WO2023247510A1 - G-quadruplex ligands for the treatment of liposarcomas - Google Patents

G-quadruplex ligands for the treatment of liposarcomas Download PDF

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Publication number
WO2023247510A1
WO2023247510A1 PCT/EP2023/066590 EP2023066590W WO2023247510A1 WO 2023247510 A1 WO2023247510 A1 WO 2023247510A1 EP 2023066590 W EP2023066590 W EP 2023066590W WO 2023247510 A1 WO2023247510 A1 WO 2023247510A1
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compound
cdk4
cells
treatment
liposarcomas
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PCT/EP2023/066590
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French (fr)
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Sara RICHTER
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Universita' Degli Studi Di Padova
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol

Definitions

  • the present invention originates in the pharmaceutical field and relates in particular to the use of a compound, 2, 7-bis(3-morpholinopropyl)-4-((2-(pyrrolidin-1 - yl)ethyl)amino)-9-(4-(pyrrolidin-1 -yl- methyl) phenyl) benzo[lmn][3,8]phenanthroline- 1 ,3,6,8- (2H,7H)-tetraone, for the treatment of liposarcomas.
  • De-differentiated liposarcomas represent the progression of well-differentiated liposarcomas (WDLS) from an indolent, sometimes locally aggressive, lesion to a faster-growing disease with metastatic potential.
  • Five-year disease survival in patients with DDLS is estimated to be around 44%, compared with 93% in patients diagnosed with pure WDLS.
  • Genomic alterations are more complex in DDLS than in WDLS.
  • Treatment of choice involves surgical removal of the tumor sometimes associated with adjuvant or precautionary chemotherapy, which aims, after surgery, to reduce the risk of recurrence both locally and as metastases.
  • the first line of treatment is usually anthracyclines
  • the second line uses trabectedin and eribulin.
  • Anthracyclines are anticancer drugs developed in the 1960s and nonspecific for liposarcomas. Trabectedin and eribulin are recently developed drugs with complex mechanism of action.
  • Other drugs under study are, for example, nutlines, which inhibit the interaction between the MDM2 protein and p53. However, all of the said drugs have demonstrated numerous limitations, including the easy onset of resistance.
  • the compound of formula (I) is thus found to decrease the levels of MDM2 and CDK4 through interaction with G4s found in the promoters of these genes and which are specifically amplified in liposarcomas, thereby inhibiting the proliferation of tumor cells. Because of this, the compound of formula (I) is effective therapy of liposarcoma, especially WDLS and DDLS liposarcomas.
  • the present invention also relates to the use of the compound of formula (I) as a ligand of a G-quadruplex structure in a promoter of an oncogene selected from MDM2 and CDK4, in the treatment of a pathology related to the amplification of an oncogene selected from MDM2 and CDK4.
  • FIG. 1 schematically shows the Chip-seq analysis with BG4 antibody performed according to Example 1 to identify the sequence of nucleotides potentially forming G4 structures within the CDK4 promoter ( Figure 1 A), in the sense strand ( Figure 1 B);
  • - Figure 3 shows the spectra obtained by circular dichroism (CD) spectroscopy on the CDK4-27, CDK4-52, CDK4-52L, and CDK4-19 sequences obtained in Example 2;
  • - Figure 4 shows the spectra obtained by circular dichroism (CD) spectroscopy on the sequences CDK4-32MBS, CDK4-35, CDK4-46, and CDK4-144 obtained in Example 2;
  • FIG. 9a and 9b show the immunofluorescence assay performed on 93T449 cells according to Example 4.
  • FIG. 10 shows the MTT assay performed with compound (I) and the analogue CM03 in WDLS cells according to Example 5;
  • FIG. 1 1 shows the MTT assay performed with compound (I) and the analogue CM03 in tumor WDLS cells and normal pre-adipocyte cells according to Example 5.
  • FIG. 12 shows the gene expression levels measured at the RNA level by RT-qPCR in 93T449 cells of the oncogenes MDM2, CDK4 and HMGA2 measured in the presence of increasing concentrations of SOP1812 and at different incubation times according to Example 5;
  • FIG. 13 shows the protein expression levels of MDM2, CDK4 and p53 after treatment of 93T449 cells with SOP1812 at different times (4 h, 7 h, 10 h and 20 h) according to Example 5.
  • a-TUBULIN is shown as loading control;
  • FIG. 14 shows yH2AX protein expression levels after treatment of 93T449 cells with SOP1812 at different times (4 h, 7 h, 10 h and 20 h) according to Example 5; yH2AX protein expression, which is the principal DNA damage marker, in liposarcoma cells WDLS after different times of exposure to compound 1 ; GAPDH was used as loading control;
  • FIG. 15 shows the levels of apoptosis activation by staining with Annexin V-FITC after treatment of 93T449 cells at increasing concentrations of SOP1812 and at different incubation times according to Example 5.
  • Data shown represent the mean ⁇ SEM of three independent experiments (*P ⁇ 0.05, **P ⁇ 0.002, ***P ⁇ 0.0002, ****P ⁇ 0.0001 ) using the t tests by GraphPad Prism 9.3.1 and
  • FIG. 16 shows the protein expression levels of apoptotic protein biomarkers such as cleaved PARP1 , inactive (Pro-Cas 3), and effector form of caspase 3 (cleaved Cas 3) after treatment of 93T449 cells with SOP1812 at different times (4 h, 7 h, 10 h and 20 h) according to Example 5; a-TUBULIN is shown as loading control.
  • apoptotic protein biomarkers such as cleaved PARP1 , inactive (Pro-Cas 3), and effector form of caspase 3 (cleaved Cas 3) after treatment of 93T449 cells with SOP1812 at different times (4 h, 7 h, 10 h and 20 h) according to Example 5; a-TUBULIN is shown as loading control.
  • the present invention relates to the use of compound of formula (I) in the treatment of liposarcoma.
