treatments of 10 min each.

[0303] Coupling of 5-hexynoic acid: To couple 5-hexynoic acid to peptides immobilized on resin, the following protocol was used: hexynoic acid (4 equivalents, 400 μmol, 45 mg), PyBOP (4 equivalents, 400 μmol, 208 mg), and HOAt (12 equivalents, 1.2 mmol, 163 mg) in DMF (1–3 mL / g resin) were added sequentially to the resin, followed by 12 equivalents of DIEA (1.2 mmol, 204 μL). The mixture was allowed to react for 1.5 h with intermittent manual stirring. The solvent was removed by aspiration, and the resin was washed with DMF (5 x 30 s) and DCM (5 x 30 s). Coupling was repeated under the same conditions. The degree of coupling was monitored using a Kaiser assay.

[0304] The OAl and Alloc groups were deprotected by adding tetrakis(triphenylphosphine)palladium(0) (0.1 equivalent, 10 μM, 12 mg) and phenylsilane (10 equivalent, 1000 μmol, 123 mg) to DCM (3 x 15 min). The resin was washed with DCM containing 0.02 M sodium diethylcarbamate (3 x 5 min).

[0305] The peptide was then cleaved and lyophilized. Product characterization. Reversed-phase HPLC: linear gradient from 10% to 60% MeCN in H2O solution over 30 min, using an Xbridge 25 cm 3.5 μm column at 1 mL / min, with MeCN (0.1% TFA) and H2O (0.1% TFA) as solvents; retention time: 11.526 min. Yield (synthesis and purification): 9%.

[0306] Example 16: Compound G2B of formula (Ib) without an amide bond between the amino group on the Dap side chain and the carboxylic acid group on the Asp side chain. Preparation of 001 linear form.

[0307] hexyne prepared as in Example 15 acid -TTDS-MiniAp4 linear starting, and using SN38-N3 as prepared in Example 3, the following scheme is followed to obtain compound of formula (Ib), which is also named G2B-001 linear:

[0308] Add 10 mL of 1.5 equivalents of (S)-4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolazino[1,2-b]quinoline-4-yl-5-azidovallotate, 1 equivalent of acetylene-TTDS-DapKAPETALD linear, 0.15 equivalents of CuTHPTA, and 0.3 equivalents of sodium ascorbate to a microwave-safe vial, and dissolve in 3 mL of DMF. Stir the mixture at 30 °C for 2 to 4 hours with MW (CEM discover SP MW) assistance throughout the reaction. Purify the crude product by semi-preparative-scale RP-HPLC. Compounds with purities greater than 95% were obtained. Product characterization: Reversed-phase HPLC-MS: linear gradient from 0% to 80% MeCN in H2O solution over 7 minutes, using a Luna 3μm C18(2)100A 50x 2.1mm column at 0.85 mL / min, with MeCN (0.1% formic acid) and H2O (0.1% formic acid) as solvents; retention time: 5.08 min. UPLC-MS [M+H] exp + : 1845.10 Da; Yield (synthesis and purification): 22%.

[0309] Example 17. Preparation of N3-Pen-SN38

[0310] In a double-necked round-bottom flask, add 1 equivalent of SN-38 (600 mg) and dissolve it in anhydrous DCM (60 mL) at a concentration of 100 mL / g. A turbid solution was observed. Cool this reaction mixture to 0–2 °C, add 6 equivalents of DIEA (2 mL), and stir for 15 min. After 15 min, add 3 equivalents of N3-Pen-Cl (700 mg) [dissolved in 500 μL of anhydrous DCM] in portions. Cool for 15 min, then continue stirring at room temperature. Monitor the reaction progress by HPLC. When the starting material is below 3%, the reaction is post-processed.

[0311] The initial volume of the reaction mixture was approximately 45 mL, which was further supplemented to 300 mL using anhydrous DCM. Extraction was performed three times with distilled water (20% by volume of the final reaction mixture volume) at 3 × 3 min × 60 mL. The DCM layer was dried over sodium sulfate (Na₂SO₄) for 30 min and evaporated on a rotary evaporator. The obtained solid was treated twice with 80:20 hexane:diethyl ether and kept dry in a desiccator. The yield after treatment was 730 mg. Product characterization: Reversed-phase HPLC: linear gradient from 10% to 90% MeCN in H₂O solution over 30 min, using an SS column (250 × 4.6 mm) packed with C-18 silica gel (5 μm) for chromatographic R, at 1 mL / min, with MeCN (0.1% TFA) and H₂O (0.1% TFA) as solvents; retention time: 19.730 min.

