All about Drugs, live, by DR ANTHONY MELVIN CRASTO, Worldpeaceambassador, Worlddrugtracker, OPEN SUPERSTAR Helping millions, 100 million hits on google, pushing boundaries,2.5 lakh plus connections worldwide, 40 lakh plus VIEWS on this blog in 227 countries, 7 CONTINENTS ……A 90 % paralysed man in action for you, I am suffering from transverse mylitis and bound to a wheel chair, With death on the horizon, I have lot to acheive
DR ANTHONY MELVIN CRASTO, Born in Mumbai in 1964 and graduated from Mumbai University, Completed his Ph.D from ICT, 1991,Matunga, Mumbai, India, in Organic Chemistry, The thesis topic was Synthesis of Novel Pyrethroid Analogues, Currently he is working with AFRICURE PHARMA, ROW2TECH, NIPER-G, Department of Pharmaceuticals, Ministry of Chemicals and Fertilizers, Govt. of India as ADVISOR, earlier assignment was
with GLENMARK LIFE SCIENCES LTD, as CONSUlTANT, Retired from GLENMARK in Jan2022 Research Centre as Principal Scientist, Process Research (bulk actives) at Mahape, Navi Mumbai, India. Total Industry exp 32 plus yrs, Prior to joining Glenmark, he has worked with major multinationals like Hoechst Marion Roussel, now Sanofi, Searle India Ltd, now RPG lifesciences, etc. He has worked with notable scientists like Dr K Nagarajan, Dr Ralph Stapel, Prof S Seshadri, etc, He did custom synthesis for major multinationals in his career like BASF, Novartis, Sanofi, etc., He has worked in Discovery, Natural products, Bulk drugs, Generics, Intermediates, Fine chemicals, Neutraceuticals, GMP, Scaleups, etc, he is now helping millions, has 9 million plus hits on Google on all Organic chemistry websites. His friends call him Open superstar worlddrugtracker. His New Drug Approvals, Green Chemistry International, All about drugs, Eurekamoments, Organic spectroscopy international,
etc in organic chemistry are some most read blogs He has hands on experience in initiation and developing novel routes for drug molecules
and implementation them on commercial scale over a 32 PLUS year tenure till date Feb 2023, Around 35 plus products in his career. He has good knowledge of IPM, GMP, Regulatory aspects, he has several International patents published worldwide . He has good proficiency in Technology transfer, Spectroscopy, Stereochemistry, Synthesis, Polymorphism etc., He suffered a paralytic stroke/ Acute Transverse mylitis in Dec 2007 and is 90 %Paralysed, He is bound to a wheelchair, this seems to have injected feul in him to help chemists all around the world, he is more active than before and is pushing boundaries, He has 100 million plus hits on Google, 2.5 lakh plus connections on all networking sites, 100 Lakh plus views on dozen plus blogs, 227 countries, 7 continents, He makes himself available to all, contact him on +91 9323115463, email amcrasto@gmail.com, Twitter, @amcrasto , He lives and will die for his family, 90% paralysis cannot kill his soul., Notably he has 38 lakh plus views on New Drug Approvals Blog in 227 countries......https://newdrugapprovals.wordpress.com/ , He appreciates the help he gets from one and all, Friends, Family, Glenmark, Readers, Wellwishers, Doctors, Drug authorities, His Contacts, Physiotherapist, etc
He has total of 32 International and Indian awards
An autologous T lymphocyte-enriched cell transduced ex vivo with an anti-BCMA CAR lentiviral vector encoding a chimeric antigen receptor CAR, comprising a CD8 hinge and TM domain, 4-1BB costimulatory domain and CD3ζ signaling domain, targeting human B cell maturation antigen for cancer immunotherapy (Celgene Corp., NJ)
Dendritic cells (DCs) are antigen-presenting cells (APCs) that process antigens and display them to other cells of the immune system. Specifically, dendritic cells are capable of capturing and presenting antigens on their surfaces to activate T cells such as cytotoxic T cells (CTLs). Further, activated dendritic cells are capable of recruiting additional immune cells such as macrophages, eosinophils, natural killer cells, and T cells such as natural killer T cells.
Despite major advances in cancer treatment, cancer remains one of the leading causes of death globally. Hurdles in designing effective therapies include cancer immune evasion, in which cancer cells escape destructive immunity, as well as the toxicity of many conventional cancer treatments such as radiation therapy and chemotherapy, which significantly impacts a patient’s ability to tolerate the therapy and/or impacts the efficacy of the treatment.
Given the important role of dendritic cells in immunity, derailed dendritic cell functions have been implicated in diseases such as cancer and autoimmune diseases. For example, cancer cells may evade immune detection and destruction by crippling dendritic cell functionality through prevention of dendritic cell recruitment and activation. In addition, dendritic cells have been found in the brain during central nervous system inflammation and may be involved in the pathogenesis of autoimmune diseases in the brain.
One mechanism by which cancers evade immune detection and destruction is by crippling dendritic cell functionality through prevention of dendritic cell (DC) recruitment and activation. Accordingly, there remains a need for cancer therapies that can effectively derail tumor evasion and enhance anti-tumor immunity as mediated, for example, by dendritic cells.
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DESCRIPTION
ABECMA is a BCMA-directed genetically modified autologousT cellimmunotherapy product consisting of a patient’s own T cells that are harvested and genetically modified ex vivo through transduction with an anti-BCMA02 chimeric antigen receptor (CAR) lentiviral vector (LVV). Autologous T cells transduced with the anti-BCMA02 CAR LVV express the anti-BCMA CAR on the T cell surface. The CAR is comprised of a murine extracellular single-chain variable fragment (scFv) specific for recognizing B cell maturation antigen (BCMA) followed by a human CD8α hinge and transmembrane domain fused to the T cell cytoplasmic signaling domains of CD137 (4-1BB) and CD3ζ chain, in tandem. Binding of ABECMA to BCMA-expressing target cells leads to signaling initiated by CD3ζ and 4-1BB domains, and subsequent CAR-positive T cell activation. Antigen-specific activation of ABECMA results in CAR-positive T cell proliferation, cytokine secretion, and subsequent cytolytic killing of BCMA-expressing cells.
ABECMA is prepared from the patient’s peripheral blood mononuclear cells (PBMCs), which are obtained via a standard leukapheresis procedure. The mononuclear cells are enriched for T cells, through activation with anti-CD3 and anti-CD28 antibodies in the presence of IL-2, which are then transduced with the replication-incompetent lentiviral vector containing the anti-BCMA CAR transgene. The transduced T cells are expanded in cell culture, washed, formulated into a suspension, and cryopreserved. The product must pass a sterility test before release for shipping as a frozen suspension in one or more patient-specific infusion bag(s). The product is thawed prior to infusion back into the patient [see DOSAGE AND ADMINISTRATION and HOW SUPPLIED/Storage And Handling].
The ABECMA formulation contains 50% Plasma-Lyte A and 50% CryoStor® CS10, resulting in a final DMSO concentration of 5%.
FDA approves idecabtagene vicleucel for multiple myeloma
On March 26, 2021, the Food and Drug Administration approved idecabtagene vicleucel (Abecma, Bristol Myers Squibb) for the treatment of adult patients with relapsed or refractory multiple myeloma after four or more prior lines of therapy, including an immunomodulatory agent, a proteasome inhibitor, and an anti-CD38 monoclonal antibody. This is the first FDA-approved cell-based gene therapy for multiple myeloma.
Idecabtagene vicleucel is a B-cell maturation antigen (BCMA)-directed genetically modified autologous chimeric antigen receptor (CAR) T-cell therapy. Each dose is customized using a patient’s own T-cells, which are collected and genetically modified, and infused back into the patient.
Safety and efficacy were evaluated in a multicenter study of 127 patients with relapsed and refractory multiple myeloma who received at least three prior lines of antimyeloma therapies; 88% had received four or more prior lines of therapies. Efficacy was evaluated in 100 patients who received idecabtagene vicleucel in the dose range of 300 to 460 x 106 CAR-positive T cells. Efficacy was established based on overall response rate (ORR), complete response (CR) rate, and duration of response (DOR), as evaluated by an Independent Response committee using the International Myeloma Working Group Uniform Response Criteria for Multiple Myeloma.
The ORR was 72% (95% CI: 62%, 81%) and CR rate was 28% (95% CI 19%, 38%). An estimated 65% of patients who achieved CR remained in CR for at least 12 months.
The idecabtagene vicleucel label carries a boxed warning for cytokine release syndrome (CRS), neurologic toxicities, hemophagocytic lymphohistiocytosis/ macrophage activation syndrome, and prolonged cytopenias. The most common side effects of idecabtagene vicleucel include CRS, infections, fatigue, musculoskeletal pain, and hypogammaglobulinemia.
Idecabtagene vicleucel is approved with a risk evaluation and mitigation strategy requiring that healthcare facilities that dispense the therapy must be specially certified to recognize and manage CRS and nervous system toxicities. To evaluate long-term safety, the FDA is requiring the manufacturer to conduct a post-marketing observational study involving patients treated with idecabtagene vicleucel.
FDA D.I.S.C.O. Burst Edition: FDA approval of ABECMA (idecabtagene vicleucel) the first FDA approved cell-based gene therapy for the treatment of adult patients with relapsed or refractory multiple myeloma
Welcome back to the D.I.S.C.O., FDA’s Drug Information Soundcast in Clinical Oncology, Burst Edition, brought to you by FDA’s Division of Drug Information in partnership with FDA’s Oncology Center of Excellence. Today we have another quick update on a recent FDA cancer therapeutic approval.
On March 26, 2021, the FDA approved idecabtagene vicleucel (brand name Abecma) for the treatment of adult patients with relapsed or refractory multiple myeloma after four or more prior lines of therapy, including an immunomodulatory agent, a proteasome inhibitor, and an anti-CD38 monoclonal antibody. This is the first FDA-approved cell-based gene therapy for multiple myeloma.
Idecabtagene vicleucel is a B-cell maturation antigen-directed genetically modified autologous chimeric antigen receptor T-cell therapy. Each dose is customized using a patient’s own T-cells, which are collected and genetically modified, and infused back into the patient.
Safety and efficacy were evaluated in a multicenter study of 127 patients with relapsed and refractory multiple myeloma who received at least three prior lines of antimyeloma therapies, 88% of whom had received four or more prior lines of therapies. Efficacy was evaluated in 100 patients who received idecabtagene vicleucel and was established based on overall response rate, complete response rate, and duration of response, as evaluated by an Independent Response committee using the International Myeloma Working Group Uniform Response Criteria for Multiple Myeloma.
The overall response rate was 72% and complete response rate was 28%. An estimated 65% of patients who achieved complete response remained in complete response for at least 12 months.
The idecabtagene vicleucel label carries a boxed warning for cytokine release syndrome, neurologic toxicities, hemophagocytic lymphohistiocytosis/ macrophage activation syndrome, and prolonged cytopenias. Idecabtagene vicleucel is approved with a risk evaluation and mitigation strategy requiring that healthcare facilities dispensing the therapy must be specially certified to recognize and manage cytokine release syndrome and nervous system toxicities. To evaluate long-term safety, the FDA is requiring the manufacturer to conduct a post-marketing observational study involving patients treated with idecabtagene vicleucel.
Full prescribing information for this approval can be found on the web at www.fda.gov, with key word search “Approved Cellular and Gene Therapy Products”.
Health care professionals should report serious adverse events to FDA’s MedWatch Reporting System at www.fda.gov/medwatch.
In various aspects, the present invention relates to XCR1 binding agents having at least one targeting moiety that specifically binds to XCR1. In various embodiments, these XCR1 binding agents bind to, but do not functionally modulate ( e.g . partially or fully neutralize) XCR1. Therefore, in various embodiments, the present XCR1 binding agents have use in, for instance, directly or indirectly recruiting a XCR1-expressing cell to a site of interest while still allowing the XCR1-expressing cell to signal via XCR1 (i.e. the binding of the XCR1 binding agent does not reduce or eliminate XCR1 signaling at the site of interest). In various embodiments, the XCR-1 binding agent functionally modulates XCR1. In an embodiment, the targeting moiety is a single domain antibody (e.g. VHH, HUMABODY, scFv, on antibody). In various embodiments, the XCR1 binding agent further comprises a signaling agent, e.g., without limitation, an interferon, an interleukin, and a tumor necrosis factor, that may be modified to attenuate activity. In various embodiments, the XCR1 binding agent comprises additional targeting moieties that bind to other targets (e.g. antigens, receptor) of interest. In an embodiment, the other targets (e.g. antigens, receptor) of interest are present on tumor cells. In another embodiment, the other targets (e.g. antigens, receptor) of interest are present on immune cells. In some embodiments, the present XCR1 binding agent may directly or indirectly recruit an immune cell (e.g. a dendritic cell) to a site of action (such as, by way of non-limiting example, the tumor microenvironment). In some embodiments, the present XCR1 binding agent facilitates the presentation of antigens (e.g., tumor antigens) by dendritic cells.
In various embodiments, the present XCR binding agent or targeting moiety of the present chimeric proteins comprises the heavy chain of SEQ ID NO: 223 and/or the light chain of SEQ ID NO: 224, or a variant thereof (e.g. an amino acid sequence having at least about 90%, or at least about 93%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, identity with SEQ ID NO: 223 and/or SEQ ID NO: 224).
In various embodiments, the present XCR binding agent or targeting moiety of the present chimeric proteins comprises a heavy chain CDR 1 of SHNLH (SEQ ID NO: 225), heavy chain CDR 2 of AIYPGNGNTAYNQKFKG (SEQ ID NO: 226), and heavy chain CDR 3 of WGSVVGDWYFDV (SEQ ID NO: 227) and/or a light chain CDR 1 of RSSLGLVHRNGNTYLH (SEQ ID NO: 228), light chain CDR 2 of KVSHRFS (SEQ ID NO: 229), and light chain CDR 3 of SQSTFIVPWT (SEQ ID NO: 230), or a variant thereof (e.g. with four or fewer amino acid substitutions, or with three or fewer amino acid substitutions, or with two or fewer amino acid substitutions, or with one amino acid substitution).
In various embodiments, the present XCR binding agent or targeting moiety of the present chimeric proteins comprises a heavy chain CDR 1 of SHNLH (SEQ ID NO: 225), heavy chain CDR 2 of AIYPGNGNTAYNQKFKG (SEQ ID NO: 226), and heavy chain CDR 3 of WGSVVGDWYFDV (SEQ ID NO: 227).
Illustrative Disease Modifying Therapies
EXAMPLES
Example 1. Identification and Characterization of Human XCR1 Ab AFNs
As used in this Example and associated figures,“AFN” is a chimera of the anti-Xcr1 5G7 antibody and human IFNa2 with an R149A mutation.
AFNs were made based on the 5G7 anti-hXcr1 Ab using the intact (full) Ab or a scFv format.
DWMTQTPLSLPVTLGNQASIFCRSSLGLVHRNGNTYLHWYLQKPGQSPKLLIYKVSHRFSGVPDRFSGSGSGT DFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 224)
5G7 Heavy chain CDR 1 is SHNLH (SEQ ID NO: 225), Heavy chain CDR 2 is AIYPGNGNTAYNQKFKG (SEQ ID NO: 226), Heavy chain CDR 3 is WGSVVGDWYFDV (SEQ ID NO: 227). 5G7 Light chain CDR 1 is RSSLGLVHRNGNTYLH (SEQ ID NO: 228), Light chain CDR 2 is KVSHRFS (SEQ ID NO: 229), and Light chain CDR 3 is SQSTHVPWT (SEQ ID NO: 230).
The sequence of hulFNa2(R149A) is:
CDLPQTHSLGSRRTLMLLAQMRKISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAA WDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMASF SLSTNLQESLRSKE (SEQ ID NO: 231).
In case of the intact Ab AFN, the 5G7 Ab heavy chain was fused to h I FN a2_R149A (human IFNal with a R149A mutation) via a flexible (GGS)2oG-linker and co-expressed with the 5G7 Ab light chain (sequences shown below). 5G7 scFv-AFN was constructed by linking the Ab VL and VH domains via a (GGGS)4 linker and followed by a (GGS)2o-linker and the sequence encoding hlFNa2_R149A. Recombinant proteins, cloned in the pcDNA3.4 expression-vector, were produced in ExpiCHO cells (Thermo Fisher Scientific) and purified on HisPUR spin plates (Thermo Fisher Scientific) according to the manufacturer’s instructions.
To test binding of the AFNs, parental HL1 16 and HL1 16 cells stably expressing hXcrl (HL116-hXcr1) were incubated with a serial dilution AFN for two hours at 4°C. Binding was detected using THE™ HIS antibody-FITC (GenScript) and measured on a MACSQuant X instrument (Miltenyi Biotec) and analysed using the FlowLogic software (Miltenyi Biotec). Data in Figures 1A and 1 B clearly show that both 5G7 Ab-AFN and 5G7 scFv bind specifically to hXcrl expressing cells.
Biological activity was measured on parental HL1 16 cells (an IFN responsive cell-line stably transfected with a p6-16 luciferase reporter) and the derived HL116-hXcr1 cells. Cells were seeded overnight and stimulated for 6 hours with a serial dilution 5G7 AFNs. Luciferase activity was measured on an EnSight Multimode Plate Reader (Perkin Elmer). Data in Figures 2A and 2B clearly illustrate that 5G7 AFNs, in the intact Ab format or as scFv, are clearly more active on cells expressing hXcrl compared to parental cells, illustrating that it is possible to restore signaling of an IFNa2 mutant by specific targeting to hXcrl .
Example 2. Identification and Characterization of Mouse Xcr1 Ab AFNs
As used in this Example and associated figures,“AFN” is a chimera of the anti-Xcr1 MAARX10 antibody and human IFNa2 with Q124R mutation.
Similar to the anti-human Xcr1 Ab, AFNs based on the MARX10 anti-mouse Xcr1 Ab were made, as intact Ab or as scFv. In case of the intact Ab AFN, the MARX10 Ab heavy chain was fused to hlFNa2_Q124R (human IFNa2 with Q124R mutation) via a flexible (GGS)2oG-linker and co-expressed with the MARX10 Ab light chain. scFv-AFN was constructed by linking the Ab VL and VH domains, in VH-VL (scFv(1 )) or VL-VH (scFv(2)) orientation, via a (GGGS)4 linker and followed by a (GGS)2o-linker and h I FN a2_Q 124R.
Selectivity of AFNs (produced and purified as described above for the human Xcr1 Ab AFNs) was tested by comparing binding at 2.5 pg/ml to MOCK or mouse Xcr1 transfected Hek293T cells. Binding was detected using THE™ HIS antibody-FITC (GenScript) and measured on a MACSQuant X instrument (Miltenyi Biotec) and analysed using the FlowLogic software (Miltenyi Biotec). Data in Figure 3 clearly show that all three specifically bind to mXcrl expressing cells.
///////////Idecabtagene vicleucel, breakthrough therapy designation, orphan drug designation, FDA 2021, APPROVALS 2021, Bb2121, Bb , ABECMA
Manufacturer: Celgene Corporation, a Bristol-Myers Squibb Company Indications:
Treatment of adult patients with relapsed or refractory multiple myeloma after four or more prior lines of therapy including an immunomodulatory agent, a proteasome inhibitor, and an anti-CD38 monoclonal antibody.
a cyclic pyranopterin monophosphate (cPMP) substrate replacement therapy, for the treatment of patients with molybdenum cofactor deficiency (MoCD) Type A.
Synthesis ReferenceClinch K, Watt DK, Dixon RA, Baars SM, Gainsford GJ, Tiwari A, Schwarz G, Saotome Y, Storek M, Belaidi AA, Santamaria-Araujo JA: Synthesis of cyclic pyranopterin monophosphate, a biosynthetic intermediate in the molybdenum cofactor pathway. J Med Chem. 2013 Feb 28;56(4):1730-8. doi: 10.1021/jm301855r. Epub 2013 Feb 19.
Fosdenopterin (or cyclic pyranopterin monophosphate, cPMP), sold under the brand name Nulibry, is a medication used to reduce the risk of death due to a rare genetic disease known as molybdenum cofactor deficiency type A (MoCD-A).[1]
The effectiveness of fosdenopterin for the treatment of MoCD-A was demonstrated in thirteen treated participants compared to eighteen matched, untreated participants.[1][6] The participants treated with fosdenopterin had a survival rate of 84% at three years, compared to 55% for the untreated participants.[1]
The U.S. Food and Drug Administration (FDA) granted the application for fosdenopterin priority review, breakthrough therapy, and orphan drug designations along with a rare pediatric disease priority review voucher.[1] The FDA granted the approval of Nulibry to Origin Biosciences, Inc., in February 2021.[1] It is the first medication approved for the treatment of MoCD-A.[1]
^ Schwahn BC, Van Spronsen FJ, Belaidi AA, Bowhay S, Christodoulou J, Derks TG, et al. (November 2015). “Efficacy and safety of cyclic pyranopterin monophosphate substitution in severe molybdenum cofactor deficiency type A: a prospective cohort study”. Lancet. 386 (10007): 1955–63. doi:10.1016/S0140-6736(15)00124-5. PMID26343839. S2CID21954888.
