WO2011014239A1 - Dérivés substitués antiprolifératifs de pyrazolo[3,4-d] pyrimidines (spp) pour inhiber l'activation immunitaire, la réplication virale et la croissance tumorale - Google Patents

Dérivés substitués antiprolifératifs de pyrazolo[3,4-d] pyrimidines (spp) pour inhiber l'activation immunitaire, la réplication virale et la croissance tumorale Download PDF

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WO2011014239A1
WO2011014239A1 PCT/US2010/002091 US2010002091W WO2011014239A1 WO 2011014239 A1 WO2011014239 A1 WO 2011014239A1 US 2010002091 W US2010002091 W US 2010002091W WO 2011014239 A1 WO2011014239 A1 WO 2011014239A1
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cells
compounds
pyrazolo
activity
cell
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Franco Lori
Davide De Forni
Michael Ray Stevens
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Virostatics Srl
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems

Definitions

  • SPP Anti-proliferative substituted pyrazolo[3,4-d]pyrimidines derivatives
  • the present invention relates to the treatment of chronic viral and bacterial infections and/or cancer by the use of selected pyrazolo[3,4-d]pyrimidine derivatives with different kinds of substitutes on the pyrimidine ring and pyrazolo ring (SPP).
  • SPP pyrazolo[3,4-d]pyrimidine derivatives with different kinds of substitutes on the pyrimidine ring and pyrazolo ring
  • Pyrazolo[3,4-d]pyrimidine derivatives are a class of compounds generally known to include members that might be useful as anticancer drugs. Some of them have been tested for AbI and Src tyrosine kinase inhibition. Some were active against both, some only against AbI and some only against Src. Si 121 was not tested (Carraro, F., et al., Pyrazolo[3,4-d]pyrimidines as potent antiproliferative and proapoptotic agents toward A431 and 8701 -BC cells in culture via inhibition of c-Src phosphorylation.
  • USPN 5,981 ,533 issued to Traxler et al. Nov. 9, 1999 discloses 4-amino-l H-pyrazolo[3,4- djpyrimidine derivatives and intermediates for their manufacture.
  • the compounds are said to inhibit especially the tyrosine kinase activity of the receptor for epidermal growth factor and can be used, for example, in the case of epidermal hyperproliferation (psoriasis) and as anti-tumor agents.
  • WO/2008/079460 discloses the use of kinase inhibitors for the treatment of acute pathogenic infections, including among others HIV infection.
  • These initiators include inhibitors of tyrosine kinase, preferably, Abelson (AbI) and/or Src-family tyrosine kinases, or pharmaceutically acceptable salts, enantiomers, analogs, esters, amides, prodrugs, metabolites, or derivatives thereof.
  • tyrosine kinase preferably, Abelson (AbI) and/or Src-family tyrosine kinases, or pharmaceutically acceptable salts, enantiomers, analogs, esters, amides, prodrugs, metabolites, or derivatives thereof.
  • AbI Abelson
  • Src-family tyrosine kinases or pharmaceutically acceptable salts, enantiomers, analogs, esters, amides, prodrugs, metabolites, or derivatives thereof.
  • WO 2006044968 re "COMBINATION THERAPY FOR TREATING VIRAL INFECTIONS” discloses a method of treating viral infections, particularly Hepatitis B (HBV) and Human Immunodeficiency Virus (HIV) infections, by administering elvucitabine and a second active agent to a patient suffering viral infection.
  • the second active agent is, for example, an immunomodulatory compound, an anti-viral agent, or a combination comprising one or more of the foregoing active agents.
  • the anti-viral agent may be a tyrosine kinase inhibitor, a CCR5 inhibitor, a non- nucleoside reverse transcriptase inhibitor, a protease inhibitor, an integrase inhibitor.
  • Tyrosine kinase inhibitors have been found to have antiviral properties and may be used as the second active agent.
  • Tyrosine kinases inhibitors included in this invention were Imatinib mesylate (GLEEVEC® or GLIVEC®, Novartis), Gefitinib (IRESSA®, Astra Zeneca), and erlotinib (TARCEVA®, OSI Pharmaceuticals). Several other tyrosine kinase inhibitors are mentioned.
  • AMN107 (TasignaTM from Novartis Pharma AG) which is now approved for use in the USA and the EU for drug-resistant chronic myelogenous leukemia (CML) and sunitinib malate (also known as SUl 1248, brand name SUTENT®, from Pfizer) which is now being marketed for the treatment of gastrointestinal and advanced renal cell carcinoma.
  • CML chronic myelogenous leukemia
  • sunitinib malate also known as SUl 1248, brand name SUTENT®, from Pfizer
  • Combinations of elvucitabine and sunitinib malate and optionally AMN107 are said to be within the scope of the invention.
  • the above invention does not disclose SPP.
