WO2025076115A1

WO2025076115A1 - Cdk inhibitors and their use as pharmaceuticals

Info

Publication number: WO2025076115A1
Application number: PCT/US2024/049630
Authority: WO
Inventors: Krishna Vaddi; Peggy SCHERLE; Neha BHAGWAT
Original assignee: Prelude Therapeutics Inc
Current assignee: Prelude Therapeutics Inc
Priority date: 2023-10-02
Filing date: 2024-10-02
Publication date: 2025-04-10
Anticipated expiration: 2026-04-02

Abstract

The present invention provides methods of reducing oncogene transcription, or of reducing phosphorylated RNA polymerase 2, in a subject in need thereof by administering to the subject of Compound (I), or a pharmaceutically acceptable salt thereof.

Description

CDK INHIBITORS AND THEIR USE AS PHARMACEUTICALS

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of United States Provisional Patent Application No. 63/587,166, filed October 2, 2023, the entirety of which is incorporated by reference herein.

TECHNICAL FIELD

[0002] The present invention relates to the field of pharmaceutical compounds for the treatment of cancer.

BACKGROUND

[0003] Cyclin-dependent kinase 9 (CDK9) is a master regulator of transcription that controls paused RNA polymerase II (RNAP2) release through phosphorylation of its carboxyterminal domain, resulting in transcription elongation.

[0004] Selective CDK9 inhibition may be a promising approach to treat transcription-addicted cancers that are dependent on oncogenic drivers with a short half-life, such as the oncogenes myelocytomatosis (MFC), myelobastosis (MYB), and myeloid leukemia cell differentiation protein (MCL1 .

[0005] Compound l is a potent and selective inhibitor of CDK9 with a biochemical half-maximal inhibitory concentration (IC50) of 0.98 nM at 1 mM adenosine triphosphate that inhibits the enzymatic activity of human CDK9/CyclinTl complex. Compound I is highly selective among CDK family members and across 177 kinases.

[0006] Intermittent intravenous (IV) administration of Compound I demonstrated strong efficacy in preclinical models of solid tumors and hematological malignancies as monotherapy and in combination with other anticancer therapies.

[0007] The chemical structure of Compound I is shown below:

Compound I.

[0008] Compound I and its synthesis have been described in U.S. Patent No.

11,673,893. Pharmaceutically acceptable salts of Compound I include those described in WO 2023064920.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] In the drawings, which are not necessarily drawn to scale, like numerals can describe similar components in different views. Like numerals having different letter suffixes can represent different instances of similar components. The drawings illustrate generally, by way of example, but not by way of limitation, various aspects discussed in the present document. In the drawings:

[0010] FIG. 1 shows an exemplary study design. DL5 (15 mg/m²) was evaluated as an intermediate DL following safety review of DL4 (18 mg/m²). Compound I was administered IV QW in a 3-week cycle until disease progression or unacceptable toxicity. (C1D1, Cycle 1 Day 1; CRPC, castrate-resistant prostate cancer; DL, dose level; ECOG PS, Eastern Cooperative Oncology Group performance status; HR+/HER2-, hormone receptor positive, human epidermal growth factor receptor 2 negative; NSCLC, non-small cell lung cancer; QW, once weekly; RECIST, Response Evaluation Criteria in Solid Tumors).

[0011] FIG. 2 is a chart showing the dosing scheme. The safety evaluable set includes all patients who received at least 1 dose of Compound I. The efficacy evaluable set includes all patients in the safety evaluable set who had >1 post-baseline response assessment or discontinued the study due to death, AE, or PD. (DCC = dose confirmation cohort).

[0012] FIG. 3 is a chart showing duration of the study follow-up including patent status. Status indicates the primary reason for treatment discontinuation. The text on the bars after baseline (SD/PD) indicate overall response assessed by the Investigator per the criteria corresponding to the underlying tumor type. The text (numbers) at the end of each row indicates PFS time, DoT, BOR, and death time. The DoT was defined as the time interval from Day 1 to the date of last IV plus 6 days. DC/CB/R at the end of each line indicates the DC response, CB response, or overall response. (BL, baseline; BOR, best overall response; CB, clinical benefit; CR, complete response; DC, disease control; DoT, duration of treatment; F, female; M, male; NA, not applicable; NE, not evaluable; NN, non-CR/non-PD; PST, prior systemic therapy).