External links
“Fosdenopterin”. Drug Information Portal. U.S. National Library of Medicine.
Molybdenum cofactor deficiency (MoCD) is an exceptionally rare autosomal recessive disorder resulting in a deficiency of three molybdenum-dependent enzymes: sulfite oxidase (SOX), xanthine dehydrogenase, and aldehyde oxidase.1 Signs and symptoms begin shortly after birth and are caused by a build-up of toxic sulfites resulting from a lack of SOX activity.1,5 Patients with MoCD may present with metabolic acidosis, intracranial hemorrhage, feeding difficulties, and significant neurological symptoms such as muscle hyper- and hypotonia, intractable seizures, spastic paraplegia, myoclonus, and opisthotonus. In addition, patients with MoCD are often born with morphologic evidence of the disorder such as microcephaly, cerebral atrophy/hypodensity, dilated ventricles, and ocular abnormalities.1 MoCD is incurable and median survival in untreated patients is approximately 36 months1 – treatment, then, is focused on improving survival and maintaining neurological function.
The most common subtype of MoCD, type A, involves mutations in MOCS1 wherein the first step of molybdenum cofactor synthesis – the conversion of guanosine triphosphate into cyclic pyranopterin monophosphate (cPMP) – is interrupted.1,3 In the past, management strategies for this disorder involved symptomatic and supportive treatment,5 though efforts were made to develop a suitable exogenous replacement for the missing cPMP. In 2009 a recombinant, E. coli-produced cPMP was granted orphan drug designation by the FDA, becoming the first therapeutic option for patients with MoCD type A.1
Fosdenopterin was approved by the FDA on Februrary 26, 2021, for the reduction of mortality in patients with MoCD type A,5 becoming the first and only therapy approved for the treatment of MoCD. By improving the three-year survival rate from 55% to 84%,7 and considering the lack of alternative therapies available, fosdenopterin appears poised to become a standard of therapy in the management of this debilitating disorder.
Fosdenopterin replaces an intermediate substrate in the synthesis of molybdenum cofactor, a compound necessary for the activation of several molybdenum-dependent enzymes including sulfite oxidase (SOX).1 Given that SOX is responsible for detoxifying sulfur-containing acids and sulfites such as S-sulfocysteine (SSC), urinary levels of SSC can be used as a surrogate marker of efficacy for fosdenopterin.7 Long-term therapy with fosdenopterin has been shown to result in a sustained reduction in urinary SSC normalized to creatinine.7
Animal studies have identified a potential risk of phototoxicity in patients receiving fosdenopterin – these patients should avoid or minimize exposure to sunlight and/or artificial UV light.7 If sun exposure is necessary, use protective clothing, hats, and sunglasses,7 in addition to seeking shade whenever practical. Consider the use of a broad-spectrum sunscreen in patients 6 months of age or older.8
Molybdenum cofactor deficiency (MoCD) is a rare autosomal-recessive disorder in which patients are deficient in three molybdenum-dependent enzymes: sulfite oxidase (SOX), xanthine dehydrogenase, and aldehyde dehydrogenase.1 The loss of SOX activity appears to be the main driver of MoCD morbidity and mortality, as the build-up of neurotoxic sulfites typically processed by SOX results in rapid and progressive neurological damage. In MoCD type A, the disorder results from a mutation in the MOCS1 gene leading to deficient production of MOCS1A/B,7 a protein that is responsible for the first step in the synthesis of molybdenum cofactor: the conversion of guanosine triphosphate into cyclic pyranopterin monophosphate (cPMP).1,4
Fosdenopterin is an exogenous form of cPMP, replacing endogenous production and allowing for the synthesis of molybdenum cofactor to proceed.7
Mechler K, Mountford WK, Hoffmann GF, Ries M: Ultra-orphan diseases: a quantitative analysis of the natural history of molybdenum cofactor deficiency. Genet Med. 2015 Dec;17(12):965-70. doi: 10.1038/gim.2015.12. Epub 2015 Mar 12. [PubMed:25764214]
Schwahn BC, Van Spronsen FJ, Belaidi AA, Bowhay S, Christodoulou J, Derks TG, Hennermann JB, Jameson E, Konig K, McGregor TL, Font-Montgomery E, Santamaria-Araujo JA, Santra S, Vaidya M, Vierzig A, Wassmer E, Weis I, Wong FY, Veldman A, Schwarz G: Efficacy and safety of cyclic pyranopterin monophosphate substitution in severe molybdenum cofactor deficiency type A: a prospective cohort study. Lancet. 2015 Nov 14;386(10007):1955-63. doi: 10.1016/S0140-6736(15)00124-5. Epub 2015 Sep 3. [PubMed:26343839]
Iobbi-Nivol C, Leimkuhler S: Molybdenum enzymes, their maturation and molybdenum cofactor biosynthesis in Escherichia coli. Biochim Biophys Acta. 2013 Aug-Sep;1827(8-9):1086-101. doi: 10.1016/j.bbabio.2012.11.007. Epub 2012 Nov 29. [PubMed:23201473]
Mendel RR: The molybdenum cofactor. J Biol Chem. 2013 May 10;288(19):13165-72. doi: 10.1074/jbc.R113.455311. Epub 2013 Mar 28. [PubMed:23539623]
FDA News Release: FDA Approves First Treatment for Molybdenum Cofactor Deficiency Type A [Link]
OMIM: MOLYBDENUM COFACTOR DEFICIENCY, COMPLEMENTATION GROUP A (# 252150) [Link]
FDA Approved Drug Products: Nulibry (fosdenopterin) for intravenous injection [Link]
Cyclic pyranopterin monophosphate (1), isolated from bacterial culture, has previously been shown to be effective in restoring normal function of molybdenum enzymes in molybdenum cofactor (MoCo)-deficient mice and human patients. Described here is a synthesis of 1 hydrobromide (1·HBr) employing in the key step a Viscontini reaction between 2,5,6-triamino-3,4-dihydropyrimidin-4-one dihydrochloride and d-galactose phenylhydrazone to give the pyranopterin (5aS,6R,7R,8R,9aR)-2-amino-6,7-dihydroxy-8-(hydroxymethyl)-3H,4H,5H,5aH,6H,7H,8H,9aH,10H-pyrano[3,2-g]pteridin-4-one (10) and establishing all four stereocenters found in 1. Compound 10, characterized spectroscopically and by X-ray crystallography, was transformed through a selectively protected tri-tert-butoxycarbonylamino intermediate into a highly crystalline tetracyclic phosphate ester (15). The latter underwent a Swern oxidation and then deprotection to give 1·HBr. Synthesized 1·HBr had in vitro efficacy comparable to that of 1 of bacterial origin as demonstrated by its enzymatic conversion into mature MoCo and subsequent reconstitution of MoCo-free human sulfite oxidase–molybdenum domain yielding a fully active enzyme. The described synthesis has the potential for scale up.
PAPER
European Journal of Organic Chemistry (2014), 2014(11), 2231-2241.
The first synthesis of an oxygen‐stable analogue of the natural product cyclic pyranopterin monophosphate (cPMP) is reported. In this approach, the hydropyranone ring is annelated to pyrazine by a sequence comprising ortho‐lithiation/acylation of a 2‐halopyrazine, followed by nucleophilic aromatic substitution. The tetrose substructure is introduced from the chiral pool, from D‐galactose or D‐arabitol.
Abstract
Molybdenum cofactor (Moco) deficiency is a lethal hereditary metabolic disease. A recently developed therapy requires continuous intravenous supplementation of the biosynthetic Moco precursor cyclic pyranopterin monophosphate (cPMP). The limited stability of the latter natural product, mostly due to oxidative degradation, is problematic for oral administration. Therefore, the synthesis of more stable cPMP analogues is of great interest. In this context and for the first time, the synthesis of a cPMP analogue, in which the oxidation‐labile reduced pterin unit is replaced by a pyrazine moiety, was achieved starting from the chiral pool materials D‐galactose or D‐arabitol. Our synthesis, 13 steps in total, includes the following key transformations: i) pyrazine lithiation, followed by acylation; ii) closure of the pyrane ring by nucleophilic aromatic substitution; and iii) introduction of phosphate.
Molybdenum cofactor (Moco) deficiency is a pleiotropic genetic disorder. Moco consists of molybdenum covalently bound to one or two dithiolates attached to a unique tricyclic pterin moiety commonly referred to as molybdopterin (MPT). Moco is synthesized by a biosynthetic pathway that can be divided into four steps, according to the biosynthetic intermediates precursor Z (cyclic pyranopterin monophosphate; cPMP), MPT, and adenylated MPT. Mutations in the Moco biosynthetase genes result in the loss of production of the molybdenum dependent enzymes sulfite-oxidase, xanthine oxidoreductase, and aldehyde oxidase. Whereas the activities of all three of these cofactor-containing enzymes are impaired by cofactor deficiency, the devastating consequences of the disease can be traced to the loss of sulfite oxidase activity. Human Moco deficiency is a rare but severe disorder accompanied by serious neurological symptoms including attenuated growth of the brain, untreatable seizures, dislocated ocular lenses, and mental retardation. Until recently, no effective therapy was available and afflicted patients suffering from Moco deficiency died in early infancy.
It has been found that administration of the molybdopterin derivative precursor Z, a relatively stable intermediate in the Moco biosynthetic pathway, is an effective means of therapy for human Moco deficiency and associated diseases related to altered Moco synthesis (see U.S. Pat. No. 7,504,095). As with most replacement therapies for illnesses, however, the treatment is limited by the availability of the therapeutic active agent.
Scheme 3.
Scheme 4.
(I).
Scheme 6.
(I).
Scheme 8.
(I).
Scheme 10.
EXAMPLESExample 1Preparation of Precursor Z (cPMP)
Experimental
Air sensitive reactions were performed under argon. Organic solutions were dried over anhydrous MgSO4 and the solvents were evaporated under reduced pressure. Anhydrous and chromatography solvents were obtained commercially (anhydrous grade solvent from Sigma-Aldrich Fine Chemicals) and used without any further purification. Thin layer chromatography (t.l.c.) was performed on glass or aluminum sheets coated with 60 F254 silica gel. Organic compounds were visualized under UV light or with use of a dip of ammonium molybdate (5 wt %) and cerium(IV) sulfate 4H2O (0.2 wt %) in aq. H2SO4 (2M), one of I2 (0.2%) and KI (7%) in H2SO4 (1M), or 0.1% ninhydrin in EtOH. Chromatography (flash column) was performed on silica gel (40-63 μm) or on an automated system with continuous gradient facility. Optical rotations were recorded at a path length of 1 dm and are in units of 10−1 deg cm2 g−1; concentrations are in g/100 mL. 1H NMR spectra were measured in CDCl3, CD3OD (internal Me4Si, δ 0 ppm) or D2O(HOD, δ 4.79 ppm), and 13C NMR spectra in CDCl3 (center line, δ 77.0 ppm), CD3OD (center line, δ 49.0 ppm) or DMSO d6 (center line δ 39.7 ppm), D2O (no internal reference or internal CH3CN, δ 1.47 ppm where stated). Assignments of 1H and 13C resonances were based on 2D (1H—1H DQF-COSY, 1H—13C HSQC, HMBC) and DEPT experiments. 31P NMR were run at 202.3 MHz and are reported without reference. High resolution electrospray mass spectra (ESI-HRMS) were recorded on a Q-TOF Tandem Mass
Spectrometer. Microanalyses were performed by the Campbell Microanalytical Department, University of Otago, Dunedin, New Zealand.
A. Preparation of (5aS,6R,7R,8R,9aR)-2-amino-6,7-dihydroxy-8-(hydroxymethyl)-3H,4H,5H,5aH,6H,7H,8H,9aH,10H-pyrano[3,2-g]pteridin-4-one mono hydrate (1)
2,5,6-Triamino-3,4-dihydropyrimidin-4-one dihydrochloride (Pfleiderer, W.; Chem. Ber. 1957, 90, 2272; Org. Synth. 1952, 32, 45; Org. Synth. 1963, Coll. Vol. 4, 245, 10.0 g, 46.7 mmol), D-galactose phenylhydrazone (Goswami, S.; Adak, A. K. Tetrahedron Lett. 2005, 46, 221-224, 15.78 g, 58.4 mmol) and 2-mercaptoethanol (1 mL) were stirred and heated to reflux (bath temp 110° C.) in a 1:1 mixture of MeOH—H2O (400 mL) for 2 h. After cooling to ambient temperature, diethyl ether (500 mL) was added, the flask was shaken and the diethyl ether layer decanted off and discarded. The process was repeated with two further portions of diethyl ether (500 mL) and then the remaining volatiles were evaporated. Methanol (40 mL), H2O (40 mL) and triethylamine (39.4 mL, 280 mmol) were successively added and the mixture seeded with a few milligrams of 1. After 5 min a yellow solid was filtered off, washed with a little MeOH and dried to give 1 as a monohydrate (5.05 g, 36%) of suitable purity for further use. An analytical portion was recrystallized from DMSO-EtOH or boiling H2O. MPt 226 dec. [α]D20 +135.6 (c1.13, DMSO). 1H NMR (DMSO d6): δ 10.19 (bs, exchanged D2O, 1H), 7.29 (d, J=5.0 Hz, slowly exchanged D2O, 1H), 5.90 (s, exchanged D2O, 2H), 5.33 (d, J=5.4 Hz, exchanged D2O, 1H), 4.66 (ddd, J˜5.0, ˜1.3, ˜1.3 Hz, 1H), 4.59 (t, J=5.6 Hz, exchanged D2O, 1H), 4.39 (d, J=10.3 Hz, exchanged D2O, 1H), 3.80 (bt, J˜1.8 Hz, exchanged D2O, 1H), 3.70 (m, 1H), 3.58 (dd, J=10.3, 3.0 Hz, 1H), 3.53 (dt, J=10.7, 6.4 Hz, 1H), 3.43 (ddd, J=11.2, 5.9, 5.9 Hz, 1H), 3.35 (t, J=6.4 Hz, 1H), 3.04 (br m, 1H). 13C NMR (DMSO d6 center line 6 39.7): δ 156.3 (C), 150.4 (C), 148.4 (C), 99.0 (C), 79.4 (CH), 76.5 (CH), 68.9 (CH), 68.6 (CH), 60.6 (CH2), 53.9 (CH). Anal. calcd. for C10H15N5O5H2O 39.60; C, 5.65; H, 23.09; N. found 39.64; C, 5.71; H, 22.83; N.
B. Preparation of Compounds 2 (a or b) and 3 (a, b or c)
Di-tert-butyl dicarbonate (10.33 g, 47.3 mmol) and DMAP (0.321 g, 2.63 mmol) were added to a stirred suspension of 1 (1.5 g, 5.26 mmol) in anhydrous THF (90 mL) at 50° C. under Ar. After 20 h a clear solution resulted. The solvent was evaporated and the residue chromatographed on silica gel (gradient of 0 to 40% EtOAc in hexanes) to give two product fractions. The first product to elute was a yellow foam (1.46 g). The product was observed to be a mixture of two compounds by 1H NMR containing mainly a product with seven Boc groups (2a or 2b). A sample was crystallized from EtOAc-hexanes to give 2a or 2b as a fine crystalline solid. MPt 189-191° C. [α]D20 −43.6 (c 0.99, MeOH). 1H NMR (500 MHz, CDCl3): δ 5.71 (t, J=1.7 Hz, 1H), 5.15 (dt, J=3.5, ˜1.0, 1H), 4.97 (t, J=3.8, 1H), 4.35 (br t, J=˜1.7, 1H), 4.09-3.97 (m, 3H), 3.91 (m, 1H), 1.55, 1.52, 1.51, 1.50, 1.45 (5s, 45H), 1.40 (s, 18H). 13C NMR (125.7 MHz, CDCl3): δ 152.84 (C), 152.78 (C), 151.5 (C), 150.9 (C), 150.7 (2×C), 150.3 (C), 149.1 (C), 144.8 (C), 144.7 (C), 118.0 (C), 84.6 (C), 83.6 (C), 83.5 (C), 82.7 (3×C), 82.6 (C), 76.3 (CH), 73.0 (CH), 71.4 (CH), 67.2 (CH), 64.0 (CH2), 51.4 (CH), 28.1 (CH3), 27.8 (2×CH3), 27.7 (CH3), 27.6 (3×CH3). MS-ESI+ for C45H72N5O19+, (M+H)+, Calcd. 986.4817. found 986.4818. Anal. calcd. for C45H71N5O19H2O 54.39; C, 7.39; H, 6.34; N. found 54.66; C, 7.17; H, 7.05; N. A second fraction was obtained as a yellow foam (2.68 g) which by 1H NMR was a product with six Boc groups present (3a, 3b or 3c). A small amount was crystallized from EtOAc-hexanes to give colorless crystals. [α]D2O −47.6 (c, 1.17, CHCl3). 1H NMR (500 MHz, CDCl3): δ 11.10 (br s, exchanged D2O, 1H), 5.58 (t, J=1.8 Hz, 1H), 5.17 (d, J=3.4 Hz, 1H), 4.97 (t, J=3.9 Hz, 1H), 4.62 (s, exchanged D2O, 1H), 4.16 (dd, J=11.3, 5.9 Hz, 1H), 4.12 (dd, J=11.3, 6.4 Hz, 1H), 3.95 (dt, J=6.1, 1.1 Hz, 1H), 3.76 (m, 1H), 1.51, 1.50, 1.49, 1.48, 1.46 (5s, 54H). 13C NMR (125.7 MHz, CDCl3): δ 156.6 (C), 153.0 (C), 152.9 (C), 151.9 (C), 150.6 (C), 149.4 (2×C), 136.2 (C), 131.8 (C), 116.9 (C), 85.0 (2×C), 83.3 (C), 82.8 (C), 82.49 (C), 82.46 (C), 73.3 (CH), 71.5 (CH), 67.2 (CH), 64.5 (CH2), 51.3 (CH), 28.0, 27.72, 27.68, 27.6 (4×CH3). MS-ESI+ for C40H64N5O17+, (M+H)+calcd. 886.4287. found 886.4289.
C. Preparation of Compound 4a, 4b or 4c
Step 1—The first fraction from B above containing mainly compounds 2a or 2b (1.46 g, 1.481 mmol) was dissolved in MeOH (29 mL) and sodium methoxide in MeOH (1M, 8.14 mL, 8.14 mmol) added. After leaving at ambient temperature for 20 h the solution was neutralized with Dowex 50WX8 (H+) resin then the solids filtered off and the solvent evaporated.
Step 2—The second fraction from B above containing mainly 3a, 3b or 3c (2.68 g, 3.02 mmol) was dissolved in MeOH (54 mL) and sodium methoxide in MeOH (1M, 12.10 mL, 12.10 mmol) added. After leaving at ambient temperature for 20 h the solution was neutralized with Dowex 50WX8 (H+) resin then the solids filtered off and the solvent evaporated.
The products from step 1 and step 2 above were combined and chromatographed on silica gel (gradient of 0 to 15% MeOH in CHCl3) to give 4a, 4b or 4c as a cream colored solid (1.97 g). 1H NMR (500 MHz, DMSO d6): δ 12.67 (br s, exchanged D2O, 1H), 5.48 (d, J=5.2 Hz, exchanged D2O, 1H), 5.43 (t, J=˜1.9 Hz, after D2O exchange became a d, J=1.9 Hz, 1H), 5.00 (br s, exchanged D2O, 1H), 4.62 (d, J=5.7 Hz, exchanged D2O, 1H), 4.27 (d, J=6.0 Hz, exchanged D2O, 1H), 3.89 (dt, J=5.2, 3.8 Hz, after D2O became a t, J=3.9 Hz, 1H), 3.62 (dd, J=6.0, 3.7 Hz, after D2O exchange became a d, J=3.7 Hz, 1H), 3.52-3.39 (m, 4H), 1.42 (s, 9H), 1.41 (s, 18H). 13C NMR (125.7 MHz, DMSO d6): δ 157.9 (C), 151.1, (C), 149.8 (2×C), 134.6 (C), 131.4 (C), 118.8 (C), 83.5 (2×C), 81.3 (C), 78.2 (CH), 76.5 (CH), 68.1 (CH), 66.8 (CH), 60.6 (CH2), 54.4 (CH), 27.9 (CH3), 27.6 (2×CH3). MS-ESI+ for C25H40N5O11+, (M+H)+ calcd. 586.2719. found 586.2717.