  • WO2005117885A1 discloses the use of imanitib for treating hepatitis C, influenza virus, rhinovirus, etc.
  • the above invention does not disclose the SPPs.
  • Other recently approved TKIs include NEXAVAR (sorafenib tosylate), which is marketed by Bayer and Bayer Schering Pharma for both liver and renal cell carcinoma, and TYKERB (lapatinib ditosylate), marketed by GSK for breast cancer.
  • SPPs 4-substituted derivatives of pyrazolo[3,4-d]pyrimidine
  • R SC2H5, or an alkylthio group
  • Rl NHC4H9, NHCH2CH2C6H5, NHCH2C6H5, NHC6H4-mCl, NHCH2C6H4-pF, NHC3H7, NHCH2CH2C6H4-mCl, 4-MORPHOLINYL, NHCH2C6H4-oCl
  • R2 CH2CHC1C6H5 , CH2CHClC6H4-pF, CH2CHClC6H4-pCl
  • Pyrazolo[3,4-d]pyrimidine derivatives with a 2-chloro-2-phenylethyl or a 2-chloro-2-(4- fluoro-phenyl)ethyl or a 2-chloro-2-(4-chloro-phenyl)ethyl side chain at Nl are particularly preferred to inhibit cell proliferation and/or viral replication.
  • Pyrazolo[3,4-d]pyrimidine derivatives with a SC2H5 or alkylthio side chain at C6 are particularly preferred to inhibit cell proliferation and/or viral replication
  • An advantage of these compounds is that they have multiple utilities, that is, as a single compound they are antiviral, or have the ability to limit viral replication, they are anti-proliferative, that is, they limit T cell hyperproliferation, and yet they have low cellular toxicity, that is, they are NOT pro-apoptotic.
  • AV-HALTS Compounds that are particularly preferred as AV-HALTS are designated herein as VSl -002,
  • SPPs 4-substituted derivatives of pyrazolo[3,4-d]pyrimidine
  • R SC2H5, SCH3, H, N(CH3)2
  • Rl NHC4H9, NHCH2CH2C6H5, NHCH2C6H5, NHC6H4-mCl, NHCH2C6H4-pF, NHC3H7, NHCH2CH2C6H4-mCl, 4-MORPHOLINYL, N(C2H5)2, NH(CH2)2OC2H5, 1-
  • R2 CH2CHC1C6H5, CH2CHClC6H4-pF, CH2CHClC6H4-pCl,
  • Such compounds can be useful for treatment of tumors and leukemia, particularly if they also tend to induce apoptosis.
  • SPPs 4-substituted derivatives of pyrazolo[3,4-d]pyrimidine
  • R H, SCH3, NH(CH2)2OH
  • Rl 1 -HEXAHYDROAZEPINYL, NHCH2CH2C6H5, 4-MORPHOLINYL, NHCYCLOHEXYL, NHCH2C6H5, NHC6H4-mCl
  • R2 CHCHC6H5
  • SPPs 4-substituted derivatives of pyrazolo[3,4-d]pyrimidine
  • R SC2H5, SCH3, H, SCH2CH2-4-MORPHOLINYL
  • Rl NHCH2CH2C6H5, NHCH2C6H5, NHC6H4-mCl, 1 -HEXAHYDROAZEPINYL, NHC3H7, 4- MORPHOLINYL, NHCH2C6H4-pCl
  • these compounds In addition to being effective for limiting viral replication and/or limiting T cell replication, these compounds also induce apoptosis, and so are particularly useful for treating tumors and leukemia as opposed to a chronic viral infection such as HIV infection.
  • Compounds particularly preferred for cancer treatment have the following designations in this application: VSl-OlO, VSl-OI l , VS1-020, VS1 -022, VS1-023, VS1-030, VS1-032, VS1-033, VSl- 034, VS1-035.
  • the advantages of the present inventions can be obtained by administering an effective amount of a compound having the desired properties to cells in order to obtain the desired results.
  • the compounds may be formulated in a pharmaceutical composition comprising the compound, or a physiologically acceptable salt thereof, having the desired effect, and one or more suitable excipients. Suitable excipients are well known in the art.
  • Fig. 1 shows the chemical structures for different compounds that could be candidates for use as antiviral, anticancer or antiviral-hyperactivation limiting therapeutics (AV-HALTS). .
