Patent Application
20210346356
This disclosure relates to treating cancer with ubiquitin specific peptidase 9X (USP9X) inhibitors alone and/or in combination with one or more immune checkpoint pathway inhibitors.
Currently 75-85% of patients who received cancer immunotherapy do not respond to it. Therefore, developing T cell-centric immunotherapy for cancer patients who fail to respond to cancer immunotherapies, such as anti-CTLA4 therapy and anti-PD1 therapy, is currently of high interest. T cell-centric immunotherapy has the potential to increase the capacity for the body's immune system to target and eliminate cancer cells. Hence, there is significant unmet need for immunomodulatory therapies that target dysfunctional T cells.
The present disclosure includes the recognition that the inhibition of USP9X has immune modulating function via activation of T cells and that this immune modulating function can reduce and/or prevent tumor growth. Accordingly, in some embodiments, USP9X inhibitors can be used to treat cancer.
The present disclosure provides methods of treating cancer in a patient in need thereof, comprising administering to the patient a USP9X Inhibitor. In some embodiments, a USP9X Inhibitor is a compound characterized in that it has an IC50 value when tested in the Biochemical Assay of Example A of:
Additionally, the present disclosure provides methods of treating cancer by administering a USP9X Inhibitor in combination with an immune checkpoint pathway inhibitor. In some embodiments, a method of treating cancer in a patient in need thereof comprises administering to the patient a USP9X Inhibitor, wherein the patient is receiving or has received an immune checkpoint pathway inhibitor. In some embodiments, a method of treating cancer in a patient in need thereof comprises administering to the patient an immune checkpoint pathway inhibitor, wherein the patient is receiving or has received a USP9X Inhibitor.
The present disclosure provides methods of treating cancer, comprising administering a USP9X Inhibitor to a patient in need thereof. The disclosure is based in part on the recognition that the inhibition of USP9X has immune modulating function via activation of T cells and that this immune modulating function can reduce and/or prevent tumor growth.
The present disclosure also provides methods of treating cancer, comprising administering a USP9X Inhibitor to a patient in need thereof, wherein the patient is receiving or has received an immune checkpoint pathway inhibitor. Additionally, the present disclosure also provides methods of treating cancer, comprising administering an immune checkpoint pathway inhibitor to a patient in need thereof, wherein the patient is receiving or has received a USP9X Inhibitor.
Without wishing to be bound by theory, USP9X Inhibitors and immune checkpoint pathway inhibitors may have separate mechanisms of action. USP9X Inhibitors, therefore, can be useful in treating cancer in a patient that is non-responsive to therapy with an immune checkpoint pathway inhibitor alone.
Methods of treating cancer are provided herein. In some embodiments, a method of treating cancer comprises administering a USP9X Inhibitor to a patient in need thereof. In some embodiments, a method of treating cancer comprises administering a USP9X Inhibitor to a patient in need thereof, wherein the patient is receiving or has received an immune checkpoint pathway inhibitor. In some embodiments, a method of treating cancer comprises administering an immune checkpoint pathway inhibitor to a patient in need thereof, wherein the patient is receiving or has received a USP9X Inhibitor. In some embodiments, the cancer is refractory or resistant to treatment. In some embodiments, the cancer has progressed after one or more previous lines of chemotherapy. In some embodiments, the cancer has progressed after two or more previous lines of chemotherapy. In some embodiments, the cancer has progressed after three or more previous lines of chemotherapy.
In some embodiments, the cancer comprises a tumor that expresses PD-L1. PD-L1 expression can be detected by an FDA-approved test, such as PD-L1 TIC 22C3 pharmDx or PD-L1 (SP142). In some embodiments, the cancer comprises a tumor that expresses CTLA-4. In some embodiments, the cancer comprises a tumor in a patient that expresses CTLA-4 in the tumor environment or draining lymphoid tissues. CTLA-4 expression can be assessed by methods known to a person skilled in the art.
In some embodiments, the cancer is selected from unresectable or metastatic melanoma, cutaneous melanoma, advanced renal cell carcinoma, microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer, metastatic squamous non-small cell lung cancer, head and neck squamous cell cancer, classical Hodgkin lymphoma, primary mediastinal large B-cell lymphoma, urothelial carcinoma, microsatellite instability-high cancer, gastric cancer, cervical cancer, hepatocellular carcinoma, or Merkel cell carcinoma.
In some embodiments, a method of treating cancer comprises administering a USP9X Inhibitor to a patient in need thereof, wherein the patient is receiving or has received an immune checkpoint pathway inhibitor, and wherein the cancer is selected from unresectable or metastatic melanoma, cutaneous melanoma, advanced renal cell carcinoma, microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer, metastatic squamous non-small cell lung cancer, head and neck squamous cell cancer, classical Hodgkin lymphoma, primary mediastinal large B-cell lymphoma, urothelial carcinoma, microsatellite instability-high cancer, gastric cancer, cervical cancer, hepatocellular carcinoma, or Merkel cell carcinoma.
In some embodiments, a method of treating cancer comprises administering an immune checkpoint pathway inhibitor to a patient in need thereof, wherein the patient is receiving or has received a USP9X Inhibitor, and wherein the cancer is selected from unresectable or metastatic melanoma, cutaneous melanoma, advanced renal cell carcinoma, microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer, metastatic squamous non-small cell lung cancer, head and neck squamous cell cancer, classical Hodgkin lymphoma, primary mediastinal large B-cell lymphoma, urothelial carcinoma, microsatellite instability-high cancer, gastric cancer, cervical cancer, hepatocellular carcinoma, or Merkel cell carcinoma.
In addition, patients can be selected to receive treatment with a USP9X Inhibitor alone, and/or with a USP9X Inhibitor in combination with an immune checkpoint pathway inhibitor. For example, patients can be selected based on their prior treatment status and/or their status in a genetic risk panel analysis of the patient, such as PD-L1.
In some embodiments, methods provided herein are useful for treating patients who have not responded to previous cancer immunotherapy. In some embodiments, provided methods are useful for treating patients who have not responded to prior therapy with an immune checkpoint pathway inhibitor, such as ipilimumab, nivolumab, or pembrolizumab.
In some embodiments, methods provided herein are useful for treating patients who have not responded to previous chemotherapy. In some embodiments, the previous chemotherapy is selected from platinum-based chemotherapy (e.g., oxaliplatin, cisplatin, or carboplatin), fluoropyrimidine therapy, irinotecan therapy, paclitaxel therapy, nab-paclitaxel therapy, HER2/neu-targeted therapy, or sorafenib therapy.
In some embodiments, methods provided herein are useful for treating patients who have received one or more prior lines of chemotherapy. In some embodiments, methods provided herein are useful for treating patients who have received two or more prior lines of chemotherapy. In some embodiments, methods provided herein are useful for treating patients who have received three or more prior lines of chemotherapy.
Patients with cancer comprising a tumor expressing PD-1 can be identified using a diagnostic test. In some embodiments, an FDA-approved diagnostic test, such as PD-L1 IHC 22C3 pharmDx (Dako North America, Inc.) is used in the detection of PD-L1 protein in cancer. Results of the test are used as an aid in the identification of cancer patients who may be considered for treatment with a therapeutic agent, such as an immune checkpoint pathway inhibitor, including pembrolizumab. In some embodiments, patients evaluated with a diagnostic test (e.g., PD-L1 IHC 22C3 pharmDx (Dako North America, Inc.)) that are determined to express PD-L1 in cancer are treated with a therapeutic agent (e.g., an immune checkpoint pathway inhibitor) in accordance with provided methods.
PD-L1 IHC 22C3 pharmDx is a qualitative immunohistochemical assay using Monoclonal Mouse Anti-PD-L1, Clone 22C3 intended for use in the detection of PD-L1 protein in formalin-fixed, paraffin-embedded (FFPE) non-small cell lung cancer (NSCLC), gastric or gastroesophageal junction (GEJ) adenocarcinoma, cervical cancer and urothelial carcinoma tissues using EnVision FLEX visualization system on Autostainer Link 48.
In some embodiments, a method of treating cancer comprises administering a USP9X Inhibitor to a patient in need thereof, wherein the patient is or has been selected for treatment using a diagnostic test, such as PD-L1 IHC 22C3 pharmDx. In some embodiments, the patient is or has been determined to have a cancer expressing PD-L1 using PD-L1 IHC 22C3 pharmDx.
In some embodiments, methods of treating cancer comprise administering two or more therapeutic regimens to a patient in need thereof (e.g., a USP9X Inhibitor and an immune checkpoint pathway inhibitor). In some embodiments, the two or more therapeutic regimens may be administered simultaneously. In some embodiments, such regimens may be administered sequentially (e.g., all “doses” of a first regimen are administered prior to administration of any doses of a second regimen). In some embodiments, such agents are administered in overlapping dosing regimens. For clarity, combination therapy does not require that individual agents be administered together in a single composition (or even necessarily at the same time). In some embodiments, two or more therapeutic agents or regimens of a combination therapy are administered to a subject separately, e.g., in separate compositions, via separate administration routes (e.g., one agent orally and another agent intravenously), and/or at different time points. In some embodiments, two or more therapeutic agents may be administered together in a combination composition.
Deubiquitylating enzymes control a number of cellular processes, including the stability of a variety of oncoproteins, by reversing ubiquitination. USP9X is a member of the USP family of DUBs and is a key regulator of protein homeostasis for protein substrates including several that are known to be oncogenic or protumorigenic. Overexpression and/or mutation of DUBs and their substrates are associated with cancer initiation and progression.
USP9X inhibition can promote antitumor T cell responses. Although USP9X is not required for T cell survival, it is required for normal T cell development and proliferation. Additionally, USP9X may have a role in T cell activation and tolerance as a regulator of the ubiquitylation and stability of ITCH, a known E3 ubiquitin ligase. ITCH, as well as Cbl-b and GRAIL, are critical for T cell activation and T cell tolerance induction, which act in part by attenuating the T cell receptor (TCR) signal. Interestingly, the co-inhibitory receptor CTLA-4, a key mediator of T cell tolerance, may exert its inhibitory T cell function, at least in part, by activating ITCH. Thus, enhanced degradation of ITCH and consequent loss of T cell tolerance could explain the spontaneous autoimmunity and lymphoproliferative diseases manifested in T cell-specific USP9X knockout (KO) mice.
By way of non-limiting example, USP9X Inhibitors that may be used in accordance with the present disclosure include those described in WO2014/172638, WO2015/054555, and WO2015/187427, each of which is hereby incorporated by reference.
In some embodiments, a USP9X Inhibitor is a compound of Formula I:
or a pharmaceutically acceptable salt thereof, wherein:
In some embodiments, a USP9X Inhibitor is a compound of Formula I-a:
or a pharmaceutically acceptable salt thereof, wherein B, R1, and R2 are as defined above for Formula I, and wherein Y2 is CH or N.
In some embodiments, a USP9X Inhibitor is a compound of Formula I-b:
or a pharmaceutically acceptable salt thereof, wherein:
In some embodiments, a USP9X Inhibitor is a compound of Formula I-c:
or a pharmaceutically acceptable salt thereof,
In some embodiments, a USP9X Inhibitor is a compound of Formula I-d:
or a pharmaceutically acceptable salt thereof,
In some embodiments, a USP9X Inhibitor is a compound of Formula I-e:
or a pharmaceutically acceptable salt thereof,
In some embodiments, a USP9X Inhibitor is a compound of Formula I-f:
or a pharmaceutically acceptable salt thereof,
In some embodiments of Formulas I, I-a, I-b, I-c, I-d, I-e, and I-f, Y1, Y2, and Y3 are each independently CRa. In some embodiments, Y1, Y2, and Y3 are each CH. In some embodiments, at least one of Y1, Y2, and Y3 is N. In some embodiments, at least one of Y1 and Y2 is N. In some embodiments, Y1 is CRa. In some embodiments, Y1 is N. In some embodiments, Y2 is CRa. In some embodiments, Y2 is N. In some embodiments, Y3 is CRa. In some embodiments, Y3 is N.
In some embodiments of Formulas I, I-c, I-d, I-e, and I-f,
In some embodiments,
In some embodiments,
In some embodiments of Formulas I, I-c, I-d, I-e, and I-f, and V, Z1 is O or S. In some embodiments, Z1 is O. In some embodiments, Z1 is S. In some embodiments, Z1 is NR. In some embodiments, Z1 is NH, NOH, or NNH2.
In some embodiments of Formulas I, I-c, I-d, I-e, and I-f, Z2 is O or NH. In some embodiments, Z2 is O. In some embodiments, Z2 is NR. In some embodiments, Z2 is NH.
In some embodiments of Formulas I, I-a, I-b, I-c, I-d, I-e, and I-f, R1 and R2 are each independently selected from the group consisting of —H, halogen, —C1-C6alkyl, —(CRbRc)nheterocyclyl, —OR, —(CRbRc)nNR2, —(CRbRc)nNRC(O)R′, or —(CRbRc)nNRC(O)NR2, wherein each heterocyclyl is optionally substituted with one or more halogen, and wherein each heterocyclyl is 3- to 14-membered and contains 1-4 heteroatoms independently selected from the group consisting of O, N, and S, and wherein the heterocyclyl does not contain an O in the γ-position relative to C(═Z1); or R1 and R2 combine with the carbon to which they are attached to form a 3- to 8-membered heterocyclyl containing 1-4 heteroatoms independently selected from O, N, and S, wherein the heterocyclyl does not contain an O in the γ-position relative to C(═Z1).
In some embodiments, R1 and R2 are each independently —H, —OR, —(CRbRc)nNR2, or —(CRbRc)nNRC(O)R′. In some embodiments, R1 and R2 are each independently —H, —OR, —CH2NR2, or —CH2NRC(O)R′. In some embodiments, R1 and R2 are each independently —H, —OH, —CH2NHMe, or —CH2NHC(O)Me. In some embodiments, R1 and R2 are each independently —H, —OH, or —CH2NHMe. In some embodiments, one of R1 and R2 is not —H. In some embodiments, R1 is —OH or —(CH2)NHMe. In some embodiments, R1 is —OH. In some embodiments, R2 is —H.
In some embodiments of Formulas I, I-a, I-b, I-c, I-d, I-e, and I-f, B is:
In some embodiments of Formulas I, I-a, I-b, I-c, I-d, I-e, and I-f, B is a phenyl ring or a bicyclic ring, wherein at least one of the rings in the bicyclic ring is a phenyl ring, wherein the phenyl ring or bicyclic ring contains 0-4 heteroatoms independently selected from the group consisting of O, N, and S, and wherein the phenyl ring or bicyclic ring is optionally substituted with one or more Rd. In some embodiments, B is a phenyl ring optionally substituted with one or more Rd. In some embodiments, B is a phenyl ring optionally substituted with one or more Rd and is fused to an aromatic, saturated, or partially unsaturated 5- to 8-membered carbocyclic or heterocyclic ring. In some embodiments, B is a phenyl ring optionally substituted with one or more Rd and is fused to a saturated or partially unsaturated 5- to 8-membered heterocyclic ring. In some embodiments, B is a monocyclic or bicyclic heteroaryl ring, wherein the ring contains 1-4 heteroatoms independently selected from the group consisting of O, N, and S, and wherein the ring is optionally substituted with one or more Rd.
In some embodiments, B is selected from:
In some embodiments of Formulas I, I-a, I-b, I-c, I-d, I-e, and I-f, each Rd is independently selected from the group consisting of halogen, —OR, —NR2 (e.g., —N(Me)(CH2CH2OMe)), —C(O)NR2, —C1-C6alkyl, —C3-C12cycloalkyl, 3- to 14-membered heterocyclyl containing 1-4 heteroatoms independently selected from the group consisting of O, N, and S, and C6-C14aryl, wherein each alkyl, heterocyclyl, or aryl is optionally substituted with one or more substituents selected from the group consisting of halogen, —C1-C6alkyl optionally substituted with one or more halogen, or —C3-C12cycloalkyl. In some embodiments, each Rd is independently selected from the group consisting of halogen, —OR, —C1-C6alkyl (e.g., methyl, ethyl, —CHF2, or —CF3), —C3-C12cycloalkyl, and 3- to 14-membered heterocyclyl containing 1-4 heteroatoms independently selected from the group consisting of O, N, and S. In some embodiments, each Rd is independently selected from the group consisting of halogen, —C1-C6alkyl, and —OR.
In some embodiments of Formulas I, I-a, I-b, I-c, I-d, I-e, and I-f, each R is independently selected from the group consisting of —H, —C1-C6alkyl, —C3-C12cycloalkyl, and 3- to 14-membered heterocyclyl containing 1-4 heteroatoms independently selected from the group consisting of O, N, and S, wherein each alkyl or heterocyclyl is optionally substituted with one or more halogen, —O—C1-C6alkyl, —NH—C1-C6alkyl, —N(C1-C6alkyl)2, —C1-C6alkyl optionally substituted with —OH, —C3-C12cycloalkyl, or 3- to 8-membered heterocyclyl containing 1-4 heteroatoms independently selected from the group consisting of O, N, and S. In some embodiments, each R is independently —H, —C1-C6alkyl, or 3- to 8-membered heterocyclyl optionally substituted with C1-C6alkyl. In some embodiments, each R is independently —H or methyl.
In some embodiments of Formulas I, I-a, I-b, I-c, I-d, I-e, and I-f, each R′ is independently —C1-C6alkyl, —C3-C12cycloalkyl, or 3- to 14-membered heterocyclyl containing 1-4 heteroatoms independently selected from the group consisting of O, N, and S. In some embodiments, each R′ is independently —C1-C6alkyl.
In some embodiments, a USP9X Inhibitor is a compound of Formula II:
or a pharmaceutically acceptable salt thereof, wherein:
In some embodiments, a USP9X Inhibitor is a compound of formula II-a:
or a pharmaceutically acceptable salt thereof,
wherein Ring A, Ring B, Y1, R1, and m are as defined above for Formula II and described in classes and subclasses of Formula II herein, both singly and in combination.
In some embodiments, a USP9X Inhibitor is a compound of Formula II-b:
or a pharmaceutically acceptable salt thereof,
wherein Y1, R1, Ra, Rb, and m are as defined above for Formula II and described in classes and subclasses of Formula II herein, both singly and in combination.
In some embodiments, a USP9X Inhibitor is a compound of Formula II-c:
or a pharmaceutically acceptable salt thereof,
wherein Ring A, Ring B, Y1, R1, and m are as defined above for Formula II and described in classes and subclasses of Formula II herein, both singly and in combination.
In some embodiments, a USP9X Inhibitor is a compound of Formula II-d:
or a pharmaceutically acceptable salt thereof,
wherein Ring A, Ring B, Y1, R1, and m are as defined above for Formula II and described in classes and subclasses of Formula II herein, both singly and in combination.
In some embodiments, a USP9X Inhibitor is a compound of Formula II-e:
or a pharmaceutically acceptable salt thereof,
wherein Y1, Ra, and Rb are as defined above for Formula II and described in classes and subclasses of Formula II herein, both singly and in combination.
In some embodiments, a USP9X Inhibitor is a compound of Formula II-f:
or a pharmaceutically acceptable salt thereof,
wherein Y1, Ra, and Rb are as defined above for Formula II and described in classes and subclasses of Formula II herein, both singly and in combination.
In some embodiments of Formulas II, II-a, II-b, II-c, II-d, II-e, and II-f, Y1 is CR7 or N. In some embodiments, Y1 is CH or N. In some embodiments, Y1 is CR7. In some embodiments, Y1 is N. In some embodiments, Y1 is CH.
In some embodiments of Formulas II, II-a, II-b, II-c, II-d, II-e, and II-f, each Ra is independently halogen, —OR, —NRC(O)R′, optionally substituted 3- to 10-membered heterocyclyl containing 1-4 heteroatoms independently selected from N, O, and S, or optionally substituted 5- to 10-membered heteroaryl containing 1-4 heteroatoms independently selected from N, O, and S, wherein an optionally substituted Ra group may be substituted with one or more halogen. In some embodiments, each Ra is independently halogen or optionally substituted 5- to 10-membered heteroaryl containing 1-4 heteroatoms independently selected from N, O, and S, wherein an optionally substituted Ra group may be substituted with one or more halogen. In some embodiments, each Ra is independently halogen or optionally substituted 5-membered heteroaryl containing 1-4 heteroatoms independently selected from N, O, and S, wherein an optionally substituted Ra group may be substituted with one or more halogen. In some embodiments, each Ra is
In some embodiments of Formulas II, II-a, II-b, II-c, II-d, II-e, and II-f, each Rb is independently selected from the group consisting of halogen, —OR, optionally substituted C1-C6 aliphatic, and optionally substituted 3- to 10-membered heterocyclyl containing 1-4 heteroatoms independently selected from N, O, and S, wherein an optionally substituted Rb group may be substituted with one or more substituents independently selected from the group consisting of —NR2 and C1-C6 aliphatic. In some embodiments, each Rb is independently selected from the group consisting of —OR, optionally substituted C1-C6 aliphatic, and optionally substituted 3- to 10-membered heterocyclyl containing 1-4 heteroatoms independently selected from N, O, and S, wherein an optionally substituted Rb group may be substituted with one or more substituents independently selected from the group consisting of —NR2 and C1-C6 aliphatic. In some embodiments, each Rb is
In some embodiments of Formulas II, II-a, II-b, II-c, II-d, II-e, and II-f, R1 is selected from the group consisting of —OR, —NR2, —CN, —C(O)NR2, and C1-C6aliphatic. In some embodiments, R1 is selected from the group consisting of —H, —OR, —CN, and C1-C6aliphatic. In some embodiments, R1 is —OR. In some embodiments, R1 is —OR, and m is 0.
In some embodiments of Formulas II, II-a, II-b, II-c, II-d, II-e, and II-f, R7 is selected from the group consisting of —H, —OR, and C1-C6aliphatic. In some embodiments, R7 is —H.
In some embodiments of Formulas II, II-a, II-b, II-c, II-d, II-e, and II-f, each R is independently selected from the group consisting of —H, optionally substituted C1-C6aliphatic, and optionally substituted 3- to 10-membered heterocyclyl containing 1-4 heteroatoms independently selected from N, O, and S, wherein an optionally substituted R group may be optionally substituted with one or more C1-C6aliphatic. In some embodiments, each R is independently selected from the group consisting of —H, methyl, and 4- to 6-membered heterocyclyl containing 1-2 heteroatoms independently selected from N, O, and S optionally substituted with methyl. In some embodiments, each R is —H.
In some embodiments of Formulas II, II-a, II-b, II-c, II-d, II-e, and II-f, each R′ is independently C1-C6aliphatic or C3-C10cycloalkyl. In some embodiments, each R′ is independently C3-C10cycloalkyl. In some embodiments, each R′ is cyclopropyl.
In some embodiments of Formulas II, II-a, II-b, II-c, II-d, II-e, and II-f, m is 0, 1, or 2. In some embodiments, m is 0. In some embodiments, m is 0 or 1. In some embodiments, m is 0 or 2. In some embodiments, m is 1 or 2.
The compounds of the present disclosure may be made by a variety of methods, including standard chemistry. Suitable synthetic routes are described in the Examples given below.
Additionally, unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures including the replacement of hydrogen by deuterium or tritium (e.g., Examples 103-46 and 103-47), or the replacement of a carbon by a 13C- or 14C-enriched carbon are within the scope of this disclosure.
Unless otherwise noted, reagents and solvents were used as received from commercial suppliers. Unless otherwise noted, reactions were conducted under an inert atmosphere of nitrogen. NMR instrument: Bruker BBFO ASCEND™400 AVANCE III 400 MHz and Bruker BBFO ULTRASHIELD™300 AVANCE III 300 MHz. Internal standard: Tetramethylsilane (TMS). MassSpec instruments and ionization method: Shimadzu LC-2020, electrospray ionization, ESI. Chromatography instruments (Reverse phase chromatography: Agela TechnologiesMP200. Preparatory HPLC (Prep-HPLC): Waters. Supercritical fluid chromatography (SFC): Shimadzu).