D. Preparation of Compound 5a, 5b or 5c
Compound 4a, 4b or 4c (992 mg, 1.69 mmol) was dissolved in anhydrous pyridine and concentrated. The residue was dissolved in anhydrous CH2Cl2 (10 mL) and pyridine (5 mL) under a nitrogen atmosphere and the solution was cooled to −42° C. in an acetonitrile/dry ice bath. Methyl dichlorophosphate (187 μL, 1.86 mmol) was added dropwise and the mixture was stirred for 2 h 20 min. Water (10 mL) was added to the cold solution which was then removed from the cold bath and diluted with ethyl acetate (50 mL) and saturated NaCl solution (30 mL). The organic portion was separated and washed with saturated NaCl solution. The combined aqueous portions were extracted twice further with ethyl acetate and the combined organic portions were dried over MgSO4 and concentrated. Purification by silica gel flash column chromatography (eluting with 2-20% methanol in ethyl acetate) gave the cyclic methyl phosphate 5a, 5b or 5c (731 mg, 65%). 1H NMR (500 MHz, CDCl3,): δ 11.72 (bs, exchanged D2O, 1H), 5.63 (t, J=1.8 Hz, 1H), 5.41 (s, exchanged D2O, 1H), 4.95 (d, J=3.2 Hz, 1H), 4.70 (dt, J=12.4, 1.8 Hz, 1H), 4.42 (dd, J=22.1, 12.1 Hz, 1H). 4.15 (q, J=3.7 Hz, 1H), 3.82 (s, 1H), 3.75 (s, 1H), 3.58 (d, J=11.7 Hz, 3H), 2.10 (bs, exchanged D20, 1H+H2O), 1.50 (s, 9H), 1.46 (s, 18H). 13C NMR (125.7 MHz, CDCl3, centre line δ 77.0): δ 157.5 (C), 151.2 (C), 149.6 (2×C), 134.5 (C), 132.3 (C), 117.6 (C), 84.7 (2×C), 82.8 (C), 77.3 (CH), 74.8 (d, J=4.1 Hz, CH), 69.7 (CH2), 68.8 (d, J=4.1 Hz, CH), 68.6 (d, J=5.9 Hz, CH), 56.0 (d, J=7.4 Hz, CH3), 51.8 (CH), 28.1 (CH3), 27.8 (CH3). MS-ESI+ for C26H40N5NaO13P+ (M+Na)+, calcd. 684.2252. found 684.2251.
E. Preparation of Compound 6a, 6b or 6c
Compound 5a, 5b or 5c (223 mg, 0.34 mmol) was dissolved in anhydrous CH2Cl2 (7 mL) under a nitrogen atmosphere. Anhydrous DMSO (104 μL, 1.46 mmol) was added and the solution was cooled to −78° C. Trifluoroacetic anhydride (104 μL, 0.74 mmol) was added dropwise and the mixture was stirred for 40 min. N,N-diisopropylethylamine (513 μL, 2.94 mmol) was added and the stirring was continued for 50 min at −78° C. Saturated NaCl solution (20 mL) was added and the mixture removed from the cold bath and diluted with CH2Cl2 (30 mL). Glacial acetic acid (170 μL, 8.75 mmol) was added and the mixture was stirred for 10 min. The layers were separated and the aqueous phase was washed with CH2Cl2 (10 mL). The combined organic phases were washed with 5% aqueous HCl, 3:1 saturated NaCl solution:10% NaHCO3 solution and saturated NaCl solution successively, dried over MgSO4, and concentrated to give compound 6a, 6b or 6c (228 mg, quant.) of suitable purity for further use. 1H NMR (500 MHz, CDCl3): δ 5.86 (m, 1 H), 5.07 (m, 1 H), 4.70-4.64 (m, 2 H), 4.49-4.40 (m, 1 H), 4.27 (m, 1 H), 3.56, m, 4 H), 1.49 (s, 9 H), 1.46 (s, 18 H) ppm. 13C NMR (500 MHz, CDCl3): δ 157.5 (C), 151.1 (C), 150.6 (2 C), 134.6 (C), 132.7 (C), 116.6 (C), 92.0 (C), 84.6 (2 C), 83.6 (C), 78.0 (CH), 76.0 (CH), 70.4 (CH2), 67.9 (CH), 56.2 (CH3) δ6.0 (CH), 28.2 (3CH3), 26.8 (6 CH3) ppm. 31P NMR (500 MHz, CDCl3): δ−6.3 ppm.
F. Preparation of compound 7: (4aR,5aR,11aR,12aS)-1,3,2-Dioxaphosphorino[4′,5′:5,6]pyrano[3,2-g]pteridin-10(4H)-one,8-amino-4-a,5a,6,9,11,11a,12,12a-octahydro-2,12,12-trihydroxy-2-oxide
Compound 6a, 6b or 6c (10 mg, 14.8 μmol was dissolved in dry acetonitrile (0.2 mL) and cooled to 0° C. Bromotrimethylsilane (19.2 μL, 148 μmol) was added dropwise and the mixture was allowed to warm to ambient temperature and stirred for 5 h during which time a precipitate formed. HCl(aq) (10 μl, 37%) was added and the mixture was stirred for a further 15 min. The mixture was centrifuged for 15 min (3000 g) and the resulting precipitate collected. Acetonitrile (0.5 mL) was added and the mixture was centrifuged for a further 15 min. The acetonitrile wash and centrifugation was repeated a further two times and the resulting solid was dried under high vacuum to give compound 7 (4 mg, 75%). 1H NMR (500 MHz, D2O): δ 5.22 (d, J=1.6 Hz, 1H), 4.34 (dt, J=13, 1.6 Hz, 1H), 4.29-4.27 (m, 1H), 4.24-4.18 (m, 1H), 3.94 (br m, 1H), 3.44 (t, J=1.4 Hz, 1H). 31P NMR (500 MHz, D2O): δ −4.8 MS-ESI+ for C10H15N5O8P+, (M+H)+calcd. 364.0653. found 364.0652.
Example 2Comparison of Precursor Z (cPMP) Prepared Synthetically to that Prepared from E. Coli in the In vitro Synthesis of Moco
In vitro synthesis of Moco was compared using samples of synthetic precursor Z (cPMP) and cPMP purified from E. coli. Moco synthesis also involved the use of the purified components E. coli MPT synthase, gephyrin, molybdate, ATP, and apo-sulfite oxidase. See U.S. Pat. No. 7,504,095 and “Biosynthesis and molecular biology of the molybdenum cofactor (Moco)” in Metal Ions in Biological Systems, Mendel, Ralf R. and Schwarz, Gunter, Informa Plc, 2002, Vol. 39, pages 317-68. The assay is based on the conversion of cPMP into MPT, the subsequent molybdate insertion using recombinant gephyrin and ATP, and finally the reconstitution of human apo-sulfite oxidase.
As shown in FIG. 1, Moco synthesis from synthetic cPMP was confirmed, and no differences in Moco conversion were found in comparison to E. coli purified cPMP.
Example 3Comparison of Precursor Z (cPMP) Prepared Synthetically to that Prepared from E. coli in the In vitro Synthesis of MPT
In vitro synthesis of MPT was compared using samples of synthetic precursor Z (cPMP) and cPMP purified from E. coli. MPT synthesis also involved the use of in vitro assembled MPT synthase from E. coli. See U.S. Pat. No. 7,504,095 and “Biosynthesis and molecular biology of the molybdenum cofactor (Moco)” in Metal Ions in Biological Systems, Mendel, Ralf R. and Schwarz, Gunter, Informa Plc, 2002, Vol. 39, pages 317-68. Three repetitions of each experiment were performed and are shown in FIGS. 2 and 3.
As shown in FIGS. 2 and 3, MPT synthesis from synthetic cPMP confirmed, and no apparent differences in MPT conversion were found when compared to E. coli purified cPMP. A linear conversion of cPMP into MPT is seen in all samples confirming the identity of synthetic cPMP (see FIG. 2). Slight differences between the repetitions are believed to be due to an inaccurate concentration determination of synthetic cPMP given the presence of interfering chromophores.
Example 4Preparation of Precursor Z (cPMP)
A. Preparation of Starting Materials
B. Introduction of the protected Phosphate
The formation of the cyclic phosphate using intermediate [10] (630 mg) gave the desired product [11] as a 1:1 mixture of diastereoisomers (494 mg, 69%).
C. Oxidation and Overall Deprotection of the Molecule
Oxidation of the secondary alcohol to the gem-diol did prove successful on intermediate [12], but the oxidized product [13] did show significant instability and could not be purified. For this reason, deprotection of the phosphate was attempted before the oxidation. However, the reaction of intermediate [11] with TMSBr led to complete deprotection of the molecule giving intermediate [14]. An attempt to oxidize the alcohol to the gem-diol using Dess-Martin periodinane gave the aromatized pteridine [15].
Oxidation of intermediate [11] with Dess-Martin periodinane gave a mixture of starting material, oxidized product and several by-products. Finally, intermediate [11] was oxidized using the method described Example 1. Upon treatment, only partial oxidation was observed, leaving a 2:1 mixture of [11]/[16]. The crude mixture was submitted to the final deprotection. An off white solid was obtained and analyzed by 1H-NMR and HPLC-MS. These analyses suggest that cPMP has been produced along with the deprotected precursor [11].
Because the analytical HPLC conditions gave a good separation of cPMP from the major impurities, this method will be repeated on a prep-HPLC in order to isolate the final material.
CLIP
BridgeBio Pharma And Affiliate Origin Biosciences Announces FDA Acceptance Of Its New Drug Application For Fosdenopterin For The Treatment Of MoCD Type A
Application accepted under Priority Review designation with Breakthrough Therapy Designation and Rare Pediatric Disease Designation previously grantedThere are currently no approved therapies for the treatment of MoCD Type A, which results in severe and irreversible neurological injury for infants and children.This is BridgeBio’s first NDA acceptanceSAN FRANCISCO, September 29, 2020 – BridgeBio Pharma, Inc. (Nasdaq: BBIO) and affiliate Origin Biosciences today announced the US Food and Drug Administration (FDA) has accepted its New Drug Application (NDA) for fosdenopterin (previously BBP-870/ORGN001), a cyclic pyranopterin monophosphate (cPMP) substrate replacement therapy, for the treatment of patients with molybdenum cofactor deficiency (MoCD) Type A.The NDA has been granted Priority Review designation. Fosdenopterin has previously been granted Breakthrough Therapy Designation and Rare Pediatric Disease Designation in the US and may be eligible for a priority review voucher if approved. It received Orphan Drug Designation in the US and Europe. This is BridgeBio’s first NDA acceptance.“We want to thank the patients, families, scientists, physicians and all others involved who helped us reach this critical milestone,” said BridgeBio CEO and founder Neil Kumar, Ph.D. “MoCD Type A is a devastating disease with a median survival of less than four years and we are eager for our investigational therapy to be available to patients, who currently have no approved treatment options. BridgeBio exists to help as many patients as possible afflicted with genetic diseases, no matter how rare. We are grateful that the FDA has accepted our first NDA for priority review and we look forward to submitting our second NDA later this year for infigratinib for second line treatment of cholangiocarcinoma.”About Fosdenopterin Fosdenopterin is being developed for the treatment of patients with MoCD Type A. Currently, there are no approved therapies for the treatment of MoCD Type A, which results in severe and irreversible neurological injury with a median survival between 3 to 4 years. Fosdenopterin is a first-in-class cPMP hydrobromide dihydrate and is designed to treat MoCD Type A by replacing cPMP and permitting the two remaining MoCo synthesis steps to proceed, with activation of MoCo-dependent enzymes and elimination of sulfites.About Molybdenum Cofactor Deficiency (MoCD) Type A MoCD Type A is an ultra-rare, autosomal recessive, inborn error of metabolism caused by disruption in molybdenum cofactor (MoCo) synthesis which is vital to prevent buildup of s-sulfocysteine, a neurotoxic metabolite of sulfite. Patients are often infants with severe encephalopathy and intractable seizures. Disease progression is rapid with a high infant mortality rate.Those who survive beyond the first few month’s experience profuse developmental delays and suffer the effects of irreversible neurological damage, including brain atrophy with white matter necrosis, dysmorphic facial features, and spastic paraplegia. Clinical presentation that can be similar to hypoxic-ischemic encephalopathy (HIE) or other neonatal seizure disorders may lead to misdiagnosis and underdiagnosis. Immediate testing for elevated sulfite levels and S-sulfocysteine in the urine and very low serum uric acid may help with suspicion of MoCD.About Origin Biosciences Origin Biosciences, an affiliate of BridgeBio Pharma, is a biotechnology company focused on developing and commercializing a treatment for Molybdenum Cofactor Deficiency (MoCD) Type A. Origin is led by a team of veteran biotechnology executives. Together with patients and physicians, the company aims to bring a safe, effective treatment for MoCD Type A to market as quickly as possible. For more information on Origin Biosciences, please visit the company’s website at www.origintx.com.
About BridgeBio Pharma BridgeBio is a team of experienced drug discoverers, developers and innovators working to create life-altering medicines that target well-characterized genetic diseases at their source. BridgeBio was founded in 2015 to identify and advance transformative medicines to treat patients who suffer from Mendelian diseases, which are diseases that arise from defects in a single gene, and cancers with clear genetic drivers. BridgeBio’s pipeline of over 20 development programs includes product candidates ranging from early discovery to late-stage development. For more information visit bridgebio.com.
The U.S. Food and Drug Administration approved Ebanga (Ansuvimab-zykl), a human monoclonal antibody, for the treatment for Zaire ebolavirus (Ebolavirus) infection in adults and children. Ebanga blocks binding of the virus to the cell receptor, preventing its entry into the cell.
Zaire ebolavirus is one of four Ebolavirus species that can cause a potentially fatal human disease. It is transmitted through blood, body fluids, and tissues of infected people or wild animals, and through surfaces and materials, such as bedding and clothing, contaminated with these fluids. Individuals who care for people with the disease, including health care workers who do not use correct infection control precautions, are at the highest risk for infection.
During an Ebola outbreak in the Democratic Republic of the Congo (DRC) in 2018-2019, Ebanga was evaluated in a clinical trial (the PALM trial). The PALM trial was led by the U.S. National Institutes of Health and the DRC’s Institut National de Recherche Biomédicale with contributions from several other international organizations and agencies.
In the PALM trial, the safety and efficacy of Ebanga was evaluated in a multi-center, open-label, randomized controlled trial. 174 participants (120 adults and 54 pediatric patients) with confirmed Ebolavirus infection received Ebanga intravenously as a single 50 mg/kg infusion and 168 participants (135 adults and 33 pediatric patients) received an investigational control. The primary efficacy endpoint was 28-day mortality. The primary analysis population was all patients who were randomized and concurrently eligible to receive either Ebanga or the investigational control during the same time period of the trial. Of the 174 patients who received Ebanga, 35.1% died after 28 days, compared to 49.4% of the 168 patients who received a control.
The most common symptoms experienced while receiving Ebanga include: fever, tachycardia (fast heart rate), diarrhea, vomiting, hypotension (low blood pressure), tachypnea (fast breathing) and chills; however, these are also common symptoms of Ebolavirus infection. Hypersensitivity, including infusion-related events, can occur in patients taking Ebanga, and treatment should be discontinued in the event of a hypersensitivity reaction.
Patients who receive Ebanga should avoid the concurrent administration of a live virus vaccine against Ebolavirus. There is the potential for Ebanga to inhibit replication of a live vaccine virus and possibly reduce the efficacy of this vaccine.
FDA granted the approval to Ridgeback Biotherapeutics, LP.
Ansuvimab, sold under the brand name Ebanga, is a monoclonal antibody medication for the treatment of Zaire ebolavirus (Ebolavirus) infection.[1][2]
The most common symptoms include fever, tachycardia (fast heart rate), diarrhea, vomiting, hypotension (low blood pressure), tachypnea (fast breathing) and chills; however, these are also common symptoms of Ebolavirus infection.[1]
Ansuvimab was approved for medical use in the United States in December 2020.[1][2]
Ansuvimab is a monoclonal antibody therapy that is infused intravenously into patients with Ebola virus disease. Ansuvimab is a neutralizing antibody,[3] meaning it binds to a protein on the surface of Ebola virus that is required to infect cells. Specifically, ansuvimab neutralizes infection by binding to a region of the Ebola virus envelope glycoprotein that, in the absence of ansuvimab, would interact with virus’s cell receptor protein, Niemann-Pick C1 (NPC1).[6][7][8] This “competition” by ansuvimab prevents Ebola virus from binding to NPC1 and “neutralizes” the virus’s ability to infect the targeted cell.[6]
Effector function
Antibodies have antigen-binding fragment (Fab) regions and constant fragment (Fc) regions. The Neutralization of virus infection occurs when the Fab regions of antibodies binds to virus antigen(s) in a manner that blocks infection. Antibodies are also able to “kill” virus particles directly and/or kill infected cells using antibody-mediated “effector functions” such as opsonization, complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity and antibody-dependent phagocytosis. These effector functions are contained in the Fc region of antibodies, but is also dependent on binding of the Fab region to antigen. Effector functions also require the use of complement proteins in serum or Fc-receptor on cell membranes. Ansuvimab has been found to be capable of killing cells by antibody-dependent cell-mediated cytotoxicity.[3] Other functional killing tests have not been performed.
Ansuvimab has also shown success with lowering the mortality rate from ~70% to about 34%. In August 2019, Congolese health authorities, the World Health Organization, and the U.S. National Institutes of Health promoted the use of ansuvimab, alongside REGN-EB3, a similar Regeneron-produced monoclonal antibody treatment, over other treatments yielding higher mortality rates, after ending clinical trials during the outbreak.[13][14]
In an experiment described in the 2016 paper, rhesus macaques were infected with Ebola virus and treated with a combination of ansuvimab and another antibody isolated from the same subject, mAb100. Three doses of the combination were given once a day starting 1 day after the animals were infected. The control animal died and the treated animals all survived.[3]
Ansuvimab monotherapy
In a second experiment described in the 2016 paper, rhesus macaques were infected with Ebola virus and only treated with ansuvimab. Three doses of ansuvimab were given once a day starting 1 day or 5 days after the animals were infected. The control animals died and the treated animals all survived.[3] Unpublished data referred to in a publication of the 2018 Phase I clinical trial results of ansuvimab, reported that a single infusion of ansuvimab provided full protection of rhesus macaques and was the basis of the dosing used for human studies.[5][4]
Experimental use in the Democratic Republic of Congo
During the 2018 Équateur province Ebola outbreak, ansuvimab was requested by the Democratic Republic of Congo (DRC) Ministry of Public Health. Ansuvimab was approved for compassionate use by the World Health OrganizationMEURI ethical protocol and at DRC ethics board. Ansuvimab was sent along with other therapeutic agents to the outbreak sites.[19][20][11] However, the outbreak came to a conclusion before any therapeutic agents were given to patients.[11]
Approximately one month following the conclusion of the Équateur province outbreak, a distinct outbreak was noted in Kivu in the DRC (2018–20 Kivu Ebola outbreak). Once again, ansuvimab received approval for compassionate use by WHO MEURI and DRC ethic boards and has been given to many patients under these protocols.[11] In November 2018, the Pamoja Tulinde Maisha (PALM [together save lives]) open-label randomized clinical control trial was begun at multiple treatment units testing ansuvimab, REGN-EB3 and remdesivir to ZMapp. Despite the difficulty of running a clinical trial in a conflict zone, investigators have enrolled 681 patients towards their goal of 725. An interim analysis by the Data Safety and Monitoring Board (DSMB) of the first 499 patient found that ansuvimab and REGN-EB3 were superior to the comparator ZMapp. Overall mortality of patients in the ZMapp and remdesivir groups were 49% and 53% compared to 34% and 29% for ansuvimab and REGN-EB3. When looking at patients who arrived early after disease symptoms appeared, survival was 89% for ansuvimab and 94% for REGN-EB3. While the study was not powered to determine whether there is any difference between REGN-EB3 and ansuvimab, the survival difference between those two therapies and ZMapp was significant. This led to the DSMB halting the study and PALM investigators dropping the remdesivir and ZMapp arms from the clinical trial. All patients in the outbreak who elect to participate in the trial will now be given either ansuvimab or REGN-EB3.[21][22][13][12]
On December 21, 2020, the US Food and Drug Administration approved Ebanga (ansuvimab-zykl) for the treatment for Zaire ebolavirus (Ebolavirus) infection in adults and children. Ebanga had been granted US Orphan Drug designation and Breakthrough Therapy designations. Ansuvimab is a human IgG1 monoclonal antibody that binds and neutralizes the virus.
The safety and efficacy of Ebanga were evaluated in the multi-center, open-label, randomized controlled PALM trial. In this study, 174 participants (120 adults and 54 pediatric patients) with confirmed Ebolavirus infection received Ebanga intravenously as a single 50 mg/kg infusion and 168 participants (135 adults and 33 pediatric patients) received an investigational control. The primary efficacy endpoint was 28-day mortality. Of the 174 patients who received Ebanga, 35.1% died after 28 days, compared to 49.4% of the 168 patients who received a control.
Ebanga is the 12th antibody therapeutic to be granted a first approval in the US or EU during 2020.