  • VSl -005 or l-styryl-6-methylthio-N-(2-phenylethyl)-lH-pyrazolo[3,4-d]pyrimidin-4-amine VSl-010 or N-benzyl-l-(2-chloro-2-phenylethyl)-lH-pyrazolo[3,4-d]pyrimidin-4-amine; VSl-01 1 or N-(3-chlorophenyl)-l-(2-chloro-2-phenylethyl)-6-(methylthio)-lH-pyrazolo [3,4- d]pyrimidin-4-amine;
  • Figs. 2 is a single table of experimental results showing relative (lack of) toxicity (expressed as viability of cells compared to untreated control), anti-proliferative activity on primary blood cells
  • HIV activity in activated and quiescent cells HIV replication is expressed as the percentage amount of p24 production compared to untreated control), and (lack of) apoptotic effects (percentage of apoptotic effects compared to untreated control), if any, for 36 compounds compared to HU.
  • "+" indicates a parameter that is better than HU
  • "-" indicates a parameter that is worse compared to HU.
  • Fig. 3 is a set of "Spider diagrams" for seven different compounds, that is, a way of visualizing the way a drug meets a group of five desired parameters (lack of toxicity, lack of apoptotic effect, anti- proliferative capacity and antiviral effect in activated and quiescent cells).
  • a "perfect” drug would meet 100% of all parameters, and in this case, would make a perfect pentagon at 100% (outer dotted line of the graph).
  • the combination of HU and ddl is used as a comparator.
  • Fig. 4 shows the relationships between substituents of the SPP and activity towards cell proliferation and viral replication. "+++" indicates excellent activity, "++" good activity and "+” poor activity.
  • Fig. 5 shows an example from the model used to find the relationships between the substituents and the activity of the SPPs.
  • Fig. 6 shows the effect of a specific compound, VSI-002, on the cell cycle.
  • a protein kinase is an enzyme that modifies other proteins chemically. They covalently attach a phosphate group to the side chain of tyrosine, serine, or threonine residues found on proteins.
  • the human genome contains about 500 protein kinase genes.
  • the family of enzymes known as protein tyrosine kinases (PTKs) act on tyrosine, and provide an essential role in the normal regulation of cell growth.
  • Various protein tyrosine kinases (TK) catalyze the phosphorylation of specific tyrosyl residues in various proteins involved in the regulation of cell growth and differentiation. Phosphorylation often results in a functional change to the target protein, or substrate.
  • PTKs are involved in the transduction and processing of extracellular and intracellular signals. They play a critical role in regulating normal cell growth and differentiation, and if they malfunction, they are also known to play a role in oncogenesis. That is, inappropriate or uncontrolled activation of many of these kinases, i.e., aberrant protein tyrosine kinase activity, for example by over-expression or mutation, has been shown to result in uncontrolled cell growth.
  • United States Patent No. 7,507,741 discloses that protein kinases have been implicated as targets in central nervous system disorders such as Alzheimer's, pain sensation, inflammatory disorders such as arthritis, bone diseases such as osteoporosis, cancer, atherosclerosis, thrombosis, metabolic disorders such as diabetes, blood vessel proliferative disorders such as angiogenesis, restenosis, autoimmune diseases and transplant rejection and infectious diseases such as viral and fungal infections.
  • PTKs Aberrant activity of PTKs has been implicated in human malignancies, such as non-small cell lung, bladder and head and neck cancers, breast, ovarian, gastric and pancreatic cancers.
  • PTK activity has also been implicated in a variety of other disorders: psoriasis, fibrosis, atherosclerosis, restenosis, autoimmune disease, allergy, asthma, transplantation rejection inflammation, thrombosis, bronchitis, and nervous system diseases.
  • Specific tyrosine kinases have been implicated in disease conditions in which T cells are hyperactive e.g., rheumatoid arthritis, autoimmune disease, allergy, asthma, and graft rejection.
  • the process of angiogenesis has been associated with a number of disease states (e.g., tumorogenesis, psoriasis, rheumatoid arthritis) and this has been shown to be controlled through the action of a number of receptor PTKs.
  • PTKs There exist two classes of PTKs: the receptor tyrosine kinases and the cytoplasmic or receptor- associated tyrosine kinases.
  • the former have a transmembrane receptor with a tyrosine kinase domain protruding into the cytoplasm.
  • the transmembrane receptor is an extracellular ligand binding domain and the tyrosine kinase domain is an intracellular catalytic domain with intrinsic tyrosine kinase activity.
  • Receptor tyrosine kinases play an important role in regulating cell division, cellular differentiation, and morphogenesis (development of shape, typically of embryos.)
  • Receptor-associated tyrosine kinases are tyrosine kinases recruited from the cytoplasm to a receptor following hormone binding, and are involved in a number of signaling cascades, principally those involved in cytokine signaling (but also others, including growth hormone).
  • One such receptor- associated tyrosine kinase is Janus kinase (JAK), many of whose effects are mediated by STAT proteins.
  • Receptor-associated tyrosine kinases transmit signals from the membrane through the interaction with the cytoplasmic domain of membrane proteins.
  • Activated Tyrosine kinases produce a variety of downstream effects that ultimately result in changes in gene expression.