In some embodiments, a USP9X Inhibitor has one or more of the following characteristics when tested in the Biochemical Assay of Example A:
In some embodiments, a USP9X Inhibitor is a compound having an IC50 value of ≤2 μM and >0.2 μM when tested in the Biochemical Assay of Example A. In some embodiments, a USP9X Inhibitor is a compound having an IC50 value of ≤0.2 μM and >0.05 μM when tested in the Biochemical Assay of Example A. In some embodiments, a USP9X Inhibitor is a compound having an IC50 value of ≤0.05 μM and >0.001 μM when tested in the Biochemical Assay of Example A. In some embodiments, a USP9X Inhibitor is a compound having an IC50 value of ≤0.1 μM and >0.001 μM when tested in the Biochemical Assay of Example A. In some embodiments, a USP9X Inhibitor is a compound having an IC50 value of ≤1 μM and >0.1 μM when tested in the Biochemical Assay of Example A. In some embodiments, a USP9X Inhibitor is a compound having an IC50 value≤10 μM and >1 μM when tested in the Biochemical Assay of Example A.
In some embodiments, a USP9X Inhibitor is selected based on various characteristics of the USP9X Inhibitor, including but not limited to the IC50 value in the Biochemical Assay of Example A.
In some embodiments, a USP9X Inhibitor is a compound, or pharmaceutically acceptable salt thereof, selected from:
In some embodiments, the amount of USP9X Inhibitor administered in methods provided herein is a therapeutically effective amount.
Checkpoint blockade therapies have produced durable clinical responses in a subset of cancers. For example, binding of the ligand PD-L1 and PD-L2 to the PD-1 receptor found on T-cells inhibits T-cell proliferation and cytokine production. Upregulation of PD-1 ligands occurs in some tumors and signaling through this pathway can contribute to inhibition of active T-cell surveillance of tumors. Therefore, therapies, such as an immune checkpoint pathway inhibitor, that bind to the PD-1 receptor and block its interaction with PD-L1 and PD-L2, prevents PD-1 pathway-mediated inhibition of the immune response and can result in decreased tumor growth.
An immune checkpoint pathway inhibitor can be selected from compounds that inhibit cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and/or programmed death 1 (PD-1). In some embodiments, the checkpoint pathway inhibitor is a CTLA-4 inhibitor. In some embodiments, the checkpoint pathway inhibitor is a PD-1 inhibitor. In some embodiments, the immune checkpoint pathway inhibitor is atezolizumab, durvalumab, ipilimumab, nivolumab, or pembrolizumab.
In some embodiments, the immune checkpoint pathway inhibitor is an antibody. In some embodiments, the immune checkpoint pathway inhibitor is anti-CTLA-4 antibody. In some embodiments, the anti-CTLA-4 antibody is imilimumab. In some embodiments, the immune checkpoint pathway inhibitor is anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody is nivolumab. In some embodiments, the anti-PD-1 antibody is pembrolizumab.
In some embodiments, the immune checkpoint pathway inhibitor is ipilimumab. Ipilimumab is a fully human IgGlK monoclonal antibody targeting CTLA-4 that inhibits the negative downstream signaling that occurs when CTLA-4 engages its ligands, CD80 and CD86, expressed on antigen presenting cells, thereby, blocking the negative down-regulation of the immune responses elicited by the interaction of these molecules. As a result, activated T cells are able to maintain their CD28 mediated signaling resulting in IL-2 secretion and proliferation of CD8 T cells in response to an antigen.
Ipilimumab is approved by the FDA for:
In some embodiments, the immune checkpoint pathway inhibitor is nivolumab. Nivolumab is a fully human IgG4 programmed death 1 (PD-1) immune checkpoint pathway inhibitor antibody that selectively blocks the interaction of the PD-1 receptor with its two known programmed death ligands, PD-L1 and PD-L2, disrupting the negative signal that regulates T-cell activation and proliferation.
Nivolumab is approved by the FDA for:
In some embodiments, the immune checkpoint pathway inhibitor is pembrolizumab. Pembrolizumab is a humanized IgG4 monoclonal antibody against programmed death receptor-1 (PD-1).
Pembrolizumab is approved by the FDA for:
In some embodiments, the immune checkpoint pathway inhibitor is atezolizumab. is a programmed cell death ligand 1 (PD-L1) blocking antibody. Atezolizumab is an Fc-engineered, humanized, non-glycosylated IgG1 kappa immunoglobulin that has a calculated molecular mass of 145 kDa.
Atezolizumab is approved by the FDA for:
In some embodiments, the immune checkpoint pathway inhibitor is durvalumab. Durvalumab is a programmed cell death ligand 1 (PD-L1) blocking antibody. Durvalumab is a human immunoglobulin G1 kappa (IgG1κ) monoclonal antibody that is produced by recombinant DNA technology in Chinese Hamster Ovary (CHO) cell suspension culture.
Durvalumab is approved by the FDA for:
The dose of an immune checkpoint pathway inhibitor and the frequency of dosing can be selected based on various characteristics of the immune checkpoint pathway inhibitor, including the pharmacokinetic properties of the inhibitor (e.g., half-life), prior dosing regimens, and patient characteristics. In some embodiments, the amount of immune checkpoint pathway inhibitor administered in methods provided herein is a therapeutically effective amount.
In some embodiments, the method of treating cancer in a patient in need thereof comprises administering to the patient a USP9X Inhibitor, wherein the patient is receiving or has received ipilimumab. In some embodiments, a method of treating cancer in a patient in need thereof comprises administering to the patient a USP9X Inhibitor, wherein the patient is receiving or has received ipilimumab in a dose of 3 mg/kg over 90 minutes every 3 weeks for a total of 4 doses. In some embodiments, a method of treating cancer in a patient in need thereof comprises administering to the patient a USP9X Inhibitor, wherein the patient is receiving or has received ipilimumab in a dose of 10 mg/kg over 90 minutes every 3 weeks for a total of 4 doses. In some embodiments, a method of treating cancer in a patient in need thereof comprises administering to the patient a USP9X Inhibitor, wherein the patient is receiving or has received ipilimumab in a dose of 10 mg/kg over 90 minutes every 3 weeks for a total of 4 doses, followed by 10 mg/kg every 12 weeks for up to 3 years. In some embodiments, a method of treating cancer in a patient in need thereof comprises administering to the patient a USP9X Inhibitor, wherein the patient is receiving or has received ipilimumab in a dose of 1 mg/kg over 30 minutes every 3 weeks for a total of 4 doses.
In some embodiments, the method of treating cancer in a patient in need thereof comprises administering to the patient a ipilimumab, wherein the patient is receiving or has received USP9X Inhibitor. In some embodiments, a method of treating cancer in a patient in need thereof comprises administering to the patient ipilimumab in a dose of 3 mg/kg over 90 minutes every 3 weeks for a total of 4 doses, wherein the patient is receiving or has received a USP9X Inhibitor. In some embodiments, a method of treating cancer in a patient in need thereof comprises administering to the patient ipilimumab in a dose of 10 mg/kg over 90 minutes every 3 weeks for a total of 4 doses, wherein the patient is receiving or has received a USP9X Inhibitor. In some embodiments, a method of treating cancer in a patient in need thereof comprises administering to the patient ipilimumab in a dose of 10 mg/kg over 90 minutes every 3 weeks for a total of 4 doses, followed by 10 mg/kg every 12 weeks for up to 3 years, wherein the patient is receiving or has received a USP9X Inhibitor. In some embodiments, a method of treating cancer in a patient in need thereof comprises administering to the patient ipilimumab in a dose of 1 mg/kg over 30 minutes every 3 weeks for a total of 4 doses, wherein the patient is receiving or has received a USP9X Inhibitor.
In some embodiments, the method of treating cancer in a patient in need thereof comprises administering to the patient a USP9X Inhibitor, wherein the patient is receiving or has received nivolumab. In some embodiments, a method of treating cancer in a patient in need thereof comprises administering to the patient a USP9X Inhibitor, wherein the patient is receiving or has received nivolumab in a dose of 3 mg/kg over 60 minutes every 2 weeks. In some embodiments, the method of treating cancer in a patient in need thereof comprises administering to the patient a USP9X Inhibitor, wherein the patient is receiving or has received nivolumab in a dose of 3 mg/kg over 30 minutes.
In some embodiments, the method of treating cancer in a patient in need thereof comprises administering to the patient a nivolumab, wherein the patient is receiving or has received USP9X Inhibitor. In some embodiments, a method of treating cancer in a patient in need thereof comprises administering to the patient nivolumab in a dose of 3 mg/kg over 60 minutes every 2 weeks, wherein the patient is receiving or has received USP9X Inhibitor. In some embodiments, a method of treating cancer in a patient in need thereof comprises administering to the patient nivolumab in a dose of 3 mg/kg over 30 minutes, wherein the patient is receiving or has received USP9X Inhibitor.
In some embodiments, the method of treating cancer in a patient in need thereof comprises administering to the patient a USP9X Inhibitor, wherein the patient is receiving or has received ipilimumab and nivolumab. In some embodiments, the method of treating cancer in a patient in need thereof comprises administering to the patient a USP9X Inhibitor, wherein the patient is receiving or has received nivolumab in a dose of 3 mg/kg over 30 minutes followed by ipilimumab in a dose of 1 mg/kg over 30 minutes on the same day, every 3 weeks for a total of 4 doses. In some embodiments, the method of treating cancer in a patient in need thereof comprises administering to the patient a USP9X Inhibitor, wherein the patient is receiving or has received nivolumab in a dose of 3 mg/kg over 30 minutes followed by ipilimumab in a dose of 1 mg/kg over 30 minutes on the same day, every 3 weeks for a total of 4 doses, then nivolumab in a dose of 240 mg every 2 weeks over 30 minutes. In some embodiments, the method of treating cancer in a patient in need thereof comprises administering to the patient a USP9X Inhibitor, wherein the patient is receiving or has received nivolumab in a dose of 3 mg/kg over 30 minutes followed by ipilimumab in a dose of 1 mg/kg over 30 minutes on the same day, every 3 weeks for a total of 4 doses, then nivolumab in a dose of 480 mg every 4 weeks over 30 minutes.
In some embodiments, the method of treating cancer in a patient in need thereof comprises administering to the patient ipilimumab and nivolumab, wherein the patient is receiving or has received a USP9X Inhibitor. In some embodiments, a method of treating cancer in a patient in need thereof comprises administering to the patient nivolumab in a dose of 3 mg/kg over 30 minutes and (e.g., followed by) ipilimumab in a dose of 1 mg/kg over 30 minutes on the same day, every 3 weeks for a total of 4 doses, wherein the patient is receiving or has received a USP9X Inhibitor. In some embodiments, a method of treating cancer in a patient in need thereof comprises administering to the patient nivolumab in a dose of 3 mg/kg over 30 minutes and (e.g., followed by) ipilimumab in a dose of 1 mg/kg over 30 minutes on the same day, every 3 weeks for a total of 4 doses, then nivolumab in a dose of 240 mg every 2 weeks over 30 minutes, wherein the patient is receiving or has received a USP9X Inhibitor. In some embodiments, a method of treating cancer in a patient in need thereof comprises administering to the patient nivolumab in a dose of 3 mg/kg over 30 minutes and (e.g., followed by) ipilimumab in a dose of 1 mg/kg over 30 minutes on the same day, every 3 weeks for a total of 4 doses, then nivolumab in a dose of 480 mg every 4 weeks over 30 minutes, wherein the patient is receiving or has received a USP9X Inhibitor.
In some embodiments, the method of treating cancer in a patient in need thereof comprises administering to the patient a USP9X Inhibitor, wherein the patient is receiving or has received pembrolizumab. In some embodiments, the method of treating cancer in a patient in need thereof comprises administering to the patient a USP9X Inhibitor, wherein the patient is receiving or has received pembrolizumab in a dose of 200 mg every 3 weeks. In some embodiments, the method of treating cancer in a patient in need thereof comprises administering to the patient a USP9X Inhibitor, wherein the patient is receiving or has received pembrolizumab in a dose of 2 mg/kg over 30 minutes every 3 weeks.
In some embodiments, the method of treating cancer in a patient in need thereof comprises administering to the patient pembrolizumab, wherein the patient is receiving or has received a USP9X Inhibitor. In some embodiments, the method of treating cancer in a patient in need thereof comprises administering to the patient pembrolizumab in a dose of 200 mg every 3 weeks, wherein the patient is receiving or has received a USP9X Inhibitor. In some embodiments, the method of treating cancer in a patient in need thereof comprises administering to the patient pembrolizumab in a dose of 2 mg/kg over 30 minutes every 3 weeks, wherein the patient is receiving or has received a USP9X Inhibitor.
In this Example, human peripheral blood mononuclear cells (PBMCs) were stimulated with Staphylococcal enterotoxin B (SEB) to induce inactive T cells in vitro. PBMCs were then washed to remove SEB and allowed to rest for 2 days in the presence or absence of an agent (e.g., a USP9X Inhibitor, anti-CTLA4 antibody, or anti-PD1 antibody). The supernatant was collected for IFN-γ measurement, and cell pellets were collected for Western blot analysis.
Production of IFNγ was restored in a concentration-dependent manner by USP9X Inhibitors 1 and 2 and was not restored by the negative control compound 5, anti-CTLA4 antibody, or anti-PD1 antibody (
As used herein a “negative control compound” is a compound with an IC50 value of >12 μM in the Biochemical Assay of Example A. In some embodiments, a negative control compound is compound 5.
Further, concentration-dependent reductions of ITCH and Cbl-b protein levels were observed in the presence of USP9X Inhibitors 1 and 2 (
In addition, USP9X Inhibitor 3 also promoted IFNγ secretion in SEB-stimulated PBMCs in a concentration-dependent manner (EC50≤1 μM and >0.1 μM), whereas negative control compound 5 did not have any significant effect (
The effect of USP9X inhibition on PBMCs that have been re-stimulated with SEB was also investigated. Naïve PBMCs produced robust increases in IFNγ (
Taken together, these results suggest USP9X inhibition can rescue T cell restimulation by upregulating IFNγ secretion and by downregulating ITCH and Cbl-b levels.
Given the reported role of ITCH and Cbl-b in T cell tolerance and as a negative regulator of T-cell receptor (TCR)/catalytic domain 28 (CD28) signaling, the effects of USP9X inhibition were evaluated in a CD3/CD28 activation assay and in a mixed lymphocyte reaction (MLR) assay. In an anti-CD3/CD28 T cell activation assay, several USP9X inhibitors demonstrated enhanced IFNγ production, with USP9X Inhibitor 3 yielding the most profound effect (
USP9X Inhibitor 3 also promoted IFNγ secretion in the MLR assay in a concentration-dependent manner (EC50≤1 μM and >0.1 μM), whereas negative control compound 5 did not have any significant effect (
Activation of allogenic CD4+ T cells cultured with allogenic dendritic cells (DCs) in the presence or absence of a USP9X Inhibitor was determined in an MLR assay. Monocytes were first isolated from healthy human PBMCs using magnetic beads and plated in RPMI 1640 medium with 10% fetal bovine serum (FBS) for dendritic cell maturation. Monocytes were then cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 (20 ng/mL; RnD systems) in order to induce formation of immature dendritic cells. Cytokines were added every other day. After 5-6 days of culture, a maturation cocktail containing 100 ng/ml tumor necrosis factor alpha (TNFa), IL-6, and IL-1β, as well as 1 μg/mL prostaglandin E2 (PGE2) was added to the culture medium for 24 hours. 145,000 matured DCs per well were added to a 96 well plate. Allogeneic CD4+ T cells were then isolated and added to 70 μL of diluted compounds in a fresh 96 well plate. 70 μL of CD4+ cells were added at a concentration of 1.45 million cells per well, resulting in a 10:1 CD4+T:DC ratio in the final experiment. CD4+ T cells were pre-incubated with agent (e.g., USP9X Inhibitor, anti-PD-1 antibody, or anti-CTLA-4 antibody) for 1 hour at 37° C. After pre-incubation, 70 μL of pre-diluted DCs were added to the CD4/compound plate. The co-culture was incubated for 4 days. The supernatant was removed and analyzed for IFNγ and IL-2 using Meso Scale Discovery Immunoassay (Meso Scale Discovery).
USP9X Inhibitors 1 and 2 enhanced IL-2 (
To determine if USP9X inhibition could potentiate immune-mediated cytotoxicity against tumor cells, human PBMCs were rendered inactive by SEB stimulation prior to incubation with A375 melanoma cells in the presence of DMSO, USP9X Inhibitor 3, or a negative control, and apoptosis of the A375 melanoma cells was evaluated. USP9X Inhibitor 3 enhanced A375 apoptosis in a concentration-dependent manner as indicated by caspase 3/7 activity (
A-375 tumor cells were cultured in 96 well plates overnight in DMEM/10% FBS such that a density of 10,000 cells per well was reached. After 24 hours, media was removed and replaced with Dulbecco's Modified Eagle Medium (DMEM) with 10% FBS containing 1× IncuCyte® Caspase 3/7 Apoptosis Assay Reagent, agent, and PBMCs from healthy human donors. PBMCs were added in a 20:1 ratio relative to A-375 cell number. As a positive control, anti-CD3 (0.1 ug/mL) and IL-2 (10 ng/mL) were added to 2 wells per plate. Plates were incubated and imaged in an IncuCyte® Live Cell Imaging System (Essen Biosciences) for 4 days. Apoptotic A-375 cells were counted via an IncuCyte® image analysis algorithm.
Both USP9X Inhibitor 1 (
Human total T cells (CD3+) were isolated using EasySep™ immunomagnetic beads (Stemcell Technologies) from healthy human PBMCs (Hemacare). Cells were stained with Cell Trace Violet proliferation dye (Invitrogen), and treated with agent or DMSO. Cells were also stimulated with a CD3/CD28 activator (Stemcell Technologies). After 4 days of incubation, cells and supernatant were collected; cells were washed with staining buffer (PBS with 2% FBS) twice and centrifuged (350 g for 5 min). Cells were stained with LIVE/DEAD stain (Invitrogen), FITC-CD4 (BD Pharmingen) and CD8 (BD Pharmingen) in 100 μL volume for 30 mins at 4° C. Cells were washed twice as described above, resuspended in 100 μL staining buffer and analyzed using a BD Canto II (BD Biosciences). Data was analyzed using FlowJo V10 (FlowJo LLC). Secreted IFNγ was measured by ELISA (Dakewe Biotech Co., Ltd). All data were analyzed by Graphpad Prism 6.0 (GraphPad Software).
IFNγ production was increased in the presence of USP9X Inhibitors 4 (
Peripheral tolerance/inactivation was induced in female BALB/c mice by a single intraperitoneal injection of 30 μg SEB. Mice were treated with a positive control (anti-CTLA4) or various doses of USP9X Inhibitor 3. Spleens and splenocytes were harvested at 24 hr after the last dose. Spleen protein levels of Cbl-b and ITCH were reduced 81% and 64%, respectively in the highest dose group, and there were no notable changes at the lower doses. No changes in IL-2 and IFNγ levels were detected in the supernatants from SEB-restimulated splenocytes. Splenocytes isolated from SEB+anti-CTLA4-treated mice showed a significant reduction of Cbl-b and ITCH and significant increase in IL-2 and IFNγ levels. These results suggest target engagement of USP9X occurs in vivo.
The effect of a combination of USP9X Inhibitor and an immune checkpoint pathway inhibitor on tumor cells was evaluated. A-375 tumor cells were cultured in 96 well plates overnight in DMEM with 10% FBS such that a density of 10,000 cells per well was reached. After 24 hours, media was removed and replaced with DMEM with 10% FBS containing 1X IncuCyte® Caspase 3/7 Apoptosis Assay Reagent, agent, and PBMCs from healthy human donors. The agent was selected from USP9X Inhibitor 2 alone, anti-CTLA-4 alone, or a combination thereof. PBMCs were added in a 5:1, 10:1, or 20:1 ratio relative to A-375 cell number. As a positive control, anti-CD3 (0.1 ug/mL) and IL-2 (10 ng/mL) were added to 2 wells per plate. Plates were incubated and imaged in an IncuCyte® Live Cell Imaging System (Essen Biosciences) for 4 days. Apoptotic A-375 cells were counted via an IncuCyte® image analysis algorithm.
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To a solution of 3-methyl-2-nitrophenol (200 g, 1.29 mol) in acetic anhydride (1600 mL) was added sulfuric acid (240 mL) and acetic acid (1620 mL). This was followed by the addition of chromium trioxide (280 g, 2.77 mol) in several batches with stirring at 0° C. The resulting mixture was stirred for 2.5 h at 0° C. and then poured into ice/water (5000 mL). The solids were collected by filtration and then washed with water (3×1 L), saturated sodium carbonate solution (3×800 mL), and water (3×1 L). The solids were dissolved in ethanol (380 mL) and concentrated hydrochloric acid (617 mL). The resulting solution was stirred for 1.5 h at 110° C. and then cooled to room temperature. The reaction mixture was concentrated under vacuum to afford 3-hydroxy-2-nitrobenzaldehyde as a yellow solid (38.0 g, 18%). LCMS (ES, m/z): 166 [M−H]−.
To a solution of 3-hydroxy-2-nitrobenzaldehyde (38.0 g, 204 mmol) in dichloromethane (500 mL) was added ZnI2 (14.5 g, 44.5 mmol). The reaction was treated with trimethylsilyl cyanide (100 mL, 708 mmol) added dropwise with stirring at 0° C. The resulting mixture was stirred for 2.5 h at 25° C. The reaction was poured into brine (200 mL) and then extracted with ethyl acetate (3×500 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to afford 2-hydroxy-2-(3-hydroxy-2-nitrophenyl)acetonitrile as a yellow solid (34.0 g, 73%). LCMS (ES, m/z): 195 [M+H]+.
To a solution of 2-hydroxy-2-(3-hydroxy-2-nitrophenyl)acetonitrile (34.0 g, 157 mmol) in methanol (80 mL) was added hydrochloric acid (80 mL, 4 N in 1,4-dioxane). The resulting solution was stirred for 45 min at 60° C. and cooled to room temperature. The reaction mixture was concentrated under vacuum and purified by silica gel chromatography (eluting with 0:100 to 35:65 ethyl acetate/petroleum ether) to afford methyl 2-hydroxy-2-(3-hydroxy-2-nitrophenyl)acetate as a yellow solid (23.0 g, 58%). LCMS (ES, m/z): 228 [M+H]+.
To a solution of methyl 2-hydroxy-2-(3-hydroxy-2-nitrophenyl)acetate (23.0 g, 0.11 mol) in methanol (500 mL) was added anhydrous palladium carbon (2.3 g, 10 wt % Pd). The resulting mixture was stirred for 16 h at 25° C. under hydrogen atmosphere (3 atm). The reaction mixture was filtered and concentrated under vacuum to afford methyl 2-(2-amino-3-hydroxyphenyl)-2-hydroxyacetate as a yellow solid (14.0 g, 60%). LCMS (ES, m/z): 198 [M+H]+.
To a solution of methyl 2-(2-amino-3-hydroxyphenyl)-2-hydroxyacetate (9.0 g, 43.4 mmol) in 1,1,1-triethoxyethane (150 mL) was added bismuth (III) trifluoromethanesulfonate (800 mg, 1.18 mmol). The resulting mixture was stirred for 10 min at 85° C. After cooling to room temperature, the reaction mixture was concentrated under vacuum. The resulting crude product was purified by silica gel chromatography (eluting with 0:100 to 50:50 ethyl acetate/petroleum ether) to afford methyl 2-hydroxy-2-(2-methyl-1,3-benzoxazol-4-yl)acetate as a white solid (6.3 g, 63%). LCMS (ES, m/z): 222 [M+H]+.