^ Jump up to:abcdef Clinical trial number NCT03478891 for “Safety and Pharmacokinetics of a Human Monoclonal Antibody, VRC-EBOMAB092-00-AB (MAb114), Administered Intravenously to Healthy Adults” at ClinicalTrials.gov
RNA, (Gm-sp-Am-sp-Cm-Um-Um-Um-(2′-deoxy-2′-fluoro)C-Am-(2′-deoxy-2′-fluoro)U-(2′-deoxy-2′-fluoro)C-(2′-deoxy-2′-fluoro)C-Um-Gm-Gm-Am-Am-Am-Um-Am-Um-Am), 3′-[[(2S,4R)-1-[29-[[2-(acetylamino)-2-deoxy-β-D-galactopyranosyl]oxy]-14,14-bis[[3-[[3-[[5-[[2-(acetylamino)-2-deoxy-β-D-galactopyranosyl]oxy]-1-oxopentyl]amino]propyl]amino]-3-oxopropoxy]methyl]-1,12,19,25-tetraoxo-16-oxa-13,20,24-triazanonacos-1-yl]-4-hydroxy-2-pyrrolidinyl]methyl hydrogen phosphate], complex with RNA (Um-sp-(2′-deoxy-2′-fluoro)A-sp-Um-Am-Um-(2′-deoxy-2′-fluoro)U-Um-(2′-deoxy-2′-fluoro)C-(2′-deoxy-2′-fluoro)C-Am-Gm-Gm-Am-(2′-deoxy-2′-fluoro)U-Gm-(2′-deoxy-2′-fluoro)A-Am-Am-Gm-Um-Cm-sp-Cm-sp-Am) (1:1)
Nucleic Acid Sequence
Sequence Length: 44, 23, 2115 a 8 c 7 g 14 umultistranded (2); modified
OXLUMO is supplied as a sterile, preservative-free, clear, colorless-to-yellow solution for subcutaneous administration containing the equivalent of 94.5 mg of lumasiran (provided as lumasiran sodium) in 0.5 Ml of water for injection and sodium hydroxide and/or phosphoric acid to adjust the pH to ~7.0.
Lumasiran An investigational RNAi Therapeutic for Primary Hyperoxaluria Type 1 (PH1)
Overview • Lumasiran (ALN-GO1) is an investigational, subcutaneously administered (under the skin) RNA interference (RNAi) therapeutic targeting glycolate oxidase (GO) in development for the treatment of primary hyperoxaluria type 1 (PH1).
• PH1 is a rare, life-threatening disease that can cause serious damage to kidneys and progressively to other organs.1
• PH1 is characterized by the pathologic overproduction of oxalate by the liver. Oxalate is an end product of metabolism that, when in excess, is toxic and accumulates in the kidneys forming calcium oxalate crystals.1,2
• Symptoms of PH1 are often associated with recurrent kidney stones and include flank pain, urinary tract infections, painful urination, and blood in the urine.2,3
• Currently, the only curative treatment is a liver transplant, to correct the metabolic defect, combined with a kidney transplant, to replace the terminally damaged kidneys.1,3 Clinical Development
• The safety and efficacy of lumasiran are being evaluated in a randomized, double-blind, placebo-controlled, global, multicenter Phase 3 study of approximately 30 PH1 patients, called ILLUMINATE-A (NCT03681184).
• The primary endpoint is percent change in 24-hour urinary oxalate excretion from baseline to Month 6.
• Key secondary and exploratory endpoints in ILLUMINATE-A will evaluate additional measures of urinary oxalate, estimated glomerular filtration rate (eGFR), safety, and tolerability.
Regulatory Designations • Breakthrough Therapy Designation by the U.S. Food and Drug Administration (FDA) • Priority Medicines (PRIME) Designation from the European Medicines Agency (EMA) • Orphan Drug Designations in both the U.S. and the European Union
Tepezza (teprotumumab-trbw) is a fully human monoclonal antibody (mAb) and a targeted inhibitor of the insulin-like growth factor 1 receptor (IGF-1R) for the treatment of active thyroid eye disease (TED).
FDA Approves Tepezza (teprotumumab-trbw) for the Treatment of Thyroid Eye Disease (TED) – January 21, 2020
Today, the U.S. Food and Drug Administration (FDA) approved Tepezza (teprotumumab-trbw) for the treatment of adults with thyroid eye disease, a rare condition where the muscles and fatty tissues behind the eye become inflamed, causing the eyes to be pushed forward and bulge outwards (proptosis). Today’s approval represents the first drug approved for the treatment of thyroid eye disease.
“Today’s approval marks an important milestone for the treatment of thyroid eye disease. Currently, there are very limited treatment options for this potentially debilitating disease. This treatment has the potential to alter the course of the disease, potentially sparing patients from needing multiple invasive surgeries by providing an alternative, non surgical treatment option,” said Wiley Chambers, M.D., deputy director of the Division of Transplant and Ophthalmology Products in the FDA’s Center for Drug Evaluation and Research. “Additionally, thyroid eye disease is a rare disease that impacts a small percentage of the population, and for a variety of reasons, treatments for rare diseases are often unavailable. This approval represents important progress in the approval of effective treatments for rare diseases, such as thyroid eye disease.”
Thyroid eye disease is associated with the outward bulging of the eye that can cause a variety of symptoms such as eye pain, double vision, light sensitivity or difficulty closing the eye. This disease impacts a relatively small number of Americans, with more women than men affected. Although this condition impacts relatively few individuals, thyroid eye disease can be incapacitating. For example, the troubling ocular symptoms can lead to the progressive inability of people with thyroid eye disease to perform important daily activities, such as driving or working.
Tepezza was approved based on the results of two studies (Study 1 and 2) consisting of a total of 170 patients with active thyroid eye disease who were randomized to either receive Tepezza or a placebo. Of the patients who were administered Tepezza, 71% in Study 1 and 83% in Study 2 demonstrated a greater than 2 millimeter reduction in proptosis (eye protrusion) as compared to 20% and 10% of subjects who received placebo, respectively.
The most common adverse reactions observed in patients treated with Tepezza are muscle spasm, nausea, alopecia (hair loss), diarrhea, fatigue, hyperglycemia (high blood sugar), hearing loss, dry skin, dysgeusia (altered sense of taste) and headache. Tepezza should not be used if pregnant, and women of child-bearing potential should have their pregnancy status verified prior to beginning treatment and should be counseled on pregnancy prevention during treatment and for 6 months following the last dose of Tepezza.
The FDA granted this application Priority Review, in addition to Fast Track and Breakthrough Therapy Designation. Additionally, Tepezza received Orphan Drug designation, which provides incentives to assist and encourage the development of drugs for rare diseases or conditions. Development of this product was also in part supported by the FDA Orphan Products Grants Program, which provides grants for clinical studies on safety and efficacy of products for use in rare diseases or conditions.
The FDA granted the approval of Tepezza to Horizon Therapeutics Ireland DAC.
Teprotumumab (RG-1507), sold under the brand name Tepezza, is a medication used for the treatment of adults with thyroid eye disease, a rare condition where the muscles and fatty tissues behind the eye become inflamed, causing the eyes to be pushed forward and bulge outwards (proptosis).[1]
The most common adverse reactions observed in people treated with teprotumumab-trbw are muscle spasm, nausea, alopecia (hair loss), diarrhea, fatigue, hyperglycemia (high blood sugar), hearing loss, dry skin, dysgeusia (altered sense of taste) and headache.[1] Teprotumumab-trbw should not be used if pregnant, and women of child-bearing potential should have their pregnancy status verified prior to beginning treatment and should be counseled on pregnancy prevention during treatment and for six months following the last dose of teprotumumab-trbw.[1]
It is a human monoclonal antibody developed by Genmab and Roche. It binds to IGF-1R.
Teprotumumab was first investigated for the treatment of solid and hematologic tumors, including breast cancer, Hodgkin’s and non-Hodgkin’s lymphoma, non-small cell lung cancer and sarcoma.[2][3] Although results of phase I and early phase II trials showed promise, research for these indications were discontinued in 2009 by Roche. Phase II trials still in progress were allowed to complete, as the development was halted due to business prioritization rather than safety concerns.
Teprotumumab was subsequently licensed to River Vision Development Corporation in 2012 for research in the treatment of ophthalmic conditions. Horizon Pharma (now Horizon Therapeutics, from hereon Horizon) acquired RVDC in 2017, and will continue clinical trials.[4] It is in phase III trials for Graves’ ophthalmopathy (also known as thyroid eye disease (TED)) and phase I for diabetic macular edema.[5] It was granted Breakthrough Therapy, Orphan Drug Status and Fast Track designations by the FDA for Graves’ ophthalmopathy.[6]
In a multicenter randomized trial in patients with active Graves’ ophthalmopathy Teprotumumab was more effective than placebo in reducing the clinical activity score and proptosis.[7] In February 2019 Horizon announced results from a phase 3 confirmatory trial evaluating teprotumumab for the treatment of active thyroid eye disease (TED). The study met its primary endpoint, showing more patients treated with teprotumumab compared with placebo had a meaningful improvement in proptosis, or bulging of the eye: 82.9 percent of teprotumumab patients compared to 9.5 percent of placebo patients achieved the primary endpoint of a 2 mm or more reduction in proptosis (p<0.001). Proptosis is the main cause of morbidity in TED. All secondary endpoints were also met and the safety profile was consistent with the phase 2 study of teprotumumab in TED.[8] On 10th of July 2019 Horizon submitted a Biologics License Application (BLA) to the FDA for teprotumumab for the Treatment of Active Thyroid Eye Disease (TED). Horizon requested priority review for the application – if so granted (FDA has a 60-day review period to decide) it would result in a max. 6 month review process.[9]
Teprotumumab-trbw was approved for use in the United States in January 2020, for the treatment of adults with thyroid eye disease.[1]
Teprotumumab-trbw was approved based on the results of two studies (Study 1 and 2) consisting of a total of 170 patients with active thyroid eye disease who were randomized to either receive teprotumumab-trbw or a placebo.[1] Of the subjects who were administered Tepezza, 71% in Study 1 and 83% in Study 2 demonstrated a greater than two millimeter reduction in proptosis (eye protrusion) as compared to 20% and 10% of subjects who received placebo, respectively.[1]
Today, the U.S. Food and Drug Administration granted approval to Givlaari (givosiran) for the treatment of adult patients with acute hepatic porphyria, a genetic disorder resulting in the buildup of toxic porphyrin molecules which are formed during the production of heme (which helps bind oxygen in the blood).
“This buildup can cause acute attacks, known as porphyria attacks, which can lead to severe pain and paralysis, respiratory failure, seizures and mental status changes. These attacks occur suddenly and can produce permanent neurological damage and death,” said Richard Pazdur, M.D., director of the FDA’s Oncology Center of Excellence and acting director of the Office of Oncologic Diseases in the FDA’s Center for Drug Evaluation and Research. “Prior to today’s approval, treatment options have only provided partial relief from the intense unremitting pain that characterizes these attacks. The drug approved today can treat this disease by helping to reduce the number of attacks that disrupt the lives of patients.”
The approval of Givlaari was based on the results of a clinical trial of 94 patients with acute hepatic porphyria. Patients received a placebo or Givlaari. Givlaari’s performance was measured by the rate of porphyria attacks that required hospitalizations, urgent health care visits or intravenous infusion of hemin at home. Patients who received Givlaari experienced 70% fewer porphyria attacks compared to patients receiving a placebo.
Common side effects for patients taking Givlaari were nausea and injection site reactions. Health care professionals are advised to monitor patients for anaphylactic (allergic) reaction and renal (kidney) function. Patients should have their liver function tested before and periodically during treatment.
The FDA granted this application Breakthrough Therapy designation and Priority Review designation. Givlaari also received Orphan Drug designation, which provides incentives to assist and encourage the development of drugs for rare diseases. The FDA granted the approval of Givlaari to Alnylam Pharmaceuticals.
Pfizer is developing ritlecitinib, an irreversible, covalent and selective dual JAK3/TEC inhibitor, for treating AA, RA, vitiligo and inflammatory bowel diseases, including UC and CD. In July 2021, this drug was reported to be in phase 3 clinical development.
PF-06651600 is a potent and selective JAK3 inhibitor. PF-06651600 is a potent and low clearance compound with demonstrated in vivo efficacy. The favorable efficacy and safety profile of this JAK3-specific inhibitor PF-06651600 led to its evaluation in several human clinical studies. JAK3 was among the first of the JAKs targeted for therapeutic intervention due to the strong validation provided by human SCID patients displaying JAK3 deficiencies
Pfizer has established a leading kinase research capability with multiple unique kinase inhibitors in development as potential medicines. PF-06651600 is a highly selective and orally bioavailable Janus Kinase 3 (JAK3) inhibitor that represents a potential immunomodulatory therapy. With the favorable efficacy, safety profile, and ADME properties, this JAK3-specific covalent inhibitor has been under clinical investigation for the treatment of alopecia areata, rheumatoid arthritis, Crohn’s disease, and ulcerative colitis. Supported by positive results from a Phase 2 study, 1 was granted Breakthrough Therapy designation by the FDA on Sept. 5, 2018 for treatment of alopecia areata.
SYN
PAPER
J. Med. Chem.2017, 60 (5), 1971–1993, DOI: 10.1021/acs.jmedchem.6b01694
A scalable process for PF-06651600 (1) has been developed through successful enabling of the first generation syntheis. The synthesis highlights include the following: (1) replacement of costly PtO2 with a less expensive 5% Rh/C catalyst for a pyridine hydrogenation, (2) identification of a diasteroemeric salt crystallization to isolate the enantiomerically pure cis-isomer directly from a racemic mixture of cis/trans isomers, (3) a high yielding amidation via Schotten–Baumann conditions, and (4) critical development of a reproducible crystallization procedure for a stable crystalline salt (1·TsOH), which is suitable for long-term storage and tablet formulation. All chromatographic purifications, including two chiral SFC chromatographic separations, were eliminated. Combined with other improvements in each step of the synthesis, the overall yield was increased from 5% to 14%. Several multikilogram batches of the API have been delivered to support clinical studies.
1 -((2S,5R)-5-((7H-Pyrrolo[2,3-d]pyrimidin-4-yl)amino)-2-methylpiperidin-1 -yl)prop-2-en-1 -one has the structural formula:
The synthesis of 1 -((2S,5R)-5-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)-2-methylpiperidin-1 -yl)prop-2-en-1 -one is described in WO2015/083028, commonly assigned to the assignee of the present invention and which is incorporated herein by reference in its entirety. 1 -((2S,5R)-5-((7H-Pyrrolo[2,3-d]pyrimidin-4-yl)amino)-2-methylpiperidin-1 -yl)prop-2-en-1 -one is useful as an inhibitor of protein kinases, such as the enzyme Janus Kinase (JAK) and as such is useful therapy as an immunosuppressive agent for organ transplants, xeno transplantation, lupus, multiple sclerosis, rheumatoid arthritis, psoriasis, Type I diabetes and complications from diabetes, cancer, asthma, atopic dermatitis, autoimmune thyroid disorders, ulcerative colitis, Crohn’s disease, alopecia, vitiligo, Alzheimer’s disease, leukemia and other indications where immunosuppression would be desirable. See ACS Chem. Biol. , 2016, 11 (12), pp 3442-3451 . The present invention relates to a novel p-toluenesulfonic acid salt and crystalline solid form of the said salt of 1 -((2S,5R)-5-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)-2-methylpiperidin-1 -yl)prop-2-en-1 -one that demonstrate improved properties for use in a pharmaceutical dosage form, particularly for oral dosage forms.
Preparations
Scheme 1. Synthesis of 1
Scheme 2. Alternate Synthesis of Intermediates 7 and 10
1. K2C03, MIBK/water 1. H2, H2O
2. EtOAc, aq. NaCI Pd(OH)2/C (wet)
3. MeOH, H20 2. NaOH, MeOH
7 + 8 – ► 9 – – 10 . H2O
89% 89%
Scheme 3. First Alternate Preparation of 1
Scheme 4. Second Alternate Preparation of 1
Preparation 1
ferf-Butyl (6-methylpyridin-3-yl)carbamate (3). To a 3000L reactor was charged 2 (72.00 kg, 665.8 mol) and THF (660 kg). A solution of NH4CI (1 .07 kg , 20 mol) in water (72 kg, 4000 mol) was added. The mixture was heated to 57 °C and Di-f-butyl dicarbonate (220.0 kg, 1003 mol) was added slowly with rinse of THF (45 kg) while maintaining the temperature between 55 – 60 °C. The mixture was stirred at 55 – 60 °C for 10 h. Upon reaction completion, the slurry was cooled to 20 °C and ethyl acetate (654 kg) and water (367 kg) were added. The organic phase was separated, washed by water (2 x 360 kg) and stirred with active carbon (22 kg) for 5 h. The mixture was filtered through a layer of diatomaceous earth (22 kg) with THF rinse and the filtrates were concentrated under vacuum at <40 °C to a residual volume of ~370 L. n-Heptane (500 kg) was added slowly over 1 h and the resulting slurry was cooled to 20 °C and stirred for 2 h. The solid was collected by centrifuge with an n-heptane wash (420 kg), then dried at 45 °C under vacuum for 20 h to give 3 (131 .15 kg, 629.7 mol) as a white powder in 94.5% yield. HPLC purity: 99.9%. 1H NMR (400 MHz, DMSO-c/6): d ppm 9.42 (brs, 1 H), 8.48 (d, J = 1 .9 Hz, 1 H), 7.75 (d, J = 8.6 Hz, 1 H), 7.13 (d, J = 8.6 Hz, 1 H), 2.38 (s, 3H), 1 .49 (s, 9H). 13C NMR (100 MHz, DMSO-d6y d ppm 153.34, 151 .56, 139.75, 134.13, 126.10, 123.09, 79.87, 28.56, 23.70. HRMS (ESI) m/z: calculated for C11H17N2O2 [M + H]+ 209.1290; observed 209.1285.
Preparation 2
ferf-Butyl (6-methylpiperidin-3-yl)carbamate (rac-4). To a 3000L reactor was charged 3 (137.0 kg, 667.8 mol), ethanol (988 kg) and acetic acid (139 kg). The reactor was purged with nitrogen three times and 5 wt% Rhodium on carbon (wet, 27.4 kg, 20 wt% loading relative to 3) was added. The reactor was purged with nitrogen three times and then with hydrogen three times. The hydrogen pressure was adjusted to 0.34 – 0.38 MPa and the reactor temperature was adjusted to 47 °C. The mixture was stirred at 45 – 60 °C under hydrogen pressure at 0.34 – 0.38 MPa for 10 h. Upon reaction completion, the reactor was cooled to 20 °C and flushed with nitrogen. The mixture was filtered through a layer of diatomaceous earth (20 kg) with an ethanol rinse (1320 kg) and the filtrates were concentrated under vacuum at <50 °C to a residual volume of ~350 L. n-Heptane (571 kg) was added and the mixture was concentrated under vacuum at <50 °C to a residual volume of~350 L. This operation was repeated twice until the residual acetic acid <8.0%. Ethanol (672 kg) was added and the mixture was concentrated under vacuum at <50 °C to a residual volume of ~350 L. This operation was repeated twice until the residual n-heptane was <0.2% and water was <0.2%. Ethanol (889 kg) was added and the solution (1254 kg) was transferred to drums for use in the subsequent classical resolution step. Achiral HPLC assay indicated that the solution contained 10.8 wt% of the total reduced product (rac-4) in 96% mass recovery and chiral SFC showed that the solution contained 36.3% of the desired stereoisomer cis-4.
Preparation 3
ferf-Butyl ((3R,6S)-6-methylpiperidin-3-yl)carbamate (R)-2-(3,5-dinitrobenzamido)-2-phenylacetic acid salt (15). To a 2000L reactor (R1 ) was charged rac-4 as a 10.8 wt% solution in ethanol (620.5 kg, ~312.7 mol. of all 4 isomers). The solution was concentrated under vacuum at <45 °C to a residual volume of ~210 L and then cooled to 20 °C. To a 3000 L reactor (R2) was charged (R)-2-(3,5-dinitrobenzamido)-2-phenylacetic acid 14 (47.0 kg, 136.1 mol) and ethanol (1 125 kg). With high speed agitation, reactor R2 was heated to 70 °C, stirred at 68 – 70 °C for ~2 h to dissolve all solid 14, and then seeded with crystalline 15 (1 1 g). The solution containing 4 in reactor R1 was slowly transferred to reactor R2 over 30 min with ethanol rinse (160 kg). Reactor R2 was stirred at ~74 °C for 3 h and then cooled to 22 °C with a linear cooling rate over a period of 5 h and stirred for 16 h. The solid was collected by centrifuge with ethanol wash (2 x 200 kg). The wet cake (with 97.1 % e.e.) was charged back to reactor R2. The slurry was heated to 74 °C and the mixture was stirred for 17 h. The mixture was then cooled to 22 °C with a linear cooling rate over a period of 5 h and stirred for 4 h. The solid was collected by centrifuge with ethanol wash (2 x 200 kg) and dried at 35 °C under vacuum for 25 h to give 15 (56.05 kg, 100.2 mol) as a white powder in 30.7% yield over 2 steps. Chiral HPLC purity: 99.1 %. 1H NMR (400 MHz, DMSO-d6): d ppm 9.46 (d, J = 7.0 Hz, 1 H), 9.07 (d, J = 2.2 Hz, 2H), 8.96 (t, J = 2.2 Hz, 1 H), 7.49 (d, J = 7.3 Hz, 2H), 7.30 (t, J = 7.3 Hz, 2H), 7.23 (t, J = 7.3, 1 H), 7.1 1 (m, 1 H), 5.31 (d, J = 7.0 Hz, 1 H), 3.66 (m, 1 H), 2.98 (m, 3H), 1 .63 (m, 2H), 1 .45 (m, 2H), 1 .40 (s, 9H), 1 .1 1 (d, J = 6.7 Hz, 3H). 13C NMR (100 MHz, DMSO-d6): d ppm 172.71 , 161 .71 , 155.42, 148.51 , 141 .27, 137.70, 128.29, 128.25, 128.02, 127.05, 121 .12, 78.49, 59.74, 50.66, 46.29, 43.34, 28.66, 26.88, 26.1 1 , 18.60.