  • cytoplasmic PTKs particularly of c-Src tyrosine kinases
  • they are active in several tumors, including breast, bone, colon, lung, pancreatic, ovarian, head and neck, bladder, neuronal cancers as well as in chronic myelogenous leukemia and multiple myeloma.
  • AbI tyrosine kinase is another cytoplasmic PTK. It is involved in the development of chronic myeloid leukemia (CML).
  • TKIs tyrosine kinase inhibitors
  • dasatinib and Imatinib for the treatment of CML
  • gefitinib and erlotinib for the treatment of non-small-cell lung cancer.
  • Many other TKIs are in development in clinical trials and they are a growing class of anticancer agents.
  • SPPs have been tested in vitro as dual Src/Abl inhibitors in several tumor cell lines. These SPPs have different substituents on the pyrimidine and pyrazolo rings. Such compounds possess the ability to interfere with the phosphorylation activity of both Src and AbI and, consequently, to show anti-cancer activity in cell lines (Carraro, F., et al., Pyrazolo[3,4-d]pyrimidines as potent anti-proliferative and proapoptotic agents toward A431 and 8701 -BC cells in culture via inhibition of c-Src phosphorylation. J Med Chem 2006, 49, 1549-1561).
  • SPPs The effects of the experimental SPPs were assessed on leukemia cell lines in order to evaluate their activity on proliferation and apoptosis, or programmed cell death.
  • SPPs showed a significant anti-proliferative activity on K-562 cells, with an IC50 spanning from 19 to 176 micromolar.
  • the compounds were also tested in MEG-01 cells with activity values similar to those obtained with the other cell lines.
  • the cell cycle or cell-division cycle, is the series of events that take place in a cell leading to its division and duplication (proliferation). In cells without a nucleus (prokaryotes), the cell cycle occurs via a process termed binary fission.
  • the cell cycle can be divided in two brief periods: interphase— during which the cell grows, accumulating nutrients needed for mitosis and duplicating its DNA— and the mitosis (M) phase, during which the cell splits itself into two distinct cells, often called "daughter cells.”
  • the cell-division cycle is a vital process by which a single-celled fertilized egg develops into a mature organism, as well as the process by which hair, skin, blood cells, and some internal organs are renewed.
  • the cell cycle consists of four distinct phases: Gl phase, S phase (synthesis), G2 phase
  • M phase is itself composed of two tightly coupled processes: mitosis, in which the cell's chromosomes are divided between the two daughter cells, and cytokinesis, in which the cell's cytoplasm divides forming distinct cells. Activation of each phase is dependent on the proper progression and completion of the previous one. Cells that have temporarily or reversibly stopped dividing are said to have entered a state of quiescence called GO phase.
  • each of the daughter cells After cell division, each of the daughter cells begins the interphase of a new cycle. Although the various stages of interphase are not usually morphologically distinguishable, each phase of the cell cycle has a distinct set of specialized biochemical processes that prepare the cell for initiation of cell division.
  • Nonproliferative cells in multicellular eukaryotes generally enter the quiescent GO state and may remain quiescent for long periods of time, possibly indefinitely (as is often the case for neurons). This is very common for cells that are fully differentiated.
  • Cellular senescence is a state that occurs in response to DNA damage or degradation that would make a cell's progeny nonviable; it is often a biochemical alternative to the self-destruction of such a damaged cell by apoptosis.
  • the first phase within interphase, from the end of the previous M phase until the beginning of DNA synthesis is called Gl (G indicating gap).
  • Gl G indicating gap
  • This phase is marked by synthesis of various enzymes that are required in S phase, mainly those needed for DNA replication. Duration of Gl is highly variable, even among different cells of the same species.
  • the ensuing S phase starts when DNA synthesis commences; when it is complete, all of the chromosomes have been replicated, i.e., each chromosome has two (sister) chromatids.
  • the cell then enters the G2 phase, which lasts until the cell enters mitosis. Again, significant protein synthesis occurs during this phase, mainly involving the production of microtubules, which are required during the process of mitosis. Inhibition of protein synthesis during G2 phase prevents the cell from undergoing mitosis.
  • M phase consists of nuclear division (karyokinesis) and cytoplasmic division (cytokinesis).
  • cytokinesis nuclear division
  • cytokinesis cytoplasmic division
  • the M phase has been broken down into several distinct phases, sequentially known as prophase, prometaphase, metaphase, anaphase, and telophase leading to cytokinesis.
  • Regulation of the cell cycle involves processes crucial to the survival of a cell, including the detection and repair of genetic damage as well as the prevention of uncontrolled cell division.
  • the molecular events that control the cell cycle are ordered and directional; that is, each process occurs in a sequential fashion and it is impossible to "reverse" the cycle.