To a solution of methyl 2-hydroxy-2-(2-methyl-1,3-benzoxazol-4-yl)acetate (500 mg, 2.26 mmol) in tetrahydrofuran (20 mL) and water (2 mL) was added lithium hydroxide (271 mg, 11.3 mmol). The resulting mixture was stirred for 1 h at room temperature. The reaction mixture was washed with diethyl ether (1×10 mL) and then acidified to pH=6 with hydrochloric acid (1 N). The resulting solution was extracted with ethyl acetate (2×20 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to afford 2-hydroxy-2-(2-methyl-1,3-benzoxazol-4-yl)acetic acid as a white solid (386 mg, 82%). LCMS (ES, m/z): 208 [M+H]+.
To a solution of n-BuLi (2.0 mL, 2.5 M in hexane) was added n-Bu2Mg (4.8 mL, 1.0 M in heptane). The resulting mixture was stirred for 10 min at room temperature. The reaction was treated with 7-bromo-2H,3H-[1,4]dioxino[2,3-b]pyridine (2.0 g, 9.26 mmol) in tetrahydrofuran (16 mL) added dropwise with stirring at −10° C. over a period of 10 min. The mixture was stirred for 1 h at −10° C. and then slowly added to a solution of sulfuryl dichloride (16 mL, 0.20 mol) in toluene (16 mL) at −10° C. and stirred for an additional 1 h. The reaction was quenched by the careful addition of saturated aqueous ammonium chloride solution (30 mL) at 0° C. The product was extracted with dichloromethane (3×50 mL). The organic layers were combined, dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The resulting crude product was purified by silica gel column (eluted with 1:3 ethyl acetate/petroleum ether) to afford 2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl chloride as a white solid (1.3 g, 60%). LCMS: (ES, m/z): 236, 238 [M+H]+.
To a solution of 1H-imidazole (14.5 g, 212 mmol) in dichloromethane (140 mL) was added 2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl chloride (25.0 g, 96 mmol) in dichloromethane (250 mL) dropwise with stirring at 0° C. The resulting mixture was stirred for 2 h at room temperature and then filtered and concentrated under vacuum. The solids were dissolved in absolute ethanol (125 mL) and added dropwise to a solution of 1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrole dihydrobromide (86.8 g, 319 mmol) in water (125 mL). The reaction was stirred for 18 h at room temperature and then 48 h at 60° C. After cooling to room temperature, the mixture was rendered basic (pH=14) with aqueous sodium hydroxide (50 wt %). The product was extracted with dichloromethane (3×300 mL). The organic layers were combined, dried over anhydrous sodium sulfate, filtered and concentrated under vacuum to afford of 2-[2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl]-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrole as a yellow solid (13 g, 39.5%). LCMS: (ES, m/z): 310 [M+H]+.
To a solution of 7-(4,5-dihydropyrrolo[3,4-c]pyrrol-2(1H,3H,4H)-ylsulfonyl)-2,3-dihydro-[1,4]dioxino[2,3-b]pyridine (B1) (1 equiv) in N,N-dimethylformamide was added 2-hydroxy-2-(2-methyl-1,3-benzoxazol-4-yl)acetic acid (A1) (1 equiv), DIEA (2 equiv), HOBt (1.1 equiv) and EDCI (1.1 equiv). The resulting mixture was stirred for 2 h at room temperature and poured into water. The resulting solution was extracted with EtOAc. The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The resulting crude product was purified by prep-TLC. 1H-NMR: (DMSO-d6, 400 MHz) δ (ppm): 8.16 (d, J=2.4 Hz, 1H), 7.63-7.58 (m, 2H), 7.33-7.32 (m, 2H), 5.70 (s, 2H), 4.52-4.50 (m, 2H), 4.34-4.32 (m, 3H), 4.09-4.05 (m, 7H), 2.63 (s, 3H). The two enantiomers were separated by Chiral Prep-HPLC (Column: CHIRALPAK IF, 5 μm, 20×250 mm; Mobile Phase, A: DCM and B: MeOH (keep 60% B over 18 min); Flow rate: 16 mL/min; Detector: UV 254/220 nm; Retention time: 1st eluting isomer (1), 11.23 min; 2nd eluting isomer (5), 15.39 min).
To a solution of 6-bromo-3,4-dihydro-2H-1,4-benzoxazine (5.00 g, 20.1 mmol) in CH3CN (150 mL) was added paraformaldehyde (3.26 g, 40.2 mmol) and sodium cyanoborohydride (2.30 g, 36.49 mmol). The resulting mixture was stirred for 15 min at 0° C. The reaction was treated with acetic acid (5 mL) and stirred for 16 h at room temperature. The reaction was quenched with water (50 mL) and then extracted with ethyl acetate (3×150 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting crude product was purified by silica gel chromatography (eluting with 0:100 to 80:20 ethyl acetate/petroleum ether) to afford 6-bromo-4-methyl-3,4-dihydro-2H-1,4-benzoxazine as a red oil (2.50 g, 55%). LCMS (ES, m/z): 228, 230 [M+H]+.
To a solution of 6-bromo-4-methyl-3,4-dihydro-2H-1,4-benzoxazine (2.50 g, 11.0 mmol) in THF (25 mL) was added n-BuLi (13.2 mL, 2.5 M in n-hexane) dropwise with stirring at −78° C. After 15 min diethyl oxalate (4.46 mL, 33.0 mmol) was added and stirring continued for 2 h. The reaction was poured into saturated aqueous ammonium chloride (10 mL). The product was extracted with ethyl acetate (3×25 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting crude product was purified by silica gel chromatography (eluting with 0:100 to 10:90 ethyl acetate/petroleum ether) to afford ethyl 2-(4-methyl-3,4-dihydro-2H-1,4-benzoxazin-6-yl)-2-oxoacetate as a yellow oil (500 mg, 18%). LCMS (ES, m/z): 250 [M+H]+.
To a solution of ethyl 2-(4-methyl-3,4-dihydro-2H-1,4-benzoxazin-6-yl)-2-oxoacetate (250 mg, 1.00 mmol) in tetrahydrofuran (10 mL) was added sodium borohydride (57 mg, 1.51 mmol). The resulting mixture was stirred for 10 min at 0° C. and then poured into water (10 mL). The product was extracted with ethyl acetate (3×10 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The crude product was purified by silica gel chromatography (eluting with 0:100 to 20:80 ethyl acetate/petroleum ether) to afford ethyl 2-hydroxy-2-(4-methyl-3,4-dihydro-2H-1,4-benzoxazin-6-yl)acetate as a colorless oil (180 mg, 71%). LCMS (ES, m/z): 252 [M+H]+.
To a solution of ethyl 2-hydroxy-2-(4-methyl-3,4-dihydro-2H-1,4-benzoxazin-6-yl)acetate (180 mg, 0.72 mmol) in tetrahydrofuran (2 mL) and water (2 mL) was added lithium hydroxide (87 mg, 3.63 mmol). The resulting mixture was stirred for 16 h at room temperature. The reaction mixture was washed with diethyl ether (1×8 mL) and then concentrated under vacuum to afford lithium 2-hydroxy-2-(4-methyl-3,4-dihydro-2H-1,4-benzoxazin-6-yl)acetate as a yellow oil (100 mg, 16%). LCMS (ES, m/z): 224 [M+H]+.
To a solution of 2,3-dimethylbut-2-ene (1000 g, 11.9 mol) in DCM (1000 mL) in a 4 L 4-necked round bottom flask was added aqueous hydrogen bromide solution (150 mL, 48%) with stirring at 10-15° C. To the reaction was added bromine (9.90 kg, 62.0 mol) with stirring at 0° C. The resulting mixture was stirred for 2 days at 45° C. in an oil bath. After cooling to room temperature, the reaction mixture was carefully poured into saturated aqueous sodium hydrogen sulfite solution (10 L). The precipitate was collected by filtration and dried in oven to afford 1,4-dibromo-2,3-bis(bromomethyl)but-2-ene as a light yellow solid (3000 g, 44%). GCMS: (EI, m/z): 398, 400, 402 [M]+.
To a solution of 1,4-dibromo-2,3-bis(bromomethyl)but-2-ene (2000 g, 3.50 mol) in DMF (20 L) was added 4-methylbenzene-1-sulfonamide (2137 g, 12.5 mol), and potassium carbonate (5175 g, 37.4 mol). The resulting mixture was stirred for 2 days at room temperature. The reaction mixture was slowly poured into water/ice (20 L). The precipitate was collected by filtration, washed with ethanol and dried in an oven to afford 2,5-ditosyl-1,2,3,4,5,6-hexahydropyrrolo[3,4-c]pyrrole as a light yellow solid (1345 g, 78%). LCMS: (ES, m/z): 419 [M+H]+.
To a solution of 2,5-ditosyl-1,2,3,4,5,6-hexahydropyrrolo[3,4-c]pyrrole (1345 g, 2.73 mol) in aqueous hydrogen bromide solution (4500 mL, 48%) in 10 L 4-necked round-bottom flask, was added phenol (1270 g, 13.5 mol). The resulting mixture was stirred for 2 days at 120° C. After cooling to room temperature, the aqueous layer was collected and concentrated under vacuum. The resulting solids were washed with DCM/MeOH (v:v=10:1, 3×300 mL) and dried in an oven to afford 1,2,3,4,5,6-hexahydropyrrolo[3,4-c]pyrrole hydrogen bromide salt as a yellow solid (480 g, 61%). L CMS: (ES, m/z): 111 [M+H]+.
To a suspension of 1,2,3,4,5,6-hexahydropyrrolo[3,4-c]pyrrole hydrogen bromide salt (458 g, 1.52 mol) in water (4 L) was added sodium bicarbonate (424 g, 5.05 mol) followed by dropwise addition of a solution of di-tert-butyl dicarbonate (807 g, 3.70 mol) in methanol (500 mL) with stirring at 0° C. The resulting solution was stirred for 16 h at 25° C. The precipitate was collected by filtration and dried in an oven to afford di-tert-butyl pyrrolo[3,4-c]pyrrole-2,5(1H,3H,4H,6H)-dicarboxylate as a white solid (300 g, 61%). LCMS (ES, m/z): 311[M+H]+.
To a solution of di-tert-butyl pyrrolo[3,4-c]pyrrole-2,5(1H,3H,4H,6H)-dicarboxylate (200 g, 612 mmol) in propan-2-yl acetate (5 L) was added 4-methylbenzene-1-sulfonic acid (123 g, 647 mmol) in portions at 0° C. The resulting mixture was stirred for 16 h at 55° C. in an oil bath. After cooling to room temperature, the precipitate was collected by filtration and dried in an oven to afford tert-butyl 4,5-dihydropyrrolo[3,4-c]pyrrole-2(1H,3H,4H)-carboxylate 4-methylbenzene-1-sulfonic acid salt as a yellow solid (197 g, 80%). LCMS: (ES, m/z): 211[M+H]+.
To a suspension of tert-butyl 4,5-dihydropyrrolo[3,4-c]pyrrole-2(1H,3H,4H)-carboxylate 4-methylbenzene-1-sulfonic acid salt (61 g, 142 mmol) in water (100 mL) and tetrahydrofuran (30 mL) was added sodium hydroxide (13 g, 325 mmol) followed by portion-wise addition of 2,3-dihydro-1,4-benzodioxine-6-sulfonyl chloride (25 g, 95.9 mmol) at 0° C. The resulting mixture was stirred for 2 h at 25° C. The product was extracted with ethyl acetate (3×200 mL). The organic layers were combined, dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The resulting material was purified by silica gel chromatography (eluting with 1:10 ethyl acetate/petroleum ether) to afford tert-butyl 5-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)-4,5-dihydropyrrolo[3,4-c]pyrrole-2(1H,3H,4H)-carboxylate as a white solid (30 g, 73%). LCMS: (ES, m/z): 409 [M+H]+.
To a solution of tert-butyl 5-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)-4,5-dihydropyrrolo[3,4-c]pyrrole-2(1H,3H,4H)-carboxylate (30.0 g, 69.8 mmol) in 1,4-dioxane (100 mL) was added hydrochloric acid (200 mL, 4 M in 1,4-dioxane). The resulting solution was stirred for 2 h at 25° C. and then concentrated under vacuum to afford 2-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)-1,2,3,4,5,6-hexahydropyrrolo[3,4-c]pyrrole hydrochloric salt (B2) as a yellow solid (20 g, 79%). LCMS: (ES, m/z): 309 [M+H]+.
To a solution of lithium 2-hydroxy-2-(4-methyl-3,4-dihydro-2H-1,4-benzoxazin-6-yl)acetate (A2) (1 equiv) in DMF was added HATU (1.2 equiv), 2-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)-1,2,3,4,5,6-hexahydropyrrolo[3,4-c]pyrrole hydrochloric salt (1B2) (1 equiv) and DIEA (3 equiv). The resulting mixture was stirred for 1 h at room temperature. The reaction mixture was poured into water and then extracted with ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting crude product was purified by Prep-HPLC. The product fractions were concentrated under vacuum. 1H-NMR: (CDCl3, 400 MHz) δ (ppm): 7.37-7.27 (m, 2H), 7.02-6.96 (m, 1H), 6.73 (d, J=7.6 Hz, 1H), 6.63-6.61 (m, 2H), 4.89-4.87 (m, 1H), 4.33-4.25 (m, 7H), 4.15-4.01 (m, 6H), 3.73-3.71 (m, 1H), 3.29-3.27 (m, 2H), 2.90 (s, 3H). The two enantiomers were further separated by Chiral Prep-HPLC (Column: CHIRALPAK IF, 5 μm, 100×460 mm; Mobile Phase, A: DCM, and B: MeOH (containing 0.1% DEA) (keep 75% B over 16 min); Flow rate: 20 mL/min; Detector: UV 254/220 nm; Retention time: 1st eluting isomer, 2.182 min; 2nd eluting isomer (2), 2.988 min).
To a solution of 2-(3-chlorophenyl)acetic acid (23.2 g, 0.14 mol) in toluene (300 mL) was added (4S)-4-benzyl-1,3-oxazolidin-2-one (20 g, 0.11 mol) followed by the slow addition of TEA (46 g, 0.45 mol) with stirring at 15° C., and then slow addition of 2,2-dimethylpropanoyl chloride (17.4 g, 0.14 mol) with stirring at 30° C. The resulting mixture was stirred for 3 h at 110° C., and then cooled to room temperature. The mixture was concentrated under vacuum and the resulting crude product was purified by silica gel chromatography (eluting with 0:100 to 5:95 ethyl acetate/petroleum ether) to afford (4S)-4-benzyl-3-[2-(3-chlorophenyl)acetyl]-1,3-Oxazolidin-2-one as a yellow solid (18 g, 40%). 1H-NMR (CDCl3, 400 MHz) δ (ppm): 7.43-7.12 (m, 9H), 4.76-4.64 (m, 1H), 4.41-4.15 (m, 4H), 3.35-3.23 (m, 1H), 2.87-2.74 (m, 1H). LCMS (ES, m/z) 330, 332 [M+H]+.
To a solution of (4S)-4-benzyl-3-[2-(3-chlorophenyl)acetyl]-1,3-oxazolidin-2-one (15 g, 0.41 mol) in dichloromethane (180 mL) was added a solution of titanium(IV) chloride (48.4 mL, 1 M in DCM) dropwise with stirring at −20° C. After stirring for 2 h at −20° C., a solution of DIEA (5.1 mL, 0.31 mol) in DCM (10 mL) was added slowly with stirring. After 1.5 h at −20° C., tert-butyl N-(methoxymethyl)-N-methylcarbamate (10.4 g, 0.59 mol) in dichloromethane (10 mL) was added dropwise. The reaction mixture was stirred for 2 h at −20° C. and then treated with saturated ammonium chloride solution (100 mL). The product was extracted with ethyl acetate (3×300 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to afford tert-butyl N-[(2R)-3-[(4S)-4-benzyl-2-oxo-1,3-oxazolidin-3-yl]-2-(3-chlorophenyl)-3-oxopropyl]-N-methylcarbamate as a yellow solid (18 g, 84%).
To a solution of lithium hydroxide (2.3 g, 0.09 mol) in water (125 mL) was added THF (170 mL) followed by the sequential addition of a solution of hydrogen peroxide (9.2 mL, 30% in water) and a solution of tert-butyl N-[(2R)-3-[(4S)-4-benzyl-2-oxo-1,3-oxazolidin-3-yl]-2-(3-chlorophenyl)-3-oxopropyl]-N-methylcarbamate (18 g, 0.04 mol) in tetrahydrofuran (10 mL) dropwise with stirring at 0° C. The resulting mixture was stirred for 3 h at 0° C. The reaction was carefully quenched with aqueous sodium sulfite solution (100 mL, 12.5 wt %) while maintaining reaction temperature<10° C. After stirring for 30 min at room temperature, the mixture (pH=14) was concentrated to remove organic solvent and then washed with diethyl ether (3×200 mL). The aqueous layer was then acidified to pH=2˜3 with aqueous potassium bisulfate solution (27 wt %) while maintaining temperature<15° C. The resulting solution was extracted with ethyl acetate (3×300 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under vacuum to afford (2R)-3-{[(tert-butoxy)carbonyl](methyl)amino}-2-(3-chlorophenyl)propanoic acid as a yellow oil (10 g, 84%). Note: the material contains about 20 wt % of (4S)-4-benzyl-1,3-oxazolidin-2-one based on HNMR determination, and its ee value is about 96%.
The crude material (4.5 g) was dissolved in MeCN (5 mL) and N-cyclohexylcyclohexanamine (3 g, 16.5 mmol) was added. The reaction was heated to 60° C. for 3 h and cooled to room temperature slowly over 16 h without stirring. The solids were collected by filtration and dried under vacuum to afford N-cyclohexylcyclohexanamine (2R)-3-{[(tert-butoxy)carbonyl](methyl)amino}-2-(3-chlorophenyl) propanoic acid complex as a white solid (5 g). The complex was then dissolved with aqueous solution of KHSO4 (50 mL, 27 wt %) and EtOAc (50 mL). The resulting solution was stirred for 1.5 h at rt and extracted with EtOAc (3×50 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under vacuum to afford pure (2R)-3-{[(tert-butoxy)carbonyl](methyl)amino}-2-(3-chlorophenyl)propanoic acid as a white solid (2.30 g, 99% purity, >99% ee). 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 7.43-7.21 (m, 4H), 4.07-3.90 (m, 1H), 3.94-3.89 (m, 2H), 2.84-2.70 (m, 3H), 1.39 (s, 9H). LCMS (ES, m/z) 314,316 [M+H]+.
To a solution of (2R)-3-{[(tert-butoxy)carbonyl](methyl)amino}-2-(3-chlorophenyl)propanoic acid (A3) (2.30 g, 7.34 mmol) in N,N-dimethylformamide (20 mL) was added HATU (3.07 g, 8.08 mmol), 2-(2,3-dihydro-1,4-benzodioxine-6-sulfonyl)-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrole hydrochloride (B2) (2.52 g, 7.34 mmol), and DIEA (3.82 mL, 22.1 mmol). The resulting solution was stirred for 2 h at rt. The reaction mixture was poured into water (100 mL) and then extracted with ethyl acetate (3×100 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The crude product was purified by silica gel chromatography (eluting with 1:15 ethyl acetate/dichloromethane) to afford tert-butyl N-[(2R)-2-(3-chlorophenyl)-3-[5-(2,3-Dihydro-1,4-benzodioxine-6-sulfonyl)-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl]-3-oxopropyl]-N-methylcarbamate as a white solid (3.5 g, 79%). 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 7.40-7.18 (m, 6H), 7.07-7.06 (m, 1H), 4.45-4.22 (m, 5H), 4.11-3.88 (m, 7H), 3.88-3.58 (m, 2H), 3.44-3.36 (m, 1H), 2.75-2.67 (m, 3H), 1.27-1.16 (m, 9H). LCMS (ES, m/z) 604, 606 [M+H]+.
To a solution of tert-butyl N-[(2R)-2-(3-chlorophenyl)-3-[5-(2,3-Dihydro-1,4-benzodioxine-6-sulfonyl)-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl]-3-oxopropyl]-N-methylcarbamate (1.5 g, 2.49 mmol) in ethyl acetate (10 mL) was added a solution of hydrochloric acid (10 mL, 4 N in 1,4-dioxane). The resulting solution was stirred for 3 h at 25° C. The mixture was concentrated under vacuum to about ⅓ volume and the solids were collected by filtration. The solids were treated with EtOAc (10 mL) at 70° C., filtered at room temperature, and dissolved with saturated potassium carbonate solution/EA (1:1, 10 mL). The resulting solution was stirred for 3 h and then extracted with EtOAc (3×5 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to afford (2R)-2-(3-chlorophenyl)-1-[5-(2,3-dihydro-1,4-benzodioxine-6-sulfonyl)-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl]-3-(methylamino)propan-1-one (3) as a white solid (1 g, 80%). 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 7.36-7.25 (m, 6H), 7.07-7.06 (m, 1H), 4.40-4.29 (m, 5H), 4.06-3.88 (m, 8H), 3.06-3.01 (m, 1H), 2.60-2.50 (m, 1H), 2.23 (s, 3H), 1.66 (s, 1H). LCMS (ES, m/z) 504, 506 [M+H]+.
To a solution of 4-bromoaniline (1 equiv) in 1,4-dioxane was added Pd(dppf)Cl2 (10 mol %) and 2-(tributylstannyl)-1,3-oxazole (1 equiv). The resulting mixture was stirred for 48 h at 100° C. and then cooled to room temperature. The reaction mixture was poured into water and then extracted with ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting crude product was purified by silica gel chromatography to afford 4-(1,3-oxazol-2-yl)aniline.
Into glacial acetic acid was bubbled in SO2 gas for 1 h at room temperature. Then CuCl2 (25 mol %) was added and SO2 gas was bubbled in for additional 2 h to afford solution A. To a pre-cooled solution of 4-(1,3-oxazol-2-yl)aniline (1 equiv) in acetic acid and concentrated hydrochloric acid was added a solution of sodium nitrite (1.1 equiv) in distilled water dropwise with stirring at −10° C. After stirring for 15 min, solution A was added to this diazonium salt solution at −10° C. The resulting solution was allowed to warm to room temperature naturally and stirred for 16 h. The reaction mixture was treated with water and then extracted with EtOAc. The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting crude product was purified by silica gel chromatography to afford 4-(1,3-oxazol-2-yl)benzene-1-sulfonyl chloride (Cl). LCMS (ES, m/z) 244 [M+H]+.
To a solution of tert-butyl 6-chloro-1H,2H,3H-pyrrolo[3,4-c]pyridine-2-carboxylate (2.0 g, 7.79 mmol) in tetrahydrofuran (20 mL) was added NaNH2 (2.0 g, 7.87 mmol), and 2-(3-bromophenyl)acetonitrile (2.32 g, 11.8 mmol). The resulting mixture was stirred for 16 h at 50° C. and then cooled to room temperature. The reaction mixture was poured into water (20 mL) and then extracted with ethyl acetate (3×20 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting crude product was purified by silica gel chromatography (eluting with 0:100 to 20:80 ethyl acetate/petroleum ether) to afford tert-butyl 6-[(3-bromophenyl)(cyano)methyl]-1H,2H,3H-pyrrolo[3,4-c]pyridine-2-carboxylate (1.1 g, 34%). LCMS (ES, m/z) 414, 416 [M+H]+.