Preparation 4
Benzyl (2S,5R)-5-amino-2-methylpiperidine-1 -carboxylate hydrochloride (7»HCI) -telescoped process. To a 2000L reactor was charged 15 (70.0 kg, 125 mol) and MTBE (500 kg). The mixture was cooled to 12 °C and 6.9 wt% aqueous NaOH solution (378 kg, 652 mol) was added slowly while maintaining the temperature between 10 – 25 °C. The mixture was stirred at 18 °C for 1 h . The organic phase was separated and washed with 3.8 wt% aqueous NaOH solution (2 x 221 kg) and then 25 wt% aqueous NaCI solution (2 x 220 kg). The organic layer (containing the free base cis-4) was concentrated under vacuum at <40 °C to a residual volume of ~300 L and then cooled to 20 °C. NaHCOs (53 kg, 632 mol) and water (200 kg) were added and the mixture was cooled to 7 °C. Benzyl chloroformate (32.30 kg, 189.3 mol) was added slowly while maintaining the temperature between 5 – 20 °C. The mixture was stirred at 17 °C for 20 h. Upon reaction completion, the mixture was cooled to 12 °C, 25 wt% aqueous ammonium hydroxide solution (79 kg, 1 160 mol) was added slowly while maintaining the temperature between 10 – 20 °C, and the mixture was stirred at 15 °C for 1 h. The organic phase was separated and washed with 25 wt% aqueous NaCI solution (3 x 90 kg). The organic layer (containing 5) was concentrated under vacuum at <45 °C to a residual volume of ~150 L. Isopropyl acetate (310 kg) was added and the mixture was concentrated under vacuum at <45 °C to a residual volume of ~150 L. This operation was repeated twice to meetthe criteria of water <0.1 % (by KF). Isopropyl acetate (130 kg) was then added and the mixture was cooled to -3 °C. 4-5N HCI in methanol (181 kg, ~730 mol) was added slowly while maintaining the temperature between -5 to 5 °C, and the mixture was stirred at 3 °C for 12 h. Upon reaction completion, the mixture was cooled to -3 °C and MTBE (940 kg) was added slowly while maintaining the temperature between -5 to 5 °C. The resulting slurry was stirred at 3 °C for 3 h. The solid was collected by centrifuge with MTBE washes (4 x 70 kg), and then dried at 45 °C under vacuum for 20 h to give 7»HCI (28.60 kg, 100.4 mol) as a white powder in 80.3% yield. Achiral HPLC purity: 100%. Chiral SFC purity: 99.8% e.e. 1H NMR (400 MHz, DMSO-d6): d ppm 8.36 (brs, 3H), 7.37 (m, 5H), 5.09 (s, 2H), 4.31 (m, 1 H), 4.16 (d, J = 8.2 Hz, 1 H), 3.00 (m, 2H), 1 .82 (m, 2H), 1 .59 (m, 2H), 1 .1 1 (d, J = 7.0 Hz, 3H). 13C NMR (100 MHz, DMSO-d6): d ppm 154.71 , 137.24, 128.92, 128.34, 128.00, 66.89, 47.20, 45.66, 40.68, 28.16, 23.02, 15.67. HRMS (ESI) m/z. calculated for C H N O [M + H]+ 249.1603; observed 249.1598.
Preparation 5
Benzyl (2S, 5R)-5-((2-chloro-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)-2 -methyl-piperidine-1 -carboxylate (9). To a 2000L reactor was charged 7»HCI (88.6 kg, 31 1 .12 mol), 8 (56.0 kg, 298 mol), K2C03 (133.0 kg, 962.3 mol), water (570 kg) and MIBK (101 kg). The mixture was heated to 90 °C and stirred at this temperature for 22 h. Upon reaction completion, the mixture was cooled to 56 °C and ethyl acetate (531 kg) was added. After cooling the mixture to 22 °C, the organic phase was separated, washed with water (570 kg) and concentrated under vacuum at <40 °C to a residual volume of ~220 L. Methanol (360 kg) was added slowly over a period of 1 h and the mixture concentrated under vacuum at <50 °C to a residual volume of ~220 L. This operation was repeated three times until residual MIBK reached <5 wt%. Methanol (270 kg) was added, followed by seeding with 9 (120 g). The mixture was stirred at 22 °C for >4 h and water (286 kg) was added slowly over 4 h. The slurry was stirred for 10 h and the solid was then collected by centrifuge. The wet cake (165.6 kg) was charged back to a clean reactor and water (896 kg) was added. The slurry was heated to 55 °C and stirred at this temperature for 7 h; and then cooled to 22 °C and stirred at this temperature for 2 h. The solid was collected by centrifuge with water wash (3 x 170 kg) and dried at 55 °C under vacuum for 20 h to give 9 (106.62 kg, 266.6 mol) as a white powder in 89.5% yield. Achiral HPLC purity: 99.7%. 1H NMR (400 MHz, DMSO-d6): d ppm 1 1 .71 (brs, 1 H), 7.72 (d, J = 7.9 Hz, 1 H), 7.38 (m, 5H), 7.10 (s, 1 H), 6.57 (d, J = 2.7 Hz, 1 H), 5.1 1 (m, 2H), 4.39 (m, 1 H), 4.17 (m, 1 H), 4.01 (m, 1 H), 3.36 (s, 2H), 2.77 (m, 1 H), 1 .73-1 .81 (m, 4H), 1 .16 (d, J = 6.6 Hz, 3H). 13C NMR (100 MHz, DMSO-d6): d ppm 156.65, 154.74, 153.04, 151 .31 , 137.43, 128.89, 128.27, 127.96, 122.13, 101 .65, 99.51 , 66.75, 49.10, 47.32, 45.64, 42.98, 29.05, 25.08. HRMS (ESI) m/z. calculated for C20H22CIN5O2 [M + H]+ 400.1540; observed 400.1535.
Preparation 6
N-((3R,6S)-6-methylpiperidin-3-yl)-7H-pyrrolo[2,3-dlpyrimidin-4-amine monohydrate (10·H2O) To a 1600L reactor was charged water (570 kg). The reactor was purged with nitrogen three times. 10% Pd(OH)2/C (wet, 3.2 kg) and 9 (53.34 kg, 133.2 mol) were added with water rinses (2 x 55 kg). The reactor was purged with nitrogen three times and then with hydrogen three times. The hydrogen pressure was adjusted to 0.34 – 0.38 MPa and the reactor temperature was adjusted to 77 °C. The mixture was stirred at 75 – 80 °C under a hydrogen pressure of 0.34 -0.38 MPa for 10 h. Upon reaction completion, the reactor was cooled to 20 °C and purged with nitrogen. The mixture was filtered through a layer of diatomaceous earth (8 kg) with a water rinse (460 kg), and the filtrates were transferred to a 3000L reactor. Methanol (260 kg) was added, followed by slow addition of 50 wt% aqueous sodium hydroxide (12.0 kg , 150 mol) while maintaining the temperature between 15 – 25 °C. The slurry was heated to 55 °C and stirred for 2 h; then cooled to 22 °C and stirred for 10 h. The solid was collected by centrifuge with a 10:1 water/methanol wash (3 x 1 10 kg) and then dried at 55 °C under vacuum for 20 h to give 10·H2O (30.90 kg, 266.6 mol) as a white powder in 89.1 % yield. Achiral HPLC purity: 99.7%. Chiral SFC
1 -((2S,5R)-5-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)-2-methylpiperidin-1 -yl)prop-2-en-1 -one (1 ). To a 100L reactor was charged water (18.0 L), 10·H2q (3.60 kg, 14.4 mol) and THF (36.0 L). The mixture was heated to 53 °C and stirred for 15 min to dissolve all the solids. The solution was then cooled to 18 °C and K3PO4 (6.38 kg, 30.1 mol) was added. The mixture was stirred at 18 °C for 10 min to dissolve all the solids, and then cooled to 10 °C. 3-Chloropropionyl chloride (2.20 kg, 17.3 mol) was added while maintaining the temperature <20 °C. The mixture was then stirred at 20 °C for 2 h. Upon reaction completion, 2 N aqueous NaOH solution (23.50 kg, 43.76 mol) was added while maintaining the temperature <25 °C. The mixture was stirred at 22 °C for >12 h until the elimination reaction was complete (11 <0.2%). KH2PO4 (10.32 kg, 75.8 mol) was added and the mixture was stirred at 20 °C for 10 min. The organic phase was separated and then washed with 23.5 wt% aqueous NaCI solution (2 x 8.5 kg). The isolated organic phase was concentrated under vacuum at <30 °C to a residual volume of ~10 L, whereupon MEK (39.6 L) was added. This operation was repeated once or twice until residual THF was <1 % and water was <2%. MgS04 (0.96 kg), Silica gel (4.90 kg) and Darco™ G-60 (0.48 kg) were added to the MEK solution, and the mixture was stirred at 20 °C for 1 h, then filtered through a layer of Diatomaceous Earth with a MEK rinse (76 L). The combined filtrates were concentrated under vacuum at <30 °C to a residual volume of ~8 L. The concentration of the residual solution was measured by qNMR, and the solution was transferred to a container with a rinse using the calculated amount of MEK to adjust the final concentration to 30 wt%. Thus, a 30 wt% solution of 1 in MEK (1 1 .09 kg, 1 1 .66 mol of 1) with 98.7% purity was obtained in 81 % yield, which was stored in a cold room (2 – 8 °C) for the next step.
Preparation 8
1 -((2S,5R)-5-((7H-pyrrolo[2,3-cdpyrimidin-4-yl)amino)-2-methylpiperidin-1 -yl)prop-2-en-1 -one p-toluenesulfonate (1»TsOH). To a 20L reactor was charged a 30 wt% solution of 1 in MEK (9.80 kg, 10.30 mol of 1) and silica gel (0.74 kg). The mixture was stirred at 22 °C for 15 min and filtered through a 0.45 micron Teflon cartridge filter with a MEK rinse (7.89 kg, 9.8 L), collecting in a 100L reactor. Water (1 .27 L) was added, followed by a solution of p-toluenesulfonic acid monohydrate (2.18 kg, 1 1 .3 mol) in MEK (4.75 kg, 5.9 L) with a MEK rinse (3.14 kg, 3.9 L), followed by the addition of 1 »TsOH seed (188 g, 0.41 mol). The mixture was stirred at 22 °C for
4 h to form a slurry and MEK (31 .56 kg, 39.2 L) was added slowly over a period of 3 h. The slurry was stirred at 22 °C for an additional 2 h and then filtered. The cake was washed with MEK (4.02 kg, 5 L) and then dried at 50 °C under vacuum for 10 h to give 1 »TsOH (4.41 kg, 9.64 mol) as a white powder in 89.6% yield (accounting for the amount of seed charged). Achiral HPLC purity: 99.6% with 0.22% of dimer 15. Chiral SFC purity: >99.7%. m.p. 199 °C. Rotomers observed for NMR spectroscopies. Ή NMR (400 MHz, DMSO-d6): d ppm 12.68 (brs, 1 H), 9.22 (brs, 1 H), 8.40 (s, 1 H), 7.50 (d, J = 8.2 Hz, 2H), 7.45 (m, 1 H), 7.12 (d, J = 8.2 Hz, 2H), 6.94 (d, J = 1 .2 Hz, 1 H), 6.84 (m, 1 H), 6.13 (m, 1 H), 5.70 (m, 1 H), 4.81 (m, 0.5H), 4.54 (m, 0.5H), 4.41 (m, 0.5H), 4.12 (m, 0.5H), 3.99 (m, 1 H), 3.15 (m, 0.5H), 2.82 (m, 0.5H), 2.29 (s, 3H), 1 .91 -1 .72 (m, 4H), 1 .24-1 .17 (m, 3H). 13C NMR (100 MHz, DMSO-c/6): d ppm 165.52, 165.13, 150.50, 145.64, 143.06, 138.48, 129.51 , 129.24, 128.67, 127.99, 127.73, 125.97, 125.02, 102.30, 49.53, 48.92, 47.27, 43.83, 42.96, 29.37, 28.41 , 25.22, 21 .28, 16.97, 15.51 . HRMS (ESI) m/z: calculated for Ci5H2oN50 [M + H]+ 286.1668; observed 286.1692.
Comparative Example
Preparation of 1 -((2S,5R)-5-((7H-Pyrrolo[2,3-d]pyrimidin-4-yl)amino)-2-methyl-piperidin-1 -yl)prop-2-en-1 -one Malonic Acid Salt (Form 1 )
A 250 ml_ round bottom flask was charged with 1 -((2S,5R)-5-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)-2-methylpiperidin-1 -yl)prop-2-en-1 -one (4.10 g, 14.4 mmol), MEK (Methyl Ethyl Ketone (15.0 ml_/g, 687 mmol, 49.5 g, 61 .5 ml_)). To the solution, malonic acid (0.950 equiv. 13.7 mmol, 1 .42 g) was added in one portion. The mixture was heated to 50 °C and stirred at 50 °C for 15min. The heating was turned off and the slurry was stirred for 16 hours. The resulting white slurry was filtered. The filter cake was washed with MEK (2 X 5 ml_) and dried in a vacuum oven (40 °C) for 2 hours give 1 -((2S,5R)-5-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)-2-methylpiperidin-1 -yl)prop-2-en-1 -one malonic acid salt (Form 1) (4.48 g, 1 1 .5 mmol, 4.48 g, 80.1 % Yield) as white powder.
PATENT
WO-2021136482
Improved process for preparing ritlecitinib (PF06651600), useful for treating alopecia areata (AA), rheumatoid arthritis (RA), Crohn’s disease (CD) and ulcerative colitis (UC). Also claims novel intermediates of ritlecitinib and their preparation method.
PATENT
CN111732591
crystalline polymorphic form of ritlecitinib L-tartrate. Jiangsu Alicorn focuses on developing generic, innovative and first-line drugs, whose website lists an undisclosed JAK inhibitor under its product’s list.
REFERENCES
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2: Robinette ML, Cella M, Telliez JB, Ulland TK, Barrow AD, Capuder K, Gilfillan S, Lin LL, Notarangelo LD, Colonna M. Jak3 deficiency blocks innate lymphoid cell development. Mucosal Immunol. 2018 Jan;11(1):50-60. doi: 10.1038/mi.2017.38. Epub 2017 May 17. PubMed PMID: 28513593; PubMed Central PMCID: PMC5693788.
3: Thorarensen A, Dowty ME, Banker ME, Juba B, Jussif J, Lin T, Vincent F, Czerwinski RM, Casimiro-Garcia A, Unwalla R, Trujillo JI, Liang S, Balbo P, Che Y, Gilbert AM, Brown MF, Hayward M, Montgomery J, Leung L, Yang X, Soucy S, Hegen M, Coe J, Langille J, Vajdos F, Chrencik J, Telliez JB. Design of a Janus Kinase 3 (JAK3) Specific Inhibitor 1-((2S,5R)-5-((7H-Pyrrolo[2,3-d]pyrimidin-4-yl)amino)-2-methylpiperidin-1-yl)prop -2-en-1-one (PF-06651600) Allowing for the Interrogation of JAK3 Signaling in Humans. J Med Chem. 2017 Mar 9;60(5):1971-1993. doi: 10.1021/acs.jmedchem.6b01694. Epub 2017 Feb 16. PubMed PMID: 28139931.
4: Telliez JB, Dowty ME, Wang L, Jussif J, Lin T, Li L, Moy E, Balbo P, Li W, Zhao Y, Crouse K, Dickinson C, Symanowicz P, Hegen M, Banker ME, Vincent F, Unwalla R, Liang S, Gilbert AM, Brown MF, Hayward M, Montgomery J, Yang X, Bauman J, Trujillo JI, Casimiro-Garcia A, Vajdos FF, Leung L, Geoghegan KF, Quazi A, Xuan D, Jones L, Hett E, Wright K, Clark JD, Thorarensen A. Discovery of a JAK3-Selective Inhibitor: Functional Differentiation of JAK3-Selective Inhibition over pan-JAK or JAK1-Selective Inhibition. ACS Chem Biol. 2016 Dec 16;11(12):3442-3451. Epub 2016 Nov 10. PubMed PMID: 27791347.
5: Walker G, Croasdell G. The European League Against Rheumatism (EULAR) – 17th Annual European Congress of Rheumatology (June 8-11, 2016 – London, UK). Drugs Today (Barc). 2016 Jun;52(6):355-60. doi: 10.1358/dot.2016.52.6.2516435. PubMed PMID: 27458612.
The U.S. Food and Drug Administration today approved Zulresso (brexanolone) injection for intravenous (IV) use for the treatment of postpartum depression (PPD) in adult women. This is the first drug approved by the FDA specifically for PPD.
The U.S. Food and Drug Administration today approved Zulresso (brexanolone) injection for intravenous (IV) use for the treatment of postpartum depression (PPD) in adult women. This is the first drug approved by the FDA specifically for PPD.
“Postpartum depression is a serious condition that, when severe, can be life-threatening. Women may experience thoughts about harming themselves or harming their child. Postpartum depression can also interfere with the maternal-infant bond. This approval marks the first time a drug has been specifically approved to treat postpartum depression, providing an important new treatment option,” said Tiffany Farchione, M.D., acting director of the Division of Psychiatry Products in the FDA’s Center for Drug Evaluation and Research. “Because of concerns about serious risks, including excessive sedation or sudden loss of consciousness during administration, Zulresso has been approved with a Risk Evaluation and Mitigation Strategy (REMS) and is only available to patients through a restricted distribution program at certified health care facilities where the health care provider can carefully monitor the patient.”
PPD is a major depressive episode that occurs following childbirth, although symptoms can start during pregnancy. As with other forms of depression, it is characterized by sadness and/or loss of interest in activities that one used to enjoy and a decreased ability to feel pleasure (anhedonia) and may present with symptoms such as cognitive impairment, feelings of worthlessness or guilt, or suicidal ideation.
Zulresso will be available only through a restricted program called the Zulresso REMS Program that requires the drug be administered by a health care provider in a certified health care facility. The REMS requires that patients be enrolled in the program prior to administration of the drug. Zulresso is administered as a continuous IV infusion over a total of 60 hours (2.5 days). Because of the risk of serious harm due to the sudden loss of consciousness, patients must be monitored for excessive sedation and sudden loss of consciousness and have continuous pulse oximetry monitoring (monitors oxygen levels in the blood). While receiving the infusion, patients must be accompanied during interactions with their child(ren). The need for these steps is addressed in a Boxed Warning in the drug’s prescribing information. Patients will be counseled on the risks of Zulresso treatment and instructed that they must be monitored for these effects at a health care facility for the entire 60 hours of infusion. Patients should not drive, operate machinery, or do other dangerous activities until feelings of sleepiness from the treatment have completely gone away.
The efficacy of Zulresso was shown in two clinical studies in participants who received a 60-hour continuous intravenous infusion of Zulresso or placebo and were then followed for four weeks. One study included patients with severe PPD and the other included patients with moderate PPD. The primary measure in the study was the mean change from baseline in depressive symptoms as measured by a depression rating scale. In both placebo controlled studies, Zulresso demonstrated superiority to placebo in improvement of depressive symptoms at the end of the first infusion. The improvement in depression was also observed at the end of the 30-day follow-up period.
The most common adverse reactions reported by patients treated with Zulresso in clinical trials include sleepiness, dry mouth, loss of consciousness and flushing. Health care providers should consider changing the therapeutic regimen, including discontinuing Zulresso in patients whose PPD becomes worse or who experience emergent suicidal thoughts and behaviors.
Approval of Zulresso was granted to Sage Therapeutics, Inc.