[0013] FIG. 4 is a graph showing pharmacokinetics profiles of the tested doses of Compound I.

[0014] FIG. 5 depicts results showing Compound I-associated inhibition of CDK9 transcriptional target MYC.

[0015] FIG. 6 depicts results showing Compound I-associated inhibition of CDK9 transcriptional target MCL1.

[0016] FIG. 7 depicts results showing Compound I-associated inhibition of phosphorylation of PNAP2 at 2 hours in PBMCs.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0017] The present disclosure may be understood more readily by reference to the following detailed description of desired embodiments and the examples included therein.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0019] The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. [0020] As used in the specification and in the claims, the term "comprising" can include the embodiments "consisting of and "consisting essentially of.” The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that require the presence of the named ingredients/steps and permit the presence of other ingredients/steps. However, such description should be construed as also describing compositions or processes as "consisting of and "consisting essentially of the enumerated ingredients/steps, which allows the presence of only the named ingredients/steps, along with any impurities that might result therefrom, and excludes other ingredients/steps.

[0021] As used herein, the terms “about” and “at or about” mean that the amount or value in question can be the value designated some other value approximately or about the same. It is generally understood, as used herein, that it is the nominal value indicated ±10% variation unless otherwise indicated or implied. The term is intended to convey that similar values promote equivalent results or effects recited in the claims. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but can be approximate and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about” or “approximate” whether or not expressly stated to be such. It is understood that where “about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0022] Unless indicated to the contrary, the numerical values should be understood to include numerical values which are the same when reduced to the same number of significant figures and numerical values which differ from the stated value by less than the experimental error of conventional measurement technique of the type described in the present application to determine the value.

[0023] All ranges disclosed herein are inclusive of the recited endpoint and independently of the endpoints. The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value; they are sufficiently imprecise to include values approximating these ranges and/or values. [0024] In some aspects, the disclosure provides methods of reducing oncogene transcription in a subject in need thereof, comprising administering to the subject Compound I, or a pharmaceutically acceptable salt thereof.

[0025] In other aspects, the disclosure provides methods of reducing phosphorylated RNA polymerase 2 (p-RNAP2) in a subject in need thereof, comprising administering to the subject Compound I, or a pharmaceutically acceptable salt thereof.

[0026] In some embodiments of the disclosed methods, the subject is a mammal, such as, for example, a human, canine, equine, bovine, murine, rattus, ovine, feline, nonhuman primate, and the like.

[0027] In some embodiments, the subject is a human.

[0028] In some embodiments of the disclosed methods, the subject is administered Compound I.

[0029] In other embodiments of the disclosed methods, the subject is administered a pharmaceutically acceptable salt of Compound I.

[0030] The chemical structure of Compound I is shown below:

Compound I.

[0031] Compound I and its synthesis have been described in U.S. Patent No. 11,673,893. Pharmaceutically acceptable salts of Compound I include those described in WO 2023064920.

[0032] In some aspects, the disclosed methods reduce oncogene transcription. As used herein, the term “oncogene” refers to a mutated gene that induces abnormal cellular proliferation, and tumor development and growth. As used herein, the term “transcription” has its normal biological meaning, i.e., the process of making an RNA chain - the transcript - that has a nucleotide sequence that is exactly complementary to the strand of DNA used as the template.

[0033] In some embodiments, the oncogene is MYC, MYB, o MCLl.

[0034] In some embodiments, the oncogene is MYC o MCLl.

[0035] In some embodiments, the oncogene is MYC.

[0036] In some embodiments, the oncogene is MYB.

[0037] In other embodiments, the oncogene C CI.l.

[0038] In some aspects of the methods of the disclosure, Compound 1, or a pharmaceutically acceptable salt thereof, is administered to the subject in an amount of from 3 mg/m² - 18 mg/m², such as, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 mg/m². It will be understood by a person of skill in the art that the “mg/m²” dosing units refers to the mass of Compound I (or a pharmaceutically acceptable salt of Compound I on a Compound I basis) administered per square meter of the subject’s body surface area.