  • the compound of formula (I), whose chemical name is 2,7-bis(3-morpholinopropyl)-4- ( (2- (pyrrolidin- 1 -yl)ethyl)amino)-9-(4-(pyrrolidin-1 -yl- methyl) phenyl)benzo[lmn][3,8]phenanthroline-1 ,3,6,8- (2H,7H)-tetraone, has indeed been shown to bind to and stabilize guanine-rich regions that can fold to form G-quadruplex (G4) structures present in the promoters of the MDM2 and CDK4 oncogenes, the amplification of which is characteristic of liposarcomas, particularly the well- differentiated (WDLS) and de-differentiated (DDLS) types.
  • WDLS well- differentiated
  • DDLS de-differentiated
  • the compound of formula (I) is thus found to decrease the levels of MDM2 and CDK4 through interaction with the G4s found in the promoters of these genes and which are specifically amplified in liposarcomas, thereby inhibiting the proliferation of tumor cells. Because of this, the compound of formula (I) is effective therapy of liposarcoma, especially WDLS and DDLS liposarcomas.
  • the present invention may be disclosed in one or more of the aspects disclosed below in the present invention.
  • the compound of formula (I) is suitable for use in the treatment of liposarcomas.
  • said liposarcoma is a well-differentiated liposarcoma (WDLS) or a dedifferentiated liposarcoma (DDLS).
  • WDLS well-differentiated liposarcoma
  • DDLS dedifferentiated liposarcoma
  • an oncogene selected from MDM2 and CDK4 is amplified in said liposarcoma.
  • the compound of formula (I) according to the present invention can be administered by systemic administration, including oral administration and parenteral administration.
  • the compound of formula (I) according to the present invention can be administered once or according to a dosing regimen wherein a number of doses are administered at varying intervals over a given period of time.
  • Suitable dosing regimens for a compound of the invention depend on the pharmacokinetic properties of that compound, such as absorption, distribution, and half-life, which can be determined by the expert.
  • suitable dosage regimens, including the duration for which these regimens are administered, for the compound of the invention depend on the condition being treated, the severity of the condition being treated, the age and physical condition of the patient being treated, the medical history of the patient to be treated, the nature of concurrent therapy, the desired therapeutic effect, and similar factors within the expert's knowledge and experience.
  • the compound of the invention may advantageously, but not necessarily, be formulated into a pharmaceutical composition prior to administration to a patient.
  • the pharmaceutical compositions of the invention are prepared using techniques and methods known to experts in the field.
  • the compound of the invention and the pharmaceutically acceptable excipient(s) are typically formulated in a dosage form adapted for patient administration by the desired route of administration.
  • the compound of formula (I) specifically interacts with G4s formed at the promoters of the MDM2 and CDK4 genes thus resulting in the ability to decrease the levels of MDM2 and CDK4 through interaction with G4s found in the promoters of these genes. Therefore, in a further aspect the present invention further relates to the use of the compound of formula (I) as a ligand of a G-quadruplex structure in a promoter of a gene selected from MDM2 and CDK4, in the treatment of a pathology related to the amplification of a gene selected from MDM2 and CDK4.
  • the purified oligonucleotide was labeled at the 5' end with FITCH (Eurofins Scientifics, Germany), resuspended in 10 mM lithium cacodylate buffer pH 7.4 with or without 100 mM KCI, denatured under heat for 5 min at 95°C and folded at room temperature overnight.
  • the sample solutions were then treated with dymethyl sulfate (DMS, 0.5% in ethanol) for 4 min and blocked by addition of 10% glycerol and [3-mercaptoethanol.
  • DMS dymethyl sulfate
  • the samples were then loaded onto native 15% polyacrylamide gels (19:1 acrylamide/bis solution) and run until the desired resolution was obtained. DNA bands were localized by autoradiography, excised and eluted overnight.
  • Membranes were blocked with 5% skim milk or 5% bovine serum albumin in TBS. The membranes were incubated with the respective primary antibody at 4°C overnight. After three washes in TBST (0.1 % Tween 20 in TBS), the membranes were incubated with the respective HRP-conjugated secondary antibody. Images were captured on the Uvitec.
  • WDLS cells were seeded (2 x 105) in a 6-well plate. After 16 h, cells were treated with SOP1812 for 4, 7 and 10 h as specified. Apoptosis was assessed using the Annexin V-FITC apoptosis detection kit (eBioscience, ThermoFisher Scientific, Monza, Italy) according to the manufacturer's instructions.
  • Example 1 Identification of potential G4 forming sequences (potential G4 forming sequences, PQS) in the CDK4 promoter
  • Figure 1 A shows specifically the results obtained by Chip-seq using BG4 antibody on the CDK4 promoter region.
  • the peak at the immunoprecipitation (IP) of BG4 represents where the putative G4 structure was detected by BG4 Ab (which is a FLAG- labeled single-chain variable fragment antibody (scFv)).
  • the sequence obtained (175 nts), relative to the peak, is shown in the panel below.
  • the CDK4 promoter region (443 bp) including sense and antisense strands is shown in Figure 1 B.
  • red is the 175 nt sequence within the entire CDK4 promoter region (GenBank: AF224272.1 ), which is located in the sense strand.
  • QGRS Mapper was used to identify putative G4s within the 175 nt sequence ( Figure 1 A) obtained from the Chip-seq experiment. This is an online bioinformatics tool that predicts and maps putative G4 sequences. Each identified sequence was associated with a score (G-score) as described in the "Materials and Methods" section above. Three putative standard G4 sequences, named CDK4-27, -52 and -144, were thus identified. In parallel, other sequences potentially forming alternative non- canonical G4 structures within the 175-nt sequence were manually identified, named -52L, -19, -32MBS, -35, -46. The sequences potentially forming G4 structures thus identified are shown in the Table shown in Figure 2.