[0312] The observed mass is 518.20 Da, and the calculated mass is 517.19 Da.

[0313]

[0314] Example 18. Preparation of compound G2B-003 of formula (Ic)

[0315] hexyne prepared as in Example 6 acid Starting with -TTDS-MiniAp4 and using N3-Pen-SN38 prepared as in Example 17, the following scheme was followed to obtain compound of formula (Ic), which was also named G2B-003.

[0316] Add 10 ml of 1.5 equivalents of N3-Pen-SN38, 1 equivalent of hexynic acid-TTDS-MiniAp4, 0.15 equivalents of CuTHPTA, and 0.3 equivalents of sodium ascorbate to the vial and dissolve it in 3 ml of DMF.

[0317] Throughout the reaction, the mixture was stirred at 30°C for 2 to 4 hours with microwave (CEM discover SP MW) assistance. The crude product was purified by semi-preparative-scale RP-HPLC. Compounds with purities higher than 95% were obtained. Product characterization: Reversed-phase HPLC: linear gradient from 20% to 70% MeCN in H2O solution over 30 min, using an Xbridge 25 cm 3.5 μm column at 1 mL / min, with MeCN (0.1% TFA) and H2O (0.1% TFA) as solvents; retention time: 12.00 min. UPLC-MS [M+H] exp + : 1826.15 Da; Yield (synthesis and purification): 17%.

[0318] Example 19. Preparation of SN38-O-CO-NH-Gly-COOH

[0319] glycine-COOBu t A solution of (1 equivalent) and DMAP (2 equivalents) in anhydrous DCM (15 mL) was added dropwise to a solution of bis(4-nitrophenyl) carbonate (1.3 equivalents) in anhydrous DCM (15 mL), and the resulting solution was stirred overnight at 50 °C. The reaction mixture was then diluted in DCM (150 mL) and washed with 0.5 N HCl (100 mL). The aqueous layer was washed with DCM (5 × 100 mL), and all organic fractions were collected, dried over MgSO4, and filtered. The solvent was evaporated under reduced pressure, and the residue was purified by rapid chromatography (hexane:DCM:Et2O:5:4:1).

[0320] The obtained PNP-Gly-COOBu t (1 equivalent) and SN38 (1.2 equivalent) were reacted in anhydrous DCM in the presence of DMAP (2 equivalent). The reaction was stirred overnight at 50 °C. The solvent was evaporated under reduced pressure, and the residue was purified by rapid chromatography.

[0321] By processing SN38-O-CO-NH-Gly-COOBu with TFA:DCM:TIS(40:40:20) t The tert-butyl ester groups were removed over 6 hours. The solvent was then evaporated. Toluene was added twice to remove trace amounts of TFA. No further purification was performed.

[0322] Example 20. Preparation of Boc-Val-Cit-PAB-SN38

[0323] In a round-bottom flask, 1 equivalent of 7-ethyl-10-hydroxycamptothecin (SN38), 1 equivalent of Boc-Val-Cit-PAB-PNP (from iris biotech), 2 equivalents of DIEA, and catalytic DMAP were stirred in anhydrous DMF at room temperature for 16 h. The reaction mixture was then diluted with AcOEt and washed with HCl (0.5 M) (×3) and brine (×4). It was then dried over MgSO4, the evaporation was evaporated, and the crude mixture was purified by silica chromatography (AcOEt:MeOH; 20:1; 15:1:9:1). The desired compound was obtained in 52% yield with a purity greater than 90%. Reversed-phase UPLC-PDA: Linear gradient from 0% to 100% MeCN in H2O solution over 2 min, using an Acquity BEH C18 (50 x 2 mm x 1.7 μm) column at 0.61 mL / min, with MeCN (0.036% TFA) and H2O (0.045% TFA) as solvents; retention time: 1.42 min. [M+H] exp+ : 898.4Da.

[0324] Example 21. Preparation of H2N-Val-Cit.PAB-SN38

[0325] In a round-bottom flask, one equivalent of Boc-Val-Cit-PAB-SN38, as prepared in Example 20, was stirred for 5 minutes in a mixture of DCM:TFA:H2O (44.75:49.75:0.5). The solvent was then evaporated. Toluene was added twice to remove trace amounts of TFA. No further purification was performed.