To a solution of sodium hydroxide (128 mg, 0.012 mmol) in water (0.13 mL) was added DMSO (8 mL), benzyltriethylammonium chloride (27 mg, 0.12 mmol) and a solution of tert-butyl 6-[(3-bromophenyl)(cyano)methyl]-1H,2H,3H-pyrrolo[3,4-c]pyridine-2-carboxylate (1.1 g, 2.66 mmol) in DMSO (10 mL). The resulting solution was stirred for 3 h at room temperature while oxygen was bubbling in. The reaction mixture was poured into water (20 mL) and then extracted with EtOAc (3×20 mL). The combined organic layers were washed by brine, dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The resulting crude product was purified by silica gel chromatography (eluting with 0:100 to 20:80 ethyl acetate/petroleum ether) to afford tert-butyl 6-(3-bromobenzoyl)-1H,2H,3H-pyrrolo[3,4-c]pyridine-2-carboxylate (640 mg, 60%). LCMS (ES, m/z) 403, 405 [M+H]+.
To a solution of tert-butyl 6-(3-bromobenzoyl)-1H,2H,3H-pyrrolo[3,4-c]pyridine-2-carboxylate (500 mg, 1.24 mmol) in tetrahydrofuran (10 mL) was added NaBH4 (95 mg, 2.49 mmol) at 0° C. The resulting mixture was stirred for 1 h at 0° C. The reaction mixture was poured into water (10 mL) and then extracted with EA (3×20 mL). The combined organic layers were washed with brine (3×20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting crude product was purified by silica gel chromatography (eluting with 0:100 to 25:80 ethyl acetate/petroleum ether) to afford tert-butyl 6-[(3-bromophenyl)(hydroxy)methyl]-1H,2H,3H-pyrrolo[3,4-c]pyridine-2-carboxylate (400 mg, 80%). LCMS (ES, m/z) 405, 407 [M+H]+.
To a solution of tert-butyl 6-[(3-bromophenyl)(hydroxy)methyl]-1H,2H,3H-pyrrolo[3,4-c]pyridine-2-carboxylate (1 equiv) in DCM was added TFA. The resulting solution was stirred for 1 h at rt and then concentrated under vacuum. The resulting mixture was then basified to pH 8 with saturated aqueous potassium carbonate solution. The resulting mixture was extracted with DCM. The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under vacuum to afford (3-bromophenyl)(2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-6-yl)methanol (D1). LCMS (ES, m/z) 305 [M+H]+.
To a solution of 4-(1,3-oxazol-2-yl)benzene-1-sulfonyl chloride (Cl) (1.5 equiv) in DCM was added (3-bromophenyl)(2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-6-yl)methanol (D1) (1 equiv) and TEA (3.0 equiv). The resulting solution was stirred for 2 h at 25° C. The reaction mixture was poured into water and then extracted with ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting crude product was purified by silica gel chromatography to afford (3-bromophenyl)(2-((4-(oxazol-2-yl)phenyl)sulfonyl)-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-6-yl)methanol. LCMS (ES, m/z) 512, 514 [M+H]+.
To a solution of (3-bromophenyl)(2-((4-(oxazol-2-yl)phenyl)sulfonyl)-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-6-yl)methanol (1 equiv) in toluene was added N-Boc-piperidine (1.4 equiv), XPhos (25 mol %), Cs2CO3 (3.3 equiv) and Pd2(dba)3.CHCl3 (12 mol %). The resulting mixture was stirred for 16 h at 100° C. and then cooled to room temperature. The reaction mixture was filtered and concentrated under vacuum. The resulting crude product was purified by silica gel chromatography to afford tert-butyl 4-(3-(hydroxy(2-((4-(oxazol-2-yl)phenyl)sulfonyl)-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-6-yl)methyl)phenyl)piperazine-1-carboxylate. LCMS (ES, m/z) 618 [M+H]+.
To a solution of HCl in 1,4-dioxane (4 N) was added tert-butyl 4-(3-(hydroxy(2-((4-(oxazol-2-yl)phenyl)sulfonyl)-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-6-yl)methyl)phenyl)piperazine-1-carboxylate (1 equiv) and water. The resulting mixture was stirred for 4 h at room temperature. The reaction mixture was concentrated and lyophilized. The resulting crude product was purified by reverse phase chromatography. The product fractions were concentrated and lyophilized to afford tert-butyl 4-(3-(hydroxy(2-((4-(oxazol-2-yl)phenyl)sulfonyl)-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-6-yl)methyl)phenyl)piperazine-1-carboxylate as a white solid (79%). 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 9.09 (br s, 2H), 8.33 (d, J=2.8 Hz, 2H), 8.16 (d, J=8.4 Hz, 2H), 8.01 (d, J=8.4 Hz, 2H), 7.48 (d, J=3.2 Hz, 2H), 7.12-7.08 (m, 1H), 7.03-7.01 (m, 1H), 6.80-6.77 (m, 2H), 6.07 (br s, 1H), 5.62 (s, 1H), 4.69-4.58 (m, 4H), 3.52-3.35 (m, 4H), 3.30-3.19 (m, 4H). LCMS (ES, m/z) 518 [M+H]+. The two enantiomers were further separated by Chiral Prep-HPLC (Column: CHIRALPAK IF, 5 μm, 20×250 mm; Mobile Phase, A: MTBE (containing 0.2% IPA) and B: EtOH (keep 50% B over 25 min); Detector: UV 254/220 nm; Retention time: 1st eluting isomer, 10.759 min; 2nd eluting isomer (4), 17.742 min).
In some embodiments, the USP9X inhibitor may be a compound of Table 1:
To a solution of 2,3-dimethylbut-2-ene (1000 g, 11.9 mol) in DCM (1000 mL) in a 4 L 4-necked round bottom flask was added aqueous hydrogen bromide solution (150 mL, 48%) with stirring at 10-15° C. To the reaction was added bromine (9.90 kg, 62.0 mol) with stirring at 0° C. The resulting mixture was stirred for 2 days at 45° C. in an oil bath. After cooling to room temperature, the reaction mixture was carefully poured into saturated aqueous sodium hydrogen sulfite solution (10 L). The precipitate was collected by filtration and dried in oven to afford 1,4-dibromo-2,3-bis(bromomethyl)but-2-ene as a light yellow solid (3000 g, 44%). GCMS: (EI, m/z): 398, 400, 402 [M]+.
To a solution of 1,4-dibromo-2,3-bis(bromomethyl)but-2-ene (2000 g, 3.50 mol) in DMF (20 L) was added 4-methylbenzene-1-sulfonamide (2137 g, 12.5 mol), and potassium carbonate (5175 g, 37.4 mol). The resulting mixture was stirred for 2 days at room temperature. The reaction mixture was slowly poured into water/ice (20 L). The precipitate was collected by filtration, washed with ethanol and dried in an oven to afford 2,5-ditosyl-1,2,3,4,5,6-hexahydropyrrolo[3,4-c]pyrrole as a light yellow solid (1345 g, 78%). LCMS: (ES, m/z): 419 [M+H]+.
To a solution of 2,5-ditosyl-1,2,3,4,5,6-hexahydropyrrolo[3,4-c]pyrrole (1345 g, 2.73 mol) in aqueous hydrogen bromide solution (4500 mL, 48%) in 10 L 4-necked round-bottom flask, was added phenol (1270 g, 13.5 mol). The resulting mixture was stirred for 2 days at 120° C. After cooling to room temperature, the aqueous layer was collected and concentrated under vacuum. The resulting solids were washed with DCM/MeOH (v:v=10:1, 3×300 mL) and dried in an oven to afford 1,2,3,4,5,6-hexahydropyrrolo[3,4-c]pyrrole hydrogen bromide salt as a yellow solid (480 g, 61%). L CMS: (ES, m/z): 111 [M+H]+.
To a suspension of 1,2,3,4,5,6-hexahydropyrrolo[3,4-c]pyrrole hydrogen bromide salt (458 g, 1.52 mol) in water (4 L) was added sodium bicarbonate (424 g, 5.05 mol) followed by dropwise addition of a solution of di-tert-butyl dicarbonate (807 g, 3.70 mol) in methanol (500 mL) with stirring at 0° C. The resulting solution was stirred for 16 h at 25° C. The precipitate was collected by filtration and dried in an oven to afford di-tert-butyl pyrrolo[3,4-c]pyrrole-2,5(1H,3H,4H,6H)-dicarboxylate as a white solid (300 g, 61%). LCMS (ES, m/z): 311[M+H]+.
To a solution of di-tert-butyl pyrrolo[3,4-c]pyrrole-2,5(1H,3H,4H,6H)-dicarboxylate (200 g, 612 mmol) in propan-2-yl acetate (5 L) was added 4-methylbenzene-1-sulfonic acid (123 g, 647 mmol) in portions at 0° C. The resulting mixture was stirred for 16 h at 55° C. in an oil bath. After cooling to room temperature, the precipitate was collected by filtration and dried in an oven to afford tert-butyl 4,5-dihydropyrrolo[3,4-c]pyrrole-2(1H,3H,4H)-carboxylate 4-methylbenzene-1-sulfonic acid salt as a yellow solid (197 g, 80%). LCMS: (ES, m/z): 211[M+H]+.
To a suspension of tert-butyl 4,5-dihydropyrrolo[3,4-c]pyrrole-2(1H,3H,4H)-carboxylate 4-methylbenzene-1-sulfonic acid salt (61 g, 142 mmol) in water (100 mL) and tetrahydrofuran (30 mL) was added sodium hydroxide (13 g, 325 mmol) followed by portion-wise addition of 2,3-dihydro-1,4-benzodioxine-6-sulfonyl chloride (25 g, 95.9 mmol) at 0° C. The resulting mixture was stirred for 2 h at 25° C. The product was extracted with ethyl acetate (3×200 mL). The organic layers were combined, dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The resulting material was purified by silica gel chromatography (eluting with 1:10 ethyl acetate/petroleum ether) to afford tert-butyl 5-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)-4,5-dihydropyrrolo[3,4-c]pyrrole-2(1H,3H,4H)-carboxylate as a white solid (30 g, 73%). LCMS: (ES, m/z): 409 [M+H]+.
To a solution of tert-butyl 5-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)-4,5-dihydropyrrolo[3,4-c]pyrrole-2(1H,3H,4H)-carboxylate (30.0 g, 69.8 mmol) in 1,4-dioxane (100 mL) was added hydrochloric acid (200 mL, 4 M in 1,4-dioxane). The resulting solution was stirred for 2 h at 25° C. and then concentrated under vacuum to afford 2-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)-1,2,3,4,5,6-hexahydropyrrolo[3,4-c]pyrrole hydrochloric salt as a yellow solid (20 g, 79%). LCMS: (ES, m/z): 309 [M+H]+.
To a solution of 4-bromo-2-methyl-1,3-benzothiazole (3.00 g, 12.9 mmol) in 1,4-dioxane (20 mL) was added 4,4,5,5-tetramethyl-2-(tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3,2-dioxaborolane (4.01 g, 15.5 mmol), Pd(dppf)Cl2 (960 mg, 1.29 mmol) and potassium acetate (2.58 g, 25.8 mmol). The resulting mixture was stirred for 16 h at 100° C. and then cooled to room temperature. The reaction mixture was poured into water (30 mL) and then extracted with ethyl acetate (3×30 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting crude product was purified by silica gel chromatography (eluting with 0:100 to 20:80 ethyl acetate/petroleum ether) to afford 2-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3-benzothiazole as a light yellow oil (2.00 g, 46%). LCMS (ES, m/z) 276 [M+H]+.
To a solution of 2-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3-benzothiazole (600 mg, 1.86 mmol) in 1,4-dioxane (10 mL) was added methyl 2-bromoprop-2-enoate (447 mg, 2.66 mmol), XPhos 3G (80 mg, 0.11 mmol), potassium phosphate (1.4 g, 6.46 mmol) and water (1 mL). The resulting mixture was stirred for 16 h at 100° C. and then cooled to room temperature. The reaction mixture was poured into water (10 mL) and then extracted with ethyl acetate (3×30 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting crude product was purified by silica gel chromatography (eluting with 0:100 to 20:80 ethyl acetate/petroleum ether) to afford methyl 2-(2-methyl-1,3-benzothiazol-4-yl)prop-2-enoate as light yellow oil (280 mg, 55%). LCMS (ES, m/z) 234 [M+H]+.
The Intermediates in Table 2 were synthesized according to the procedure described for Intermediate 2-4 above.
In a 250 mL round-bottom flask was placed methyl 2-(3-methoxyphenyl)acetate (5 g, 27.2 mmol), paraformaldehyde (3 g, 33.3 mmol), n-BU4NI (1 g, 2.7 mmol), potassium carbonate (9.6 g, 69.5 mmol) and N,N-dimethylformamide (60 mL). The resulting solution was stirred for 10 min at 60° C. in an oil bath. After cooling to room temperature, the solution was diluted with 100 mL of water and extracted with ethyl acetate (3×100 mL). The organic layers were combined, dried over anhydrous sodium sulfate, filtered and concentrated under vacuum to afford 3 g (570%) of methyl 2-(3-methoxyphenyl)prop-2-enoate as a yellow oil. MS: (ESI, m/z): 193 [M+H]+.
The Intermediate in Table 3 was synthesized according to the procedure described for Intermediate 3-1 above.
To a solution of 2-bromo-4-chloro-5-methylphenol (2.0 g, 8.1 mmol) in acetone (20 mL) was added potassium carbonate (2.5 g, 16 mmol), and iodomethane (0.66 mL, 9.5 mmol). The resulting mixture was stirred for 2 h at 25° C. The reaction mixture was poured into water (30 mL) and then extracted with ethyl acetate (3×30 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting crude product was purified by silica gel chromatography (eluting with 0:100 to 20:80 ethyl acetate/petroleum ether) to afford 1-bromo-5-chloro-2-methoxy-4-methylbenzene as a light yellow oil (1.87 g, 82%). GCMS (EI, m/z): 234, 236 [M]+.
The Intermediates in Table 4 were synthesized according to the procedure described for Intermediate 5-1 above.
To a solution of tert-buty 4-bromo-2,3-dihydro-1H-isoindole-2-carboxylatein (2.0 g, 6.4 mmol) in THF (20 mL) was added a solution of n-BuLi (2.6 mL, 2.5 M in THF) dropwise with stirring at −78° C. After stirring for 15 min at −78° C., diethyl oxalate (3.1 mL, 32 mmol) was added in. The resulting mixture was stirred for 1 h at −60° C. The reaction mixture was poured into saturated ammonium chloride solution (20 mL) and then extracted with ethyl acetate (3×20 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The resulting crude product was purified by silica gel chromatography (eluting with 0:100 to 20:80 ethyl acetate/petroleum ether) to afford tert-butyl 4-(2-ethoxy-2-oxoacetyl)-2,3-dihydro-1H-isoindole-2-carboxylate as a light yellow solid (1.12 g, 47%). LCMS (ES, m/z) 320 [M+H]+.
To a solution of tert-butyl 4-(2-ethoxy-2-oxoacetyl)-2,3-dihydro-1H-isoindole-2-carboxylate (1.12 g, 2.98 mmol) in tetrahydrofuran (6 mL) was added water (6 mL) and LiOH (421 mg, 16.70 mmol). The resulting mixture was stirred for 16 h at room temperature. The reaction mixture was washed with diethyl ether (1×10 mL) and then acidified to pH=5 with saturated citric acid. The resulting solution was extracted with ethyl acetate (2×10 mL). The combined organic layers were combined, dried over anhydrous sodium sulfate, filtered and concentrated under vacuum to afford 2-[2-[(tert-butoxy)carbonyl]-2,3-dihydro-1H-isoindol-4-yl]-2-oxoacetic acid as a light yellow solid (1.0 g, crude). LCMS (ES, m/z) 292 [M+H]+.
The Intermediate in Table 5 was synthesized according to the procedure described for Intermediate 7-1 above.
To a solution of methyl 3-{[(tert-butoxy)carbonyl](methyl)amino}-2-(4-fluoro-2-methoxyphenyl)propanoate (180 mg, 0.53 mmol) in dichloromethane (3 mL) was added NCS (211 mg, 1.58 mmol). The resulting solution was stirred for 16 h at room temperature. The reaction mixture were filtered, and concentrated under vacuum. The resulting crude product was purified by prep-TLC (eluting with 1:5 ethyl acetate/petroleum ether) to afford methyl 3-{[(tert-butoxy)carbonyl](methyl)amino}-2-(3-chloro-4-fluoro-2-methoxyphenyl)propanoate as a light yellow oil (160 mg, 81%). LCMS (ES, m/z) 376, 378 [M+H]+.
To a solution of 3-bromo-4,5-difluorobenzoic acid (2.50 g, 10.6 mmol) in toluene (15 mL), was added thionyl chloride (15 mL). The resulting solution was refluxed for 3 h, then cooled to room temperature and concentrated under vacuum. The resulting mixture was dissolved in THF (15 mL) and treated with triethylamine (2.47 mL, 17.9 mmol) and (diazomethyl)trimethylsilane (8.8 mL, 2.0 M in THF) at 0° C. The resulting mixture was stirred for 16 h at room temperature and then poured into saturated aqueous sodium bicarbonate (50 mL) and extracted with ethyl acetate (3×100 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting mixture was dissolved in methanol (40 mL) and treated with triethylamine (2.47 mL, 17.9 mmol) and silver (I) benzoate (1.40 g, 6.33 mmol) at 0° C. The mixture was stirred for 16 h at room temperature and then concentrated under vacuum. The crude product was purified by silica gel chromatography (eluting with 0:100 to 10:90 ethyl acetate/petroleum ether) to afford methyl 2-(3-bromo-4,5-difluorophenyl)acetate as a colorless oil (0.98 g, 35%). LCMS (ES, m/z): 265, 267 [M+H]+.
To a solution of methyl 2-(3-bromo-4,5-difluorophenyl)acetate (1.70 g, 6.72 mmol) in 1,4-dioxane (40 mL) was added cyclopropylboronic acid (865 mg, 10.1 mmol), potassium phosphate (4.20 g, 20.1 mmol), Pd(dppf)Cl2 (246 mg, 0.34 mmol) and water (8 mL). The mixture was stirred for 16 h at 90° C. and cooled to room temperature. The reaction mixture was poured into water (50 mL) and then extracted with ethyl acetate (3×50 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The crude product was purified by silica gel chromatography (eluting with 0:100 to 30:70 ethyl acetate/petroleum ether) to afford methyl 2-(3-cyclopropyl-4,5-difluorophenyl)acetate as a colorless oil (550 mg, 36%). LCMS (ES, m/z): 227 [M+H]+.
To a solution of methyl 2-(3-cyclopropyl-4,5-difluorophenyl)acetate (550 mg, 2.43 mmol) in DMF (15 mL), was added potassium carbonate (840 mg, 6.08 mmol), tetrabutylammonium iodide (90 mg, 0.24 mmol) and paraformaldehyde (263 mg, 2.92 mmol). The resulting mixture was stirred for 10 min at 60° C. and then cooled to room temperature. The reaction mixture was poured into water (30 mL) and then extracted with ethyl acetate (3×30 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting crude product was purified by silica gel chromatography (eluting with 0:100 to 30:70 ethyl acetate/petroleum ether) to afford methyl 2-(3-cyclopropyl-4,5-difluorophenyl)prop-2-enoate as a colorless oil (159 mg, 27%). LCMS (ES, m/z): 239 [M+H]+.
To a solution of methyl 2-(3-chlorophenyl)acetate (5.00 g, 25.7 mmol) in CH3CN (50 mL) was added 1,8-diazabicyclo[5.4.0]undec-7-ene (4.87 mL, 32.6 mmol) dropwise with stirring at 0° C. followed by the addition of 4-methylbenzene-1-sulfonyl azide (6.40 g, 32.5 mmol) added dropwise with stirring at 0° C. The solution was stirred for 4 h at 25° C. The reaction mixture was treated with water (50 mL) and extracted with ethyl acetate (3×50 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting crude product was purified by silica gel chromatography (eluting with 0:100 to 10:90 ethyl acetate/dichloromethane) to afford methyl 2-(3-chlorophenyl)-2-diazoacetate as a yellow solid (5.00 g, 83%). LCMS (ES, m/z): 211, 213 [M+H]+.
To a solution of tert-butyl pyrrolidine-1-carboxylate (894 mg, 5.22 mmol) in hexane (150 mL) was added tetrakis [(R)-(+)-N-(P-dodecylphenylsulfonyl)prolinato]dirhodium (II) (49 mg, 0.026 mmol) followed by treatment with methyl 2-(3-chlorophenyl)-2-diazoacetate (550 mg, 2.61 mmol) as a solution in hexane (100 mL) over 60 min with stirring at −50° C. The resulting solution was stirred for 10 h at −50° C. and then 16 h at room temperature. The reaction was poured into saturated ammonium chloride solution (100 mL) and then extracted with ethyl acetate (3×150 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting crude product was purified by silica gel chromatography (eluting with 0:100 to 10:90 ethyl acetate/petroleum ether) to afford tert-butyl 2-[1-(3-chlorophenyl)-2-methoxy-2-oxoethyl]pyrrolidine-1-carboxylate as a yellow solid (400 mg, 39%). LCMS (ES, m/z): 354, 356 [M+H]+.
To a solution of tert-butyl 2-[1-(3-chlorophenyl)-2-methoxy-2-oxoethyl]pyrrolidine-1-carboxylate (400 mg, 1.13 mmol) in tetrahydrofuran (20 mL) and water (5 mL) was added lithium hydroxide (135 mg, 5.65 mmol). The resulting mixture was stirred for 18 h at room temperature. The reaction mixture was washed with diethyl ether (1×10 mL) and then acidified to pH=6 with saturated citric acid. The resulting solution was extracted with ethyl acetate (3×20 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to afford 2-{1-[(tert-butoxy)carbonyl]pyrrolidin-2-yl}-2-(3-chlorophenyl)acetic acid as yellow oil (300 mg, 78%). LCMS (ES, m/z): 340, 342 [M+H]+.
To a solution of 2,3-dimethylbut-2-ene (1000 g, 11.9 mol) in DCM (500 mL) in 4 L 4-necked round bottom flask was added aqueous hydrogen bromide solution (150 mL, 48%). The reaction was treated with bromine (9.90 kg, 62.0 mol) while stirring at 0° C. and then heated to 45° C. in an oil bath and stirred for an additional 2 days. After cooling to room temperature, the reaction mixture was carefully poured into saturated sodium hydrogen sulfite solution (10 L). The precipitate was collected by filtration and dried in oven to afford 1,4-dibromo-2,3-bis(bromomethyl)but-2-ene as a light yellow solid (3345 g, 49%). GCMS: (EI, m/z): 398, 400, 402 [M]+.
To a solution of 1,4-dibromo-2,3-bis(bromomethyl)but-2-ene (2000 g, 3.50 mol) in DMF (20 L) was added 4-methylbenzene-1-sulfonamide (2137 g, 12.5 mol), and potassium carbonate (5175 g, 37.4 mol). The resulting mixture was stirred for 2 days at room temperature. The reaction mixture was then slowly poured into water/ice (20 L). The precipitate was collected by filtration, washed with ethanol and dried in oven to afford 2,5-ditosyl-1,2,3,4,5,6-hexahydropyrrolo[3,4-c]pyrrole as a light yellow solid (1345 g, 78%). LCMS: (ES, m/z): 419 [M+H]+.
To a solution of 2,5-ditosyl-1,2,3,4,5,6-hexahydropyrrolo[3,4-c]pyrrole (1345 g, 2.73 mol) in aqueous hydrogen bromide solution (4500 mL, 48%) in 10 L 4-necked round-bottom flask, was added phenol (1270 g, 13.5 mol). The resulting mixture was stirred for 2 days at 120° C. After cooling to room temperature, the aqueous layer was collected and concentrated under vacuum. The resulting solids were washed with DCM/MeOH (v:v=10:1, 3×300 mL) and dried in an oven to afford 1,2,3,4,5,6-hexahydropyrrolo[3,4-c]pyrrole hydrogen bromide salt as a yellow solid (480 g, 61%). L CMS: (ES, m/z): 111 [M+H]+.