Allopregnanolone, also known as 5α-pregnan-3α-ol-20-one or 3α,5α-tetrahydroprogesterone (3α,5α-THP), as well as brexanolone (USAN),[1] is an endogenousinhibitorypregnaneneurosteroid[2] which has been approved by the FDA as a treatment for post-partum depression. It is synthesized from progesterone, and is a potent positive allosteric modulator of the action of γ-aminobutyric acid (GABA) at GABAA receptor.[2] Allopregnanolone has effects similar to those of other positive allosteric modulators of the GABA action at GABAA receptor such as the benzodiazepines, including anxiolytic, sedative, and anticonvulsant activity.[2][3][4] Endogenously produced allopregnanolone exerts a pivotal neurophysiological role by fine-tuning of GABAA receptor and modulating the action of several positive allosteric modulators and agonists at GABAA receptor.[5] The 21-hydroxylated derivative of this compound, tetrahydrodeoxycorticosterone (THDOC), is an endogenous inhibitory neurosteroid with similar properties to those of allopregnanolone, and the 3β-methyl analogue of allopregnanolone, ganaxolone, is under development to treat epilepsy and other conditions, including post-traumatic stress disorder (PTSD).[2]
Allopregnanolone acts as a highly potent positive allosteric modulator of the GABAA receptor.[2] While allopregnanolone, like other inhibitory neurosteroids such as THDOC, positively modulates all GABAA receptor isoforms, those isoforms containing δ subunitsexhibit the greatest potentiation.[7] Allopregnanolone has also been found to act as a positive allosteric modulator of the GABAA-ρ receptor, though the implications of this action are unclear.[8][9] In addition to its actions on GABA receptors, allopregnanolone, like progesterone, is known to be a negative allosteric modulator of nACh receptors,[10] and also appears to act as a negative allosteric modulator of the 5-HT3 receptor.[11] Along with the other inhibitory neurosteroids, allopregnanolone appears to have little or no action at other ligand-gated ion channels, including the NMDA, AMPA, kainate, and glycine receptors.[12]
Unlike progesterone, allopregnanolone is inactive at the nuclear progesterone receptor (nPR).[12] However, allopregnanolone can be intracellularly oxidized into 5α-dihydroprogesterone, which is an agonist of the nPR, and thus/in accordance, allopregnanolone does appear to have indirect nPR-mediated progestogenic effects.[13] In addition, allopregnanolone has recently been found to be an agonist of the newly discovered membrane progesterone receptors (mPR), including mPRδ, mPRα, and mPRβ, with its activity at these receptors about a magnitude more potent than at the GABAA receptor.[14][15] The action of allopregnanolone at these receptors may be related, in part, to its neuroprotective and antigonadotropic properties.[14][16] Also like progesterone, recent evidence has shown that allopregnanolone is an activator of the pregnane X receptor.[12][17]
Similarly to many other GABAA receptor positive allosteric modulators, allopregnanolone has been found to act as an inhibitor of L-type voltage-gated calcium channels (L-VGCCs),[18] including α1 subtypesCav1.2 and Cav1.3.[19] However, the threshold concentration of allopregnanolone to inhibit L-VGCCs was determined to be 3 μM (3,000 nM), which is far greater than the concentration of 5 nM that has been estimated to be naturally produced in the human brain.[19] Thus, inhibition of L-VGCCs is unlikely of any actual significance in the effects of endogenous allopregnanolone.[19] Also, allopregnanolone, along with several other neurosteroids, has been found to activate the G protein-coupled bile acid receptor (GPBAR1, or TGR5).[20] However, it is only able to do so at micromolar concentrations, which, similarly to the case of the L-VGCCs, are far greater than the low nanomolar concentrations of allopregnanolone estimated to be present in the brain.[20]
Increased levels of allopregnanolone can produce paradoxical effects, including negative mood, anxiety, irritability, and aggression.[29][30][31] This appears to be because allopregnanolone possesses biphasic, U-shaped actions at the GABAA receptor – moderate level increases (in the range of 1.5–2 nM/L total allopregnanolone, which are approximately equivalent to luteal phase levels) inhibit the activity of the receptor, while lower and higher concentration increases stimulate it.[29][30] This seems to be a common effect of many GABAA receptor positive allosteric modulators.[26][31] In accordance, acute administration of low doses of micronized progesterone (which reliably elevates allopregnanolone levels) has been found to have negative effects on mood, while higher doses have a neutral effect.[32]
Allopregnanolone is a pregnane (C21) steroid and is also known as 5α-pregnan-3α-ol-20-one, 3α-hydroxy-5α-pregnan-20-one, or 3α,5α-tetrahydroprogesterone (3α,5α-THP). It is very closely related structurally to 5-pregnenolone (pregn-5-en-3β-ol-20-dione), progesterone (pregn-4-ene-3,20-dione), the isomers of pregnanedione (5-dihydroprogesterone; 5-pregnane-3,20-dione), the isomers of 4-pregnenolone (3-dihydroprogesterone; pregn-4-en-3-ol-20-one), and the isomers of pregnanediol (5-pregnane-3,20-diol). In addition, allopregnanolone is one of four isomers of pregnanolone (3,5-tetrahydroprogesterone), with the other three isomers being pregnanolone (5β-pregnan-3α-ol-20-one), isopregnanolone(5α-pregnan-3β-ol-20-one), and epipregnanolone (5β-pregnan-3β-ol-20-one).
Derivatives
A variety of syntheticderivatives and analogues of allopregnanolone with similar activity and effects exist, including alfadolone (3α,21-dihydroxy-5α-pregnane-11,20-dione), alfaxolone (3α-hydroxy-5α-pregnane-11,20-dione), ganaxolone (3α-hydroxy-3β-methyl-5α-pregnan-20-one), hydroxydione (21-hydroxy-5β-pregnane-3,20-dione), minaxolone (11α-(dimethylamino)-2β-ethoxy-3α-hydroxy-5α-pregnan-20-one), Org 20599 (21-chloro-3α-hydroxy-2β-morpholin-4-yl-5β-pregnan-20-one), Org 21465 (2β-(2,2-dimethyl-4-morpholinyl)-3α-hydroxy-11,20-dioxo-5α-pregnan-21-yl methanesulfonate), and renanolone (3α-hydroxy-5β-pregnan-11,20-dione).
Allopregnanolone and the other endogenous inhibitory neurosteroids have short terminal half-lives and poor oral bioavailability, and for these reason, have not been pursued for clinical use as oral therapies, although development as a parenteral therapy for multiple indications has been carried out. However, syntheticanalogs with improved pharmacokineticprofiles have been synthesized and are being investigated as potential oral therapeutic agents.
In other studies of compounds related to allopregnanolone, exogenous progesterone, such as oral micronized progesterone (OMP), elevates allopregnanolone levels in the body with good dose-to-serum level correlations.[35] Due to this, it has been suggested that OMP could be described as a prodrug of sorts for allopregnanolone.[35] As a result, there has been some interest in using OMP to treat catamenial epilepsy,[36] as well as other menstrual cycle-related and neurosteroid-associated conditions. In addition to OMP, oral pregnenolonehas also been found to act as a prodrug of allopregnanolone,[37][38][39] though also of pregnenolone sulfate.[40]
^Bullock AE, Clark AL, Grady SR, et al. (June 1997). “Neurosteroids modulate nicotinic receptor function in mouse striatal and thalamic synaptosomes”. J. Neurochem. 68 (6): 2412–23. doi:10.1046/j.1471-4159.1997.68062412.x. PMID9166735.
^Wetzel CH, Hermann B, Behl C, et al. (September 1998). “Functional antagonism of gonadal steroids at the 5-hydroxytryptamine type 3 receptor”. Mol. Endocrinol. 12 (9): 1441–51. doi:10.1210/mend.12.9.0163. PMID9731711.
^Lamba V, Yasuda K, Lamba JK, et al. (September 2004). “PXR (NR1I2): splice variants in human tissues, including brain, and identification of neurosteroids and nicotine as PXR activators”. Toxicol. Appl. Pharmacol. 199 (3): 251–65. doi:10.1016/j.taap.2003.12.027. PMID15364541.
^Hu AQ, Wang ZM, Lan DM, et al. (July 2007). “Inhibition of evoked glutamate release by neurosteroid allopregnanolone via inhibition of L-type calcium channels in rat medial prefrontal cortex”. Neuropsychopharmacology. 32 (7): 1477–89. doi:10.1038/sj.npp.1301261. PMID17151597.
^ Jump up to:abKeitel V, Görg B, Bidmon HJ, et al. (November 2010). “The bile acid receptor TGR5 (Gpbar-1) acts as a neurosteroid receptor in brain”. Glia. 58 (15): 1794–805. doi:10.1002/glia.21049. PMID20665558.
^Rougé-Pont F, Mayo W, Marinelli M, Gingras M, Le Moal M, Piazza PV (July 2002). “The neurosteroid allopregnanolone increases dopamine release and dopaminergic response to morphine in the rat nucleus accumbens”. Eur. J. Neurosci. 16 (1): 169–73. doi:10.1046/j.1460-9568.2002.02084.x. PMID12153544.
^Patte-Mensah C, Meyer L, Taleb O, Mensah-Nyagan AG (February 2014). “Potential role of allopregnanolone for a safe and effective therapy of neuropathic pain”. Prog. Neurobiol. 113: 70–8. doi:10.1016/j.pneurobio.2013.07.004. PMID23948490.
^ Jump up to:abBäckström T, Andersson A, Andreé L, et al. (December 2003). “Pathogenesis in menstrual cycle-linked CNS disorders”. Ann. N. Y. Acad. Sci. 1007: 42–53. doi:10.1196/annals.1286.005. PMID14993039.
^Finocchi C, Ferrari M (May 2011). “Female reproductive steroids and neuronal excitability”. Neurol. Sci. 32 Suppl 1: S31–5. doi:10.1007/s10072-011-0532-5. PMID21533709.
^ Jump up to:abBäckström T, Haage D, Löfgren M, et al. (September 2011). “Paradoxical effects of GABA-A modulators may explain sex steroid induced negative mood symptoms in some persons”. Neuroscience. 191: 46–54. doi:10.1016/j.neuroscience.2011.03.061. PMID21600269.
^ Jump up to:abAndréen L, Nyberg S, Turkmen S, van Wingen G, Fernández G, Bäckström T (September 2009). “Sex steroid induced negative mood may be explained by the paradoxical effect mediated by GABAA modulators”. Psychoneuroendocrinology. 34 (8): 1121–32. doi:10.1016/j.psyneuen.2009.02.003. PMID19272715.
^Andréen L, Sundström-Poromaa I, Bixo M, Nyberg S, Bäckström T (August 2006). “Allopregnanolone concentration and mood–a bimodal association in postmenopausal women treated with oral progesterone”. Psychopharmacology. 187 (2): 209–21. doi:10.1007/s00213-006-0417-0. PMID16724185.
^Mellor DJ, Diesch TJ, Gunn AJ, Bennet L (2005). “The importance of ‘awareness’ for understanding fetal pain”. Brain Res. Brain Res. Rev. 49 (3): 455–71. doi:10.1016/j.brainresrev.2005.01.006. PMID16269314.
^ Jump up to:abAndréen L, Spigset O, Andersson A, Nyberg S, Bäckström T (June 2006). “Pharmacokinetics of progesterone and its metabolites allopregnanolone and pregnanolone after oral administration of low-dose progesterone”. Maturitas. 54 (3): 238–44. doi:10.1016/j.maturitas.2005.11.005. PMID16406399.
^Saudan C, Desmarchelier A, Sottas PE, Mangin P, Saugy M (2005). “Urinary marker of oral pregnenolone administration”. Steroids. 70 (3): 179–83. doi:10.1016/j.steroids.2004.12.007. PMID15763596.
^Piper T, Schlug C, Mareck U, Schänzer W (2011). “Investigations on changes in ¹³C/¹²C ratios of endogenous urinary steroids after pregnenolone administration”. Drug Test Anal. 3(5): 283–90. doi:10.1002/dta.281. PMID21538944.
The U.S. Food and Drug Administration today approved Poteligeo (mogamulizumab-kpkc) injection for intravenous use for the treatment of adult patients with relapsed or refractory mycosis fungoides (MF) or Sézary syndrome (SS) after at least one prior systemic therapy. This approval provides a new treatment option for patients with MF and is the first FDA approval of a drug specifically for SS.
The U.S. Food and Drug Administration today approved Poteligeo (mogamulizumab-kpkc) injection for intravenous use for the treatment of adult patients with relapsed or refractory mycosis fungoides (MF) or Sézary syndrome (SS) after at least one prior systemic therapy. This approval provides a new treatment option for patients with MF and is the first FDA approval of a drug specifically for SS.
“Mycosis fungoides and Sézary syndrome are rare, hard-to-treat types of non-Hodgkin lymphoma and this approval fills an unmet medical need for these patients,” said Richard Pazdur, M.D., director of the FDA’s Oncology Center of Excellence and acting director of the Office of Hematology and Oncology Products in the FDA’s Center for Drug Evaluation and Research. “We are committed to continuing to expedite the development and review of this type of targeted therapy that offers meaningful treatments for patients.”
Non-Hodgkin lymphoma is a cancer that starts in white blood cells called lymphocytes, which are part of the body’s immune system. MF and SS are types of non-Hodgkin lymphoma in which lymphocytes become cancerous and affect the skin. MF accounts for about half of all lymphomas arising from the skin. It causes itchy red rashes and skin lesions and can spread to other parts of the body. SS is a rare form of skin lymphoma that affects the blood and lymph nodes.
Poteligeo is a monoclonal antibody that binds to a protein (called CC chemokine receptor type 4 or CCR4) found on some cancer cells.
The approval was based on a clinical trial of 372 patients with relapsed MF or SS who received either Poteligeo or a type of chemotherapy called vorinostat. Progression-free survival (the amount of time a patient stays alive without the cancer growing) was longer for patients taking Poteligeo (median 7.6 months) compared to patients taking vorinostat (median 3.1 months).
The most common side effects of treatment with Poteligeo included rash, infusion-related reactions, fatigue, diarrhea, musculoskeletal pain and upper respiratory tract infection.
Serious warnings of treatment with Poteligeo include the risk of dermatologic toxicity, infusion reactions, infections, autoimmune problems (a condition where the immune cells in the body attack other cells or organs in the body), and complications of stem cell transplantation that uses donor stem cells (allogeneic) after treatment with the drug.
The FDA granted this application Priority Review and Breakthrough Therapydesignation. Poteligeo also received Orphan Drug designation, which provides incentives to assist and encourage the development of drugs for rare diseases.
The FDA granted this approval to Kyowa Kirin, Inc.
///////////////// Poteligeo, mogamulizumab-kpkc, fda 2018, Kyowa Kirin, Priority Review, Breakthrough Therapy designation, Orphan Drug designation
In 2013, Array Biopharma licensed the product to Loxo Oncology for development and commercialization in the U.S. In 2016, breakthrough therapy designation was received in the U.S. for the treatment of unresectable or metastatic solid tumors with NTRK-fusion proteins in adult and pediatric patients who require systemic therapy and who have either progressed following prior treatment or who have no acceptable alternative treatments. In 2017, Bayer acquired global co-development and commercialization rights from Loxo Oncology.
Originator Array BioPharma
Developer Array BioPharma; Loxo Oncology; National Cancer Institute (USA)
Class Antineoplastics; Pyrazoles; Pyrimidines; Pyrrolidines; Small molecules
Mechanism of Action Tropomyosin-related kinase antagonists
Orphan Drug Status Yes – Solid tumours; Soft tissue sarcoma
LOXO-101 is a small molecule that was designed to block the ATP binding site of the TRK family of receptors, with 2 to 20 nM cellular potency against the TRKA, TRKB, and TRKC kinases. IC50 value: 2 – 20 nM Target: TRKA/B/C in vitro: LOXO-101 is an orally administered inhibitor of the TRK kinase and is highly selective only for the TRK family of receptors. LOXO-101 is evaluated for off-target kinase enzyme inhibition against a panel of 226 non-TRK kinases at a compound concentration of 1,000 nM and ATP concentrations near the Km for each enzyme. In the panel, LOXO-101 demonstrates greater than 50% inhibition for only one non-TRK kinase (TNK2 IC50, 576 nM). Measurement of proliferation following treatment with LOXO-101 demonstrates a dose-dependent inhibition of cell proliferation in all three cell lines. The IC50 is less than 100 nM for CUTO-3.29 and less than 10 nM for KM12 and MO-91, consistent with the known potency of this drug for the TRK kinase family. [1] LOXO-101 demonstrates potent and highly-selective inhibition of TRKA, TRKB, and TRKC over other kinase- and non-kinase targets. LOXO-101 is a potent, ATP-competitive TRK inhibitor with IC50s in low nanomolar range for inhibition of all TRK family members in binding and cellular assays, with 100x selectivity over other kinases. [2] in vivo: Athymic nude mice injected with KM12 cells are treated with LOXO-101 orally daily for 2 weeks. Dose-dependent tumor inhibition is observed, demonstrating the ability of this selective compound to inhibit tumor growth in vivo. [1]
N-Boc-pyrrolidine as starting material The method involves enantioselective deprotonation, transmetalation with ZnCl2, Negishi coupling with 2-bromo-1,4-difluorobenzene,
N-arylation with 5-chloropyrazolo[1,5-a]pyrimidine, nitration, nitro reduction and condensation with CDI and 3(S)-pyrrolidinol.
[00423] To a DCM (0.8 mL) solution of (R)-5-(2-(2,5-difiuorophenyl)pyrrolidin-l-yl)pyrazolo[l,5-a]pyrimidin-3-amine (Preparation B; 30 mg, 0.095 mmol) was added CDI (31 mg, 0.19 mmol) at ambient temperature in one portion. After stirring two hours, (S)-pyrrolidin-3-ol (17 mg, 0.19 mmol) [purchased from Suven Life Sciences] was added in one portion. The reaction was stirred for 5 minutes before it was concentrated and directly purified by reverse-phase column chromatography, eluting with 0 to 50% acetonitrile/water to yield the final product as a yellowish foamy powder (30 mg, 74% yield). MS (apci) m/z = 429.2 (M+H).
[00424] To a solution of (S)-N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-l-yl)pyrazolo [ 1 ,5 -a]pyrimidin-3 -yl)-3 -hydroxypyrrolidine- 1 -carboxamide (4.5 mg, 0.011 mmol) in methanol (1 mL) at ambient temperature was added sulfuric acid in MeOH (105 μL, 0.011 mmol). The resulting solution was stirred for 30 minutes then concentrated to provide (S)-N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-l-yl)pyrazolo[l,5-a]pyrimidin-3-yl)-3 -hydroxypyrrolidine- 1 -carboxamide sulfate (5.2 mg, 0.0099 mmol, 94 % yield) as a yellow solid.
(R,E)-N-(2,5-difluorobenzylidene)-2-methylpropane-2-sulfinamide (17): Compound 16 and (R)-2-methylpropane-2-sulfinamide (1.05 eq.) were charged to a reactor outfitted with a mechanical stirrer, reflux condensor, J-Kem temperature probe under N2. DCM (3 mL/g of 14) was added (endothermic from 22 °C to about 5 °C) followed by addition of cesium carbonate (0.70 eq.) (exothermic to -50 °C). Once the addition was complete, the reaction mixture was stirred at room temperature for 3 h (slowly cools from about 40 °C). When the reaction was called complete (HPLC) the mixture was filtered through Celite. The Celite pad (0.3 wt eq) was equilibrated with DCM (1 mL/g of 16), and the reaction mixture was poured through the pad. The Celite cake was washed with DCM (2 x 1 mL/g), and the filtrate concentrated partially to leave about 0.5 to 1 mL/g DCM remaining. The orange solution was stored at room temperature (generally overnight) and used directly in the next reaction. (100% yield was assumed).
2)
(R)-N-((R)-l-(2,5-difluorophenyl)-3-(l,3-dioxan-2-yl)propyl)-2-methylpropane-2-sulfinamide (19): To a reactor equipped with overhead stirring, reflux condensor, under
nitrogen, was added magnesium turnings (2.0 eq), and THF (8 mL/g of 17). The mixture was heated to 40 °C. Dibal-H (25% wt in toluene, 0.004 eq) was added to the solution, and the suspension heated at 40 °C for 25 minutes. A solution of 2-(2-bromoethyl)-l,3-dioxane (18) (2 eq) in THF (4.6 mL/g of 17) was added dropwise to the Mg solution via addition funnel. The solution temperature was maintained < 55 °C. The reaction progress was monitored by GC. When the Grignard formation was judged complete, the solution was cooled to -30 °C, and 17 (1.0 eq, in DCM) was added dropwise via addition funnel. The temperature was kept between -30 °C and -20 °C and the reaction was monitored for completion (FIPLC). Once the reaction was called complete, the suspension (IT = -27.7 °C) was vacuum transferred to a prepared and cooled (10 °C) 10% aqueous citric acid solution (11 mL/g of 17). The mixture temperature rose to 20 °C during transfer. The milky solution was allowed to stir at ambient temperature overnight. MTBE (5.8 mL/g) was added to the mixture, and it was transferred to a separatory funnel. The layers were allowed to separate, and the lower aqueous layer was removed. The organic layer was washed with sat. NaHC03 (11 mL/g) and then sat. NaCl (5.4 mL/g). The organic layer was removed and concentrated to minimum volume via vacuum distillation. MTBE (2 mL/g) was added, and the mixture again concentrated to minimum volume. Finally MTBE was added to give 2 mL/g total MTBE (GC ratio of MTBE:THF was about 9: 1), and the MTBE mixture was heated to 50 °C until full dissolution occurred. The MTBE solution was allowed to cool to about 35 °C, and heptane was added portion -wise. The first portion (2 mL/g) is added, and the mixture allowed to stir and form a solid for 1-2 h, and then the remainder of the heptane is added (8 mL/g). The suspension was allowed to stir for >lh. The solids were collected via filtration through polypropylene filter cloth (PPFC) and washed with 10% MTBE in heptane (4 mL/g. The wet solid was placed in trays and dried in a vacuum oven at 55 °C until constant weight (3101 g, 80.5%, dense white solid, 100a% and 100wt%).