[0326] Example 22. Preparation of G2B-004(Id)

[0327] G2B-004 was prepared starting with diethylene glycol-MiniAp4 (prepared using the standard coupling method described above, method 4) and 2 equivalents of H2N-Val-Cit-PAB-SN38 (prepared as in Example 21), using PyBOP (4 equivalents), HOAt (12 equivalents) dissolved in DMF (1-3 mL / g resin), followed by the addition of DIEA (12 equivalents). The mixture was reacted for 1 h with intermittent manual stirring. The solvent was removed by suction and the resin was washed with DMF (5 × 30 s) and DCM (5 × 30 s). The coupling reaction was performed twice (1 h and overnight).

[0328] G2B-004 was then cut and lyophilized. Product characterization. Reversed-phase UPLC: linear gradient from 0 to 100% MeCN in H2O solution over 2 min, using an Acquity BEH C18 (50 × 2 mm × 1.7 μm) column at 0.61 mL / min, with MeCN (0.036% TFA) and H2O (0.045% TFA) as solvents; retention time: 1.17 min. UPLC-MS [M+H] exp + 1807.84 Da. Yield (synthesis and purification): 21%.

[0329] Example 23. Preparation of G2B-005(Ie)

[0330] Nval-Pro-Gly-MiniAp4 was prepared using the standard method described above (coupling method 4).

[0331] G2B-005 was prepared starting with Nval-Pro-Gly-MiniAp4 and 2 equivalents of SN38-O-CO-NH-Gly-OH (prepared as in Example 19), using PyBOP (4 equivalents), HOAt (12 equivalents) dissolved in DMF (1-3 mL / g resin), followed by the addition of DIEA (12 equivalents). The mixture was reacted for 1 h with intermittent manual stirring. The solvent was removed by suction and the resin was washed with DMF (5 × 30 s) and DCM (5 × 30 s). The coupling reaction was performed twice (1 h and overnight).

[0332] The G2B-005 was then cut and lyophilized. Product characterization. Reversed-phase UPLC: linear gradient from 0 to 100% MeCN in H2O solution over 2 min, using an Acquity BEH C18 (50 × 2 mm × 1.7 μm) column at 0.61 mL / min, with MeCN (0.036% TFA) and H2O (0.045% TFA) as solvents; retention time: 1.51 min. UPLC-MS [M+H] exp + 1640.77 Da. Yield (synthesis and purification): 18%.

[0333] Example 24. Stability of G2B-001 linear (Ib) in human plasma at 37°C in vitro relative to time.

[0334] In order to evaluate G2B-001 Linear (Ib) (Example 16) Stability in human plasma at 37°C: The compound was prepared in triplicate at 200 μM in plasma samples at each time point (0 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, and 24 h). Once incubation was complete, plasma proteins were immediately precipitated by adding cold methanol (ratio 4:1, methanol:plasma) and vortexing the tubes for a few seconds. The samples were centrifuged at 3,000 rpm for 30 min at 4°C, the supernatant was collected, transferred to a sample plate, and injected into an LC-MS system. Compound recoveries were determined by comparing the analyte response in the analyte-spiked and treated biological sample with the response in the analyte-spiked and treated methanol sample.

[0335] Plasma stability was determined using blank plasma from untreated volunteers. K2-EDTA was used as an anticoagulant. The human blank plasma used was obtained from Hospital Sant Pau.

[0336] The half-life of G2B-001 linear (Ib) in humans at 200 μM is longer than 24 hours.

[0337] ( Figure 26 ).

[0338] Table 7. Percentage recovery of G2B-001 linear (Ib) in human plasma after 24 h.

[0339] G2B001 linear % recovery rate people 69.9

[0340] Example 25. A novel linear compound (Ib) named G2B-001 targets diffuse endogenous pontine glial cells in cancer cell lines. Antitumor activity of sclerotum

[0341] The inventors compared the activity of the novel compound (Ib) G2B-001 linear (Example 16) with that of one of the closely related drugs, G2B-001 (Ia) and SN38.

[0342] The cancer cell line used in these experiments was HSJD-DIPG-007. In short, 3000 cancer cells were cultured in 96-well plates and, after 24 hours, were exposed to compounds G2B-001 (Ia), G2B-001 linear (Ib), and SN-38 (each at concentrations ranging from 1 to 0.001 μM). Cell viability after 72 hours of incubation with the drugs was determined using MTS assays. G2B-001 (Ia) and G2B-001 linear (Ib) showed similar activity. Figure 27 Therefore, after chemical modification of compound G2B-001(Ib), the in vitro activity of compound G2B-001(Ia) was maintained. As an internal reference for the experiments, SN-38 showed activity similar to that reported in Example 9. The determined IC50 values ​​are shown in Table 8.