  • Example 1 To assess whether the putative sequences identified in Example 1 are capable of organizing into a G4 structure, an analysis by circular dichroism (CD) spectroscopy was conducted. The selected sequences were analyzed in the presence of 100 mM KCI (physiological intracellular potassium concentrations) over a wavelength range of 230-320 nm. The starting temperature was set at 20°C with an increase of 5°C/min up to 90°C to assess the melting temperature (Tm).
  • the Tm is an indicator of structure stability that provides information about the two-state transition from a folded to an open state of a G4 structure. The higher the Tm, the greater the stability of the G4 structure.
  • the guanine bands showed a lower signal due to protection by DMS (Figure 8).
  • the "M" band corresponds to the CDK4-G4 split at G and A after treatment with formic acid and hot piperidine.
  • DMS and formic acid were instead used to treat CDK4- 52L and the protected guanines were identified in the presence or absence of 100 mM K+.
  • the protected guanines are indicated with a black line.
  • the visible oligonucleotide sequence is shown at the bottom of the figure.
  • Example 5 Test with compound of formula (I) according to the invention in LPS cell line as an anticancer agent.
  • the Compound of formula (I) (“SOP1812”) was tested on well-differentiated human liposarcoma cells (WDLS). First, the cytotoxicity of the compound on cells was evaluated by MTT assay according to the method described in "Materials and Methods "section above. While the compound of formula (I) showed a CC50 of about 3 pM after 24 hours of treatment, said value decreased to about 0.2 pM after 48 hours ( Figure 10 A).

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Abstract

The present invention relates to the use of the compound 2,7-bis(3-morpholinopropyl)- 4-((2-(pyrrolidin-1-yl)ethyl)amino)-9-(4-(pyrrolidin-1-yl- methyl) phenyl)benzo[lmn][3,8]phenanthroline-1,3,6,8- (2H,7H)-tetraone for the treatment of liposarcomas.

Description

G-QUADRUPLEX LIGANDS FOR THE TREATMENT OF LIPOSARCOMAS
DESCRIPTION
FIELD OF THE INVENTION
The present invention originates in the pharmaceutical field and relates in particular to the use of a compound, 2, 7-bis(3-morpholinopropyl)-4-((2-(pyrrolidin-1 - yl)ethyl)amino)-9-(4-(pyrrolidin-1 -yl- methyl) phenyl) benzo[lmn][3,8]phenanthroline- 1 ,3,6,8- (2H,7H)-tetraone, for the treatment of liposarcomas.
STATE OF THE ART
Sarcomas are a rare and heterogeneous type of tumors, predominantly originating from mesenchymal cells. Among the most common forms are bone sarcomas and soft-tissue sarcomas, with about 28,000 new cases per year in Europe (Stiller, C. A. et al. Descriptive epidemiology of sarcomas in Europe: Report from the RARECARE project. Eur. J. Cancer 49, 684-695 (2013)).
Liposarcoma (LPS) is one of the most common types of soft tissue sarcoma (STS), rappresenting 50% of retroperitoneal STS and 25% of STS of the extremities. Liposarcomas are rare malignant tumors that affect fatty or adipose tissue. These neoplasms are very aggressive and tend to recur at the same site of origin or at a distance (metastasis).
There are several separate biological groups of LPS comprising different subhistological subtypes. Each group is characterized by specific genetic alterations that are presumed to drive tumor initiation. Well-differentiated (WDLS) and dedifferentiated (DDLS) liposarcomas account for more than 60% of all liposarcomas and are almost universally associated with cells that contain supernumerary ring or giant marker chromosomes characterized by amplification of chromosomal segment 12q13-15, which carries the oncogenes MDM2(12q15), CDK4(12q14. 1 ) and HMGA2(12q14.3). Currently, the genetic diagnosis of this type of tumor is based on the presence of these amplifications. De-differentiated liposarcomas (DDLS) represent the progression of well-differentiated liposarcomas (WDLS) from an indolent, sometimes locally aggressive, lesion to a faster-growing disease with metastatic potential. Five-year disease survival in patients with DDLS is estimated to be around 44%, compared with 93% in patients diagnosed with pure WDLS. Genomic alterations are more complex in DDLS than in WDLS. Treatment of choice involves surgical removal of the tumor sometimes associated with adjuvant or precautionary chemotherapy, which aims, after surgery, to reduce the risk of recurrence both locally and as metastases. For cases in which the tumor, because of its location, is not removable, the first line of treatment is usually anthracyclines, the second line uses trabectedin and eribulin. Anthracyclines are anticancer drugs developed in the 1960s and nonspecific for liposarcomas. Trabectedin and eribulin are recently developed drugs with complex mechanism of action. Other drugs under study are, for example, nutlines, which inhibit the interaction between the MDM2 protein and p53. However, all of the said drugs have demonstrated numerous limitations, including the easy onset of resistance.
Therefore, the need to find new treatments for liposarcomas, especially for liposarcomas of the well-differentiated and dedifferentiated types, is pressing and strongly felt.
SUMMARY OF THE INVENTION
An object of the present invention, therefore, is the identification of new compounds capable of being used in the treatment of liposarcomas, especially for liposarcomas of the well-differentiated and de-differentiated types.
The present invention therefore relates to the use of compound of formula (I)
Figure imgf000003_0001
in the treatment of a liposarcoma.
The compound of formula (I), whose chemical name is 2,7-bis(3-morpholinopropyl)-4- ((2-(pyrrolidin-1 -yl)ethyl)amino)-9-(4-(pyrrolidin-1 -yl-methyl) phenyl)benzo[lmn][3,8]phenanthroline-1 ,3,6,8- (2H,7H)-tetraone, has indeed been shown to bind to and stabilize guanine-rich regions that can fold to form G-quadruplex (G4) structures present in the promoters of the MDM2 and CDK4 oncogenes, the amplification of which is characteristic of liposarcomas, particularly the well- differentiated (WDLS) and de-differentiated (DDLS) types. The compound of formula (I) is thus found to decrease the levels of MDM2 and CDK4 through interaction with G4s found in the promoters of these genes and which are specifically amplified in liposarcomas, thereby inhibiting the proliferation of tumor cells. Because of this, the compound of formula (I) is effective therapy of liposarcoma, especially WDLS and DDLS liposarcomas.