To a solution of 3-methyl-2-nitrophenol (200 g, 1.29 mol) in acetic anhydride (1600 mL) was added sulfuric acid (240 mL) and acetic acid (1620 mL). This was followed by the addition of chromium trioxide (280 g, 2.77 mol) in several batches with stirring at 0° C. The resulting mixture was stirred for 2.5 h at 0° C. and then poured into ice/water (5000 mL). The solids were collected by filtration and then washed with water (3×1 L), saturated sodium carbonate solution (3×800 mL), and water (3×1 L). The solids were dissolved in ethanol (380 mL) and concentrated hydrochloric acid (617 mL). The resulting solution was stirred for 1.5 h at 110° C. and then cooled to room temperature. The reaction mixture was concentrated under vacuum to afford 3-hydroxy-2-nitrobenzaldehyde as a yellow solid (38.0 g, 18%). LCMS (ES, m/z): 166 [M−H]−.
To a solution of 3-hydroxy-2-nitrobenzaldehyde (38.0 g, 204 mmol) in dichloromethane (500 mL) was added ZnI2 (14.5 g, 44.5 mmol). The reaction was treated with trimethylsilyl cyanide (100 mL, 708 mmol) added dropwise with stirring at 0° C. The resulting mixture was stirred for 2.5 h at 25° C. The reaction was poured into brine (200 mL) and then extracted with ethyl acetate (3×500 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to afford 2-hydroxy-2-(3-hydroxy-2-nitrophenyl)acetonitrile as a yellow solid (34.0 g, 73%). LCMS (ES, m/z): 195 [M+H]+.
To a solution of 2-hydroxy-2-(3-hydroxy-2-nitrophenyl)acetonitrile (34.0 g, 157 mmol) in methanol (80 mL) was added hydrochloric acid (80 mL, 4 N in 1,4-dioxane). The resulting solution was stirred for 45 min at 60° C. and cooled to room temperature. The reaction mixture was concentrated under vacuum and purified by silica gel chromatography (eluting with 0:100 to 35:65 ethyl acetate/petroleum ether) to afford methyl 2-hydroxy-2-(3-hydroxy-2-nitrophenyl)acetate as a yellow solid (23.0 g, 58%). LCMS (ES, m/z): 228 [M+H]+.
To a solution of methyl 2-hydroxy-2-(3-hydroxy-2-nitrophenyl)acetate (23.0 g, 0.11 mol) in methanol (500 mL) was added anhydrous palladium carbon (2.3 g, 10 wt % Pd). The resulting mixture was stirred for 16 h at 25° C. under hydrogen atmosphere (3 atm). The reaction mixture was filtered and concentrated under vacuum to afford methyl 2-(2-amino-3-hydroxyphenyl)-2-hydroxyacetate as a yellow solid (14.0 g, 60%). LCMS (ES, m/z): 198 [M+H]+.
To a solution of methyl 2-(2-amino-3-hydroxyphenyl)-2-hydroxyacetate (9.0 g, 43.4 mmol) in 1,1,1-triethoxyethane (150 mL) was added bismuth (III) trifluoromethanesulfonate (800 mg, 1.18 mmol). The resulting mixture was stirred for 10 min at 85° C. After cooling to room temperature, the reaction mixture was concentrated under vacuum. The resulting crude product was purified by silica gel chromatography (eluting with 0:100 to 50:50 ethyl acetate/petroleum ether) to afford methyl 2-hydroxy-2-(2-methyl-1,3-benzoxazol-4-yl)acetate as a white solid (6.3 g, 63%). LCMS (ES, m/z): 222 [M+H]+.
To a solution of methyl 2-hydroxy-2-(2-methyl-1,3-benzoxazol-4-yl)acetate (500 mg, 2.26 mmol) in tetrahydrofuran (20 mL) and water (2 mL) was added lithium hydroxide (271 mg, 11.3 mmol). The resulting mixture was stirred for 1 h at room temperature. The reaction mixture was washed with diethyl ether (1×10 mL) and then acidified to pH=6 with hydrochloric acid (1 N). The resulting solution was extracted with ethyl acetate (2×20 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to afford 2-hydroxy-2-(2-methyl-1,3-benzoxazol-4-yl)acetic acid as a white solid (386 mg, 82%). LCMS (ES, m/z): 208 [M+H]+.
To a solution of 4-bromo-2-methyl-1,3-benzothiazole (2.10 g, 9.21 mmol) in 1,4-dioxane (70 mL) was added (tributylstannyl)methanol (3.84 g, 12.0 mmol), and Pd(PPh3)4 (1.60 g, 1.38 mmol). The resulting mixture was stirred for 16 h at 100° C. and then cooled to room temperature. The reaction mixture was poured into water (50 mL) and then extracted with ethyl acetate (3×70 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The crude product was purified by silica gel chromatography (eluting with 0:100 to 80:20 ethyl acetate/petroleum ether) to afford (2-methyl-1,3-benzothiazol-4-yl)methanol as a yellow oil (1.20 g, 73%). LCMS (ES, m/z): 180 [M+H]+.
To a solution of oxalyl chloride (1.39 mL, 13.39 mmol) in dichloromethane (30 mL) was added DMSO (1.43 mL, 20.1 mmol) dropwise with stirring at −78° C. The resulting mixture was stirred for 30 min at −78° C. The reaction was treated with (2-methyl-1,3-benzothiazol-4-yl)methanol (1.20 g, 6.69 mmol) in dichloromethane (10 mL) added slowly at −78° C. After 2 h TEA (5.58 mL, 40.1 mmol) was added and the mixture was warmed to room temperature and stirred for 2 h. The reaction was poured into brine (30 mL) and then extracted with ethyl acetate (3×50 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to afford 2-methyl-1,3-benzothiazole-4-carbaldehyde as a yellow oil (900 mg, 76%). LCMS (ES, m/z): 178 [M+H]+.
In a dry 25 ml RBF under N2 was added 2-((2,3-dihydrobenzo[b][1,4]dioxin-6-yl)sulfonyl)-1,2,3,4,5,6-hexahydropyrrolo[3,4-c]pyrrole (200 mg, 0.649 mmol), (R)-3-((tert-butoxycarbonyl)amino)-2-phenylpropanoic acid (189 mg, 0.713 mmol), DMF (1 mL), DIEA (170 μl, 0.973 mmol) and HATU (271 mg, 0.713 mmol). After 3 h, the reaction was diluted with 50 ml of saturated aqueous bicarbonate solution (50 mL) and extracted with EtOAc (4×20 mL). The extracts were dried over Na2SO4, filtered and the solvent was removed in vacuo to afford 524 mg of a brown gummy solid. The crude material was purified by a Biotage SNAP-25 Silica column and eluted with an EtOAc/Hexane gradient (0-100% EtOAc). The desired product was isolated affording 309 mg of a white solid.
The Intermediate in Table 6 was synthesized according to the procedure described for Intermediate 63-1 above.
To a solution of (2S)-2-(3-bromophenyl)-1-[5-(2,3-dihydro-1,4-benzodioxine-6-sulfonyl)-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl]-2-hydroxyethan-1-one (100 mg, 0.20 mmol) in toluene (5 mL) was added tert-butyl octahydropyrrolo[3,4-c]pyrrole-2-carboxylate (609 mg, 2.87 mmol), RuPhos 2G (15 mg, 0.02 mmol), RuPhos (18 mg, 0.04 mmol), and cesium carbonate (189 mg, 0.58 mmol). The resulting mixture was stirred overnight at 100° C. After cooling to room temperature, the reaction was concentrated under vacuum. The crude product was purified by silica gel chromatography (eluting with 1:25 MeOH/DCM) to afford tert-butyl 5-[3-[(1S)-2-[5-(2,3-dihydro-1,4-benzodioxine-6-sulfonyl)-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl]-1-hydroxy-2-oxoethyl]phenyl]-octahydropyrrolo-[3,4-c]pyrrole-2-carboxylate as a light yellow solid (100 mg, 80%). LCMS (ES, m/z): 653 [M+H]+.
The Intermediates in Table 7 were synthesized according to the procedure described for Intermediate 66 above.
To a solution of methyl 2-(3,5-dichlorophenyl)acetate (2.00 g, 8.67 mmol) in tetrahydrofuran (20 mL) was added LDA (5.5 mL, 2 M in THF) dropwise with stirring at −78° C. The solution was stirred for 30 min at −78° C. The reaction was treated with 2-iodoacetonitrile (2.30 g, 13.8 mmol) and stirred for 1 h at −78° C. The contents were poured into saturated aqueous ammonium chloride solution (50 mL) and then extracted with ethyl acetate (3×50 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The crude product was purified by silica gel chromatography (eluting with 0:100 to 20:80 ethyl acetate/petroleum ether) to afford methyl 3-cyano-2-(3,5-dichlorophenyl)propanoate as a yellow oil (1.30 g, 58%). LCMS (ES, m/z): 258, 260 [M+H]+.
To a solution of methyl 3-cyano-2-(3,5-dichlorophenyl)propanoate (1.50 g, 5.52 mmol) in methanol (20 mL) was added Raney Ni (946 mg, 11.0 mmol), and di-tert-butyl dicarbonate (6.03 g, 27.6 mmol). The resulting mixture was stirred for 4 h at room temperature under hydrogen (2-3 atm). The reaction mixture was filtered and concentrated under vacuum. The resulting crude product was purified by silica gel chromatography (eluting with 0:100 to 20:80 ethyl acetate/petroleum ether) to afford methyl 4-[[(tert-butoxy)carbonyl]amino]-2-(3,5-dichlorophenyl)butanoate as a yellow oil (1.80 g, 90%). LCMS (ES, m/z): 362, 364 [M+H]+.
To a solution of methyl 4-[[(tert-butoxy)carbonyl]amino]-2-(3,5-dichlorophenyl)butanoate (300 mg, 0.79 mmol) in tetrahydrofuran (2 mL) and water (2 mL) was added lithium hydroxide (94 mg, 3.93 mmol). The resulting mixture was stirred for 16 h at room temperature. The reaction mixture was washed with diethyl ether (1×10 mL) and then acidified to pH=7 with saturated aqueous citric acid. The product was extracted with ethyl acetate (3×20 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to afford 4-[[(tert-butoxy)carbonyl]amino]-2-(3,5-dichlorophenyl)butanoic acid as a yellow oil (180 mg, 66%). LCMS (ES, m/z): 348, 350 [M+H]+.
To a solution of methyl 2-(2-methyl-1,3-benzothiazol-4-yl)prop-2-enoate (200 mg, 0.86 mmol) in tetrahydrofuran (2 mL) was added azetidine (98 mg, 1.72 mmol). The resulting mixture was stirred for 2 h at room temperature. The reaction mixture was concentrated under vacuum. The resulting crude product was purified by prep-TLC (eluting with 20:1 dichloromethane/methanol) to afford methyl 3-(azetidin-1-yl)-2-(2-methyl-1,3-benzothiazol-4-yl)propanoate as a brown oil (170 mg, 68%). LCMS (ES, m/z): 305 [M+H]+.
To a solution of 2-(3-bromophenyl)-1-[5-(2,3-dihydro-1,4-benzodioxine-6-sulfonyl)-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl]-2-hydroxyethan-1-one (800 mg, 1.53 mmol) in DMF (10 mL) was added imidazole (314 mg, 4.61 mmol), tert-butyl(chloro)dimethylsilane (0.43 mL, 2.30 mmol) and DMAP (20 mg, 0.164 mmol). The resulting solution was stirred for 2 h at 70° C. and then cooled to room temperature. The reaction mixture was poured into water (10 mL) and then extracted with ethyl acetate (3×20 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting crude product was purified by silica gel chromatography (eluting with 0:100 to 50:50 ethyl acetate/petroleum ether) to afford 2-(3-bromophenyl)-2-[(tert-butyldimethylsilyl)oxy]-1-[5-(2,3-dihydro-1,4-benzodioxine-6-sulfonyl)-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl]ethan-1-one as a light yellow oil (750 mg, 69%). LCMS (ES, m/z): 635, 637 [M+H]+.
The Intermediates in Table 8 were synthesized according to the procedure described for Intermediate 83 above.
To 2-[(tert-butyldimethylsilyl)oxy]-2-(3-{9,9-difluoro-3,7-diazabicyclo[3.3.1]nonan-3-yl}phenyl)-1-[5-(2,3-dihydro-1,4-benzodioxine-6-sulfonyl)-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl]ethan-1-one (280 mg, 0.34 mmol) was added TBAF (5 mL, 1 M in TIF). The resulting solution was stirred for 30 min at room temperature. The reaction mixture was poured into water (5 mL) and then extracted with ethyl acetate (3×10 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting crude product was purified by prep-TLC (eluting with 3:1 ethyl acetate/petroleum ether) to afford tert-butyl 7-(3-[2-[5-(2,3-dihydro-1,4-benzodioxine-6-sulfonyl)-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl]-1-hydroxy-2-oxoethyl]phenyl)-9,9-difluoro-3,7-diazabicyclo[3.3.1]nonane-3-carboxylate as a yellow oil (220 mg, 82%). LCMS (ES, m/z): 703 [M+H]+.
To a solution of tert-butyl 4-(3-[1-[(tert-butyldimethylsilyl)oxy]-2-[5-(2,3-dihydro-1,4-benzodioxine-6-sulfonyl)-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl]-2-oxoethyl]phenyl)piperazine-1-carboxylate (320 mg, 0.43 mmol) in dichloromethane (5 mL), was added Lawesson Reagent (88 mg, 0.22 mmol). The resulting mixture was stirred for 16 h at room temperature. The reaction mixture was concentrated under vacuum. The resulting crude product was purified by Prep-TLC eluting with 1:1 ethyl acetate/petroleum ether to give the product as a light yellow oil (130 mg, 36%). LCMS (ES, m/z) 757 [M+H]+.
To a solution of n-BuLi (5.6 mL, 2.5 M in THF) was added n-Bu2Mg (14 mL, 1 M in THF) at room temperature. The resulting mixture was stirred for 10 min at room temperature and treated with 6-bromo-2,3-dihydro(2,2,3,3-2H4)-1,4-benzodioxine (2.0 g, 9.04 mmol) in tetrahydrofuran (10 mL) added dropwise with stirring at −10° C. The resulting mixture was stirred for 1 h and then added to a solution of sulfuryl chloride (16 mL) in toluene (8 mL) with stirring at −10° C. The resulting mixture was stirred for 0.5 h and quenched with saturated aqueous ammonium chloride solution (30 mL). The product was extracted with ethyl acetate (3×30 mL). The organic layers were combined, dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The resulting crude product was purified by silica gel chromatography (eluting with 0 to 50% ethyl acetate/petroleum ether) to afford 2,3-dihydro(2,2,3,3-2H4)-1,4-benzodioxine-6-sulfonyl chloride as a yellow oil (1.3 g, 60%).
To a solution of 2,3-dihydro(2,2,3,3-H)-1,4-benzodioxine-6-sulfonyl chloride (1.3 g, 5.01 mmol) in DCM (20 mL) was added 1H-imidazole (742 mg, 10.9 mmol). The resulting solution was stirred for 2 h at room temperature. Then the reaction mixture was filtered and concentrated under vacuum to afford 1-[2,3-dihydro(2,2,3,3-2H4)-1,4-benzodioxine-6-sulfonyl]-1H-imidazole as a white solid (1.2 g, 89%). LCMS (ES, m/z): 271 [M+H]+.
To a solution of 1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrole dihydrobromide (3.6 g, 13.2 mmol) in water (15 mL) and ethanol (15 mL) was added 1-[2,3-dihydro(2,2,3,3-2H4)-1,4-benzodioxine-6-sulfonyl]-1H-imidazole (1.2 g, 4.08 mmol). The resulting solution was stirred for 18 h at room temperature and then 48 h at 60° C. After cooling to room temperature, the solution was basified to pH=14 with sodium hydroxide and then extracted with DCM (3×20 mL). The organic layers were combined, dried over anhydrous sodium sulfate, filtered and concentrated under vacuum to afford 2-[2,3-dihydro(2,2,3,3-2H4)-1,4-benzodioxine-6-sulfonyl]-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrole as a yellow solid (500 mg, 39%). LCMS (ES, m/z): 313 [M+H]+.
To a solution of 5-[2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl]-1H,2H,3H,5H-pyrrolo[3,4-c]pyrrole (180 mg, 0.43 mmol) in N,N-dimethylformamide (10 mL) was added 2-hydroxy-2-(2-methyl-1,3-benzoxazol-4-yl)acetic acid (89 mg, 0.43 mmol), DIEA (110.4 mg, 0.86 mmol), HOBt (63.5 mg, 0.47 mmol) and EDCI (90 mg, 0.47 mmol). The resulting mixture was stirred for 2 h at room temperature and poured into water (50 mL). The resulting solution was extracted with EtOAc (3×30 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The resulting crude product was purified by prep-TLC (eluting with 1:10 MeOH/DCM). The two enantiomers were separated by Chiral Prep-HPLC (Column: CHIRALPARK IC, 5 μm, 20×250 mm; Mobile Phase, A: DCM and B: MeOH (hold 85% B for 25 min); flow rate: 20 mL/min; Detector: UV 254/220 nm; RT: A (1st), 16.24 min; B (2nd), 21.61 min). The fractions of A were concentrated and lyophilized to afford 1-(5-[2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl]-1H,2H,3H,5H-pyrrolo[3,4-c]pyrrol-2-yl)-2-hydroxy-2-(2-methyl-1,3-benzoxazol-4-yl)ethan-1-one, 1st eluting isomer, as a white solid (39.8 mg, 19%). The fractions of B were concentrated and lyophilized to afford 1-(5-[2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl]-1H,2H,3H,5H-pyrrolo[3,4-c]pyrrol-2-yl)-2-hydroxy-2-(2-methyl-1,3-benzoxazol-4-yl)ethan-1-one, 2nd eluting isomer, as a white solid (31.7 mg, 15%).
To a solution of methyl 2-(3-cyclopropyl-4-methoxyphenyl)prop-2-enoate (300 mg, 1.29 mmol) in tetrahydrofuran (10 mL) was added methylamine (2 mL). The resulting mixture was stirred for 30 min at room temperature and concentrated under vacuum to remove excess methylamine. The residue was dissolved in tetrahydrofuran (5 mL) and treated with di-tert-butyl dicarbonate (423 mg, 1.94 mmol). The reaction stirred for 16 h and was concentrated under vacuum. The resulting crude product was purified by silica gel chromatography (eluting with 0:100 to 5:95 ethyl acetate/petroleum ether) to afford methyl 3-{[(tert-butoxy)carbonyl](methyl)amino}-2-(3-cyclopropyl-4-methoxyphenyl)propanoate as a light yellow oil (200 mg, 43%). LCMS (ES, m/z) 364 [M+H]+.
To a solution of methyl 3-{[(tert-butoxy)carbonyl](methyl)amino}-2-(3-cyclopropyl-4-methoxyphenyl)propanoate (200 mg, 0.55 mmol) in tetrahydrofuran (5 mL) was added water (5 mL), and lithium hydroxide (66 mg, 2.75 mmol). The resulting mixture was stirred for 16 h at room temperature. The reaction mixture was washed with diethyl ether (1×5 mL) and then acidified to pH=5 with saturated citric acid. The resulting solution was extracted with ethyl acetate (3×5 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to afford 3-{[(tert-butoxy)carbonyl](methyl)amino}-2-(3-cyclopropyl-4-methoxyphenyl)propanoic acid as a light yellow oil (150 mg, 87%). LCMS (ES, m/z) 350 [M+H]+.
To a solution of 3-{[(tert-butoxy)carbonyl](methyl)amino}-2-(3-cyclopropyl-4-methoxyphenyl)propanoic acid (150 mg, 0.43 mmol) in DMF (5 mL) was added 2-(2,3-dihydro-1,4-benzodioxine-6-sulfonyl)-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrole (177 mg, 0.51 mmol), DIEA (0.23 mL, 1.29 mmol) and HATU (196 mg, 0.51 mmol). The resulting solution was stirred for 1 h and poured into water (5 mL). The product was extracted with ethyl acetate (3×5 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting crude product was purified by prep-TLC (eluting with 1:1 ethyl acetate/petroleum ether) to afford tert-butyl N-[2-(3-cyclopropyl-4-methoxyphenyl)-3-[5-(2,3-dihydro-1,4-benzodioxine-6-sulfonyl)-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl]-3-oxopropyl]-N-methylcarbamate as alight yellow oil (150 mg, 55%). LCMS (ES, m/z) 640 [M+H]+.
To a solution of tert-butyl N-[2-(3-cyclopropyl-4-methoxyphenyl)-3-[5-(2,3-dihydro-1,4-benzodioxine-6-sulfonyl)-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl]-3-oxopropyl]-N-methylcarbamate (150 mg, 0.16 mmol) in dichloromethane (4 mL) was added trifluoroacetic acid (1 mL). The resulting solution was stirred for 2 h at room temperature and concentrated under vacuum. The reaction was quenched with saturated potassium carbonate solution (5 mL) and then extracted with dichloromethane (3×5 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The crude product was purified by Prep-HPLC (Column: XBridge Shield RP18 OBD Column, 5 μm, 19×150 mm; Mobile Phase, A: water (containing 10 mmol/L NH4HCO3) and B: CH3CN (30% to 58% over 7 min); Flow rate: 20 mL/min; Detector: UV 254 nm). The product fractions were concentrated under vacuum. The two enantiomers were further separated by (Column: CHIRALPAK IC, 5 μm, 20×250 mm; Mobile Phase, A: MTBE (containing 0.1% DEA) and B: EtOH (keep 50% B over 18 min); Detector: UV 254/220 nm; Retention time: A (1st), 9.54 min; B (2nd), 12.96 min). The product fractions were concentrated and lyophilized to afford 2-(3-cyclopropyl-4-methoxyphenyl)-1-[5-(2,3-dihydro-1,4-benzodioxine-6-sulfonyl)-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl]-3-(methylamino)propan-1-one), 1st eluting isomer, as a white solid (44.3 mg, 70%), and 2-(3-cyclopropyl-4-methoxyphenyl)-1-[5-(2,3-dihydro-1,4-benzodioxine-6-sulfonyl)-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl]-3-(methylamino)propan-1-one), 2nd eluting isomer, as a white solid (30.2 mg 48%).
To a solution of 3-[[(tert-butoxy)carbonyl]amino]-2-(3-chlorophenyl)-2-fluoropropanoic acid (120 mg, 0.34 mmol) in DMF (2 mL) was added HATU (155 mg, 0.41 mmol), 2-(2,3-dihydro-1,4-benzodioxine-6-sulfonyl)-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrole (110 mg, 0.34 mmol) and DIEA (132 mg, 1.02 mmol). The resulting solution was stirred for 1 h at room temperature. The reaction mixture was poured into water (10 mL) and then extracted with ethyl acetate (3×20 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting crude product was purified by prep-TLC (eluting with 2:1 ethyl acetate/petroleum ether) to afford tert-butyl N-[2-(3-chlorophenyl)-3-[5-(2,3-dihydro-1,4-benzodioxine-6-sulfonyl)-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl]-2-fluoro-3-oxopropyl]carbamate as a yellow oil (120 mg, 58%). LCMS (ES, m/z) 608, 610 [M+H]+.
To a solution of tert-butyl N-[2-(3-chlorophenyl)-3-[5-(2,3-dihydro-1,4-benzodioxine-6-sulfonyl)-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl]-2-fluoro-3-oxopropyl]carbamate (120 mg, 0.20 mmol) in THF (2 mL) was added sodium hydride (10 mg, 0.25 mmol, 60% dispersion in mineral oil) at 0° C. The resulting solution was stirred for 30 min at 0° C. and then treated with iodomethane (28 mg, 0.20 mmol). The resulting mixture was stirred for 6 h at room temperature. The reaction mixture was poured into aqueous ammonium chloride solution (10 mL) and then extracted with ethyl acetate (3×20 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to afford tert-butyl N-[2-(3-chlorophenyl)-3-[5-(2,3-dihydro-1,4-benzodioxine-6-sulfonyl)-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl]-2-fluoro-3-oxopropyl]-N-methylcarbamate as a yellow oil (120 mg, 98%). LCMS (ES, m/z) 622, 624 [M+H]+.