[0039] In some embodiments, Compound 1, or a pharmaceutically acceptable salt thereof, is administered to the subject in an amount of from 3 mg/m².

[0040] In some embodiments, Compound 1, or a pharmaceutically acceptable salt thereof, is administered to the subject in an amount of from 6 mg/m².

[0041] In some embodiments, Compound 1, or a pharmaceutically acceptable salt thereof, is administered to the subject in an amount of from 12 mg/m².

[0042] In some embodiments, Compound 1, or a pharmaceutically acceptable salt thereof, is administered to the subject in an amount of from 15 mg/m².

[0043] In some embodiments, Compound 1, or a pharmaceutically acceptable salt thereof, is administered to the subject in an amount of from 18 mg/m².

[0044] In some aspects of the disclosed methods, Compound I, or a pharmaceutically acceptable salt thereof, is administered to the subject intravenously.

[0045] In some aspects of the disclosed methods, Compound I, or a pharmaceutically acceptable salt thereof, is administered once weekly.

[0046] In some aspects of the disclosed methods, administration of Compound I, or a pharmaceutically acceptable salt thereof, results in reduction of oncogene transcription relative to pre-dose levels. In some aspects of the disclosed methods, administration of Compound I, or a pharmaceutically acceptable salt thereof, results in a dose-dependent reduction in oncogene transcription relative to pre-dose levels. [0047] In some embodiments of the disclosed methods, administration of Compound I, or a pharmaceutically acceptable salt thereof, results in oncogene transcription that is decreased relative to pre-dose levels by 0.05-fold to 0.95-fold, such as, for example, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, or 0.95-fold.

[0048] In some embodiments of the disclosed methods, administration of Compound I, or a pharmaceutically acceptable salt thereof, results in oncogene transcription that is decreased relative to pre-dose levels by about 0.05-fold.

[0049] In some embodiments of the disclosed methods, administration of Compound I, or a pharmaceutically acceptable salt thereof, results in oncogene transcription that is decreased relative to pre-dose levels by about 0.1 -fold.

[0050] In some embodiments of the disclosed methods, administration of Compound I, or a pharmaceutically acceptable salt thereof, results in oncogene transcription that is decreased relative to pre-dose levels by about 0.15-fold.

[0051] In some embodiments of the disclosed methods, administration of Compound I, or a pharmaceutically acceptable salt thereof, results in oncogene transcription that is decreased relative to pre-dose levels by about 0.2-fold.

[0052] In some embodiments of the disclosed methods, administration of Compound I, or a pharmaceutically acceptable salt thereof, results in oncogene transcription that is decreased relative to pre-dose levels by about 0.25-fold.

[0053] In some embodiments of the disclosed methods, administration of Compound I, or a pharmaceutically acceptable salt thereof, results in oncogene transcription that is decreased relative to pre-dose levels by about 0.3-fold.

[0054] In some embodiments of the disclosed methods, administration of Compound I, or a pharmaceutically acceptable salt thereof, results in oncogene transcription that is decreased relative to pre-dose levels by about 0.35-fold.

[0055] In some embodiments of the disclosed methods, administration of Compound I, or a pharmaceutically acceptable salt thereof, results in oncogene transcription that is decreased relative to pre-dose levels by about 0.4-fold.

[0056] In some embodiments of the disclosed methods, administration of Compound I, or a pharmaceutically acceptable salt thereof, results in oncogene transcription that is decreased relative to pre-dose levels by about 0.45-fold. [0057] In some embodiments of the disclosed methods, administration of Compound I, or a pharmaceutically acceptable salt thereof, results in oncogene transcription that is decreased relative to pre-dose levels by about 0.5-fold.

[0058] In some embodiments of the disclosed methods, administration of Compound I, or a pharmaceutically acceptable salt thereof, results in oncogene transcription that is decreased relative to pre-dose levels by about 0.55-fold.