The fact that the compound of formula (I) specifically interacts with G4s that are formed at the promoters of the MDM2 and CDK4 genes makes it specific and noncritical in terms of toxicity to other cellular targets, thus further increasing the therapeutic utility of the compound itself.
In a further aspect, the present invention also relates to the use of the compound of formula (I) as a ligand of a G-quadruplex structure in a promoter of an oncogene selected from MDM2 and CDK4, in the treatment of a pathology related to the amplification of an oncogene selected from MDM2 and CDK4.
BRIEF DESCRIPTION OF FIGURES
In the drawings:
- Figure 1 schematically shows the Chip-seq analysis with BG4 antibody performed according to Example 1 to identify the sequence of nucleotides potentially forming G4 structures within the CDK4 promoter (Figure 1 A), in the sense strand (Figure 1 B);
- Figure 2 shows the Table showing the sequences potentially forming G4 structures identified in Example 1 ;
- Figure 3 shows the spectra obtained by circular dichroism (CD) spectroscopy on the CDK4-27, CDK4-52, CDK4-52L, and CDK4-19 sequences obtained in Example 2; - Figure 4 shows the spectra obtained by circular dichroism (CD) spectroscopy on the sequences CDK4-32MBS, CDK4-35, CDK4-46, and CDK4-144 obtained in Example 2;
- Figure 5 shows the base-pair sequence named CDK4-llb amplified in Example 3;
- Figure 6 shows the PCR stop assay according to Example 3;
- Figure 7 shows the comparison of the identified G4 structures and their correlation with the pausing found in the amplification of the CDK4-llb fragment according to Example 3;
- Figure 8 shows the bands obtained in the dimethyl sulfate footprinting assay (DMS) according to Example 3;
- Figures 9a and 9b show the immunofluorescence assay performed on 93T449 cells according to Example 4;
- Figure 10 shows the MTT assay performed with compound (I) and the analogue CM03 in WDLS cells according to Example 5;
A) Cell viability assay of compound 1 at 24 and 48 h of treatment.
B) Cell viability assay of compound 1 , one analogue of compound 1 (CM03) and two unrelated G4 ligands (PhenDC3 and PDS) at 24 of treatment. Data shown represent the mean ± SEM of three independent experiments;
- Figure 1 1 shows the MTT assay performed with compound (I) and the analogue CM03 in tumor WDLS cells and normal pre-adipocyte cells according to Example 5. Cell viability assay of compound 1 (A) and the analogue CM03 (B) at 24 h of treatment on liposarcoma cells WDLS and normal pre-adipocyte cells PCS-210-010;
- Figure 12 shows the gene expression levels measured at the RNA level by RT-qPCR in 93T449 cells of the oncogenes MDM2, CDK4 and HMGA2 measured in the presence of increasing concentrations of SOP1812 and at different incubation times according to Example 5;
- Figure 13 shows the protein expression levels of MDM2, CDK4 and p53 after treatment of 93T449 cells with SOP1812 at different times (4 h, 7 h, 10 h and 20 h) according to Example 5.
A) MDM2 and CDK4 and B) P53 protein expression in liposarcoma cells WDLS at increasing concentration of compound 1 , tested at different time points (4, 7 and 10 hours). a-TUBULIN is shown as loading control;
- Figure 14 shows yH2AX protein expression levels after treatment of 93T449 cells with SOP1812 at different times (4 h, 7 h, 10 h and 20 h) according to Example 5; yH2AX protein expression, which is the principal DNA damage marker, in liposarcoma cells WDLS after different times of exposure to compound 1 ; GAPDH was used as loading control;
- Figure 15 shows the levels of apoptosis activation by staining with Annexin V-FITC after treatment of 93T449 cells at increasing concentrations of SOP1812 and at different incubation times according to Example 5. Data shown represent the mean ± SEM of three independent experiments (*P < 0.05, **P < 0.002, ***P < 0.0002, ****P < 0.0001 ) using the t tests by GraphPad Prism 9.3.1 and
- Figure 16 shows the protein expression levels of apoptotic protein biomarkers such as cleaved PARP1 , inactive (Pro-Cas 3), and effector form of caspase 3 (cleaved Cas 3) after treatment of 93T449 cells with SOP1812 at different times (4 h, 7 h, 10 h and 20 h) according to Example 5; a-TUBULIN is shown as loading control.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to the use of compound of formula (I)
Figure imgf000006_0001
in the treatment of liposarcoma.
The compound of formula (I), whose chemical name is 2,7-bis(3-morpholinopropyl)-4- ( (2- (pyrrolidin- 1 -yl)ethyl)amino)-9-(4-(pyrrolidin-1 -yl- methyl) phenyl)benzo[lmn][3,8]phenanthroline-1 ,3,6,8- (2H,7H)-tetraone, has indeed been shown to bind to and stabilize guanine-rich regions that can fold to form G-quadruplex (G4) structures present in the promoters of the MDM2 and CDK4 oncogenes, the amplification of which is characteristic of liposarcomas, particularly the well- differentiated (WDLS) and de-differentiated (DDLS) types. The compound of formula (I) is thus found to decrease the levels of MDM2 and CDK4 through interaction with the G4s found in the promoters of these genes and which are specifically amplified in liposarcomas, thereby inhibiting the proliferation of tumor cells. Because of this, the compound of formula (I) is effective therapy of liposarcoma, especially WDLS and DDLS liposarcomas.
The fact that the compound of formula (I) specifically interacts with G4s and that G4s formed at the level of the MDM2 and CDK4 gene promoters are amplified in WDLSe DDLS, makes it specific and non-critical in terms of toxicity to other cellular targets, thus further increasing the therapeutic utility of the compound itself. The present invention may be disclosed in one or more of the aspects disclosed below in the present invention.