3)
(R)-2-(2,5-difluorophenyl)pyrrolidine (R)-2-hydroxysuccinate (10): To a flask containing 4: 1 TFA:water (2.5 mL/g, pre-mixed and cooled to <35 °C before adding 19) was added (R)-N-((R)-l-(2,5-difluorophenyl)-3-(l,3-dioxan-2-yl)propyl)-2-methylpropane-2-sulfinamide (19) (1 eq). The mixture temperature rose from 34 °C to 48 °C and was stirred at ambient temperature for 1 h. Additional TFA (7.5 mL/g) was added, followed by triethylsilane (3 eq) over 5 minutes. The biphasic mixture was stirred vigorously under nitrogen for 21 h until judged complete (by GC, <5% of imine). The mixture was then concentrated under vacuum until -10 kg target mass (observed 10.8 kg after concentration). The resulting concentrate was transferred to a separatory funnel and diluted with MTBE (7.5 mL/g), followed by water (7.5 mL/g). The layers were separated. The MTBE layer was back-extracted with 1M HC1 (3 mL/g). The layers were separated, and the aqueous layers were combined in a round-bottomed flask with DCM (8 mL/g). The mixture was cooled in an ice bath and 40% NaOH was charged to adjust the pH to >12 (about 0.5 mL/g; the temperature went from 24 °C to 27 °C, actual pH was 13), and the layers separated in the separatory funnel. The aqueous layer was back-extracted twice with DCM (2 x 4 mL/g). The organic layers were concentrated to an oil (<0.5 mL/g) under vacuum (rotovap) and EtOH (1 mL/g based on product) was added. The yellow solution was again concentrated to an oil (81% corrected yield, with 3% EtOH, 0.2% imine and Chiral HPLC showed 99.7%ee).
Salt formation: To a solution of (R)-2-(2,5-difluorophenyl)pyrrolidine 10 (1 eq) in EtOH (15 mL/g) was added Z)-(+)-Malic Acid (1 eq). The suspension was heated to 70 °C for 30 minutes (full dissolution had occurred before 70 °C was reached), and then allowed to cool to room temperature slowly (mixture was seeded when the temperature was < 40 °C). The slurry was stirred at room temperature overnight, then cooled to <5 °C the next morning. The suspension was stirred at <5 °C for 2h, filtered (PPFC), washed with cold EtOH (2 x 2 mL/g), and dried (50-55 °C) under vacuum to give the product as a white solid (96% based on 91% potency, product is an EtOH solvate or hemi- solvate).
Compound 5 and 10 (1.05 eq) were charged to a reactor outfitted with a mechanical stirrer, J-Kem temperature probe, under N2. EtOH and THF (4: 1, 10 mL/g of 5) were added and the mixture was cooled to 15-25 °C. Triethylamine (3.5 eq) was added and the internal temp generally rose from 17.3 – 37.8 °C. The reaction was heated to 50 – 60 °C and held at that temperature for 7 h. Once the reaction is judged complete (HPLC), water (12 mL/g of 5) is added maintaining the temperature at 50 – 60 °C. The heat is removed and the suspension was slowly cooled to 21 °C over two h. After stirring at -21 °C for 2 h, the suspension was centrifuged and the cake was washed with water (3 x 3 mL/g of 5). The solid was transferred to drying trays and placed in a vacuum oven at 50 – 55 °C to give 11.
2)
(R)-5-(2-(2,5-difluorophenyl)pyrrolidin-l-yl)pyrazolo[l,5-a]pyrimidin-3-amine fumarate Pt/C hydrogenation (12 fumarate): To a Parr reactor was charged 11 (1.0 eq), 5% Pt/C ~ 50 wt% water (2 mol% Pt / Johnson Matthey B 103018-5 or Sigma Aldrich 33015-9), and MeOH (8 mL/g). The suspension was stirred under hydrogen at 25-30 psi and the temperature was maintained below 65 °C for ~8 h. When the reaction was called complete (HPLC), the reaction was cooled to 15 – 25 °C and the hydrogen atmosphere was replaced with a nitrogen atmosphere. The reaction mixture was filtered through a 2 micron bag filter and a 0.2 micron line filter in series. The filtrate from the Pt/C hydrogenation was transferred to a reactor under nitrogen with mechanical stirring and then MTBE (8 mL/g) and fumaric acid (1.01 eq) were charged. The mixture was stirred under nitrogen for 1 h and solids formed after -15 min. The mixture was cooled to -10 to -20 °C and stirred for 3 h. The suspension was filtered (PPFC), washed with MTBE (-2.5 mL/g), and the solids was dried under vacuum at 20-25 °C with a nitrogen bleed to yield an off-white solid (83% yield).
3)
Phenyl (5-((R)-2-(2,5-difluorophenyl)pyrrolidin-l-yl)-3,3a-dihydropyrazolo[l,5-a]pyrimidin-3-yl)carbamate (13): To a 5 to 15°C solution of 12-fumarate (1.0 eq) in 2-MeTHF (15 mL/g) was added a solution of potassium carbonate (2.0 eq.) in water (5 mL/g) followed by phenyl chloroformate (1.22 eq.) (over 22 min, an exotherm from 7 °C to 11 °C occurred). The mixture was stirred for 2 h and then the reaction was called complete (HPLC). The stirring ceased and the aqueous layer was removed. The organic layer was washed with brine (5 mL/g) and concentrated to ca. 5 mL/g of 2-MeTHF under vacuum and with heating to 40 °C. To the 2-MeTHF solution was added heptanes (2.5 mL/g) followed by seeds (20 mg, 0.1 wt%). This mixture was allowed to stir at room temperature for 2 h (until a solid formed), and then the remainder of the heptanes (12.5 mL/g) was added. The mixture was stirred at ambient temperature for 2 h and then the solids were collected via filtration (PPFC), washed with 4: 1 heptanes :MeTHF (2 x 2 mL/g), and dried to give 13 (96%).
4)
(S)-N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-l-yl)pyrazolo[l,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-l-carboxamide hydrogen sulfate: To a flask containing 13 (1.0 eq) was added a solution of (S)-pyrrolidin-3-ol (1.1 eq.) in EtOH (10 mL/g). The mixture was heated at 50 – 60 °C for 5 h, called complete (HPLC), and then cooled to 20-35 °C. Once <35°C, the reaction was polish-filtered (0.2 micron) into a clean reaction vessel and the mixture was cooled to -5 to 5 °C. Sulfuric acid (1.0 eq.) was added over 40 minutes, the temperature rose to 2 °C and the mixture was seeded. A solid formed, and the mixture was allowed to stir at -5 to 5 °C for 6.5 h. Heptanes (10 mL/g) was added, and the mixture stirred for 6.5 h. The
suspension was filtered (PPFC), washed with 1 : 1 EtOH:heptanes (2 x 2 mL/g), and dried (under vacuum at ambient temperature) to give Formula I (92.3%).
Preparation of the hydrogen sulfate salt of the compound of Formula I:
Concentrated sulfuric acid (392 mL) was added to a solution of 3031 g of (S)-N-(5- ((R)-2-(2,5-difluorophenyl)pyrrolidin-l-yl)-pyrazolo[l,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-l-carboxamide in 18322 mL EtOH to form the hydrogen sulfate salt. The solution was seeded with 2 g of (,S)-N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-l-yl)-pyrazolo[l,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-l-carboxamide hydrogen sulfate and the solution was stirred at room temperature for at least 2 hours to form a slurry of the hydrogen sulfate salt. Heptane (20888 g) was added and the slurry was stirred at room temperature for at least 60 min. The slurry was filtered and the filter cake was washed with 1 : 1 heptane/EtOH. The solids were then dried under vacuum at ambient temperature (oven temperature set at 15° Celsius).
The dried hydrogen sulfate salt (6389 g from 4 combined lots) was added to a 5 :95 w/w solution of water/2-butanone (total weight 41652 g). The mixture was heated at about 68° Celsius with stirring until the weight percent of ethanol was about 0.5%, during which time a slurry formed. The slurry was filtered, and the filter cake was washed with a 5 :95 w/w solution of water/2-butanone. The solids were then dried under vacuum at ambient temperature (oven temperature set at 15° Celsius) to provide the crystalline form of (S)-N-(5-((R)-2-(2,5-difluorophenyl)-pyrrolidin-l-yl)-pyrazolo[l,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-l-carboxamide hydrogen sulfate.
Provided herein is a novel crystalline form of the compound of Formula I:
[0000]
also known as (S)—N-(5-((R)-2-(2, 5-difluorophenyl)-pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide. In particular, the novel crystalline form comprises the hydrogen sulfate salt of the compound of Formula I in a stable polymorph form, hereinafter referred to as crystalline form (I-HS) and LOXO-101, which can be characterized, for example, by its X-ray diffraction pattern—the crystalline form (I-HS) having the formula:
[0000]
In some embodiments of the above step (c), the base is an alkali metal base, such as an alkali metal carbonate, such as potassium carbonate.
Preparation of 5-chloro-3-nitropyrazolo[1,5-a]pyrimidine Step A—Preparation of sodium pyrazolo[1,5-a]pyrimidin-5-olate
A solution of 1H-pyrazol-5-amine and 1,3-dimethylpyrimidine-2,4(1H,3H)-dione (1.05 equiv.) were charged to a round bottom flask outfitted with a mechanical stirrer, a steam pot, a reflux condenser, a J-Kem temperature probe and an N2 adaptor for positive N2 pressure control. Under mechanical stirring the solids were suspended with 4 vol. (4 mL/g) of absolute EtOH under a nitrogen atmosphere, then charged with 2.1 equivalents of NaOEt (21 wt % solution in EtOH), and followed by line-rinse with 1 vol. (1 mL/g) of absolute EtOH. The slurry was warmed to about 75° Celsius and stirred at gentle reflux until less than 1.5 area % of 1H-pyrazol-5-amine was observed by TRK1PM1 HPLC to follow the progression of the reaction using 20 μL of slurry diluted in 4 mL deionized water and 5 μL injection at 220 nm.
After 1 additional hour, the mixture was charged with 2.5 vol. (2.5 mL/g) of heptane and then refluxed at 70° Celsius for 1 hour. The slurry was then cooled to room temperature overnight. The solid was collected by filtration on a tabletop funnel and polypropylene filter cloth. The reactor was rinsed and charged atop the filter cake with 4 vol. (4 mL/g) of heptane with the cake pulled and the solids being transferred to tared drying trays and oven-dried at 45° Celsius under high vacuum until their weight was constant. Pale yellow solid sodium pyrazolo[1,5-a]-pyrimidin-5-olate was obtained in 93-96% yield (corrected) and larger than 99.5 area % observed by HPLC (1 mg/mL dilution in deionized water, TRK1PM1 at 220 nm).
Step B—Preparation of 3-nitropyrazolo[1,5-a]pyrimidin-5(4H)-one
A tared round bottom flask was charged with sodium pyrazolo[1,5-a]pyrimidin-5-olate that was dissolved at 40-45° Celsius in 3.0 vol. (3.0 mL/g) of deionized water, and then concentrated under high vacuum at 65° Celsius in a water-bath on a rotary evaporator until 2.4× weight of starting material was observed (1.4 vol/1.4 mL/g deionized water content). Gas chromatography (GC) for residual EtOH (30 μL of solution dissolved in ˜1 mL MeOH) was performed showing less than 100 ppm with traces of ethyl nitrate fumes being observed below upon later addition of HNO3. In some cases, the original solution was charged with an additional 1.5 vol. (1.5 mL/g) of DI water, then concentrated under high vacuum at 65° Celsius in a water-bath on a rotary evaporator until 2.4× weight of starting material was observed (1.4 vol/1.4 mL/g DI water content). Gas chromatograph for residual EtOH (30 μL of solution dissolved in about 1 mL MeOH) was performed showing <<100 ppm of residual EtOH without observing any ethyl nitrate fumes below upon later addition of HNO3.
A round bottom vessel outfitted with a mechanical stirrer, a steam pot, a reflux condenser, a J-Kem temperature probe and an N2 adaptor for positive N2 pressure control was charged with 3 vol. (3 mL/g, 10 equiv) of >90 wt % HNO3 and cooled to about 10° Celsius under a nitrogen atmosphere using external ice-water cooling bath under a nitrogen atmosphere. Using a pressure equalizing addition funnel, the HNO3solution was charged with the 1.75-1.95 volumes of a deionized water solution of sodium pyrazolo[1,5-a]pyrimidin-5-olate (1.16-1.4 mL DI water/g of sodium pyrazolo[1,5-a]pyrimidin-5-olate) at a rate to maintain 35-40° Celsius internal temperature under cooling. Two azeotropes were observed without any ethyl nitrate fumes. The azeotrope flask, the transfer line (if applicable) and the addition funnel were rinsed with 2×0.1 vol. (2×0.1 mL/g) deionized water added to the reaction mixture. Once the addition was complete, the temperature was gradually increased to about 45-50° Celsius for about 3 hours with HPLC showing >99.5 area % conversion of sodium pyrazolo[1,5-a]pyrimidin-5-olate to 3-nitropyrazolo[1,5-a]pyrimidin-5(4H)-one.
Step C—Preparation of 5-chloro-3-nitropyrazolo[1,5-a]pyrimidine
3-nitropyrazolo[1,5-a]pyrimidin-5(4H)-one was charged to a round bottom flask outfitted with a mechanical stirrer, a heating mantle, a reflux condenser, a J-Kem temperature probe and an N2 adaptor for positive N2pressure control. Under mechanical stirring the solids were suspended with 8 volumes (8 mL/g) of CH3CN, and then charged with 2,6-lutitine (1.05 equiv) followed by warming the slurry to about 50° Celsius. Using a pressure equalizing addition funnel, the mixture was dropwise charged with 0.33 equivalents of POCl3. This charge yielded a thick, beige slurry of a trimer that was homogenized while stirring until a semi-mobile mass was observed. An additional 1.67 equivalents of POCl3 was charged to the mixture while allowing the temperature to stabilize, followed by warming the reaction mixture to a gentle reflux (78° Celsius). Some puffing was observed upon warming the mixture that later subsided as the thick slurry got thinner.
The reaction mixture was allowed to reflux until complete dissolution to a dark solution and until HPLC (20 μL diluted in 5 mL of CH3CN, TRK1PM1 HPLC, 5 μL injection, 268 nm) confirmed that no more trimer (RRT 0.92) was present with less than 0.5 area % of 3-nitropyrazolo[1,5-a]pyrimidin-5(4H)-one (RRT 0.79) being observed by manually removing any interfering and early eluting peaks related to lutidine from the area integration. On a 1.9 kg scale, 0 area % of the trimer, 0.25 area % of 3-nitropyrazolo[1,5-a]pyrimidin-5(4H)-one, and 99.5 area % of 5-chloro-3-nitropyrazolo[1,5-a]pyrimidine was observed after 19 hours of gentle reflux using TRK1PM1 HPLC at 268 [0000]
Preparation of (R)-2-(2,5-difluorophenyl)-pyrrolidine (R)-2-hydroxysuccinate Step A—Preparation of tert-butyl(4-(2,5-difluorophenyl)-4-oxobutyl)-carbamate
2-bromo-1,4-difluorobenzene (1.5 eq.) was dissolved in 4 volumes of THF (based on weight of tert-butyl 2-oxopyrrolidine-1-carboxylate) and cooled to about 5° Celsius. A solution of 2.0 M iPrMgCl in THF (1.4 eq.) was added over 2 hours to the mixture while maintaining a reaction temperature below 25° Celsius. The solution was allowed to cool to about 5° Celsius and stirred for 1 hour (GC analysis confirmed Grignard formation). A solution of tert-butyl 2-oxopyrrolidine-1-carboxylate (1.0 eq.) in 1 volume of THF was added over about 30 min while maintaining a reaction temperature below 25° Celsius. The reaction was stirred at about 5° Celsius for 90 min (tert-butyl 2-oxopyrrolidine-1-carboxylate was confirmed to be less than 0.5 area % by HPLC). The reaction was quenched with 5 volumes of 2 M aqueous HCl while maintaining a reaction temperature below 45° Celsius. The reaction was then transferred to a separatory funnel adding 10 volumes of heptane and removing the aqueous layer. The organic layer was washed with 4 volumes of saturated aqueous NaCl followed by addition of 2×1 volume of saturated aqueous NaCl. The organic layer was solvent-switched to heptane (<1% wt THF confirmed by GC) at a distillation temperature of 35-55° Celsius and distillation pressure of 100-200 mm Hg for 2×4 volumes of heptane being added with a minimum distillation volume of about 7 volumes. The mixture was then diluted to 10 volumes with heptane while heating to about 55° Celsius yielded a denser solid with the mixture being allowed to cool to room temperature overnight. The slurry was cooled to less than 5° Celsius and filtered through polypropylene filter cloth. The wet cake was washed with 2×2 volumes of heptane. The solids were dried under vacuum at 55° Celsius until the weight was constant, yielding tert-butyl(4-(2,5-difluorophenyl)-4-oxobutyl)-carbamate as a white solid at about 75% to 85% theoretical yield.
Step B—Preparation of 5-(2,5-difluorophenyl)-3,4-dihydro-2H-pyrrole
tert-butyl(4-(2,5-difluorophenyl)-4-oxobutyl)-carbamate was dissolved in 5 vol. of toluene with 2.2 eq. of 12M HCl being added observing a mild exotherm and gas evolution. The reaction was heated to 65° Celsius for 12-24 hours and monitored by HPLC. Upon completion the reaction was cooled to less than 15° Celsius with an ice/water bath. The pH was adjusted to about 14 with 3 equivalents of 2M aqueous NaOH (4.7 vol.). The reaction was stirred at room temperature for 1-2 hours. The mixture was transferred to a separatory funnel with toluene. The aqueous layer was removed and the organic layer was washed with 3 volumes of saturated aqueous NaCl. The organic layer was concentrated to an oil and redissolved in 1.5 volumes of heptane. The resulting suspension was filtered through a GF/F filter paper and concentrated to a light yellow oil of 5-(2,5-difluorophenyl)-3,4-dihydro-2H-pyrrole with a 90% to 100% theoretical yield.
Step C—Preparation of (R)-2-(2,5-difluorophenyl)-pyrrolidine
Chloro-1,5-cyclooctadiene iridium dimer (0.2 mol %) and (R)-2-(2-(diphenylphosphino)phenyl)-4-isopropyl-4,5-dihydrooxazole (0.4 mol %) were suspended in 5 volumes of MTBE (based on 5-(2,5-difluorophenyl)-3,4-dihydro-2H-pyrrole) at room temperature. The mixture was stirred for 1 hour and most of the solids dissolved with the solution turning dark red. The catalyst formation was monitored using an HPLC/PDA detector. The reaction was cooled to less than 5° Celsius and 5-(2,5-difluorophenyl)-3,4-dihydro-2H-pyrrole (1.0 eq.) was added using a 0.5 volumes of MTBE rinse. Diphenylsilane (1.5 eq.) was added over about 20 minutes while maintaining a reaction temperature below 10° Celsius. The reaction was stirred for 30 minutes below 10° Celsius and then allowed to warm to room temperature. The reaction was stirred overnight at room temperature. The completion of the reaction was confirmed by HPLC and then cooled to less than 5° Celsius. The reaction was quenched with 5 volumes of 2M aqueous HCl maintaining temperature below 20° Celsius. After 10 minutes the ice/water bath was removed and the reaction temperature was allowed to increase to room temperature while stirring for 2 hours. The mixture was transferred to a separatory funnel with 3 volumes of MTBE. The aqueous layer was washed with 3.5 volumes of MTBE followed by addition of 5 volumes of MTBE to the aqueous layer while adjusting the pH to about 14 by adding 0.75 volumes of aqueous 50% NaOH. The organic layer was washed with 5 volumes of aqueous saturated NaCl, then concentrated to an oil, and diluted with 3 volumes of MTBE. The solution was filtered through a polypropylene filter cloth and rinsed with 1 volume of MTBE. The filtrate was concentrated to an oil of (R)-2-(2,5-difluorophenyl)-pyrrolidine with a 95% to 100% theoretical yield and with 75-85% ee.