[0343] Table 8. IC50 values ​​(μM) of compounds G2B-001 (Ia), G2B-001 linear (Ib), and SN38.

[0344] cell lines G2B-001(Ia) G2B-001(Ib) SN-38 HSJD-DIPG-007 0.0278 0.0368 0.000739

[0345] Example 26. G2B-003(Ic) and irinotecan in patient-derived neuroblastoma xenografts The antitumor activity of the tumor cells was compared. The tumor model used was HSJD-NB-013.

[0346] We subcutaneously inserted freshly excised tumors (obtained from one mouse) into the lateral ventral regions of three athymic nude mice. Post-implantation, tumor volumes ranged from 100 to 500 mm. 3 Mice were treated with irinotecan, G2B-003 (Ib), or saline solution (control) via intravenous injection. One mouse received 10 doses of irinotecan at 10 mg / kg on days 1, 2, 3, 4, 5, 8, 9, 10, 11, and 12. One mouse received 6 doses of G2B-003 (Ib) at 100 mg / kg on days 1, 2, 3, 4, 5, and 8. One mouse received saline solution using the same regimen as irinotecan. Tumor volume was measured until day 44 or if the volume was greater than 1500 mm². 3Irinotecan and G2B-003 treatment achieved measurable and durable antitumor responses. Figure 28 The control tumor grew faster. Therefore, in this tumor model, irinotecan and G2B-003(Ic) showed similar in vivo activities.

[0347] List of cited references

[0348] Patent documents

[0349] US8299089B2

[0350] WO2015 / 051307A1

[0351] WO2017 / 113687A2

[0352] WO2018 / 064683A1

[0353] WO2015 / 001015A1

[0354] Non-patent literature

[0355] F.Koizumi et al. in "Novel SN38-incorporating polymeric micelles,NK012, eradicate vascular endothelial growth factor-secreting bulky tumors", CancerRes2006, vol.66(20)pp.10048-56

[0356] F Meyer-Losic et al. in "DTS-108, A novel Peptidic profurg of DN38: In vivoEfficacy and Toxicokinetic Studies", Clinical Cancer Research 2008, vol.14, issue 7, pp.2145-2153

[0357] B.Oller et al., "MiniAp-4: A Venom-Inspired Peptidomimetic for BrainDelivery"; Angew Chem Int Ed 2016, vol.55, pp.572-575

[0358] M.Amblard,Eds.,"Methods and Protocols of Modern Solid-Phase Peptide Synthesis”Molecular Biotechnology 2006,Vol.33,pp.239-254

[0359] JHLee.Eur.J.Biochem.2001.vol.268.pp.2004-2012

[0360] MJHatfield and PMPotter,Exp.Opin.Ther.Pat.2011,21(8):1159-1171

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[0363] A.Madder Chem.,Eur.J.Org.Chem.1999,pp.2787-2791

[0364] Greene's Protective Groups in Organic Synthesis,Protective Effects.PeterG.M.Wuts.2014John Wiley&Sons,Inc

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[0366] here J.Org.Chem.1998,63,vol.20,pp.6878-6885

[0367] MTNguyen Chem.,J.Org.Chem.1998,63,vol.20,pp.6878-6885

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[0369] https: / / www.cliffsnotes.com / study-guides / chemistry / organic-chemistry-ii / aldehydes-and-ketones / reactions-of-aldehydes-and-ketones

[0370] SFMvan Dongen et al.; Bioconjugate Chem.2009, vol.20, pp.20-23.https: / / www.lumiprobe.com / protocols / click-chemistry-dna-labelingMol.Pharmaceutics,2016,13,379-390

[0371] M. Azzolini et al., Eur J Pharm Biopharm, 2017, vol. 115, pp. 149-158.