The compound of formula (I) is suitable for use in the treatment of liposarcomas. Preferably, said liposarcoma is a well-differentiated liposarcoma (WDLS) or a dedifferentiated liposarcoma (DDLS).
In a preferred embodiment, an oncogene selected from MDM2 and CDK4 is amplified in said liposarcoma.
The compound of formula (I) according to the present invention can be administered by systemic administration, including oral administration and parenteral administration.
The compound of formula (I) according to the present invention can be administered once or according to a dosing regimen wherein a number of doses are administered at varying intervals over a given period of time.
Suitable dosing regimens for a compound of the invention depend on the pharmacokinetic properties of that compound, such as absorption, distribution, and half-life, which can be determined by the expert. In addition, suitable dosage regimens, including the duration for which these regimens are administered, for the compound of the invention depend on the condition being treated, the severity of the condition being treated, the age and physical condition of the patient being treated, the medical history of the patient to be treated, the nature of concurrent therapy, the desired therapeutic effect, and similar factors within the expert's knowledge and experience. The compound of the invention may advantageously, but not necessarily, be formulated into a pharmaceutical composition prior to administration to a patient. The pharmaceutical compositions of the invention are prepared using techniques and methods known to experts in the field.
The pharmaceutical compositions of the invention can be prepared and packaged in bulk form wherein an effective amount of the compound of the invention can be extracted and then given to the patient as with powders, syrups and solutions for injection. Alternatively, the pharmaceutical compositions of the invention may be prepared and packaged in unit dosage form. One dose of the pharmaceutical composition contains at least one therapeutically effective amount of the compound of this invention.
The compound of the invention and the pharmaceutically acceptable excipient(s) are typically formulated in a dosage form adapted for patient administration by the desired route of administration.
Conventional dosage forms include those adapted for (1 ) oral administration such as tablets, capsules, oval tablets, pills, lozenges, powders, syrups, elixirs, suspensions, solutions, emulsions, sachets, and caches; (2) parenteral administration such as solutions, suspensions, and sterile reconstruction powders.
Pharmaceutically suitable excipients include the following types of excipients: diluents, fillers, binders, disintegrants, lubricants, granulating agents, coating agents, wetting agents, suspending agents, emulsifiers, sweeteners, flavor-masking agents, coloring agents, anti-caking agents, humectants, plasticizers, viscosity-enhancing agents, antioxidants, preservatives, stabilizers, surfactants, and buffering agents. Suitable diluents and fillers include lactose, sucrose, dextrose, mannitol, sorbitol, starch, cellulose, calcium sulfate, and dibasic calcium phosphate. The oral solid dosage form may also include a binder. Suitable binders include starch, gelatin, sodium alginate, alginic acid, guar gum, povidone, and cellulose and its derivatives (e.g., microcrystalline cellulose). The oral solid dosage form may also include a disintegrant. Suitable disintegrants include crospovidone, sodium starch glycolate, alginic acid, and carboxymethyl cellulose sodium. The oral solid dosage form may also include a lubricant. Suitable lubricants include stearic acid, magnesium stearate, calcium stearate, and talc. Suitable vehicle lubricants for oral dosage form include, but are not limited to, magnesium carbonate, magnesium stearate, talc, lactose, pectin, dextrin, starch, methylcellulose, sodium carboxymethyl cellulose, and the like. The techniques used to prepare oral formulations are conventional mixing, granulation, and capsule compression or filling.
The compound of formula (I) according to the present invention can also be formulated for parenteral administration with suitable carriers, including aqueous vehicle solutions (i.e.: saline, dextrose) and/or oily emulsions.
The compound of formula (I) specifically interacts with G4s formed at the promoters of the MDM2 and CDK4 genes thus resulting in the ability to decrease the levels of MDM2 and CDK4 through interaction with G4s found in the promoters of these genes. Therefore, in a further aspect the present invention further relates to the use of the compound of formula (I) as a ligand of a G-quadruplex structure in a promoter of a gene selected from MDM2 and CDK4, in the treatment of a pathology related to the amplification of a gene selected from MDM2 and CDK4.
Further features and advantages of the invention will emerge more clearly from the following description of some related preferred forms of embodiment, produced below in the present by way of non-limiting example with reference to the following illustrative examples.
EXPERIMENTAL PART
Materials and Methods
Cell lines, oligonucleotides and compounds
Human WD-Liposarcoma cell line 93T449 (ATCC® CRL-3043™) was cultured in RPMI medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS. Desalted oligonucleotides were purchased from Sigma-Aldrich (Milan, Italy).
The compound 2,7-bis(3-morpholinopropyl)-4-((2-(pyrrolidin-1 -yl)ethyl)amino)-9-(4- (pyrrolidin-1 -yl- methyl) phenyl) benzo[lmn][3,8]phenanthroline-1 ,3,6,8- (2H,7H)- tetraone("SOP1812") of formula (I)
Figure imgf000010_0001
was synthesized according to the method described in the Supporting Information of the publication Ahmed A. Ahmed et al. "Asymmetrically Substituted Quadruplex- Binding Naphthalene Diimide Showing Potent Activity in Pancreatic Cancer Models," ACS Med. Chem. Lett. 2020, 1 1 , 8, 1634-1644, https://doi.Org/10.1021/acsmedchemlett.0c00317.
Bioinformatics analysis by QGRS Mapper
The promoter region of the human CDK4 gene was analyzed by QGRS Mapper (//bioinformatics. ramapo.edu/QGRS/index.php) for prediction of the number of G4- forming sequences of guanine (G) tetrads and Ny the length of the rings connecting the G 124 tetrads. The following restrictions were applied: i) the number of tetrads had to be > 2; ii) the maximum length of the QGRS was set at 40 bases; iii) at most one of the loops could be zero in length; iv) loop size from 0 to 15. The QGRSs found were ranked according to G-score, which is the probability of forming a stable G4, according to the following principles: a) shorter loops are more common than longer loops; b) G4s tend to have loops of roughly equal size; c) the higher the number of G tetrads, the more stable the G4.