[0059] In some embodiments of the disclosed methods, administration of Compound I, or a pharmaceutically acceptable salt thereof, results in oncogene transcription that is decreased relative to pre-dose levels by about 0.6-fold.

[0060] In some embodiments of the disclosed methods, administration of Compound I, or a pharmaceutically acceptable salt thereof, results in oncogene transcription that is decreased relative to pre-dose levels by about 0.65-fold.

[0061] In some embodiments of the disclosed methods, administration of Compound I, or a pharmaceutically acceptable salt thereof, results in oncogene transcription that is decreased relative to pre-dose levels by about 0.7-fold.

[0062] In some embodiments of the disclosed methods, administration of Compound I, or a pharmaceutically acceptable salt thereof, results in oncogene transcription that is decreased relative to pre-dose levels by about 0.75-fold.

[0063] In some embodiments of the disclosed methods, administration of Compound I, or a pharmaceutically acceptable salt thereof, results in oncogene transcription that is decreased relative to pre-dose levels by about 0.8-fold.

[0064] In some embodiments of the disclosed methods, administration of Compound I, or a pharmaceutically acceptable salt thereof, results in oncogene transcription that is decreased relative to pre-dose levels by about 0.85-fold.

[0065] In some embodiments of the disclosed methods, administration of Compound I, or a pharmaceutically acceptable salt thereof, results in oncogene transcription that is decreased relative to pre-dose levels by about 0.9-fold.

[0066] In some embodiments of the disclosed methods, administration of Compound I, or a pharmaceutically acceptable salt thereof, results in oncogene transcription that is decreased relative to pre-dose levels by about 0.95-fold. [0067] In other aspects of the disclosed methods, administration of Compound I, or a pharmaceutically acceptable salt thereof, results in reduction in phosphorylation of RNA polymerase 2 (p-RNAP2) in the subject.

[0068] In other aspects of the disclosed methods, administration of Compound I, or a pharmaceutically acceptable salt thereof, results in a dose-dependent reduction in phosphorylation of RNA polymerase 2 (p-RNAP2) in the subject.

[0069] In some embodiments, administration of Compound I, or a pharmaceutically acceptable salt thereof, results in reduction in phosphorylation of RNA polymerase 2 (p- RNAP2) in the subject of 20-90% relative to pre-dose levels, such as, for example, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%.

[0070] In some embodiments, administration of Compound I, or a pharmaceutically acceptable salt thereof, results in reduction in phosphorylation of RNA polymerase 2 (p- RNAP2) in the subject of 20-80% relative to pre-dose levels.

[0071] In some embodiments, administration of Compound I, or a pharmaceutically acceptable salt thereof, results in reduction in phosphorylation of RNA polymerase 2 (p- RNAP2) in the subject of 20-70% relative to pre-dose levels.

[0072] In some embodiments, administration of Compound I, or a pharmaceutically acceptable salt thereof, results in reduction in phosphorylation of RNA polymerase 2 (p- RNAP2) in the subject of 20-60% relative to pre-dose levels.

[0073] In some embodiments, administration of Compound I, or a pharmaceutically acceptable salt thereof, results in reduction in phosphorylation of RNA polymerase 2 (p- RNAP2) in the subject of 20-50% relative to pre-dose levels.

[0074] In some embodiments, administration of Compound I, or a pharmaceutically acceptable salt thereof, results in reduction in phosphorylation of RNA polymerase 2 (p- RNAP2) in the subject of 30-80% relative to pre-dose levels.

[0075] In some embodiments, administration of Compound I, or a pharmaceutically acceptable salt thereof, results in reduction in phosphorylation of RNA polymerase 2 (p- RNAP2) in the subject of 30-70% relative to pre-dose levels.

[0076] In some embodiments, administration of Compound I, or a pharmaceutically acceptable salt thereof, results in reduction in phosphorylation of RNA polymerase 2 (p- RNAP2) in the subject of 30-60% relative to pre-dose levels. [0077] In some embodiments, administration of Compound I, or a pharmaceutically acceptable salt thereof, results in reduction in phosphorylation of RNA polymerase 2 (p- RNAP2) in the subject of 30-50% relative to pre-dose levels.