  • T cells are immune system cells that can develop the capacity to kill infected or neoplastic cells. When T cells are contacted by antigens they become activated, or sensitized, and proliferate, that is, appear in greater numbers. This is a normal physiological process, which is useful to protect the host from the "sick cells" (tumor cells and infected cells).
  • T cell hyperactivation or -hyperproliferation can contribute to disease progression and is considered a key pathogenetic factor in several chronic diseases such as cancer, and prolonged infectious diseases including HIV/ AIDS infection.
  • HIV infects T cells and depends on actively dividing or proliferating cells to serve as a means of replication.
  • the infected cells produce HIV particles, which is an antigen.
  • Antigenic stimulation by HIV particles in turn, sustains further T cell activation or proliferation, as mentioned before.
  • GALT gut-associated lymphoid tissues
  • T cell activation/proliferation Brenchley, J.M., et al., Microbial translocation is a cause of systemic immune activation in chronic HIV infection. Nat Med, 2006. 12(12): p. 1365-71). This chronic cycle of events, over time, exhausts the immune system. Therefore, limiting cell T cell hyperactivation and hyperproliferation will suppress HIV replication, limit the loss of functional CD4 T helper cells and slow disease progression.
  • HU hydroxyurea
  • Imatinib tyrosine kinase inhibitor
  • HU Hydroxyurea
  • HU Hydroxyurea
  • HU is an antiproliferative agent indicated to treat different neoplastic as well as non-neoplastic diseases such as sickle cell anemia and psoriasis.
  • HU has been used for the treatment of HIV infected individuals, especially in combination with antiretroviral drugs, such as ddl.
  • HU inhibits the cellular enzyme ribonucleotide reductase, thus blocking the transformation of ribonucleotides into deoxyribonucleotides, depleting the intracellular deoxynucleotide triphosphate
  • HU strongly inhibits viral deoxyribonucleic acid (DNA) synthesis and has synergistic anti-HIV activity when combined with nucleoside reverse transcriptase inhibitors (NRTIs). Clinical trials have shown that HU-containing regimens effective in patients with varying degrees of treatment experience at different stages of infection.
  • HU can suppress virus replication by slowing down the rate of T cell proliferation (because
  • the present invention discloses such novel antiviral/anti-proliferative compounds (AV-HALTS) from the family of SPPs designated VS1-002, VS1-003, VS1-004, VS1-005, VSl-OlO, VSl-011, VS1-029, VS1-031, VS1-036 that meet all the following characteristics.
  • AV-HALTS novel antiviral/anti-proliferative compounds
  • HU at lOO ⁇ M concentration was incorporated in all experimental studies as a comparator ( Figure 2). This concentration was not toxic, that is, the viability of the cells treated with this compound at this concentration was above 40 percent.
  • HU 100 ⁇ M did substantially decrease cell proliferation but, HU alone was not effective in inhibiting HIV replication in activated T cells.
  • potent antiviral activity of HU has been demonstrated on quiescent cells, which are one of the major reservoirs of HIV.
  • HU did not substantially affect the percentage of total apoptotic events, a feature that is useful in antiviral compounds.
  • many known potent anticancer compounds operate by inhibiting cell proliferation and inducing apoptosis.
  • VSl-001 did not have toxicity, was less effective than HU in decreasing cell proliferation; however, more importantly, it was a very effective inhibitor of HIV replication in both activated and quiescent T cells and similarly to HU, did not induce apoptosis. This compound is best characterized as an antiviral drug.”
  • VS 1-002 10 ⁇ M was not toxic, had an anti-proliferative effect comparable to that of 100 ⁇ M HU and superior to that of Imatinib.
  • This compound has anti-proliferative activity and antiviral activity in both quiescent and in hyperactivated cells. Differently from 100 ⁇ MHU, even at 10 ⁇ M it inhibited HIV replication in activated T cells. The compound inhibited the production of HIV-I indirectly by reducing cellular proliferation (the majority of HIV-I replication occurs in actively dividing cells) and also had a direct anti-HIV activity. It was not proapoptotic.
  • This compound is an antiviral drug with anti-proliferative capacity, and we define it as an AV-HALT (AntiViral- Hyperactivation Limiting Therapeutic) drug.
  • VS 1-003 was about as effective as VS 1-002 in inhibiting HIV, with anti-proliferative capacity somewhat less than that of VS 1-002. It did not induce apoptosis. This compound is an AV-
  • VS 1-004 10 ⁇ M was not toxic, had good antiviral potency in both activated and quiescent cells, was as anti-proliferative as 100 ⁇ M HU, and only induced a small apoptotic effect. This compound is classified as an AV-HALT.
  • VS1-005 was not toxic and had anti-proliferative activity but slightly lower compared to HU.
  • VS 1-006 was not effective either against proliferation or HIV in activated T cells.