Circular dichroism (CD) spectroscopy
To evaluate the folding of the tested DNA oligonucleotides into G4 structures and determine their conformation and melting temperature, CD spectroscopy was performed. The oligonucleotides were diluted to a final concentration of 4 pM in lithium cacodylate buffer (10 mM, pH 7.4) with or without KCL After thermal denaturation at 95°C for 5 min, the samples were folded at room temperature for overnight. CD spectra were recorded on a Chirascan-Plus (Applied Photophysics, Leatherhead, UK) equipped with a Peltier temperature controller using a quartz cell with an optical path length of 5 mm. The temperature was set at 20°C, and spectra were recorded over a wavelength range of 230-320 nm. For Tm determination, spectra were recorded over a temperature range of 20-90°C, with a temperature rise of 5°C/min. Tm values were calculated according to the van't Hoff equation, applied for a two-state transition from a folded to an unfolded state. The acquired spectra were corrected at baseline for the signal contribution due to the buffer, and the observed ellipticities were converted to mean residual ellipticity (9) = degrees x cm2 x dmol-1 (molar ellipticity).
Dimethyl sulfate footprinting (DMS)
The purified oligonucleotide was labeled at the 5' end with FITCH (Eurofins Scientifics, Luxembourg), resuspended in 10 mM lithium cacodylate buffer pH 7.4 with or without 100 mM KCI, denatured under heat for 5 min at 95°C and folded at room temperature overnight. The sample solutions were then treated with dymethyl sulfate (DMS, 0.5% in ethanol) for 4 min and blocked by addition of 10% glycerol and [3-mercaptoethanol. The samples were then loaded onto native 15% polyacrylamide gels (19:1 acrylamide/bis solution) and run until the desired resolution was obtained. DNA bands were localized by autoradiography, excised and eluted overnight. Supernatants were recovered, precipitated with ethanol and treated with 1 M piperidine for 30 min at 90°C. The samples were speed-vac dried, washed with water, dried again and resuspended in formamide gel loading buffer. Reaction products were analyzed on 20% denaturing polyacrylamide gels (19:1 acrylamide/bis solution) containing 8 M urea, visualized by fluorescence analysis on a TyphoonFLA 9000 (GE Healthcare Europe, Milan, Italy) and quantified by ImageQuant TL software30 (GE Healthcare Europe, Milan, Italy).
MTT cytotoxicity assay
The cytotoxicity of compound of formula (I) was determined by MTT assay. 10000 cells/well were plated in a 96-well plate and grown for 24 hours. Serial dilutions of compounds were dispensed to the cells in triplicate. Twenty-four hours after treatment, the cells were supplemented with freshly diluted (5 mg/mL) 3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyltetrazolium bromide solution (MTT) (Sigma-Aldrich, Milan, Italy) and incubated 4 h at 37°C. MTT crystals were solubilized by the addition of 100 pL of solubilization solution (10% SDS and 0.01 M HCI) O/N h at 37°C, and absorbance was measured by Sunrise Tecan plate reader (Mannendorf, Switzerland) at 570 nm. The cytotoxic concentration of 50% (CC50) was defined as the concentration of compound required to reduce cell growth by 50%.
PCR arrest assay
Genomic DNA from 93T449 cells was extracted and purified using the Quick-DNA Universal Kit (Zymo Research # ZRC186999). 100 pmol of primer pairs covering the entire MDM2 and CDK4 promoter regions were labeled at the 5' end with [y-32P]ATP by polynucleotide kinase T4 for 30 min at 37°C and purified using MicroSpin G-25 columns (GE Healthcare Europe, Milan, Italy). PCR amplification of 600 ng of genomic DNA was carried out in the presence of 15 pmol radiolabeled forward and reverse primers, respectively, and an equivalent amount of the complementary primer, with the following cycling conditions: 1 x5 min 95°C, 35x 30 sec 95°C - 45 sec 56°C - 60 sec 72°C. Part of the PCR product was subjected to Maxam and Gilbert treatment for visualization of Gs and As bases. The G+A markers, intact primers and PCR product were then loaded onto 10% denaturing polyacrylamide gel (acrylamide/bis solution 19:1 ) containing 7 M urea and visualized by phosphorimaging analysis on a Typhoon FLA 9000 (GEHealthcare Europe, Milan, Italy).
Real-time PCR
93T449 cells were seeded in 6 wells and treated as indicated. Total RNA was isolated using TRIzol reagent (Life Technologies, Monza, Italy) according to the manufacturer's instructions and treated with DNase I without RNase (Ambion Turbo DNA free, Life Technologies, Monza, Italy). The extracted RNA (500 ng) was subjected to reverse transcription and then real time PCR using SYBr Green PCRMaster Mix (Applied Byosystems). Experiments were performed using the LightCycler 480 system (Roche) under the following conditions: 95 °C for 10 min followed by 45 cycles of 1 min at 95 °C and 30 s at 60 °C, 30 s min at 72 °C. The mRNA transcript levels were standardized against GAPDH and ACTB gene. Untreated retrotranscribed RNA was used as the mRNA-expressed control. Each sample was analyzed in duplicate.
Confocal fluorescence microscopy
WDLS cells were seeded (6 x 104) on coverslips in a 12-well plate. After 16 h, the cells were fixed with 4% paraformaldehyde for 10 min permeabilized with 0.5% Triton-X for 10 min. Then, the cells were treated with RNase A (40 pg/ml) for 1 hr at 37°C and consequently blocked for 1 hr with 2% bovine serum albumin. Incubation with BG4 (1 :100 dilution, Merck KGaA, Darmstadt, Germany) was conducted at 37°C for 1 hour and then was detected by murine anti-FLAG antibody (1 :2000 dilution, Merck KGaA, Darmstadt, Germany) for 1 hour at 37°C. Final incubation of the antibody with antimouse Alexa Fluor-488 (1 :1000 dilution, ThermoFisher Scientific, Monza, Italy) was conducted at room temperature for 1 hour. TOTO-3 was used as nuclear staining, and coverslips were mounted with Glycergel (Agilent Technologies, Inc., Santa Clara, USA). All washes were performed in 1 xPBS - 0.1 % Tween-20.