[0078] In some embodiments, administration of Compound I, or a pharmaceutically acceptable salt thereof, results in reduction in phosphorylation of RNA polymerase 2 (p- RNAP2) in the subject of 40-80% relative to pre-dose levels.

[0079] In some embodiments, administration of Compound I, or a pharmaceutically acceptable salt thereof, results in reduction in phosphorylation of RNA polymerase 2 (p- RNAP2) in the subject of 40-70% relative to pre-dose levels.

[0080] In some embodiments, administration of Compound I, or a pharmaceutically acceptable salt thereof, results in reduction in phosphorylation of RNA polymerase 2 (p- RNAP2) in the subject of 40-60% relative to pre-dose levels.

[0081] In some embodiments, administration of Compound I, or a pharmaceutically acceptable salt thereof, results in reduction in phosphorylation of RNA polymerase 2 (p- RNAP2) in the subject of 40-50% relative to pre-dose levels.

[0082] In some embodiments, administration of Compound I, or a pharmaceutically acceptable salt thereof, results in reduction in phosphorylation of RNA polymerase 2 (p- RNAP2) in the subject of 27% relative to pre-dose levels.

[0083] In some embodiments, administration of Compound I, or a pharmaceutically acceptable salt thereof, results in reduction in phosphorylation of RNA polymerase 2 (p- RNAP2) in the subject of 27% relative to pre-dose levels.

[0084] In some embodiments, administration of Compound I, or a pharmaceutically acceptable salt thereof, results in reduction in phosphorylation of RNA polymerase 2 (p- RNAP2) in the subject of 77% relative to pre-dose levels.

[0085] The disclosure is also directed to the following aspects:

[0086] Aspect 1. A method of treating a transcriptionally active cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a CDK9 inhibitor.

[0087] Aspect 2. The method of aspect 1, wherein the CDK9 inhibitor comprises Compound 1, or a pharmaceutically acceptable salt thereof.

[0088] Aspect 3. The method of any one of the preceding aspects, wherein the

CDK9 inhibitor is administered using a dosing regimen and schedule disclosed herein. [0089] Aspect 4. The method of any one of the preceding aspects, wherein the transcriptionally active cancer comprises one or more transcriptional dependent cancers.

[0090] Aspect 5. The method of any one of the preceding aspects, wherein the transcriptionally active cancer comprises prostate cancer.

[0091] Aspect 6. The method of aspect 5, wherein the prostate cancer comprises castration-resistant prostate cancer.

EXAMPLE

[0092] This is an open-label, multicenter, phase 1 dose-escalation study of Compound I in participants with select advanced solid tumors.

[0093] Dose escalation employed a Bayesian optimal interval design to evaluate the safety, tolerability, and incidence of dose-limiting toxicities (DLTs).

[0094] Key inclusion criteria include:

• Adults (aged >18 years) with previously treated selected sarcomas, castrate-resistant prostate cancer (CRPC), HR+/HER2- (hormone receptor positive, human epidermal growth factor receptor 2 negative) breast cancer, NSCLC (non-small cell lung cancer), or MFC-amplified solid tumors.

• Prior systemic therapy

• Measurable (per RECIST vl .1 ; Response Evaluation Criteria in Solid Tumors) or evaluable (CRPC or sarcoma only) disease

• ECOG PS 0-1 (Eastern Cooperative Oncology Group performance status)

• Adequate organ function

• Central nervous system disease is allowed if stable and adequately controlled.

[0095] Key exclusion criteria include:

• Impaired cardiac function

• Chronic inflammatory bowel disease

• Prior exposure to CDK9 inhibitor

• Prior anti-cancer therapy within 5 half-lives or 28 days (whichever is shorter) prior to Cycle 1 Day 1 (C1D1). [0096] Study Endpoints

[0097] The primary endpoint was to identify dose-limiting toxicities (DLTs) and establish the maximum tolerated dose and the recommended phase 2 dose (RP2D) of Compound I based on incidence of DLTs, adverse events (AEs), changes in laboratory parameters, and changes in dosing.