Step D—Preparation of (R)-2-(2,5-difluorophenyl)-pyrrolidine (R)-2-hydroxy-succinate
(R)-2-(2,5-difluorophenyl)-pyrrolidine (1.0 eq.) was transferred to a round bottom flask charged with 15 volumes (corrected for potency) of EtOH (200 prf). D-malic acid (1.05 eq.) was added and the mixture was heated to 65° Celsius. The solids all dissolved at about 64° Celsius. The solution was allowed to cool to RT. At about 55° Celsius the solution was seeded with (R)-2-(2,5-difluorophenyl)-pyrrolidine (R)-2-hydroxy-succinate (about 50 mg, >97% ee) and stirred at room temperature overnight. The suspension was then filtered through a polypropylene filter cloth and washed with 2×1 volumes of EtOH (200 prf). The solids were dried under vacuum at 55° Celsius, yielding (R)-2-(2,5-difluorophenyl)-pyrrolidine (R)-2-hydroxy-succinate with a 75% to 90% theoretical yield and with >96% ee.
Referring to Scheme 1, suitable bases include tertiary amine bases, such as triethylamine, and K2CO3. Suitable solvents include ethanol, heptane and tetrahydrofuran (THF). The reaction is conveniently performed at temperatures between 5° Celsius and 50° Celsius. The reaction progress was generally monitored by HPLC TRK1PM1.
[0247]
Compounds II (5-chloro-3-nitropyrazolo[1,5-a]pyrimidine) and III ((R)-2-(2,5-difluorophenyl)-pyrrolidine (R)-2-hydroxysuccinate, 1.05 eq.) were charged to a round bottom flask outfitted with a mechanical stirrer, a J-Kem temperature probe and an N2 adaptor for positive N2 pressure control. A solution of 4:1 EtOH:THF (10 mL/g of compound II) was added and followed by addition of triethylamine (NEt3, 3.50 eq.) via addition funnel with the temperature reaching about 40° Celsius during addition. Once the addition was complete, the reaction mixture was heated to 50° Celsius and stirred for 0.5-3 hours to yield compound IV.
To a round bottom flask equipped with a mechanical stirrer, a J-Kem temperature probe, and an N2 inlet compound IV was added and followed by addition of tetrahydrofuran (10 mL/g of compound IV). The solution was cooled to less than 5° Celsius in an ice bath, and Zn (9-10 eq.) was added. 6M HCl (9-10 eq.) was then added dropwise at such a rate to keep the temperature below 30° Celsius (for 1 kg scale the addition took about 1.5 hours). Once the exotherm subsided, the reaction was allowed to warm to room temperature and was stirred for 30-60 min until compound IV was not detected by HPLC. At this time, a solution of potassium carbonate (K2CO3, 2.0 eq.) in water (5 mL/g of compound IV) was added all at once and followed by rapid dropwise addition of phenyl chloroformate (PhOCOCl, 1.2 eq.). Gas evolution (CO2) was observed during both of the above additions, and the temperature increased to about 30° Celsius after adding phenyl chloroformate. The carbamate formation was stirred at room temperature for 30-90 min. HPLC analysis immediately followed to run to ensure less than 1 area % for the amine being present and high yield of compound VI in the solution.
To the above solution amine VII ((S)-pyrrolidin-3-ol, 1.1 eq. based on theoretical yield for compound VI) and EtOH (10 mL/g of compound VI) was added. Compound VII was added before or at the same time as EtOH to avoid ethyl carbamate impurities from forming. The above EtOH solution was concentrated to a minimum volume (4-5 mL/g) using the batch concentrator under reduced pressure (THF levels should be <5% by GC), and EtOH (10 mL/g of compound VI) was back-added to give a total of 10 mL/g. The reaction was then heated at 50° Celsius for 9-19 hours or until HPLC shows that compound VI is less than 0.5 area %. The reaction was then cooled to room temperature, and sulfuric acid (H2SO4, 1.0 eq. to compound VI) was added via addition funnel to yield compound I-HS with the temperature usually exotherming at about 30° Celsius.
Example 1 Preparation of Crystalline Form (I-HS) (Method 1)
(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide (0.500 g, 1.17 mmol) was dissolved in EtOH (2.5 mL) and cooled to about 5° Celsius. Concentrated sulfuric acid (0.0636 mL, 1.17 mmol) was added to the cooled solution and stirred for about 10 min, while warming to room temperature. Methyl tert-butyl ether (MTBE) (2 mL) was slowly added to the mixture, resulting in the product gumming out. EtOH (2.5 mL) was then added to the mixture and heated to about reflux until all solids were dissolved. Upon cooling to room temperature and stirring for about 1 hour, some solids formed. After cooling to about 5° Celsius, the solids were filtered and washed with MTBE. After filtration and drying at air for about 15 minutes, (S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide hydrogen sulfate was isolated as a solid.
Example 2 Preparation of Crystalline Form (I-HS) (Method 2)
Concentrated sulfuric acid (392 mL) was added to a solution of 3031 g of (S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide in 18322 mL EtOH to form the hydrogen sulfate salt. The solution was seeded with 2 g of (S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide hydrogen sulfate and the solution was stirred at room temperature for at least 2 hours to form a slurry of the hydrogen sulfate salt. Heptane (20888 g) was added and the slurry was stirred at room temperature for at least 60 min. The slurry was filtered and the filter cake was washed with 1:1 heptane/EtOH. The solids were then dried under vacuum at ambient temperature (oven temperature set at 15° Celsius).
The dried hydrogen sulfate salt (6389 g from 4 combined lots) was added to a 5:95 w/w solution of water/2-butanone (total weight 41652 g). The mixture was heated at about 68° Celsius with stirring until the weight percent of ethanol was about 0.5%, during which time a slurry formed. The slurry was filtered, and the filter cake was washed with a 5:95 w/w solution of water/2-butanone. The solids were then dried under vacuum at ambient temperature (oven temperature set at 15° Celsius) to provide the crystalline form of (S)—N-(5-((R)-2-(2,5-difluorophenyl)-pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide hydrogen sulfate.
Example 3 Preparation of Amorphous Form AM(HS)
To a solution of (S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide (9.40 g, 21.94 mmol) in MeOH (220 mL) was slowly added sulfuric acid (0.1 M in MeOH, 219.4 mL, 21.94 mmol) at ambient temperature under rapid stirring. After 30 minutes, the reaction was first concentrated by rotary evaporator to near dryness, then on high vacuum for 48 h to provide amorphous form of (S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide sulfate (11.37 g, 21.59 mmol, 98.43% yield). LCMS (apci m/z 429.1, M+H).
WO 2010/048314 discloses in Example 14A a hydrogen sulfate salt of (S)—N-(5-((R)-2-(2,5-difluorophenyl)-pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide. WO 2010/048314 does not disclose the particular form of the hydrogen sulfate salt described herein when prepared according to the method of Example 14A in that document. In particular, WO 2010/048314 does not disclose crystalline form (l-HS) as described below.
(S)—N-(5-((R)-2-(2,5-difluorophenyl)-pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide having the formula (I):
Example 1 Preparation of Crystalline Form (I-HS) (Method 1)
(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide (0.500 g, 1.17 mmol) was dissolved in EtOH (2.5 mL) and cooled to about 5° Celsius. Concentrated sulfuric acid (0.0636 mL, 1.17 mmol) was added to the cooled solution and stirred for about 10 min, while warming to room temperature. Methyl tert-butyl ether (MTBE) (2 mL) was slowly added to the mixture, resulting in the product gumming out. EtOH (2.5 mL) was then added to the mixture and heated to about reflux until all solids were dissolved. Upon cooling to room temperature and stirring for about 1 hour, some solids formed. After cooling to about 5° Celsius, the solids were filtered and washed with MTBE. After filtration and drying at air for about 15 minutes, (S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidi n-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide hydrogen sulfate was isolated as a solid.
Example 2 Preparation of Crystalline Form (I-HS) (Method 2)
Concentrated sulfuric acid (392 mL) was added to a solution of 3031 g of (S)—N-(5-((R)-2-(2, 5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1, 5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide in 18322 mL EtOH to form the hydrogen sulfate salt. The solution was seeded with 2 g of (S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide hydrogen sulfate and the solution was stirred at room temperature for at least 2 hours to form a slurry of the hydrogen sulfate salt. Heptane (20888 g) was added and the slurry was stirred at room temperature for at least 60 min. The slurry was filtered and the filter cake was washed with 1:1 heptane/EtOH. The solids were then dried under vacuum at ambient temperature (oven temperature set at 15° Celsius).
The dried hydrogen sulfate salt (6389 g from 4 combined lots) was added to a 5:95 w/w solution of water/2-butanone (total weight 41652 g). The mixture was heated at about 68° Celsius with stirring until the weight percent of ethanol was about 0.5%, during which time a slurry formed. The slurry was filtered, and the filter cake was washed with a 5:95 w/w solution of water/2-butanone. The solids were then dried under vacuum at ambient temperature (oven temperature set at 15° Celsius) to provide the crystalline form of (S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide hydrogen sulfate.
Example 3 Preparation of Amorphous Form AM(HS)
To a solution of (S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide (9.40 g, 21.94 mmol) in MeOH (220 mL) was slowly added sulfuric acid (0.1 M in MeOH, 219.4 mL, 21.94 mmol) at ambient temperature under rapid stirring. After 30 minutes, the reaction was first concentrated by rotary evaporator to near dryness, then on high vacuum for 48 h to provide amorphous form of (S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide sulfate (11.37 g, 21.59 mmol, 98.43% yield). LCMS (apci m/z 429.1, M+H).
CRYSTALLINE FORM OF (S)-N-(5-((R)-2-(2, 5-DIFLUOROPHENYL)-PYRROLIDIN-1-YL)-PYRAZOLO[1, 5-A]PYRIMIDIN-3-YL)-3-HYDROXYPYRROLIDINE-1-CARBOXAMIDE HYDROGEN SULFATE
CRYSTALLINE FORM OF (S)-N-(5-((R)-2-(2, 5-DIFLUOROPHENYL)-PYRROLIDIN-1-YL)-PYRAZOLO[1, 5-A]PYRIMIDIN-3-YL)-3-HYDROXYPYRROLIDINE-1-CARBOXAMIDE HYDROGEN SULFATE
CRYSTALLINE FORM OF (S)-N-(5-((R)-2-(2, 5-DIFLUOROPHENYL)-PYRROLIDIN-1-YL)-PYRAZOLO[1, 5-A]PYRIMIDIN-3-YL)-3-HYDROXYPYRROLIDINE-1-CARBOXAMIDE HYDROGEN SULFATE
CRYSTALLINE FORM OF (S)-N-(5-((R)-2-(2, 5-DIFLUOROPHENYL)-PYRROLIDIN-1-YL)-PYRAZOLO[1, 5-A]PYRIMIDIN-3-YL)-3-HYDROXYPYRROLIDINE-1-CARBOXAMIDE HYDROGEN SULFATE
STAMFORD, Conn., May 29, 2018 (GLOBE NEWSWIRE) — Loxo Oncology, Inc. (Nasdaq:LOXO), a biopharmaceutical company innovating the development of highly selective medicines for patients with genetically defined cancers, today announced that the U.S. Food and Drug Administration (FDA) has accepted the company’s New Drug Application (NDA) and granted Priority Review for larotrectinib for the treatment of adult and pediatric patients with locally advanced or metastatic solid tumors harboring an NTRK gene fusion. The FDA has set a target action date of November 26, 2018, under the Prescription Drug User Fee Act (PDUFA).
“We are excited the larotrectinib NDA has been accepted by FDA and granted Priority Review status,” said Josh Bilenker, M.D., chief executive officer of Loxo Oncology. “Larotrectinib marks an important shift towards treating cancer based on the tumor’s genetics rather than its site of origin in the body.”
The FDA grants Priority Review for the applications of medicines that, if approved, would provide significant improvements in the safety or effectiveness of the treatment, diagnosis, or prevention of serious conditions when compared to standard applications. Larotrectinib has also been granted Breakthrough Therapy Designation, Rare Pediatric Disease Designation and Orphan Drug Designation by the FDA.
Loxo Oncology and Bayer are engaged in a collaboration for the development and commercialization of larotrectinib. Bayer plans to submit a Marketing Authorization Application (MAA) in the European Union in 2018.
About Larotrectinib (LOXO-101)
Larotrectinib is an oral and highly selective investigational tropomyosin receptor kinase (TRK) inhibitor in clinical development for the treatment of patients with cancers that harbor a neurotrophic tyrosine receptor kinase (NTRK) gene fusion. Growing research suggests that the NTRK genes, which encode for TRKs, can become abnormally fused to other genes, resulting in growth signals that can lead to cancer in many sites of the body. In clinical trials, larotrectinib demonstrated anti-tumor activity in patients with tumors harboring NTRK gene fusions, regardless of patient age or tumor type. In an analysis of 55 RECIST-evaluable adult and pediatric patients with NTRK gene fusions, larotrectinib demonstrated a 75 percent centrally-assessed confirmed overall response rate (ORR) and an 80 percent investigator-assessed confirmed ORR, across many different types of solid tumors. The majority of all adverse events were grade 1 or 2.
Larotrectinib has been granted Priority Review, Breakthrough Therapy Designation, Rare Pediatric Disease Designation and Orphan Drug Designation by the U.S. FDA.
In November 2017, Loxo Oncology and Bayer entered into an exclusive global collaboration for the development and commercialization of larotrectinib and LOXO-195, a next-generation TRK inhibitor. Bayer and Loxo Oncologywill jointly develop the two products with Loxo Oncology leading the ongoing clinical studies as well as the filing in the U.S., and Bayer leading ex-U.S. regulatory activities and worldwide commercial activities. In the U.S., Loxo Oncology and Bayer will co-promote the products.
For additional information about the larotrectinib clinical trials, please refer to www.clinicaltrials.gov. Interested patients and physicians can contact the Loxo Oncology Physician and Patient Clinical Trial Hotline at 1-855-NTRK-123 or visit www.loxooncologytrials.com/trk-trials.
About TRK Fusion Cancer
TRK fusion cancer occurs when a neurotrophic tyrosine receptor kinase (NTRK) gene fuses with another unrelated gene, producing an altered tropomyosin receptor kinase (TRK) protein. The altered protein, or TRK fusion protein, is constantly active, triggering a permanent signal cascade. These proteins become the primary driver of the spread and growth of tumors in patients with TRK fusion cancer. TRK fusion cancer is not limited to certain types of cells or tissues and can occur in any part of the body. NTRK gene fusions occur in various adult and pediatric solid tumors with varying prevalence, including appendiceal cancer, breast cancer, cholangiocarcinoma, colorectal cancer, GIST, infantile fibrosarcoma, lung cancer, mammary analogue secretory carcinoma of the salivary gland, melanoma, pancreatic cancer, thyroid cancer, and various sarcomas. It may affect greater than 60 percent of both adult and pediatric patients with certain rare tumor types, such as secretory breast, secretory salivary gland and infantile fibrosarcoma. Only sensitive and specific tests can reliably detect TRK fusion cancer. Next-generation sequencing (NGS) can provide a comprehensive view of genomic alterations across a large number of genes. Fluorescence in situ hybridization (FISH) can also be used to test for TRK fusion cancer, and immunohistochemistry (IHC) can be used to detect the presence of TRK protein
About Loxo Oncology
Loxo Oncology is a biopharmaceutical company innovating the development of highly selective medicines for patients with genetically defined cancers. Our pipeline focuses on cancers that are uniquely dependent on single gene abnormalities, such that a single drug has the potential to treat the cancer with dramatic effect. We believe that the most selective, purpose-built medicines have the highest probability of maximally inhibiting the intended target, with the intention of delivering best-in-class disease control and safety. Our management team seeks out experienced industry partners, world-class scientific advisors and innovative clinical-regulatory approaches to deliver new cancer therapies to patients as quickly and efficiently as possible. For more information, please visit the company’s website at www.loxooncology.com.
larotrectinib
larotrectinib
Treatment for Solid Tumors
FDA Accepts Larotrectinib New Drug Application and Grants Priority Review
WHIPPANY, N.J., May 29, 2018 /PRNewswire/ — Bayer announced today that the U.S. Food and Drug Administration (FDA) has accepted the New Drug Application (NDA) submitted by its collaboration partner Loxo Oncology, Inc. (NASDAQ: LOXO), and granted Priority Review for larotrectinib for the treatment of adult and pediatric patients with locally advanced or metastatic solid tumors harboring a neurotrophic tyrosine receptor kinase (NTRK) gene fusion. The FDA has set a target action date of November 26, 2018, under the Prescription Drug User Fee Act (PDUFA).
NTRK gene fusions are genetic alterations that result in production of tropomyosin receptor kinase (TRK) fusion proteins, and lead to the development of tumor growth. Bayer and Loxo Oncology are jointly developing larotrectinib, which is being studied globally for the treatment of patients across a wide range of cancers that harbor an NTRK gene fusion. Bayer plans to submit a Marketing Authorization Application (MAA) in the European Union in 2018.
“TRK fusion cancer is not limited to any organ or site of the body and occurs in both adults and children,” said Scott Fields, MD, senior vice president and head of oncology development at Bayer’s Pharmaceutical Division. “The Priority Review designation for larotrectinib may help bring this treatment option to patients, facing a high unmet medical need, as soon as possible.”
The FDA grants Priority Review for the applications of medicines that, if approved, would provide significant improvements in the safety or effectiveness of the treatment, diagnosis, or prevention of serious conditions when compared to standard applications. Larotrectinib has also been granted Breakthrough Therapy Designation, which is a process designed to expedite the development and review of drugs that are intended to treat a serious condition and preliminary clinical evidence indicates that the medicine may demonstrate substantial improvement over available therapies on a clinically significant endpoint, Rare Pediatric Disease Designation and Orphan Drug Designation by the U.S. FDA.
About Larotrectinib (LOXO-101)
Larotrectinib is an investigational tropomyosin receptor kinase (TRK) inhibitor in clinical development for the treatment of patients with cancers that harbor a neurotrophic tyrosine receptor kinase (NTRK) gene fusion. Growing research suggests that the NTRK genes can become abnormally fused to other genes, producing a TRK fusion protein that can lead to the development of solid tumors across multiple sites of the body.
In November 2017, Bayer and Loxo Oncology entered into an exclusive global collaboration for the development and commercialization of larotrectinib and LOXO-195, a TRK inhibitor in clinical development. Bayer and Loxo Oncology will jointly develop the two products with Loxo Oncology leading the ongoing clinical studies as well as the filing in the U.S., and Bayer leading ex-U.S. regulatory activities and worldwide commercial activities. In the U.S., Bayer and Loxo Oncology will co-promote the products.
For additional information about the larotrectinib clinical trials, please refer to http://www.clinicaltrials.gov or visit http://www.loxooncologytrials.com. Larotrectinib has not been approved by the U.S. Food and Drug Administration, the European Medicines Agency or any other health authority.
About TRK Fusion Cancer
TRK fusion cancer occurs when a neurotrophic tyrosine receptor kinase (NTRK) gene fuses with another unrelated gene, producing an altered tropomyosin receptor kinase (TRK) protein. The altered protein, or TRK fusion protein, becomes active and triggers a signal cascade. These proteins become the primary oncogenic driver of the spread and growth of tumors. NTRK gene fusion has been identified in various adult and pediatric solid tumors with varying frequencies.
About Oncology at Bayer
Bayer is committed to delivering science for a better life by advancing a portfolio of innovative treatments. The oncology franchise at Bayer now includes four oncology products and several other compounds in various stages of clinical development. Together, these products reflect the company’s approach to research, which prioritizes targets and pathways with the potential to impact the way that cancer is treated.
About Bayer
Bayer is a global enterprise with core competencies in the Life Science fields of health care and agriculture. Its products and services are designed to benefit people and improve their quality of life. At the same time, the Group aims to create value through innovation, growth and high earning power. Bayer is committed to the principles of sustainable development and to its social and ethical responsibilities as a corporate citizen. In fiscal 2017, the Group employed around 99,800 people and had sales of EUR 35.0 billion. Capital expenditures amounted to EUR 2.4 billion, R&D expenses to EUR 4.5 billion. For more information, go to http://www.bayer.us.
///////////Larotrectinib, UNII:PF9462I9HX, ларотректиниб , 拉罗替尼 , ARRY-470, LOXO-101, PF9462I9HX, phase 3, Array BioPharma, Loxo Oncology, National Cancer Institute, BAYER, orphan drug designation, breakthrough therapy designation
DRUG APPROVALS BY DR ANTHONY MELVIN CRASTO
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