To a solution of tert-butyl N-[2-(3-chlorophenyl)-3-[5-(2,3-dihydro-1,4-benzodioxine-6-sulfonyl)-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl]-2-fluoro-3-oxopropyl]-N-methyl carbamate (120 mg, 0.18 mmol) in dichloromethane (2 mL) was added TFA (0.4 mL). The resulting solution was stirred for 1 h at room temperature and concentrated under vacuum. The resulting crude product was purified by Prep-HPLC (Column: XBridge Shield RP18 OBD Column, 5 μm, 19×150 mm; Mobile Phase, A: water (containing 10 mmol/L NH4HCO3) and B: CH3CN (28% to 50% over 15 min); Flow rate: 20 mL/min; Detector: UV 254 nm). The two enantiomers were further separated by (Column: CHIRALPAK IF, 5 μm, 20×250 mm; Mobile Phase, A: methanol (containing 0.1% DEA) and B: DCM (hold 50% B over 15 min); Detector: UV 254/220 nm; Retention time: A (1st), 8.817 min; B (2nd), 11.059 min). The product fractions of A were concentrated and lyophilized to afford 2-(3-chlorophenyl)-1-[5-(2,3-dihydro-1,4-benzodioxine-6-sulfonyl)-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl]-2-fluoro-3-(methylamino)propan-1-one, 1st eluting isomer, as a white solid (24.4 mg, 26%). The product fractions of B were concentrated and lyophilized to afford 2-(3-chloro-4-cyclopropoxyphenyl)-1-[5-(2,3-Dihydro-1,4-benzodioxine-6-sulfonyl)-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl]-3-(methylamino)propan-1-one, 2nd eluting isomer, as a white solid (11.9 mg, 12%).
To a solution of tert-butyl 4-[2-[5-(2,3-dihydro-1,4-benzodioxine-6-sulfonyl)-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl]-1-hydroxy-2-oxoethyl]-2,3-dihydro-1H-isoindole-2-carboxylate (780 mg, 1.34 mmol) in dichloromethane (6 mL) was added hydrochloric acid (6 mL, 4 N in 1,4-dioxane). The resulting solution was stirred for 2 h at room temperature and concentrated under vacuum to afford 1-[5-(2,3-dihydro-1,4-benzodioxine-6-sulfonyl)-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl]-2-(2,3-dihydro-1H-isoindol-4-yl)-2-hydroxyethan-1-one HCl salt as a dark red solid (680 mg, 62%). LCMS (ES, m/z) 484 [M+H]+.
To a solution of 1-[5-(2,3-dihydro-1,4-benzodioxine-6-sulfonyl)-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl]-2-(2,3-dihydro-1H-isoindol-4-yl)-2-hydroxyethan-1-one (680 mg, 1.40 mmol) in methanol (7 mL) was added formaldehyde (7 mL, 40 wt % in water). The resulting solution was stirred for 2 h at room temperature and then treated with sodium triacetoxyborohydride (893 mg, 4.21 mmol). The resulting mixture was stirred for 16 h at room temperature and concentrated under vacuum. The crude product was purified by prep-TLC (eluting with 1:10 MeOH/DCM). The enantiomers were separated by prep-Chiral HPLC (Column: CHIRAL ART Cellulose-SB, 5 μm, 20×250 mm; Mobile Phase, A: DCM and B: EtOH (0.1% DEA) (keep 40% B over 10 min); Detector: UV 254/220 nm; Retention time: 1st, 6.63 min; 2nd, 8.63 min. The product fractions were concentrated and lyophilized to afford 1-[5-(2,3-dihydro-1,4-benzodioxine-6-sulfonyl)-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl]-2-hydroxy-2-(2-methyl-2,3-dihydro-1H-isoindol-4-yl)ethan-1-one, 1st eluting isomer, as a white solid (41.2 mg, 6%), and 1-[5-(2,3-dihydro-1,4-benzodioxine-6-sulfonyl)-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl]-2-hydroxy-2-methyl-2,3-dihydro-1H-isoindol-4-yl)ethan-1-one, 2nd eluting isomer, as a white solid (42.4 mg, 5%).
To a solution of 2-(2,3-dihydro-1,4-benzodioxine-6-sulfonyl)-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrole (529 mg, 1.63 mmol) in DMF (3 mL) was added 2-[2-[(tert-butoxy)carbonyl]-2,3-dihydro-1H-isoindol-4-yl]-2-oxoacetic acid (500 mg, 1.46 mmol), DIEA (665 mg, 4.89 mmol) and HATU (783 mg, 1.96 mmol). The resulting solution was stirred for 1 h at room temperature. The reaction mixture was poured into water (5 mL) and then extracted with EtOAc (3×5 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting crude product was purified by prep-TLC (eluting with 3:1 ethyl acetate/petroleum ether) to afford tert-butyl 4-[2-[5-(2,3-dihydro-1,4-benzodioxine-6-sulfonyl)-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl]-2-oxoacetyl]-2,3-dihydro-1H-isoindole-2-carboxylate as a yellow oil (300 mg, 30%). LCMS (ES, m/z) 582 [M+H]+.
To a solution of tert-butyl 4-[2-[5-(2,3-dihydro-1,4-benzodioxine-6-sulfonyl)-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl]-2-oxoacetyl]-2,3-dihydro-1H-isoindole-2-carboxylate (300 mg, 0.45 mmol) in DCM (6 mL) was added TFA (1.5 mL). The resulting mixture was stirred for 2 h at room temperature and concentrated under vacuum. The resulting mixture was basified to pH=8 with saturated potassium carbonate solution and then extracted with dichloromethane (2×5 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under vacuum to afford 1-[5-(2,3-dihydro-1,4-benzodioxine-6-sulfonyl)-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl]-2-(2,3-dihydro-1H-isoindol-4-yl)ethane-1,2-dione as a yellow solid (200 mg, crude). LCMS (ES, m/z) 482 [M+H]+.
To a solution of 1-[5-(2,3-dihydro-1,4-benzodioxine-6-sulfonyl)-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl]-2-(2,3-dihydro-1H-isoindol-4-yl)ethane-1,2-dione (217 mg, 0.38 mmol) in 1,2-dichloroethane (15 ml) was added copper (II) acetate (90 mg, 0.43 mmol), 2,2′-bipyridine (70 mg, 0.43 mmol), cyclopropylboronic acid (77 mg, 0.85 mmol) and sodium carbonate (95 mg, 0.86 mmol). The resulting mixture was stirred for 16 h at 70° C. under air atmosphere and cooled to room temperature. The reaction mixture was filtered and poured into water (10 mL). The resulting solution was extracted with ethyl acetate (3×10 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The resulting crude product was purified by prep-TLC (eluting with 3:1 ethyl acetate/petroleum ether) to afford 1-(2-cyclopropyl-2,3-dihydro-1H-isoindol-4-yl)-2-[5-(2,3-dihydro-1,4-benzodioxine-6-sulfonyl)-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl]ethane-1,2-dione as a white solid (60 mg, 26%). LCMS (ES, m/z) 522 [M+H]+.
To a solution of 1-(2-cyclopropyl-2,3-dihydro-1H-isoindol-4-yl)-2-[5-(2,3-dihydro-1,4-benzodioxine-6-sulfonyl)-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl]ethane-1,2-dione (60 mg, 0.10 mmol) in methanol (1.5 mL) was added sodium borohydride (9 mg, 0.23 mmol). The resulting solution was stirred for 2 h at room temperature. The reaction mixture was poured into water (5 mL) and then extracted with EtOAc (3×5 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The resulting crude product was purified by silica gel chromatography (eluting with 10:1 DCM/MeOH) and further purified by Prep-HPLC (Column: XBridge Prep C18 OBD Column (19×150 mm) 5 um; Mobile Phase A: Water (10 mmoL/L NH4HCO3), Mobile Phase B: MeCN (30% B to 55% B over 7 min); Flow rate: 20 mL/min; Detector: 254/220 nm). The two enantiomers were further separated by Chiral-Prep-HPLC (Column: CHIRALPAK IE, 2×25 cm, 5 μm; Mobile Phase A: MeOH (containing 0.1% DEA), Mobile Phase B: DCM (Hold 35% B over 14 min); Flow rate: 19 mL/min; Detector: 220/254 nm; A: 9.39 min; B: 12.4 min). The fractions of A were concentrated and lyophilized to afford 2-(2-cyclopropyl-2,3-dihydro-1H-isoindol-4-yl)-1-[5-(2,3-dihydro-1,4-benzodioxine-6-sulfonyl)-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl]-2-hydroxyethan-1-one, 1st eluting isomer, as a white solid (5.0 mg, 10%). The fractions of B were concentrated and lyophilized to afford 2-(2-cyclopropyl-2,3-dihydro-1H-isoindol-4-yl)-1-[5-(2,3-dihydro-1,4-benzodioxine-6-sulfonyl)-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl]-2-hydroxyethan-1-one, 2nd eluting isomer, as a white solid (5.3 mg, 10%).
To a solution of (2R)-3-{[(tert-butoxy)carbonyl](methyl)amino}-2-(3-chlorophenyl)propanoic acid (2.30 g, 7.34 mmol) in N,N-dimethylformamide (20 mL) was added HATU (3.07 g, 8.08 mmol), 2-(2,3-dihydro-1,4-benzodioxine-6-sulfonyl)-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrole hydrochloride (2.52 g, 7.34 mmol), and DIEA (3.82 mL, 22.1 mmol). The resulting solution was stirred for 2 h at rt. The reaction mixture was poured into water (100 mL) and then extracted with ethyl acetate (3×100 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The crude product was purified by silica gel chromatography (eluting with 1:15 ethyl acetate/dichloromethane) to afford tert-butyl N-[(2S)-2-(3-chlorophenyl)-3-[5-(2,3-Dihydro-1,4-benzodioxine-6-sulfonyl)-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl]-3-oxopropyl]-N-methylcarbamate as a white solid (3.5 g, 79%). 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 7.40-7.18 (m, 6H), 7.07-7.06 (m, 1H), 4.45-4.22 (m, 5H), 4.11-3.88 (m, 7H), 3.88-3.58 (m, 2H), 3.44-3.36 (m, 1H), 2.75-2.67 (m, 3H), 1.27-1.16 (m, 9H). LCMS (ES, m/z) 604, 606 [M+H]+.
To a solution of tert-butyl N-[(2S)-2-(3-chlorophenyl)-3-[5-(2,3-Dihydro-1,4-benzodioxine-6-sulfonyl)-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl]-3-oxopropyl]-N-methylcarbamate (1.5 g, 2.49 mmol) in ethyl acetate (10 mL) was added a solution of hydrochloric acid (10 mL, 4 N in 1,4-dioxane). The resulting solution was stirred for 3 h at 25° C. The mixture was concentrated under vacuum to about ⅓ volume and the solids were collected by filtration. The solids were treated with EtOAc (10 mL) at 70° C., filtered at room temperature, and dissolved with saturated potassium carbonate solution/EA (1:1, 10 mL). The resulting solution was stirred for 3 h and then extracted with EA (3×5 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to afford (2S)-2-(3-chlorophenyl)-1-[5-(2,3-dihydro-1,4-Benzodioxine-6-sulfonyl)-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl]-3-(methylamino)propan-1-one as a white solid (1 g, 80%). 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 7.36-7.25 (m, 6H), 7.07-7.06 (m, 1H), 4.40-4.29 (m, 5H), 4.06-3.88 (m, 8H), 3.06-3.01 (m, 1H), 2.60-2.50 (m, 1H), 2.23 (s, 3H), 1.66 (s, 1H). LCMS (ES, m/z) 504, 506 [M+H]+.
To a solution of 3-[(oxetan-3-yl)amino]-2-phenylpropanoic acid (60 mg, 0.27 mmol) in DMF (10 mL) was added HATU (123 mg, 0.32 mmol), 2-(2,3-dihydro-1,4-benzodioxine-6-sulfonyl)-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrole hydrochloride (93 mg, 0.27 mmol) and DIEA (0.13 mL, 0.81 mmol). The resulting mixture was stirred for 1 h at room temperature. The reaction mixture was poured into water (5 mL) and then extracted with ethyl acetate (3×10 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting crude product was purified by Prep-HPLC (Column: XBridge Shield RP18 OBD Column, 5 μm, 30×150 mm; Mobile Phase, A: water (containing 10 mmol/L NH4HCO3) and B: CH3CN (25% to 45% over 7 min); Flow rate: 60 mL/min; Detector: UV 254 nm). The product fractions were concentrated under vacuum. The two enantiomers were further separated by Chiral Prep-HPLC (Column: CHIRALPAK IF, 5 μm, 20×250 mm; Mobile Phase, A: MeOH (containing 0.1% DEA) and B: DCM (keep 10% B over 16 min); Flow rate: 20 mL/min; Detector: UV 254/220 nm; Retention time: 1st, 17.285 min; 2nd, 21.532 min). The product fractions were concentrated and lyophilized to afford 1-[5-(2,3-dihydro-1,4-benzodioxine-6-sulfonyl)-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl]-3-[(oxetan-3-yl)amino]-2-phenylpropan-1-one, 1st eluting isomer, as a white solid (1 mg, 1.4%), and 1-[5-(2,3-dihydro-1,4-benzodioxine-6-sulfonyl)-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl]-3-[(oxetan-3-yl)amino]-2-phenylpropan-1-one, 2nd eluting isomer, as a white solid (1 mg, 1.4%).
To a solution of 2-(2,3-dihydro-1-benzofuran-7-yl)-2-oxoacetic acid (250 mg, 1.30 mmol) in DMF (4 mL) was added 2-[2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl]-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrole (450 mg, 1.30 mmol), DIEA (0.43 mL, 2.60 mmol), and HATU (544 mg, 1.43 mmol). The resulting solution was stirred for 1 h at room temperature. The reaction mixture was poured into water (10 mL) and then extracted with ethyl acetate (3×10 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting crude product was purified by prep-TLC (eluting with 1:1 ethyl acetate/petroleum ether) to afford 1-(2,3-dihydro-1-benzofuran-7-yl)-2-(5-{2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl}-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl)ethane-1,2-dione as a light yellow solid (200 mg, 32%). LCMS (ES, m/z): 484 [M+H]+.
To a solution of 1-(2,3-dihydro-1-benzofuran-7-yl)-2-(5-{2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl}-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl)ethane-1,2-dione (200 mg, 0.41 mmol) in tetrahydrofuran (2 mL) was added sodium borohydride (8 mg, 0.21 mmol). The resulting solution was stirred for 30 min at 0° C. The reaction mixture was poured into water (10 mL) and then extracted with ethyl acetate (3×10 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting crude product was purified by Prep-HPLC (Column: XBridge Shield RP18 OBD Column, 5 μm, 19×150 mm; Mobile Phase, A: water (containing 10 mmol/L NH4HCO3) and B: MeCN (15% to 45% over 10 min); Flow rate: 20 mL/min; Detector: UV 254 nm). The product fractions were concentrated under vacuum. The two enantiomers were separated by Chiral Prep-HPLC (Column: CHIRALPAK IF, 5 μm, 20×250 mm; Mobile Phase, A: MeOH (containing 0.1% DEA) and B: DCM (keep 40% B over 50 min); Flow rate: 15 mL/min; Detector: UV 254/220 nm; Retention time: 1st, 19.223 min; 2nd, 29.404 min). The product fractions were concentrated and lyophilized to afford 2-(2,3-dihydro-1-benzofuran-7-yl)-1-(5-{2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl}-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl)-2-hydroxyethan-1-one, 1st eluting isomer, as a white solid (30.5 mg, 15%), and 2-(2,3-dihydro-1-benzofuran-7-yl)-1-(5-{2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl}-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl)-2-hydroxyethan-1-one, 2′ eluting isomer, as a white solid (33.5 mg, 17%).
To tert-butyl (R)-(3-(5-((2,3-dihydrobenzo[b][1,4]dioxin-6-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-oxo-2-phenylpropyl)carbamate (180 μl, 36.0 μmol; 0.2M in dioxane) was added acetic acid (150 μl, 30.0 μmol; 0.2M in dioxane) and DCE, cyclopentanecarbaldehyde (180 μl, 36.0 μmol; 0.2M in dioxane) and sodium triacetoxyborohydride (300 μl, 60.0 μmol; 0.2M in dioxane). The reaction was heated at 50° C. for 4 h. The reaction was run through an SCX-SPE cartridge and eluted with 2 ml of 10% MeOH/EtOAc (ETW) followed by 2 ml of 2M Ammonia/MeOH (ETC). The basic eluent was dried under a stream of N2 and the product was purified by reverse phase HPLC.
As set forth in Table 10, IC50 values are defined as follows. ≤25 μM and >2 μM (+); ≤2 μM and >0.2 μM (++); ≤0.2 μM and >0.05 μM (+++); ≤0.05 μM and >0.001 μM (++++); and not tested (−−), based upon the Biochemical Assay of Example A.
In Tables 1 and 10, absolute stereochemistry has not been determined for some Examples. Accordingly, assignment of any Examples as the “R” or “S” stereoisomer is arbitrary, unless otherwise noted. In some cases, Examples are labeled with “1st eluting isomer”, “2nd eluting isomer”, etc. based on the purification method used to separate the stereoisomers (see Table 9).
1H NMR
2H4)-1,4-benzodioxine-6-
2H4)-1,4-benzodioxine-6-
In some embodiments, the USP9X inhibitor may be a compound of Table 11:
To a solution of 4-bromo-3-fluoroaniline (474 mg, 2.51 mmol) in 1,4-dioxane (10 mL) was added Pd(dppf)Cl2 (183 mg, 0.25 mmol) and 2-(tributylstannyl)-1,3-oxazole (900 mg, 2.52 mmol). The resulting mixture was stirred for 48 h at 100° C. and then cooled to room temperature. The reaction mixture was poured into water (10 mL) and then extracted with ethyl acetate (3×10 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting crude product was purified by silica gel chromatography (eluting with 0:100 to 50:50 ethyl acetate/petroleum ether) to afford 3-fluoro-4-(1,3-oxazol-2-yl)aniline (180 mg, 41%). LCMS (ES, m/z) 179 [M+H]+.
Into glacial acetic acid (10 mL) was bubbled in SO2 gas for 1 h at room temperature. Then CuCl2 (34 mg, 0.25 mmol) was added and SO2 gas was bubbled in for additional 2 h to afford solution A. To a pre-cooled solution of 3-fluoro-4-(1,3-oxazol-2-yl)aniline (180 mg, 1.01 mmol) in acetic acid (2 mL) and concentrated hydrochloric acid (6 mL) was added a solution of sodium nitrite (77 mg, 1.11 mmol) in distilled water (0.5 mL) dropwise with stirring at −10° C. After stirring for 15 min, solution A was added to this diazonium salt solution at −10° C. The resulting solution was allowed to warm to room temperature naturally and stirred for 16 h. The reaction mixture was treated with water (10 mL) and then extracted with EA (3×10 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting crude product was purified by silica gel chromatography (eluting with 0:100 to 12:88 ethyl acetate/petroleum ether) to afford 3-fluoro-4-(1,3-oxazol-2-yl)benzene-1-sulfonyl chloride (120 mg, 45%). LCMS (ES, m/z) 262, 264 [M+H]+.
The Intermediate in Table 12 was synthesized according to the procedure described for Intermediate 2′-1 above.
To a solution of 6-[(3-chlorophenyl)carbonyl]-2-(2,3-dihydro-1,4-benzodioxine-6-sulfonyl)-1H,2H,3H-pyrrolo[3,4-c]pyridine (80 mg, 0.16 mmol) in toluene (8 mL) was added 1-tert-butyl-1lambda3,3,6-oxadiazocan-2-one (42 mg, 0.22 mmol), XPhos (19 mg, 0.04 mmol), Cs2CO3 (171 mg, 0.52 mmol) and Pd2(dba)3.CHCl3 (18 mg, 0.02 mmol). The resulting mixture was stirred for 16 h at 100° C. and then cooled to room temperature. The reaction mixture was filtered and concentrated under vacuum. The resulting crude product was purified by silica gel chromatography (eluting with 1:10 EA/PE) to afford 1-tert-butyl-6-(3-[[2-(2,3-dihydro-1,4-benzodioxine-6-sulfonyl)-1H,2H,3H-pyrrolo[3,4-c]pyridin-6-yl]carbonyl]phenyl)-1lambda3,3,6-oxadiazocan-2-one as a yellow solid (80 mg, 84%). LCMS (ES, m/z) 607 [M+H]+.
The Intermediate in Table 13 was synthesized according to the procedure described for Intermediate 3′-1 above.
aRuphos 3G, Ruphos, K3PO4, dioxane, 100° C., 16 h;
To a solution of tert-butyl 4-(3-(2-((2,3-dihydrobenzo[b][1,4]dioxin-6-yl)sulfonyl)-2,3-dihydro-1H-pyrrolo[3,4-c]pyridine-6-carbonyl)phenyl)piperazine-1-carboxylate (80 mg, 0.12 mmol) in DCM (3 mL) was added TFA (1 mL). The resulting solution was stirred for 1 h at rt and then concentrated under vacuum. The resulting mixture was then basified to pH 8 with saturated aqueous potassium carbonate solution. The resulting mixture was extracted with DCM (3×5 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under vacuum to afford 1-(3-[[2-(2,3-dihydro-1,4-benzodioxine-6-sulfonyl)-1H,2H,3H-pyrrolo[3,4-c]pyridin-6-yl]carbonyl]phenyl)piperazine as a yellow solid (50 mg, 83%). LCMS (ES, m/z) 507 [M+H]+.
The Intermediates in Table 14 were synthesized according to the procedure described for Intermediate 4′-1 above.
To a solution of 4-(1H-imidazol-1-yl)benzene-1-sulfonyl chloride (200 mg, 0.82 mmol) in DCM (2 mL) was added 6-benzoyl-1H,2H,3H-pyrrolo[3,4-c]pyridine (124 mg, 0.55 mmol) and TEA (0.22 mL, 1.60 mmol). The resulting solution was stirred for 2 h at 25° C. The reaction mixture was poured into water (10 mL) and then extracted with ethyl acetate (3×10 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting crude product was purified by silica gel chromatography (eluting with 0:100 to 10:90 ethyl acetate/petroleum ether) to afford 1-[4-([6-benzoyl-1H,2H,3H-pyrrolo[3,4-c]pyridin-2-yl]sulfonyl)phenyl]-1H-imidazole as a white solid (95 mg, 220%). LCMS (ES, m/z) 431 [M+H]+.
The Intermediates in Table 15 were synthesized according to the procedure described for Intermediate 8′-1 above.
Into a high pressure tank was placed a solution of tert-butyl 6-chloro-1H,2H,3H-pyrrolo[3,4-c]pyridine-2-carboxylate (2 g, 6.99 mmol) in MeOH (30 mL), Pd(dppf)Cl2.CH2Cl2 (640 mg, 0.78 mmol) and TEA (3.28 mL, 23.7 mmol). Then CO (30 atm) was introduced. The resulting mixture was stirred for 16 h at 120° C. and cooled to room temperature. The reaction mixture was filtered and concentrated under vacuum. The resulting crude product was purified by silica gel chromatography (eluting with 1:5 EA/PE) to afford 2-tert-butyl 6-methyl 1H,2H,3H-pyrrolo[3,4-c]pyridine-2,6-dicarboxylate as a yellow solid (1.2 g, 56%). LCMS (ES, m/z) 279 [M+H]+.