  • VSl- 007 at a concentration of 10 ⁇ M was the weakest compound tested to date. It showed poor performance against cell proliferation and it had no effect on HIV replication in activated T cells.
  • VS 1-008 had no toxicity, showed good activity against proliferation (even better than HU though 10 times less concentrated) and HIV in both activated and quiescent cells. It did not induce apoptosis.
  • This compound is an AV-HALT.
  • VS1-009 had very little activity with a profile similar to VS1-006 and VS1-007.
  • VSl-010 and VSl-Ol 1 10 ⁇ M had acceptable toxicity and both had increased anti-proliferative capacity compared to 100 ⁇ M HU and were active as antivirals in both activated and quiescent cells. They induced apoptosis.
  • the combination of anti-proliferative, antiviral, and apoptosis-inducing qualities suggests that these compounds will be potent anticancer drugs.
  • VS 1-012 through VS 1-016 showed poor or no anti-proliferative activity. Nonetheless, VSl- 012, VS 1-015 and VS 1-016 showed better anti-HIV activity than HU. These compounds are characterized as antiviral drugs.
  • VS1-017, VS1-018, VS1-019, VS1-021 , and VS1-025 had poor activity both versus proliferation and HIV.
  • VS1-020 and VS1-022 had no toxicity, were as anti-proliferative or even better than HU. They were effective against HIV either in both quiescent and activated T cells. They were both proapoptotic and can be suggested as anticancer drugs.
  • VS1-023 was not toxic, showed anti-proliferative and antiviral activity similar to HU though it was 10 times less concentrated. It was not proapoptotic. Because it is similar to HU in our system but less toxic, it could be suitable to replace HU as an anti-hyperproliferation and anticancer drug.
  • VS 1-024 and VS 1-026 showed a good toxicity profile, were equal to or a little less antiproliferative than HU and slightly less potent against HIV. They were not proapoptotic. These compounds are AV-HALTs.
  • VS 1-027, VS 1-028 and VS 1-029 10 ⁇ M showed a good toxicity profile, good anti- proliferative capacity (superior to 100 ⁇ M HU) and good anti-HIV activity in both activated and quiescent cells. They were no more apoptotic than HU. These compounds are AV-HALTs.
  • VS1-030 had a good toxicity profile, at 10 ⁇ M it was greater than 100 ⁇ M HU and 10 ⁇ M Imatinib in inhibiting cell proliferation, and it was proapoptotic. This is a potentially potent anticancer drug.
  • VS1-032, VS1-033 and VS1-034 had anti-proliferative activity comparable or even greater than HU. They could be anticancer drugs.
  • VS1-035 had good anti-proliferative activity, similar to HU, and proapoptotic activity. The toxicity profile was good. This compound is a potential anticancer drug.
  • VS1-031 and VS1-036 were not toxic, at 10 ⁇ M had anti-proliferative capacity greater or equal to that of 100 ⁇ M HU, were effective against HIV in activated and quiescent cells. They did not induce apoptosis. These compounds are AV-HALTs.
  • Results are shown graphically in Fig. 3 for seven representative compounds.
  • Each spider graph shows the results for five parameters measured for the compound (10 ⁇ M), in comparison with the combination of HU (100 ⁇ M) + ddl (2 ⁇ M).
  • a "perfect" drug would meet 100% of all parameters, and would make a perfect pentagon at 100% (outer dotted line of the graph).
  • SPPs are represented with a solid line
  • HU+ddl combination is represented with a dashed line.
  • VSl -002 at 10 ⁇ M has the same activity of HU 100 ⁇ M+ddl 2 ⁇ M combination, 20% less toxicity and more than 20% less proapoptotic effect.
  • VS1-003 has an activity profile similar to HU+ddl (it is slightly less potent against proliferation and HIV in activated cells), with less toxicity and no effect on apoptosis.
  • VS 1-004 is as effective as HU+ddl in inhibiting cell proliferation and HIV, with the same level of toxicity and apoptosis. These three compounds show a graph comparable to that of HU+ddl and can be classified as AV-HALTs.
  • VS 1-005 has the same toxicity of HU+ddl, no effect on apoptosis, has the same antiviral power but lacks anti-proliferative capacity (50% less than HU+ddl). This compound has a profile of a purely antiviral drug.
  • VSl-OlO has a good activity against HIV, is 20% more anti-proliferative than HU+ddl, it is more toxic and more proapoptotic compared to the two drug combination.
  • VSl-OI l has activity comparable to that of HU+ddl but it is extremely proapoptotic, as shown graphically by the fact that the point on the "lack of apoptotic effect" arm is on the negative scale. These two compounds have a profile of anticancer drugs. VS1-029 is again similar to HU+ddl, even more anti-proliferative (about 20% more), and is another AV-HALT candidate.