[0372] S.-J. et al. J. Med. Chem. 2008, vol. 51, pp. 6916-6926. sequence list <110> The International Research Foundation for Biomedical Research (IRB Barcelona) University of Barcelona San Juan Deshen Hospital <120> SN38 peptide conjugates that can be used to treat cancer <130> P5482EP00 <150> EP20382854.6 <151> 2020-09-28 <160> 14 <170> PatentIn version 3.5 <210> 1 <211> 9 <212> PRT <213> Artificial sequence <220> <223> peptides <220> <221> misc_feature <222> (1)..(1) <223> Xaa is selected from the group consisting of Dpr and Dbu, and it is bound to the 9th amino acid via a peptide lactam bond. <220> <221> misc_feature <222> (9)..(9) <223> Xaa is selected from the group consisting of Asp and Glu, and it is bound to the first amino acid via a peptide lactam bond. <400> 1 Xaa Lys Ala Pro Glu Thr Ala Leu Xaa 1 5 <210> 2 <211> 12 <212> PRT <213> Artificial sequence <220> <223> peptides <220> <221> misc_feature <222> (1)..(1) <223> Xaa is the same amino acid as the 9th amino acid, and it is selected from cysteine, selenocysteine, and penicillamine. <220> <221> misc_feature <222> (1)..(1) <223> It binds to the 9th amino acid via intrapeptide disulfide bonds or diselenide bonds. <220> <221> misc_feature <222> (9)..(9) <223> Xaa is the same amino acid as the first amino acid, and it is selected from cysteine, selenocysteine, and penicillamine. <220> <221> misc_feature <222> (9)..(9) <223> It binds to the amino acid at position 1 via intrapeptide disulfide bonds or diselenide bonds. <400> 2 Xaa Lys Ala Pro Glu Thr Ala Leu Xaa Ala Ala Ala 1 5 10 <210> 3 <211> 9 <212> PRT <213> Artificial sequence <220> <223> peptides <220> <221> misc_feature <222> (1)..(1) <223> Xaa is the same amino acid as the 9th amino acid, and it is selected from cysteine, selenocysteine, and penicillamine. <220> <221> misc_feature <222> (1)..(1) <223> It binds to the 9th amino acid via intrapeptide disulfide bonds or diselenide bonds. <220> <221> misc_feature <222> (9)..(9) <223> Xaa is the same amino acid as the first amino acid, and it is selected from cysteine, selenocysteine, and penicillamine. <220> <221> misc_feature <222> (9)..(9) <223> It binds to the amino acid at position 1 via intrapeptide disulfide bonds or diselenide bonds. <400> 3 Xaa Lys Ala Pro Glu Thr Ala Leu Xaa 1 5 <210> 4 <211> 10 <212> PRT <213> Artificial sequence <220> <223> peptides <220> <221> misc_feature <222> (1)..(1) <223> Xaa is the same amino acid as the 9th amino acid, and it is selected from cysteine, selenocysteine, and penicillamine. <220> <221> misc_feature <222> (1)..(1) <223> It binds to the 9th amino acid via intrapeptide disulfide bonds or diselenide bonds. <220> <221> misc_feature <222> (9)..(9) <223> Xaa is the same amino acid as the first amino acid, and it is selected from cysteine, selenocysteine, and penicillamine. <220> <221> misc_feature <222> (9)..(9) <223> It binds to the amino acid at position 1 via intrapeptide disulfide bonds or diselenide bonds. <400> 4 Xaa Lys Ala Pro Glu Thr Ala Leu Xaa Ala 1 5 10 <210> 5 <211> 11 <212> PRT <213> Artificial sequence <220> <223> peptides <220> <221> misc_feature <222> (1)..(1) <223> Xaa is the same amino acid as the 9th amino acid, and it is selected from cysteine, selenocysteine, and penicillamine. <220> <221> misc_feature <222> (1)..(1) <223> It binds to the 9th amino acid via intrapeptide disulfide bonds or diselenide bonds. <220> <221> misc_feature <222> (9)..(9) <223> Xaa is the same amino acid as the first amino acid, and it is selected from cysteine, selenocysteine, and penicillamine. <220> <221> misc_feature <222> (9)..(9) <223> It binds to the amino acid at position 1 via intrapeptide disulfide bonds or diselenide bonds. <400> 5 Xaa Lys Ala Pro Glu Thr Ala Leu Xaa Ala Ala 1 5 10 <210> 6 <211> 16 <212> PRT <213> Artificial sequence <220> <223> peptides <220> <221> misc_feature <222> (1)..