Western blotting
93T449 cells were seeded in 6-well plates, cultured overnight and then treated as indicated. Cells were lysed in RIPA buffer (NaCI 150 mM, IGEPAL 1 %, SDS 0.1 %, Tris-HCI pH 7.5 50 mM, Na-deoxycholate 0.5%). Protein concentration was quantified using the Pierce® BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA) and samples were stored at -80°C. Each sample was electrophoresed on 10% SDS-PAGE and transferred to an absorbent nitrocellulose membrane (Amersham TM Protan TM, GE Healtcare Lifescience, Milan, Italy) by Mini trans-blot Cell (Bio-Rad Laboratories, Milan, Italy). Membranes were blocked with 5% skim milk or 5% bovine serum albumin in TBS. The membranes were incubated with the respective primary antibody at 4°C overnight. After three washes in TBST (0.1 % Tween 20 in TBS), the membranes were incubated with the respective HRP-conjugated secondary antibody. Images were captured on the Uvitec.
Annexin V-FITC Assay
WDLS cells were seeded (2 x 105) in a 6-well plate. After 16 h, cells were treated with SOP1812 for 4, 7 and 10 h as specified. Apoptosis was assessed using the Annexin V-FITC apoptosis detection kit (eBioscience, ThermoFisher Scientific, Monza, Italy) according to the manufacturer's instructions.
Example 1. - Identification of potential G4 forming sequences (potential G4 forming sequences, PQS) in the CDK4 promoter
By Chip-seq analysis with BG4 antibody, a 175 nucleotide (nt) sequence potentially forming G4 structures within the CDK4 promoter (Figure 1A), in the sense strand (Figure 1 B), was identified.
Figure 1 A shows specifically the results obtained by Chip-seq using BG4 antibody on the CDK4 promoter region. The peak at the immunoprecipitation (IP) of BG4 represents where the putative G4 structure was detected by BG4 Ab (which is a FLAG- labeled single-chain variable fragment antibody (scFv)). The sequence obtained (175 nts), relative to the peak, is shown in the panel below. In contrast, the CDK4 promoter region (443 bp) including sense and antisense strands is shown in Figure 1 B. In red is the 175 nt sequence within the entire CDK4 promoter region (GenBank: AF224272.1 ), which is located in the sense strand.
To identify putative G4s within the 175 nt sequence (Figure 1 A) obtained from the Chip-seq experiment, QGRS Mapper was used. This is an online bioinformatics tool that predicts and maps putative G4 sequences. Each identified sequence was associated with a score (G-score) as described in the "Materials and Methods" section above. Three putative standard G4 sequences, named CDK4-27, -52 and -144, were thus identified. In parallel, other sequences potentially forming alternative non- canonical G4 structures within the 175-nt sequence were manually identified, named -52L, -19, -32MBS, -35, -46. The sequences potentially forming G4 structures thus identified are shown in the Table shown in Figure 2.
Example 2 - Biophysical characterization of the G4 structures identified in the CDK4 promoter
To assess whether the putative sequences identified in Example 1 are capable of organizing into a G4 structure, an analysis by circular dichroism (CD) spectroscopy was conducted. The selected sequences were analyzed in the presence of 100 mM KCI (physiological intracellular potassium concentrations) over a wavelength range of 230-320 nm. The starting temperature was set at 20°C with an increase of 5°C/min up to 90°C to assess the melting temperature (Tm). The Tm is an indicator of structure stability that provides information about the two-state transition from a folded to an open state of a G4 structure. The higher the Tm, the greater the stability of the G4 structure. The results (Figures 3 and 4) showed that 4 out of 8 sequences did not assume a classical G4 topology, while the remaining sequences showed CD spectra with parallel topology (characterized by a negative peak at 240 nm and a positive peak at 260 nm), as shown schematically in Table 1 below.
Figure imgf000015_0001
Table 1
Example 3 - Experimental verification of polymerase stalling caused by the presence of the G4 structures characterized in Example 2 To experimentally verify that the presence of the G4 structures identified in Example 2 causes polymerase stalling, genomic DNA was extracted from 93T449 cells characterized by CDK4 gene amplification. The extracted double-stranded DNA was then subjected to standard PCR according to the method described in the "Materials and Methods" section above to test whether the template G-rich strand could induce G4-mediated stalling during polymerase amplification.
Since no more than 200 base pairs per sample can be resolved at the single nucleotide level with this approach (Patel, R. B. et al. Recent translational research into targeted therapy for liposarcoma. Stem Cell Investig. 4, 21 -21 (2017)), a 122-base-pair long region, called CDK4-llb, was amplified, which overlaps with the sequence immunoprecipitated from BG4 (Figure 5).
The PCR elongation step was performed at 56°C to allow detection of only the most stable G4s in the CDK4 promoter and to avoid artifacts due to G4 structureindependent effects. The sensitive strand was used as a template for elongation. Several breaks were detected at the G stretches, suggesting the presence of G4 structures causing DNA polymerase stalling (Figure 6 UREA(7M)-PAGE 10% gel images of CDK4-llb with associated stops [indicated by triangle] caused by G4 formation along the sequence). The detected stops coincided with the four parallel sequences previously analyzed by CD spectroscopy according to Example 2 (Figure 7).