To a solution of 2-tert-butyl 6-methyl 1H,2H,3H-pyrrolo[3,4-c]pyridine-2,6-dicarboxylate (2 g, 6.47 mmol) in THF (20 mL) was added water (15 mL) and LiOH (863 mg, 36.0 mmol). The resulting solution was stirred for 16 h at rt. The resulting mixture was washed with Et2O (1×10 mL) and then acidified to pH 5 with hydrochloric acid solution (2 N). The resulting mixture was extracted with EA (3×25 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The resulting crude product was purified by reversed phase chromatography (eluting with 1:1 water/MeCN). The collected fractions were combined and concentrated under vacuum to afford 2-(tert-butoxycarbonyl)-2,3-dihydro-1H-pyrrolo[3,4-c]pyridine-6-carboxylic acid as yellow oil (1.0 g, 53%). LCMS (ES, m/z) 265 [M+H]+.
To a solution of 2-[(tert-butoxy)carbonyl]-1H,2H,3H-pyrrolo[3,4-c]pyridine-6-carboxylic acid (1 g, 3.41 mmol) in DMF (15 mL) was added methoxy(methyl)amine hydrochloride (441 mg, 4.52 mmol), HATU (2.88 g, 7.57 mmol) and DIEA (1.98 mL, 11.37 mmol). The resulting solution was stirred for 1 h at rt. The reaction mixture was poured into water (15 mL) and then extracted with EA (3×15 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrate under vacuum. The resulting crude product was purified by silica gel chromatography (eluting with 1:10 EA/PE) to afford tert-butyl 6-[methoxy(methyl)carbamoyl]-1H,2H,3H-pyrrolo[3,4-c]pyridine-2-carboxylate as light yellow oil (700 mg, 67%). LCMS (ES, m/z) 308 [M+H]+.
To a solution of tert-butyl 6-[methoxy(methyl)carbamoyl]-1H,2H,3H-pyrrolo[3,4-c]pyridine-2-carboxylate (100 mg, 0.29 mmol) in THF (1 mL) was added a solution of bromo(3-chlorophenyl)magnesium (0.78 mL, 0.5 M in THF) dropwise with stirring at 0° C. The resulting solution was stirred for 1 h at rt and then poured into saturated ammonium chloride solution (5 mL). The resulting mixture was extracted with EA (3×5 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The resulting crude product was purified by silica gel chromatography (eluting with 1:3 EA/PE) to afford tert-butyl 6-[(3-chlorophenyl)carbonyl]-1H,2H,3H-pyrrolo[3,4-c]pyridine-2-carboxylate as yellow oil (80 mg, 76%). LCMS (ES, m/z) 359, 361 [M+H]+.
To a solution of tert-butyl 6-[(3-chlorophenyl)carbonyl]-1H,2H,3H-pyrrolo[3,4-c]pyridine-2-carboxylate (500 mg, 1.25 mmol) in dichloromethane (8 mL) was added TFA (2 mL). The resulting solution was stirred for 1 h at rt. The resulting mixture was concentrated under vacuum to afford 6-[(3-chlorophenyl)carbonyl]-1H,2H,3H-pyrrolo[3,4-c]pyridine (TFA salt) as brown oil (500 mg, crude). LCMS (ES, m/z) 259, 261 [M+H]+.
To a solution of tert-butyl 2,4-dichloro-5H,6H,7H-pyrrolo[3,4-d]pyrimidine-6-carboxylate (6.00 g, 17.6 mmol) in methanol (48 mL) was added zinc powder (1.80 g, 26.4 mmol) and acetic acid (10.6 mL, 176 mmol). The resulting mixture was stirred for 16 h at 50° C. and cooled to room temperature. The resulting mixture was concentrated under vacuum. The resulting crude product was purified by silica gel chromatography (eluting with 1:1 ethyl acetate/petroleum ether) to afford tert-butyl 2-chloro-5H,6H,7H-pyrrolo[3,4-d]pyrimidine-6-carboxylate as a white solid (2.90 g, 54%). LCMS (ES, m/z): 256, 258 [M+H]+.
To a solution of tert-butyl 2-chloro-5H,6H,7H-pyrrolo[3,4-d]pyrimidine-6-carboxylate (1.50 g, 4.99 mmol) in DMF (15 mL) was added Zn(CN)2 (868 mg, 7.48 mmol) and Pd(dppf)Cl2 (364 mg, 0.50 mmol). The resulting mixture was irradiated with microwave for 3 h at 140° C. After cooling to rt, the reaction mixture was poured into water (10 mL) and then extracted with ethyl acetate (3×15 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting crude product was purified by silica gel chromatography (eluting with 1:1 ethyl acetate/petroleum ether) to afford tert-butyl 2-cyano-5H,6H,7H-pyrrolo[3,4-d]pyrimidine-6-carboxylate as a yellow oil (500 mg, 34%). LCMS (ES, m/z): 247 [M+H]+.
To a solution of tert-butyl 2-cyano-5H,6H,7H-pyrrolo[3,4-d]pyrimidine-6-carboxylate (200 mg, 0.69 mmol) in THF (2 mL) was added bromo(phenyl)magnesium (1.38 mL, 1 M in THF) dropwise at 0° C. The resulting mixture was stirred for 1 h at rt. Then 1 N hydrochloric acid (2 mL) was added. The resulting mixture was stirred for 30 min at rt and then extracted with EA (3×5 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting crude product was purified by silica gel chromatography (eluting with 4:5 ethyl acetate/petroleum ether) to afford tert-butyl 2-benzoyl-5H,6H,7H-pyrrolo[3,4-d]pyrimidine-6-carboxylate as a yellow oil (90 mg, 34%). LCMS (ES, m/z): 326 [M+H]+.
To a solution of tert-butyl 2-benzoyl-5H,6H,7H-pyrrolo[3,4-d]pyrimidine-6-carboxylate (90 mg, 0.24 mmol) in methanol (1 mL) was added sodium borohydride (18.7 mg, 0.47 mmol). The resulting mixture stirred for 1 h at rt. The reaction mixture was poured into water (5 mL) and then extracted with EA (3×5 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting crude product was purified by Prep-TLC (eluting with 2:5 ethyl acetate/petroleum ether) to afford tert-butyl 2-[hydroxy(phenyl)methyl]-5H,6H,7H-pyrrolo[3,4-d]pyrimidine-6-carboxylate as a yellow solid (60 mg, 77%). LCMS (ES, m/z): 328 [M+H]+.
To a solution of tert-butyl 2-[hydroxy(phenyl)methyl]-5H,6H,7H-pyrrolo[3,4-d]pyrimidine-6-carboxylate (60 mg, 0.18 mmol) in DCM (6 mL) was added trifluoroacetic acid (2 mL). The resulting mixture was stirred for 1 h at rt and concentrated under vacuum. The resulting mixture was basified to pH 8 with saturated potassium carbonate solution and extracted with DCM (3×5 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to afford phenyl({5H,6H,7H-pyrrolo[3,4-d]pyrimidin-2-yl})methanol as a yellow solid (35 mg, 85%). LCMS (ES, m/z): 228 [M+H]+.
The Intermediate in Table 16 was synthesized according to the procedure described for Intermediate 20′-1 above.
To a solution of 4-bromo-2,3-dihydro-1H-isoindole hydrochloride (3.00 g, 12.2 mmol) and TEA (5.10 mL, 36.5 mmol) in dichloromethane (50 mL) was added CbzCl (4.10 g, 24.3 mmol) in portions at 0° C. The resulting solution was stirred for 5 h at room temperature. The reaction mixture was poured into water (50 mL) and then extracted with ethyl acetate (3×50 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The resulting crude product was purified by silica gel chromatography (eluting with 0:100 to 20:80 ethyl acetate/petroleum ether) to afford benzyl 4-bromo-2,3-dihydro-1H-isoindole-2-carboxylate as a pink solid (3.50 g, 87%). LCMS (ES, m/z): 332, 334[M+H]+.
To a solution of tert-butyl 4-bromo-2,3-dihydro-1H-isoindole-2-carboxylate (1.50 g, 4.78 mmol) in mesitylene (20 mL) was added Pd(allyl)2Cl2 (46 mg, 0.10 mmol), SPhos (118 mg, 0.29 mmol) and sodium 2-cyanoacetate (808 mg, 7.17 mmol). The resulting mixture was stirred for 5 h at 140° C. After cooling to room temperature, the reaction mixture was filtered and concentrated under vacuum. The resulting crude product was purified by silica gel chromatography (eluting with 0:100 to 50:50 ethyl acetate/petroleum ether) to afford tert-butyl 4-(cyanomethyl)-2,3-dihydro-1H-isoindole-2-carboxylate as a brown solid (1.00 g, 81%). LCMS (ES, m/z): 293[M+H]+.
To a solution of tert-butyl 6-chloro-1H,2H,3H-pyrrolo[3,4-c]pyridine-2-carboxylate (500 mg, 1.86 mmo) in THF (10 mL) was added benzyl 4-(cyanomethyl)-2,3-dihydro-1H-isoindole-2-carboxylate (861 mg, 2.80 mmol) and sodium amide (146 mg, 3.74 mmol). The resulting solution was stirred for 4 h at 50° C. After cooling to room temperature, the reaction mixture was poured into water (20 mL) and then extracted with ethyl acetate (3×20 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting crude product was purified by silica gel chromatography (eluting with 0:100 to 30:60 ethyl acetate/petroleum ether) to afford benzyl 4-([2-[(tert-butoxy)carbonyl]-1H,2H,3H-pyrrolo[3,4-c]pyridin-6-yl](cyano)methyl)-2,3-dihydro-1H-isoindole-2-carboxylate as yellow oil (300 mg, 32%). LCMS (ES, m/z): 511[M+H]+.
To a solution of benzyl 4-([2-[(tert-butoxy)carbonyl]-1H,2H,3H-pyrrolo[3,4-c]pyridin-6-yl](cyano)methyl)-2,3-dihydro-1H-isoindole-2-carboxylate (300 mg, 0.56 mmol) in DMSO (5 mL) was added benzyltriethylammonium chloride (6 mg, 0.03 mmol) and sodium hydroxide (0.2 mL, 4 N in water). Then oxygen was bubbled in. The resulting mixture was stirred for 2 h at room temperature. The reaction mixture was poured into water (20 mL) and then extracted with ethyl acetate (3×20 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting crude product was purified by silica gel chromatography (eluting with 0:100 to 50:50 ethyl acetate/petroleum ether) to afford benzyl 4-[2-[(tert-butoxy)carbonyl]-1H,2H,3H-pyrrolo[3,4-c]pyridine-6-carbonyl]-2,3-dihydro-1H-isoindole-2-carboxylate as yellow oil (200 mg, 72%). LCMS (ES, m/z): 500 [M+H]+.
To a solution of benzyl 4-[2-[(tert-butoxy)carbonyl]-1H,2H,3H-pyrrolo[3,4-c]pyridine-6-carbonyl]-2,3-dihydro-1H-isoindole-2-carboxylate (200 mg, 0.38 mmol) in dichloromethane (5 mL) was added trifluoroacetic acid (1 mL). The resulting solution was stirred for 5 h at room temperature. The resulting mixture was concentrated under vacuum to afford benzyl 4-[1H,2H,3H-pyrrolo[3,4-c]pyridine-6-carbonyl]-2,3-dihydro-1H-isoindole-2-carboxylate (TFA salt) as yellow oil (180 mg, crude). LCMS (ES, m/z): 400[M+H]+.
To a solution of tert-butyl 6-[2-(4-methylpiperazin-1-yl)benzoyl]-1H, 2H, 3H-pyrrolo[3,4-c]pyridine-2-carboxylate (200 mg, 0.43 mmol) in MeOH (10 mL) was added sodium borohydride (8 mg, 0.21 mmol). The resulting mixture was stirred for 1 h at 25° C. The reaction mixture was concentrated under vacuum. The resulting crude product was purified by silica gel chromatography (eluting with 1:10 MeOH/DCM) to afford tert-butyl 6-[hydroxy[2-(4-methylpiperazin-1-yl)phenyl]methyl]-1H,2H,3H-pyrrolo[3,4-c]pyridine-2-carboxylate as yellow oil (170 mg, 85%). LCMS (ES, m/z): 425 [M+H]+.
The Intermediate in Table 17 was synthesized according to the procedure described for Intermediate 25′-1 above.
To tert-butyl 3-oxopyrrolidine-1-carboxylate (20 g, 102 mmol) was added dimethylformamide dimethyl acetal (200 mL). The resulting solution was stirred for 12 h at 140° C. After cooling to room temperature, the resulting mixture was concentrated. The residue was dissolved with a minimum amount of DCM and then treated with hexane (100 mL). The resulting solids were collected by filtration and dried under vacuum to afford tert-butyl (3E)-3-[(dimethylamino)methylidene]-4-oxopyrrolidine-1-carboxylate as a yellow solid (15 g, 58%). LCMS (ES, m/z): 241 [M+H]+.
To a solution of (methylsulfanyl)methanimidamide (17 g, 183 mmol) in EtOH (200 mL) was added sodium ethoxide (13 g, 183 mmol) at 0° C. After stirring for 10 min, to the above solution was added tert-butyl (3E)-3-[(dimethylamino)methylidene]-4-oxopyrrolidine-1-carboxylate (15 g, 61.2 mmol). The resulting mixture was stirred for 4 h at 80° C. After cooling to room temperature, the reaction mixture was concentrated under vacuum. The residue was dissolved with water (100 mL) and then extracted with ethyl acetate (3×100 mL). The organic layers were combined, dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The resulting crude product was purified by silica gel chromatography (eluting with 1:2 ethyl acetate/petroleum ether) to afford tert-butyl 2-(methylsulfanyl)-5H,6H,7H-pyrrolo[3,4-d]pyrimidine-6-carboxylate as a yellow solid (4 g, 41%). LCMS (ES, m/z): 268 [M+H]+.
To a solution of tert-butyl 2-(methylsulfanyl)-5H,6H,7H-pyrrolo[3,4-d]pyrimidine-6-carboxylate (4 g, 14.1 mmol) in DCM (80 mL) was added m-CPBA (7.5 g, 42.6 mmol). The resulting mixture was stirred for 5 h at 0° C. The resulting mixture was washed with saturated sodium bicarbonate solution (5×100 mL), dried over anhydrous sodium sulfate, filtered and concentrated. The resulting crude product was purified by silica gel chromatography (eluting with 2:1 ethyl acetate/petroleum ether) to afford tert-butyl 2-methanesulfonyl-5H,6H,7H-pyrrolo[3,4-d]pyrimidine-6-carboxylate as a yellow solid (4 g, 89%). LCMS (ES, m/z): 300 [M+H]+.
To a solution of tert-butyl 6-benzoyl-1H,2H,3H-pyrrolo[3,4-c]pyridine-2-carboxylate (340 mg, 1.05 mmol) in MeOH (10 mL), was added sodium borohydride (12 mg, 0.32 mmol). The resulting mixture was stirred for 1 h at 25° C. The reaction mixture was poured into water (15 mL) and then extracted with ethyl acetate (3×10 mL). The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting crude product was purified by silica gel chromatography (eluting with 0:100 to 10:90 ethyl acetate/petroleum ether) to afford tert-butyl 6-[hydroxy(phenyl)methyl]-1H,2H,3H-pyrrolo[3,4-c]pyridine-2-carboxylate as a yellow solid (300 mg, 88%). LCMS (ES, m/z): 327 [M+H]+.
To a solution of tert-butyl 6-[hydroxy(phenyl)methyl]-1H,2H,3H-pyrrolo[3,4-c]pyridine-2-carboxylate (280 mg, 0.86 mmol) in dichloromethane (4 mL) was added TFA (1 mL). The resulting mixture was stirred for 1 h at 25° C. The reaction mixture was concentrated under vacuum to afford phenyl({1H,2H,3H-pyrrolo[3,4-c]pyridin-6-yl})methanol (TFA salt) as light yellow oil (200 mg, crude). LCMS (ES, m/z): 227 [M+H]+.
To a solution of phenyl({1H,2H,3H-pyrrolo[3,4-c]pyridin-6-yl})methanol (TFA salt) (225 mg, 0.99 mmol) in dichloromethane (7 mL) was added TEA (0.55 mL, 3.98 mmol) and 4-cyanobenzene-1-sulfonyl chloride (200 mg, 0.99 mmol). The resulting mixture was stirred for 2 h at 25° C. and concentrated under vacuum. The resulting crude product was purified by silica gel chromatography (eluting with 0:100 to 10:90 ethyl acetate/petroleum ether) to afford 4-({6-[hydroxy(phenyl)methyl]-1H,2H,3H-pyrrolo[3,4-c]pyridin-2-yl}sulfonyl)benzonitrile as a white solid (200 mg, 51%). LCMS (ES, m/z): 392 [M+H]+.
The Intermediate in Table 18 was synthesized according to the procedure described for Intermediate 32′-1 above.
To a solution of 2-(3-bromophenyl)acetonitrile (1.96 g, 9.52 mmol) in THF (50 mL) was added tert-butyl 2-methanesulfonyl-5H,6H,7H-pyrrolo[3,4-d]pyrimidine-6-carboxylate (2 g, 6.35 mmol) and potassium bis(trimethylsilyl)amide solution (10 mL, 1 M in THF). The resulting mixture was stirred for 12 h at room temperature while oxygen was kept bubbling in. The reaction mixture was poured into water (50 mL) and then extracted with ethyl acetate (3×50 mL). The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting crude product was purified by silica gel chromatography (eluting with 0:100 to 30:70 ethyl acetate/petroleum ether) to afford tert-butyl 2-(3-bromobenzoyl)-5H,6H,7H-pyrrolo[3,4-d]pyrimidine-6-carboxylate as a yellow solid (1.20 g, 44%). LCMS (ES, m/z): 404, 406 [M+H]+.
The Intermediate in Table 19 was synthesized according to the procedure described for Intermediate 33′-1 above.
To a solution of (3-[2-[2-(2,3-dihydro-1,4-benzodioxine-6-sulfonyl)-1H,2H,3H-pyrrolo[3,4-c]pyridin-6-yl]-2-phenylethyl]phenyl)methyl N-methylcarbamate (100 mg, 0.17 mmol) in methanol (2 mL) was added palladium carbon (10 mg, 10 wt % palladium on charcoal). Then hydrogen was introduced with hydrogen balloon. The resulting mixture was stirred for 16 h at room temperature. The reaction mixture was filtered and concentrated under vacuum. The resulting crude product was purified by Prep-HPLC (Column: XBridge Shield RP18 OBD Column, 5 μm, 19×150 mm; Mobile Phase, A: water (containing 10 mmol/L NH4HCO3) and B: CH3CN (5% to 30% over 25 min); Flow rate: 20 mL/min; Detector: UV 254 nm). The two enantiomers were further separated by Chiral-Pre-HPLC (Column: CHIRALPAK IG, 5 μm, 20×250 mm; Mobile Phase, A: methanol (containing 0.1% DEA) and B: DCM (hold 50% B over 10 min); Detector: UV 254/220 nm; Retention time: 1st eluting isomer, 3.965 min; 2nd eluting isomer, 5.955 min). The product fractions of 1st eluting isomer were concentrated and lyophilized to afford a white solid (10.1 mg, 26%). 1H-NMR (Methanol-d4, 400 MHz) δ (ppm): 8.40 (s, 1H), 7.36-7.16 (m, 7H), 7.17 (s, 1H), 6.99-6.92 (m, 1H), 4.60 (s, 2H), 4.54 (s, 2H), 4.35-4.33 (m, 1H), 4.26-4.22 (m, 4H), 3.53-3.50 (m, 1H), 3.19-3.14 (m, 1H), 2.44 (s, 3H). LCMS (ES, m/z) 452 [M+H]+. The product fractions of 2nd eluting isomer were concentrated and lyophilized to a white solid (11.5 mg, 30%). 1H-NMR (Methanol-d4, 400 MHz) δ (ppm): 8.40 (s, 1H), 7.36-7.16 (m, 7H), 7.17 (s, 1H), 6.99-6.92 (m, 1H), 4.61 (s, 2H), 4.56 (s, 2H), 4.42-4.39 (m, 1H), 4.26-4.22 (m, 4H), 3.66-3.59 (m, 1H), 3.19-3.14 (m, 1H), 2.55 (s, 3H). LCMS (ES, m/z) 452 [M+H]+.
To a solution of (3-bromophenyl)([2-[3-fluoro-4-(1,3-oxazol-2-yl)benzenesulfonyl]-1H,2H,3H-pyrrolo[3,4-c]pyridin-6-yl])methanol (50 mg, 0.09 mmol) in 1,4-dioxane (2 mL) was added K3PO4 (61 mg, 0.29 mmol), tert-butyl piperazine-1-carboxylate (88 mg, 0.47 mmol), RuPhos 3G (16 mg, 0.02 mmol), and RuPhos (9 mg, 0.02 mmol). The resulting mixture was stirred for 2 h at 100° C. and then cooled to room temperature. The reaction mixture was poured into water (3 mL) and then extracted with ethyl acetate (3×10 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting crude product was purified by prep-TLC (eluting with 1:1 ethyl acetate/petroleum ether) to afford tert-butyl 4-[3-[[2-[3-fluoro-4-(1,3-oxazol-2-yl)benzenesulfonyl]-1H,2H,3H-pyrrolo[3,4-c]pyridin-6-yl](hydroxy)methyl]phenyl}piperazine-1-carboxylate (40 mg, 67%). LCMS (ES, m/z) 636 [M+H]+.
To a solution of tert-butyl 4-[3-[[2-[3-fluoro-4-(1,3-oxazol-2-yl)benzenesulfonyl]-1H,2H,3H-pyrrolo[3,4-c]pyridin-6-yl](hydroxy)methyl]phenyl}piperazine-1-carboxylate (40 mg, 0.06 mmol) in dichloromethane (4 mL) was added TFA (1 mL). The resulting solution was stirred for 2 h at room temperature and concentrated under vacuum. The resulting mixture was basified to pH 8 with saturated potassium carbonate solution and then extracted with dichloromethane (2×5 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting crude product was purified by Prep-HPLC (Column: XBridge Shield RP18 OBD Column, 5 μm, 19×150 mm; Mobile Phase, A: water (containing 10 mmol/L NH4HCO3) and B: CH3CN (25% to 45% over 7 min); Flow rate: 20 mL/min; Detector: UV 254 nm). The product fractions were concentrated and lyophilized to afford [2-[3-fluoro-4-(1,3-oxazol-2-yl)benzenesulfonyl]-1H,2H,3H-pyrrolo[3,4-c]pyridin-6-yl][3-(piperazin-1-yl)phenyl]methanol (13.1 mg, 37%). 1H-NMR (DMSO-d6, 400 MHz) δ (ppm) 8.35 (d, J=16.8 Hz, 2H), 8.24-8.22 (m, 1H), 7.90-7.83 (m, 2H), 7.52 (s, 1H), 7.47 (s, 1H), 7.06-7.02 (m, 1H), 6.95 (s, 1H), 6.76-6.67 (m, 2H), 5.98 (d, J=4.0 Hz, 1H), 5.60 (d, J=4.0 Hz, 1H), 4.73-4.62 (m, 4H), 2.98-2.81 (m, 4H), 2.80-2.70 (m, 4H), 2.57-2.54 (m, 1H). LCMS (ES, m/z) 536 [M+H]+.