  • PBMC peripheral blood mononuclear cells
  • IL-2 was then added (20 U/ml), cells were plated (10 5 cells per well in 200 ml) and drug exposure was begun.
  • Different concentrations of the compounds were tested in triplicate with HU employed as a comparator. After 7 days of incubation at 37 0 C, cells were stained with Trypan Blue and counted using a hemocytometer. The number of dead and living cells was counted at the microscope. The percentage of living cells over the total number of cells was calculated. Viability was expressed as percentage of live cells compared to the untreated control, indicated as 100%.
  • Viability of the cells was measured after treatment with SPPs for seven consecutive days. Viability was expressed as a percentage of the not treated control (NT), indicated as 100%.
  • Example 2 Study of the effects of the SPPs on cell hyperproliferation and on apoptosis in primary T cells.
  • Unstimulated (quiescent/resting, day 0 to 5) primary human CD4 T-lymphocytes were obtained by magnetic bead separation from healthy, normal donors. After five days in culture, cells were activated with PHA (5 ⁇ g/ml) to induce hyperproliferation then, at day 7, IL-2 (20 U/ml) was added (stimulated, day 6 to 10). All available compounds were tested at a concentration of 10 ⁇ M. HU 100 ⁇ M and Imatinib 10 ⁇ M were also employed as comparators and the negative control was left untreated. At day 10, cells were harvested.
  • M carboxyfluorescein diacetate-succinimidyl ester
  • T cells were cultured as described above and at day 10 cells were stained with Annexin V and 7-AAD, in order to determine the percentage of total apoptotic events. Effects of SPPs on cellular hyperproliferation and apoptosis
  • HU 100 ⁇ M strongly decreased cell proliferation (by 74% compared to not treated control). As expected HU + ddl combination had the same anti-proliferative capacity. Imatinib was also effective in reducing cell proliferation (by 62% compared to not treated control). We therefore feel confident that our model is suitable to study the activity of compounds used as anticancer (anti-leukemia) agents. All the SPPs having a 2-chloro-2-phenylethyl side chain at Nl showed anti-proliferative activity. VSl - 002 10 ⁇ M and VS1-004 were as effective as 100 ⁇ M HU in inhibiting cell proliferation and more effective than the same concentration of Imatinib.
  • VS 1-003 had a little inferior anti-proliferative capacity compared to HU, more similar to that of Imatinib. VS1-005, not bearing a 2-chloro-2- phenylethyl side chain at Nl, only reduced proliferation by 27%.
  • VSl-010, VSl-01 1 and VS1-029 were even more potent than HU since they inhibited cell proliferation by 99%, 100% and 95%, respectively, compared to an untreated control, though 10 times less concentrated.
  • HU 100 ⁇ M and Imatinib 10 ⁇ M produced no significant increases in the percentage of apoptotic events, even though it is generally viewed as favorable for a cancer drug to induce apoptosis.
  • VS 1-002, VS 1-003 and VS 1-005 did not affect the percentage of apoptotic events compared to the control.
  • VS1-004, VSl-010 and VS1-029 produced a slight increase in the percentage of apoptotic events, similar to the combination of HU 100 ⁇ M and ddl 2 ⁇ M.
  • VSl-Ol 1 was strongly proapoptotic (+182% apoptotic events), compared to the control.
  • VS1-018, VS1-020, VS1-022, VS1-030 and VSl- 035 were also proapoptotic.
  • Anti-proliferative compounds, which do not induce apoptosis are likely antiviral drugs and could be also used for the treatment of cancer, just as HU or Imatinib.
  • more potent cancer drugs are both anti-proliferative and proapoptotic, such as VS1-030 and VS1-035.
  • Example 3 Study of the antiviral activity of the SPPs on HIV-I replication in quiescent cells
  • HU 100 ⁇ M strongly affected HIV replication (13.0 ⁇ 19.4 % HIV p24 compared to untreated control).
  • HU 100 ⁇ M + ddl 2 ⁇ M had increased antiviral potency.
  • All the SPPs having a 2-chloro-2- phenylethyl side chain at Nl (R2 position) showed antiviral activity.
  • 10 ⁇ M of VS1-002, VS1-003, VS 1-004, VSl-010 and VS 1-029 were as well strong HIV inhibitors as they reduced HIV-I p24 production by 90%, 95%, 93%, 89% and 94% compared to untreated control, respectively.
  • VSl-011 was a little less potent (80% inhibition of HIV replication).
  • VS1-005, bearing a styryl group at Nl also showed a 97% inhibition of HIV replication.
  • Example 4 Study of the antiviral activity of the SPPs on HIV-I replication in activated cells
  • HU has little, if any anti-HIV activity in activated T cells because it is active against subunit R2 of ribonucleotide reductase, which is over-expressed upon cell cycle progression from Gl to S phase.
  • a good candidate compound should also be active in activated T cells.