(1) <223> Xaa is the same amino acid as the 11th amino acid, and it is selected from the group consisting of cysteine, selenocysteine, and penicillamine. <220> <221> misc_feature <222> (1)..(1) <223> It binds to the 11th amino acid via intrapeptide disulfide bonds or diselenide bonds. <220> <221> misc_feature <222> (3)..(3) <223> Xaa is the same amino acid as the 15th amino acid, and it is selected from the group consisting of cysteine, selenocysteine, and penicillamine. <220> <221> misc_feature <222> (3)..(3) <223> It binds to the amino acid at position 15 via intrapeptide disulfide bonds or diselenide bonds. <220> <221> misc_feature <222> (11)..(11) <223> Xaa is the same amino acid as the first amino acid, and it is selected from the group consisting of cysteine, selenocysteine, and penicillamine. <220> <221> misc_feature <222> (11)..(11) <223> It is linked to the amino acid at position 1 via an intrapeptide disulfide bond or a diselenide bond. <220> <221> misc_feature <222> (15)..(15) <223> Xaa is the same amino acid as the third amino acid, and it is selected from the group consisting of cysteine, selenocysteine, and penicillamine. <220> <221> misc_feature <222> (15)..(15) <223> It is linked to the amino acid at position 3 via an intrapeptide disulfide bond or a diselenide bond. <400> 6 Xaa Asn Xaa Lys Ala Pro Glu Thr Ala Leu Xaa Ala Ala Ala Xaa His 1 5 10 15 <210> 7 <211> 9 <212> PRT <213> Artificial sequence <220> <223> peptides <220> <221> MISC_FEATURE <222> (1)..(1) <223> Xaa is selected from Dpr and Dpu <220> <221> MISC_FEATURE <222> (9)..(9) <223> Xaa is selected from Glu and Asp. <400> 7 Xaa Lys Ala Pro Glu Thr Ala Leu Xaa 1 5 <210> 8 <211> 9 <212> PRT <213> Artificial sequence <220> <223> peptides <220> <221> misc_feature <222> (1)..(1) <223> Xaa represents Dap, and it is linked to the 9th amino acid via a peptide lactam bond. <400> 8 Xaa Lys Ala Pro Glu Thr Ala Leu Asp 1 5 <210> 9 <211> 12 <212> PRT <213> Artificial sequence <220> <223> peptides <220> <221> disulfides <222> (1)..(9) <400> 9 Cys Lys Ala Pro Glu Thr Ala Leu Cys Ala Ala Ala 1 5 10 <210> 10 <211> 9 <212> PRT <213> Artificial sequence <220> <223> peptides <220> <221> disulfides <222> (1)..(9) <400> 10 Cys Lys Ala Pro Glu Thr Ala Leu Cys 1 5 <210> 11 <211> 10 <212> PRT <213> Artificial sequence <220> <223> peptides <220> <221> disulfides <222> (1)..(9) <400> 11 Cys Lys Ala Pro Glu Thr Ala Leu Cys Ala 1 5 10 <210> 12 <211> 11 <212> PRT <213> Artificial sequence <220> <223> peptides <220> <221> disulfides <222> (1)..(9) <400> 12 Cys Lys Ala Pro Glu Thr Ala Leu Cys Ala Ala 1 5 10 <210> 13 <211> 16 <212> PRT <213> Artificial sequence <220> <223> peptides <220> <221> disulfides <222> (1)..(11) <220> <221> disulfides <222> (3)..(15) <400> 13 Cys Asn Cys Lys Ala Pro Glu Thr Ala Leu Cys Ala Ala Ala Cys His 1 5 10 15 <210> 14 <211> 9 <212> PRT <213> Artificial sequence <220> <223> peptides <220> <221> misc_feature <222> (1)..(1) <223> Xaa means Dpr <400> 14 Xaa Lys Wing Pro Glu Thr Wing Leu Asp 1 5

Claims

1. Choose compounds from the following groups: a) Compounds having formula (Ia): (Ia), b) Compounds having formula (Ib): (Ib), c) Compounds having formula (Ic): (Ic), d) Compounds having formula (Id): (Id), e) Compounds having formula (Ie): (Ie).

2. A pharmaceutical composition comprising a therapeutically effective amount of the compound of claim 1 and an appropriate amount of a pharmaceutically acceptable carrier or excipient.

3. Use of the compound of claim 1 in the preparation of a medicament for treating cancers in mammals, wherein the cancer is selected from the group consisting of: adult glioma, pediatric glioma, retinoblastoma, Ewing sarcoma, DIPG, neuroblastoma, and rhabdomyosarcoma.

4. The use as described in claim 3, wherein the mammal is a human.

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