[0098] Secondary endpoints include evaluation of the pharmacokinetic profde, safety, and efficacy signal of Compound I based on overall response rate, disease control rate, duration of response, and progression-free survival (PFS).

[0099] Exploratory endpoints include the pharmacodynamic assessment of the expression of CDK9 transcriptional targets (MYC, MCL1) in peripheral blood mononuclear cells (PBMCs) that may be associated with response to CDK9 inhibition. Phosphorylation of RNAP2 (p-RNAP2) was assessed by capillary electrophoresis and monitoring changes in absolute monocyte and neutrophil counts using clinical complete blood count.

[00100] Study Intervention

[00101] Compound I was administered (by intravenous infusion) once weekly for a three-week cycle.

[00102] Study Results

[00103] Patient Summary

[00104] Patient baseline characteristics are presented in Table 1 and patient disposition is presented in FIG. 2.

[00105] At the time of data cut-off (median treatment duration of 5.4 [range, 1.0- 66.0] weeks), 93.3% of patients had discontinued Compound I.

[00106] The primary reasons for discontinuation were progressive disease (PD; n=22, 78.6%), AEs (n=5, 17.9%), and withdrawal by patient (n=l, 3.6%). One treatment- related AE led to discontinuation in the 15 mg/m² cohort.

[00107] Table 1. Patient Baseline Characteristics

[00108] Safety

[00109] The most common treatment-emergent AEs (TEAEs) were nausea (46.7%), vomiting (40.7%), neutropenia (33.3%), diarrhea (23.3%), and fatigue (23.3%). Two DLTs of grade 4 neutropenia were observed during dose escalation in the 15 and 18 mg/m² QW cohorts. No grade 3/4 hepatotoxicity was observed. Nine serious AEs were reported; one (pneumonia staphylococcal) was considered related to study treatment. The most common treatment-related AEs were nausea (43.3%), vomiting (36.7%), and neutropenia (33.3%). Anti-emetic or growth factor prophylaxis was not required per protocol. Three patients received growth factor support for neutropenia. No deaths related to treatment were observed.

[00110] Table 2. Overview of TEAEs

Table 3. TEAE/Treatment-Related AEs in >10% of the Total Population

[00111] Preliminary Efficacy

[00112] Of the 29 patients with 1 post baseline scan, no objective responses (per RECIST vl.l) were observed: 8 (27.6%) patients had stable disease (SD) as the best response (FIG. 3). Prolonged SD for 66+ weeks was observed in a patient with chondrosarcoma who received Compound I at a starting dose of 6 mg/m². This patient was dose escalated to 12 mg/m². Median PFS was 1.25 (95% confidence interval, 1.02-1.64) months.

[00113] Pharmacokinetics

[00114] Compound I demonstrated dose-dependent increases in exposure (FIG. 4). The mean half-life of Compound I was 3.8 hours after a single dose of 18 mg/m². Consistent with the short half-life, no accumulation was observed with QW dosing.

[00115] Pharmacodynamics

[00116] Evaluation of CDK9 transcriptions targets in PBMCs

[00117] RNA was isolated from blood collected in PAXgene® Blood RNA tubes using the PAXgene® Blood RNA kit as per manufacturer’s instructions. cDNA was synthesized using the qScript cDNA Synthesis SuperMix (5X). A total of 32 pl of RNA was used in conjunction with 8 pl of qScript SuperMix (5X) to make a 40 ul reaction per sample. cDNA was then diluted in nuclease-free water to a final concentration of 10 ng/pl. qPCR was conducted using the PerfeCTa SYBR Green SuperMix kit (Cat. No. 95054). The following BioRad assays were used - GAPDH (qHsaCED0038674), MCL1 (qHsaCED0036603), HPRT1 (qHsaCIDOO 16375), MYC (qHsaCIDOO 12921). Data analysis was completed using the BioRad CFX Maestro software.