To a solution of 1-(3-[[2-(2,3-dihydro-1,4-benzodioxine-6-sulfonyl)-1H,2H,3H-pyrrolo[3,4-c]pyridin-6-yl]carbonyl]phenyl)piperazine (30 mg, 0.05 mmol) in THF (0.5 mL) was added NaBH4 (2 mg, 0.05 mmol) at 0° C. The resulting solution was stirred for 30 min at 0° C. The reaction mixture was poured into water (3 mL) and then extracted with DCM (3×3 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The resulting crude product was purified by silica gel chromatography (eluting with 1:10 MeOH/DCM), and further purified by Prep-HPLC (Column: XBridge Shield C18 OBD Column, 5 μm, 19×150 mm; Mobile Phase, A: water (containing 10 mmol/L NH4HCO3) and B: CH3CN (20% to 42% over 7 min); Flow rate: 20 mL/min; Detector: UV 254/220 nm). The product fractions were concentrated and lyophilized to afford [2-(2,3-dihydro-1,4-benzodioxine-6-sulfonyl)-1H,2H,3H-pyrrolo[3,4-c]pyridin-6-yl][3-(piperazin-1-yl)phenyl]methanol as a white solid (3.6 mg, 13%). 1H-NMR (Methanol-d4, 400 MHz) δ (ppm): 8.30 (s, 1H), 7.49 (s, 1H), 7.40-7.27 (m, 2H), 7.21-7.10 (m, 1H), 7.08-7.01 (m, 1H), 6.99-6.94 (m, 1H), 6.88-6.79 (m, 2H), 5.71 (s, 1H), 4.59 (s, 4H), 4.36-4.20 (m, 4H), 3.16-3.07 (m, 4H), 3.01-2.91 (m, 4H). LCMS (ES, m/z) 509 [M+H]+.
To a solution of phenyl([1H,2H,3H-pyrrolo[3,4-c]pyridine-6-yl])methanol (TFA salt) (100 mg, 0.29 mmol) in dichloromethane (10 mL) and N,N-dimethylformamide (2 mL) was added potassium carbonate (122 mg, 0.88 mmol). The resulting mixture was stirred for 30 min at room temperature. To this was added 4-(5-fluoro-1H-pyrazol-1-yl)benzene-1-sulfonyl chloride (77 mg, 0.30 mmol). The resulting solution was stirred for 1 h at room temperature. The reaction mixture was poured into water (3 mL) and then extracted with ethyl acetate (3×10 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting crude product was purified by prep-TLC (eluting with 99:1 ethyl acetate/petroleum ether), and further purified by prep-HPLC (Column: XBridge Shield RP18 OBD Column, 5 μm, 19×150 mm; Mobile Phase, A: water (containing 10 mmol/L NH4HCO3) and B: CH3CN (30% B to 62% B over 7 min); Flow rate: 20 mL/min; Detector: UV 254 nm). The product fractions were concentrated and lyophilized to afford [2-[4-(5-fluoro-1H-pyrazol-1-yl)benzenesulfonyl]-1H,2H,3H-pyrrolo[3,4-c]pyridine-6-yl](phenyl)methanol (13.6 mg, 10%). 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 8.33 (s, 1H), 8.07-7.98 (m, 2H), 7.88-7.85 (m, 2H), 7.74-7.73 (m, 1H), 7.49 (s, 1H), 7.31 (d, J=6.8 Hz, 2H), 7.24-7.21 (m, 2H), 7.17-7.14 (m, 1H). 6.30-6.29 (m, 1H), 6.08-6.06 (m, 1H), 5.64 (d, J=4.0 Hz, 1H), 4.69-4.57 (m, 4H). LCMS (ES, m/z) 451 [M+H]+.
To a solution of phenyl[2-(piperidine-4-sulfonyl)-1H,2H,3H-pyrrolo[3,4-c]pyridin-6-yl]methanol (40 mg, 0.11 mmol) in 1,4-dioxane (1 mL), was added 2-bromo-1,3-thiazole (18 mg, 0.11 mmol), Cs2CO3 (105 mg, 0.32 mmol) and RuPhos 3G (10 mg, 0.01 mmol). The resulting mixture was stirred for 16 h at 100° C. and then cooled to room temperature. The reaction mixture was poured into water (2 mL) and then extracted with ethyl acetate (2×5 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting crude product was purified by silica gel chromatography (eluting with 0:100 to 10:90 methanol/dichloromethane) and further purified by Prep-HPLC (Column: XBridge Shield C18 OBD Column, 5 μm, 19×150 mm; Mobile Phase, A: water (containing 10 mmol/L NH4HCO3) and B: CH3CN (30% to 65% over 7 min); Flow rate: 20 mL/min; Detector: UV 254/220 nm). The product fractions were concentrated and lyophilized to afford phenyl(2-[[1-(1,3-thiazol-2-yl)piperidin-4-yl]sulfonyl]-1H,2H,3H-pyrrolo[3,4-c]pyridin-6-yl)methanol as a white solid (2.0 mg, 4%). 1H-NMR (CDCl3, 400 MHz) δ (ppm): 8.51 (s, 1H), 7.47-7.35 (m, 4H), 7.32-7.31 (m, 1H), 7.19 (s, 1H), 7.10 (s, 1H), 6.62-6.60 (s, 1H), 5.79 (s, 1H), 4.98 (s, 1H), 4.87-4.83 (m, 2H), 4.79-4.69 (m, 2H), 4.19-4.16 (m, 2H), 3.29-3.22 (m, 1H), 3.07-3.01 (m, 2H), 2.23-2.20 (m, 2H), 2.06-1.96 (m, 2H). LCMS (ES, m/z) 457 [M+H]+
To a solution of [3-(azetidin-3-yloxy)phenyl]([2-[3-fluoro-4-(1,3-oxazol-2-yl)benzenesulfonyl]-1H,2H,3H-pyrrolo[3,4-c]pyridin-6-yl])methanol (TFA salt) (80 mg, 0.14 mmol) in MeOH (2 mL) was added formaldehyde (1 mL, 30% in water). The resulting solution was stirred for 30 min at room temperature. This was followed by the addition of STAB (92 mg, 0.41 mmol). The resulting solution was stirred for 12 h at room temperature. The reaction mixture was poured into water (10 mL) and then extracted with ethyl acetate (3×10 mL). The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting crude product was purified by Prep-HPLC (Column: XBridge Shield RP18 OBD Column, 5 μm, 30×150 mm; Mobile Phase, A: water (containing 10 mmol/L NH4HCO3) and B: CH3CN (25% to 50% in 7 min); Flow rate: 60 mL/min; Detector: UV 220 nm). The product fractions were concentrated under vacuum. The two enantiomers were further separated by Chiral Prep-HPLC (Column: CHIRALPAK IC, 5 μm, 20×250 mm; Mobile Phase, A: MeOH (containing 0.1% DEA) and B: DCM (keep 40% B in 15 min); Flow rate: 20 mL/min; Detector: UV 254/220 nm; Retention time: 1st eluting isomer, 10.772 min; 2nd eluting isomer, 13.314 min). The product fractions were concentrated and lyophilized to afford 1st eluting isomer as a white solid (12.4 mg, 16%). 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 8.38 (s, 1H), 8.34 (s, 1H), 8.24-8.20 (m, 1H), 7.90-7.83 (m, 2H), 7.53 (s, 1H), 7.47 (s, 1H), 7.14-7.10 (m, 1H), 6.88 (d, J=7.6 Hz, 1H), 6.81 (s, 1H), 6.60-6.57 (m, 1H), 6.10 (d, J=4.0 Hz, 1H), 5.63 (d, J=4.4 Hz, 1H), 4.70-4.63 (m, 5H), 3.71-3.68 (m, 2H), 2.92-2.89 (m, 2H), 2.27 (s, 3H). LCMS (ES, m/z): 537 [M+H]+. The product fractions were concentrated and lyophilized to afford 2nd eluting isomer as a white solid (13.3 mg, 18%). 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 8.38 (s, 1H), 8.34 (s, 1H), 8.24-8.20 (m, 1H), 7.90-7.83 (m, 2H), 7.53 (s, 1H), 7.47 (s, 1H), 7.14-7.10 (m, 1H), 6.88 (d, J=7.6 Hz, 1H), 6.81 (s, 1H), 6.60-6.58 (m, 1H), 6.10 (d, J=4.4 Hz, 1H), 5.63 (d, J=4.0 Hz, 1H), 4.69-4.65 (m, 5H), 3.74-3.71 (in 2H), 2.92-2.89 (m, 2H), 2.29 (s, 3H). LCMS (ES, m/z): 537 [M+H]+.
As set forth in Table 21, IC50 values are defined as follows: ≤25 μM and >10 μM (+); ≤10 μM and >1 μM (++); ≤1 μM and >0.1 μM (+++); ≤0.1 μM and >0.001 μM (++++); based upon the Biochemical Assay of Example A.
In Tables 11 and 21, absolute stereochemistry has not been determined for some Examples. Accordingly, assignment of any Examples as the “R” or “S” stereoisomer is arbitrary, unless otherwise noted. In some cases, Examples are labeled with “1st eluting isomer”, “2nd eluting isomer”, etc. based on the purification method used to separate the stereoisomers (see Table 20).
1H NMR
The assay was performed in a final volume of 6 μL assay buffer containing 20 mM Tris-HCl (pH 8.0, (1M Tris-HCl, pH 8.0 solution; Corning 46-031-CM)), L-Glutathione (GSH) reducing agent (1 mM, Sigma-Aldrich, G4251-100G), 0.03% Bovine Gamma Globulin (BGG) (0.22 μM filtered, Sigma, G7516-25G), and 0.01% Triton X-100 (Sigma, T9284-10L). DMSO solutions of the compounds in nanoliter quantities (10-point, 3-fold serial dilutions) were dispensed into 1536 assay plates (Corning, #3724BC) for final test concentrations of 25 μM to 1.3 nM, top to lowest dose, respectively. Concentration and incubation times were optimized for the maximal signal-to-background while maintaining initial velocity conditions at a fixed substrate concentration (<<Km). The final concentration of USP9X (Enzyme, E) was 0.025 nM, and the final concentration of Ubiquitin-Rhodamine 110 (Ub-Rh110, UbiQ-126) (Substrate, S) was 25 nM. To assay plates (pre-stamped with compound) was added 3 μL 2x Enzyme. The enzyme was preincubated for 30 minutes and then treated with 3 μL of 2x Substrate. Plates were incubated for 11 min (continuous kinetic read) at room temperature before the fluorescence was read on the Envision plate reader (Perkin Elmer) or PheraSTAR plate reader (BMG), with excitation at 485 nm and emission at 535 nm. The slope (best fit linear regression) of the five reads was used to normalize for inhibition. For all assays, data are reported as percent inhibition compared with control wells based on the following equation: % inh=100*((FLU−AveLow)/(AveHigh−AveLow)), wherein FLU is measured Fluorescence, AveLow is average Fluorescence of no enzyme control (n=64), and AveHigh is average Fluorescence of DMSO control (n=64). IC50 values are determined by curve fitting of the standard 4 parameter logistic fitting algorithm included in the Activity Base software package: IDBS XE Designer Model205. Data are fitted using the Levenburg Marquardt algorithm.
The present disclosure enables one of skill in the relevant art to make and use the inventions provided herein in accordance with multiple and varied embodiments. Various alterations, modifications, and improvements of the present disclosure that readily occur to those skilled in the art, including certain alterations, modifications, substitutions, and improvements are also part of this disclosure. Accordingly, the foregoing description and drawings are by way of example to illustrate the discoveries provided herein.
1. A method of treating cancer in a patient in need thereof, comprising administering to the patient a USP9X Inhibitor.
2. A method of treating cancer in a patient in need thereof, comprising administering to the patient an antineoplastic therapy consisting of the administration of a USP9X Inhibitor.
3. A method of treating cancer in a patient in need thereof, comprising administering to the patient a USP9X Inhibitor, wherein the patient is receiving or has received an immune checkpoint pathway inhibitor.
4. A method of treating cancer in a patient in need thereof, comprising administering to the patient an immune checkpoint pathway inhibitor, wherein the patient is receiving or has received a USP9X Inhibitor.
5. A method of treating cancer in a patient in need thereof, comprising administering to the patient an antineoplastic therapy consisting of the administration of a USP9X Inhibitor and the administration of an immune checkpoint pathway inhibitor.
6. A method of treating a patient diagnosed with a cancer, comprising administering a USP9X Inhibitor to the patient, wherein the patient is already being treated for the cancer with an immune checkpoint pathway inhibitor.
7. A method of treating a patient diagnosed with a cancer, comprising administering a USP9X Inhibitor to the patient, wherein the cancer has progressed while receiving an immune checkpoint pathway inhibitor.
8. A method of treating a patient diagnosed with a cancer, wherein the patient i) has been diagnosed with cancer that has progressed, or ii) has relapsed after previously being administered an immune checkpoint pathway inhibitor for the cancer.
9. A method of treating a patient diagnosed with a cancer, wherein the method comprises administering a USP9X Inhibitor to the patient while the patient continues to receive an immune checkpoint pathway inhibitor after being diagnosed with a cancer that is refractory to an immune checkpoint pathway inhibitor.
10. The method of any one of the preceding embodiments, wherein the cancer comprises a tumor that expresses PD-L1.
11. The method of any one of the preceding embodiments, wherein the cancer comprises a tumor that expresses PD-L1 and the PD-L1 can be detected using PD-L1 IHC 22C3 pharmDx.
12. The method of any one of embodiments 1-9, wherein the cancer comprises a tumor that expresses CTLA-4.
13. The method of any one of the preceding embodiments, wherein the cancer is selected from unresectable or metastatic melanoma, cutaneous melanoma, advanced renal cell carcinoma, microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer, metastatic squamous non-small cell lung cancer, head and neck squamous cell cancer, classical Hodgkin lymphoma, primary mediastinal large B-cell lymphoma, urothelial carcinoma, microsatellite instability-high cancer, gastric cancer, cervical cancer, hepatocellular carcinoma, or Merkel cell carcinoma.
14. The method of any one of the preceding embodiments, wherein the patient has received one or more prior lines of chemotherapy.
15. The method of any one of the preceding embodiments, wherein the patient has received two or more prior lines of chemotherapy.
16. The method of any one of the preceding embodiments, wherein the patient has received three or more prior lines of chemotherapy.
17. The method of any one of the preceding embodiments, wherein the patient has not responded to a prior line of chemotherapy.
18. The method of any one of the preceding embodiments, wherein the patient has relapsed after receiving a prior line of chemotherapy.
19. The method of any one of embodiments 14-18, wherein the prior line of chemotherapy is selected from platinum-based chemotherapy, fluoropyrimidine therapy, irinotecan therapy, paclitaxel therapy, nab-paclitaxel therapy, HER2/neu-targeted therapy, or sorafenib therapy.
20. The method of any one of the preceding embodiments, wherein the patient has not responded to prior therapy with an immune checkpoint pathway inhibitor.
21. The method of any one of the preceding embodiments, wherein the cancer is refractory or resistant to treatment with an immune checkpoint pathway inhibitor.
22. The method of any one of the preceding embodiments, wherein the cancer is unresectable or metastatic melanoma.
23. The method of any one of the preceding embodiments, wherein the cancer is cutaneous melanoma with pathologic involvement of regional lymph nodes.
24. The method of claim 23, wherein the patient has undergone complete resection and/or a total lymphadenectomy.
25. The method of any one of the preceding embodiments, wherein the cancer is previously untreated advanced renal cell carcinoma.
26. The method of any one of the preceding embodiments, wherein the cancer is microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer.
27. The method of claim 26, wherein the cancer has progressed following treatment with a fluoropyrimidine, oxaliplatin, and irinotecan.
28. The method of any one of the preceding embodiments, wherein the cancer is unresectable or metastatic melanoma that has progressed following treatment with ipilimumab.
29. The method of any one of the preceding embodiments, wherein the cancer is metastatic squamous non-small cell lung cancer.
30. The method of claim 29, wherein the cancer has progressed on or after platinum-based chemotherapy.
31. The method of any one of the preceding embodiments, wherein the cancer is melanoma with involvement of lymph node(s) following complete resection.
32. The method of any one of the preceding embodiments, wherein the cancer is metastatic nonsquamous non-small cell lung cancer with no EGFR or ALK genomic tumor aberrations.
33. The method of any one of the preceding embodiments, wherein the cancer is metastatic non-small cell lung cancer, wherein the cancer comprises a tumor with high PD-L1 expression with no EGFR or ALK genomic tumor aberrations.
34. The method of claim 33, wherein high PD-L1 expression is a Tumor Proportion Score (TPS)≥50% as determined by an FDA-approved test.
35. The method of any one of the preceding embodiments, wherein the cancer is metastatic non-small cell lung cancer, wherein the cancer comprises a tumor with PD-L1 expression of TPS≥1% as determined by an FDA-approved test, with disease progression on or after platinum-containing chemotherapy.
36. The method of any one of the preceding embodiments, wherein the cancer is recurrent or metastatic head and neck squamous cell cancer (HNSCC) with disease progression on or after platinum-containing chemotherapy.
37. The method of any one of the preceding embodiments, wherein the cancer is refractory classical Hodgkin lymphoma.
38. The method of any one of the preceding embodiments, wherein the cancer is refractory primary mediastinal large B-cell lymphoma.
39. The method of any one of the preceding embodiments, wherein the cancer is locally advanced or metastatic urothelial carcinoma, wherein the cancer comprises a tumor with PD-L1 expression of Combined Positive Score (CPS)≥10 as determined by an FDA-approved test.
40. The method of any one of the preceding embodiments, wherein the cancer is locally advanced or metastatic urothelial carcinoma, and wherein the patient is not eligible for platinum-containing chemotherapy.
41. The method of any one of the preceding embodiments, wherein the cancer is locally advanced or metastatic urothelial carcinoma.
42. The method of any one of the preceding embodiments, wherein the cancer is unresectable or metastatic, microsatellite instability-high (MSI-H) or mismatch repair deficient solid tumors that have progressed following prior treatment.
43. The method of any one of the preceding embodiments, wherein the cancer is locally advanced or metastatic gastric or gastroesophageal junction adenocarcinoma, wherein the cancer comprises a tumor with PD-L1 expression of Combined Positive Score (CPS)≥1 as determined by an FDA-approved test.
44. The method of claim 43, wherein the cancer has progressed on or after prior lines of therapy.
45. The method of any one of the preceding embodiments, wherein the cancer is recurrent or metastatic cervical cancer with disease progression on or after chemotherapy whose tumors express PD-L1 (CPS≥1) as determined by an FDA-approved test.
46. The method of any one of the preceding embodiments, wherein the cancer is hepatocellular carcinoma (HCC), and wherein the patient has previously been treated with sorafenib.
47. The method of any one of the preceding embodiments, wherein the cancer is recurrent locally advanced or metastatic Merkel cell carcinoma.
48. The method of any one of the preceding embodiments, wherein the immune checkpoint pathway inhibitor is selected from ipilimumab, nivolumab, or pembrolizumab.
49. The method of any one of the preceding embodiments, comprising administering two or more immune checkpoint pathway inhibitors.
50. The method of any one of the preceding embodiments, wherein the immune checkpoint pathway inhibitor is ipilimumab.
51. The method of any one of the preceding embodiments, wherein the patient is receiving or has received ipilimumab in a dose of any one of the following:
52. The method of any one of the preceding embodiments, comprising administering to the patient ipilimumab in a dose of any one of the following:
53. The method of any one of the preceding embodiments, wherein the immune checkpoint pathway inhibitor is nivolumab.
54. The method of any one of the preceding embodiments, wherein the patient is receiving or has received nivolumab in a dose of any one of the following:
55. The method of any one of the preceding embodiments, comprising administering to the patient nivolumab in a dose of any one of the following:
56. The method of any one of the preceding embodiments, wherein the immune checkpoint pathway inhibitor is ipilimumab and nivolumab.
57. The method of any one of the preceding embodiments, wherein the patient is receiving or has received the immune checkpoint pathway inhibitor in a dose of any one of the following:
58. The method of any one of the preceding embodiments, comprising administering to the patient the immune checkpoint pathway inhibitor in a dose of any one of the following:
59. The method of any one of the preceding embodiments, wherein the immune checkpoint pathway inhibitor is pembrolizumab.
60. The method of any one of the preceding embodiments, wherein the patient is receiving or has received pembrolizumab in a dose of any one of the following:
61. The method of any one of the preceding embodiments, comprising administering to the patient pembrolizumab in a dose of any one of the following:
62. The method of any one of the preceding embodiments, wherein the USP9X Inhibitor has an IC50 value of ≤10 μM in the Biochemical Assay of Example A.
63. The method of any one of the preceding embodiments, wherein the USP9X Inhibitor has an IC50 value of ≤2 μM in the Biochemical Assay of Example A.
64. The method of any one of the preceding embodiments, wherein the USP9X Inhibitor has an IC50 value of ≤1 μM in the Biochemical Assay of Example A.
65. The method of any one of the preceding embodiments, wherein the USP9X Inhibitor has an IC50 value of ≤0.2 μM in the Biochemical Assay of Example A.
66. The method of any one of the preceding embodiments, wherein the USP9X Inhibitor has an IC50 value of ≤0.1 μM in the Biochemical Assay of Example A.
67. The method of any one of the preceding embodiments, wherein the USP9X Inhibitor has an IC50 value of ≤0.05 μM in the Biochemical Assay of Example A.
68. The method of any one of the preceding embodiments, wherein the USP9X Inhibitor is a compound of Formula I:
or a pharmaceutically acceptable salt thereof, wherein:
69. The method of any one of the preceding embodiments, wherein the USP9X Inhibitor is a compound of Formula I-a:
or a pharmaceutically acceptable salt thereof
wherein Y2 is CH or N.
70. The method of any one of the preceding embodiments, wherein the USP9X Inhibitor is a compound of Formula I-b:
or a pharmaceutically acceptable salt thereof, wherein:
71. The method of any one of the preceding embodiments, wherein the USP9X Inhibitor is a compound Formula I-c:
or a pharmaceutically acceptable salt thereof.
72. The method of any one of the preceding embodiments, wherein the USP9X Inhibitor is a compound of Formula I-d:
or a pharmaceutically acceptable salt thereof.
73. The method of any one of the preceding embodiments, wherein the USP9X Inhibitor is a compound of Formula I-e:
or a pharmaceutically acceptable salt thereof.
74. The method of any one of the preceding embodiments, wherein the USP9X Inhibitor is a compound of Formula I-f:
or a pharmaceutically acceptable salt thereof.
75. The method of any one of the preceding embodiments, wherein the USP9X Inhibitor is a compound of Formula II:
or a pharmaceutically acceptable salt thereof, wherein:
76. The method of any one of the preceding embodiments, wherein the USP9X Inhibitor is a compound of Formula II-a:
or a pharmaceutically acceptable salt thereof.
77. The method of any one of the preceding embodiments, wherein the USP9X Inhibitor is a compound of Formula II-b:
or a pharmaceutically acceptable salt thereof.
78. The method of any one of the preceding embodiments, wherein the USP9X Inhibitor is a compound of Formula II-c:
or a pharmaceutically acceptable salt thereof.
79. The method of any one of the preceding embodiments, wherein the USP9X Inhibitor is a compound of Formula II-d:
or a pharmaceutically acceptable salt thereof.
80. The method of any one of the preceding embodiments, wherein the USP9X Inhibitor is a compound of Formula II-e:
or a pharmaceutically acceptable salt thereof.
81. The method of any one of the preceding embodiments, wherein the USP9X Inhibitor is a compound of Formula II-f:
or a pharmaceutically acceptable salt thereof.
82. The method of any one of the preceding embodiments, wherein the USP9X Inhibitor is selected from:
or a pharmaceutically acceptable salt thereof.
83. The method of any one of the preceding embodiments, wherein the USP9X Inhibitor is administered in a therapeutically effective amount.
This application claims priority to and the benefit of U.S. Provisional Application No. 62/733,595, filed Sep. 19, 2018; U.S. Provisional Application No. 62/784,981, filed Dec. 26, 2018; and U.S. Provisional Application No. 62/819,883, filed Mar. 18, 2019; the entire contents of each of which are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2019/051828 | 9/19/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62819883 | Mar 2019 | US | |
| 62784981 | Dec 2018 | US | |
| 62733595 | Sep 2018 | US |