  • the experimental setup described below allows us to detect direct antiviral activity of a compound. If a compound is not antiproliferative but it inhibits HIV in activated T cells, the compound has a direct anti-HIV activity.
  • Human PBMC obtained from healthy, normal donors, were stimulated for two days with PHA 5 ⁇ g/ml. Then cells were infected with HIV-I NL 4.3, IL-2 was added (20 U/ml), cells were plated (10 5 cells per well in 200 ⁇ l) and drug treatment started. 100 ⁇ M HU (comparator), 10 ⁇ M of Imatinib and of each SPP were tested in triplicates. The control was left untreated. After 7 days of incubation at 37°C supernatants were harvested and HIV-I p24 Ag was measured by ELISA.
  • VS1-002 and VS1-004 were strongly effective against HIV replication as % p24 was 13.6 ⁇ 13.0 and 1 1.6 ⁇ 11.7 respectively, compared to not treated control (they inhibited p24 production by 86% and 88% compared to untreated control, respectively).
  • VSl-010, VSl-Ol 1 and VS 1-029 were similarly effective as they reduced the % p24 to 1 1.8 ⁇ 5.6, 15.5 ⁇ 2.6 and 12.3 ⁇ 8.8, respectively, compared to not treated control (they reduced HIV replication by 88%, 85% and 88% compared to untreated control, respectively). Also Imatinib showed comparable antiviral capacity. VS1-005, bearing a styryl group at Nl, showed the same antiviral activity. VS 1-003 had slightly inferior anti-HtV capacity, % p24 was 23.8 ⁇ 7.6 compared to not treated control (it inhibited viral replication by 76%).
  • PBMC peripheral blood mononuclear cells
  • Stimulated cells were cultured for 2 days then treated with HU, CsA, and VS 1-002 to induce cell cycle arrest. After 24 hours cells were collected, stained with Ki-67 antibody and 7-AAD, and analyzed at the flow cytometer to detect the percentage of cells in GO, Gl , S and G2/M phases. Results are shown in Figure 6.
  • Cyclosporin a known G0/G1 inhibitor, provoked a decrease in the percentage of cells in Gl phase in parallel with an increase in the percentage of cells in GO phase, compared to the not treated control.
  • VS1-002 and HU both decreased the percentage of cells in S phase with an increase in the percentage of cells in Gl phase of the cell cycle, compared to the not treated control.
  • SPPs only 10 ⁇ M like HU 100 ⁇ M limits cell cycle progression at the Gl/S boundary.
  • Antiviral-hyperactivation limiting therapeutics (AV-HALTS)

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Abstract

L'invention porte sur une famille de dérivés de pyrazolo [3,4-D] pyrimidines (SPP) avec différents substituants sur les noyaux pyrimidine et pyrazolo qui ont été caractérisés avec un panel de tests démontrant leurs effets dans la prolifération, la toxicité et l'apoptose cellulaire et l'inhibition de la réplication virale. Nous avons identifié des composés et des structures moléculaires appropriés pour le traitement d'une infection virale en raison de leur activité antivirale, de leur activité antiproliférative ou de préférence des deux, de telle sorte que sous forme d'une molécule unique, à la fois ils limitent l'hyperactivation des lymphocytes T et inhibent la réplication virale. Ces composés ne sont pas toxiques à des concentrations efficaces et sont faiblement apoptotiques. D'autres composés non toxiques à l'intérieur de cette famille avec d'excellentes caractéristiques antiprolifératives et apoptotiques sont potentiellement efficaces comme médicaments anticancéreux.
PCT/US2010/002091 2009-07-27 2010-07-22 Dérivés substitués antiprolifératifs de pyrazolo[3,4-d] pyrimidines (spp) pour inhiber l'activation immunitaire, la réplication virale et la croissance tumorale WO2011014239A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016066755A3 (fr) * 2014-10-30 2016-06-23 Lead Discovery Siena S.R.L. Composés et leurs utilisations

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6552192B1 (en) * 1999-01-26 2003-04-22 Ustau Experimentalni Botaniky Av-Cr Substituted nitrogen heterocyclic derivatives and pharmaceutical use thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6552192B1 (en) * 1999-01-26 2003-04-22 Ustau Experimentalni Botaniky Av-Cr Substituted nitrogen heterocyclic derivatives and pharmaceutical use thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MANETTI ET AL: "Structure-Based Optimization of Pyrazolo[3,4-d]pyrimidines as Abl Inhibitors and Antiproliferative Agents toward Human Leukemia Cell Lines", JOURNAL MEDICINAL CHEMISTRY, vol. 51, 2008, pages 1252 - 1259 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016066755A3 (fr) * 2014-10-30 2016-06-23 Lead Discovery Siena S.R.L. Composés et leurs utilisations

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