[00118] Evaluation of pRNAP2 in PBMCs [00119] PBMCs were isolated from blood collected in BD Vacutainer® CPT™ Mononuclear Cell Preparation Tubes. Cells were lysed in 4% Sodium Dodecyl Sulfate solution and protein was isolated using the Omega Bio-Tek Homogenizer Mini Columns Kit (Catalog #HCR003). Protein was quantified using the Pierce™ BCA Protein Assay Kit (Catalog # 23225) and analyzed using the Jess Automated Western Blot System (ProteinSimple, part of Bio-Techne). Briefly, samples were diluted to 0.5 pg/pl using a 0.1X Sample Buffer made from the 10X Sample Buffer stock (Catalog # 042-195). One part 5X Fluorescent Master Mix (from EZ Standard Pack) was mixed with 4 parts of the diluted samples. 5 pl of sample and reagents were added to the 25 -well microplate according to manufacturer’s instructions. The following primary antibodies and dilutions were used - pRNAP2 (Abeam, Cat. No. ab 193468, 1:10), GAPDH (CST Cat. no. 2118, 1:200). 10 pl of anti-rabbit secondary HRP antibody (Catalog # 042-206) and 200 pl of BioRad Clarity MAX ECL Substrate (Cat. No. 1705062) was added to microplate. After completion of the run, peak intensities (Area Under Curve (AUC)) corresponding to each capillary were quantified using Compass for Simple Western software (ProteinSimple).

[00120] A dose-dependent downregulation of MYC and MCL1 mRNA expression in PBMCs was observed (FIGS. 5 and 6, respectively). Maximum inhibition of CDK9 transcriptional targets MYC and MCL1 at doses >12 mg/m² is consistent with the degree of target engagement required for preclinical efficacy. A dose-dependent inhibition of p- RNAP2 was observed in PBMCs at 15 and 18 mg/m² doses (FIG. 7) with a median decrease of 27.0% (n=10) at 15 mg/m² and a median decrease of 77.2% (n=5) at 18 mg/m².

[00121] Conclusions

[00122] In adults with advanced solid tumors, Compound I demonstrated favorable tolerability with manageable neutropenia and absence of significant gastrointestinal events or hepatotoxicity. The short half-life of Compound I enables acute CDK9 inhibition over a defined period, making it suitable for weekly administration without inducing significant toxicity. The observed dose-dependent downregulation of CDK9 transcriptional targets MYC and MCL1 mRNA expression in PBMCs isolated from patients treated with Compound I was consistent with the degree of target engagement required for preclinical efficacy. 12 mg/m² QW dosing and higher showed optimal target inhibition.

Claims

What is claimed:

1. A method of reducing oncogene transcription in a subject in need thereof, comprising administering to the subject Compound I, or a pharmaceutically acceptable salt thereof.

2. The method of claim 1, wherein the oncogene is MYC o MCLl.

3. The method of any one of the preceding claims, wherein Compound 1, or a pharmaceutically acceptable salt thereof, is administered in an amount of from 3 mg/m² - 18 mg/m².

4. The method of any one of the preceding claims, wherein Compound 1, or a pharmaceutically acceptable salt thereof, is administered intravenously.

5. The method of any one of the preceding claims, wherein Compound 1, or a pharmaceutically acceptable salt thereof, is administered once weekly.

6. The method of any one of claims 1-5, wherein the administration results in a 0.05-fold to 0.95-fold reduction in oncogene transcription relative to pre-dose levels.

7. A method of reducing phosphorylated RNA polymerase 2 (p-RNAP2) in a subject in need thereof, comprising administering to the subject Compound I, or a pharmaceutically acceptable salt thereof.

8. The method of claim 7, wherein Compound 1, or a pharmaceutically acceptable salt thereof, is administered in an amount of from 3 mg/m² - 18 mg/m².

9. The method of claim 7 or claim 8, wherein Compound 1, or a pharmaceutically acceptable salt thereof, is administered intravenously.

10. The method of any one of claims 7-9, wherein Compound 1, or a pharmaceutically acceptable salt thereof, is administered once weekly.

11. The method of any one of claims 7-10, wherein the administration results in reduction in phosphorylation of RNA polymerase 2 (p-RNAP2) in the subject of 20-90% relative to pre-dose levels.