The next step was to assess which guanines were involved in the formation of G4 responsible for the DNA polymerase stalling. A dimethyl sulfate footprinting (DMS) assay was performed according to the method described in the "Materials and Methods" section above to identify the guanines involved in the G4 structure. Being DMS a nitrogen alkylator at position 7 of guanines, in the presence of G4 structure it is unable to exert its function on guanines involved in G4 formation. The CDK4-46 sequence was chosen for the test, as it also partly overlapped with the CDK4-52, -52L and -19 sequences. A high signal of bands corresponding to guanines was observed in the absence of K+ ions. In contrast, in the presence of K+ ions, the guanine bands showed a lower signal due to protection by DMS (Figure 8). In Figure 8, the "M" band corresponds to the CDK4-G4 split at G and A after treatment with formic acid and hot piperidine. DMS and formic acid were instead used to treat CDK4- 52L and the protected guanines were identified in the presence or absence of 100 mM K+. The protected guanines are indicated with a black line. The visible oligonucleotide sequence is shown at the bottom of the figure. These results confirmed the ability of the guanine-rich region in CDK4 to fold into G4.
Example 4 - Visualization of G4 structures in well-differentiated human liposarcoma cells and tissues
The anti-G4 BG4 antibody was useful in confocal microscopy to visualize the presence of G4 structures in the nucleus of liposarcoma (LPS) cells. After 93T449 cells were seeded and fixed, they were subjected to immunofluorescence with BG4 antibody. As expected, the data confirmed the presence of G4 structures within the cell nucleus (Figure 9a and 9b), and the signal decreased in the presence of compound of formula (I) ("SOP1812"), an indication confirming its mechanism at the level of G4.
Example 5 - Test with compound of formula (I) according to the invention in LPS cell line as an anticancer agent.
The Compound of formula (I) ("SOP1812") was tested on well-differentiated human liposarcoma cells (WDLS). First, the cytotoxicity of the compound on cells was evaluated by MTT assay according to the method described in "Materials and Methods "section above. While the compound of formula (I) showed a CC50 of about 3 pM after 24 hours of treatment, said value decreased to about 0.2 pM after 48 hours (Figure 10 A).
The same assay was performed with one analogue of compound (I) (CM03) and two unrelated G4 ligands (PhenDC3 and PDS). The two unrelated compounds displayed much lower CC50 at 24 h of treatment (> 50 pM and 41 pM respectively). CM03 had a CC50 of 20 pM, about 7 times less than that of compound (I) (Figure 10B). To check for selectivity towards liposarcoma cells, the two compounds, i.e. compound(l) and CM03 were further tested against normal cells, i.e. normal preadipocytes. CC50 for compound (I) was > 50 pM, while CC50 for CM03 was 48 pM. These data clearly show that compound (I) is specific for liposarcoma cells and that other G4 ligands, even if they have a similar mechanism of action, are much less effective likely because of the lower affinity for the MDM2/CDK4 G4s involved in liposarcoma, or for the higher affinity towards also other cell G4s. To evaluate the effects of compound of formula (I) on the transcription of genes implicated in the pathogenesis of well-differentiated liposarcoma, RTqPCR was performed. 93T449 cells were then treated for 4 h, 7 h and 10 h at increasing concentrations of compound. The data showed that the compound stimulated a decrease in MDM2 mRNA from 4 hours that increased dramatically at 10 hours; after 10 hours of treatment, a decrease in CDK4 expression was also observed. In contrast, the expression of the HMGA2 gene, which is located in the same amplified region as MDM2 and CDK4 but does not have relevant G4s in its promoter, was not changed (Figure 12). The decrease in gene expression was also assessed at the protein level: the MDM2 protein demonstrated a decrease, but with a wave-like pattern, characteristic of the fact that this protein undergoes positive feedback from the p53 protein, which in turn is regulated by MDM2 (Figure 13A). The CDK4 protein demonstrated a decrease that increased as treatment time increased. At the same time, a drastic increase was observed in p53 whose expression leads the cell into apoptosis (Figure 13B). The decrease in p53 levels did not depend on G4-mediated DNA damage, as assessed by the expression of yH2AX protein, the principal marker for DNA damage: yH2AX was expressed only after 10 h of treatment with compound (I), hence this mechanism cannot respond for the observed decrease in p53 levels (Figure 14). A drastic increase in positive cells was also observed upon staining with Annexin-V-FITC, which, by binding to phosphatidylserine, indicated cells going into apoptosis (Figure 15), confirming what was observed with p53. The triggering of the apoptotic pathway upon treatment with compound (I) was further demonstrated by the increase in cleaved PARP1 and Cas3 levels and decrease in Pro-Cas 3 (Figure 16).

Claims

1 . A compound of formula (I)
Figure imgf000019_0001
for use in the treatment of a liposarcoma.
2. The compound of formula (I) for use according to claim 1 , wherein said liposarcoma is a well-differentiated liposarcoma or a de-differentiated liposarcoma.
3. The compound of formula (I) for use according to claim 1 or 2, wherein in said liposarcoma an oncogene selected from MDM2 and CDK4 is amplified.
4. A compound of formula (I)
Figure imgf000019_0002
for use as a ligand of a G-quadruplex structure in a promoter of an oncogene selected from MDM2 and CDK4, in the treatment of a disease related to amplification of an oncogene selected from MDM2 and CDK4.
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Publication number Priority date Publication date Assignee Title
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Title
AHMED A. AHMED ET AL.: "Asymmetrically Substituted Quadruplex-Binding Naphthalene Diimide Showing Potent Activity in Pancreatic Cancer Models", ACS MED. CHEM. LETT., vol. 11, no. 8, 2020, pages 1634 - 1644, XP093012276, Retrieved from the Internet <URL:https://doi.org/10.1021/acsmedchem!ett.0c00317> DOI: 10.1021/acsmedchemlett.0c00317
LAGO SARA ET AL: "The MDM2 inducible promoter folds into four-tetrad antiparallel G-quadruplexes targetable to fight malignant liposarcoma", vol. 49, no. 2, 7 January 2021 (2021-01-07), GB, pages 847 - 863, XP093012737, ISSN: 0305-1048, Retrieved from the Internet <URL:http://academic.oup.com/nar/article-pdf/49/2/847/36085125/gkaa1273.pdf> DOI: 10.1093/nar/gkaa1273 *
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