IACS -9571

IACS-9571

TRIM24/BRPF1 bromodomain inhibitor
IACS-9571; IACS 9571; IACS9571.

Molecular Formula:	C32H42N4O8S
Molecular Weight:	642.76288 g/mol

N-[6-[3-[4-(dimethylamino)butoxy]-5-propoxyphenoxy]-1,3-dimethyl-2-oxobenzimidazol-5-yl]-3,4-dimethoxybenzenesulfonamide
BOARD OF REGENTS, UNIVERSITY OF TEXAS SYSTEM


IACS-9571 is a potent and selective inhibitor TRIM24 and BRPF1. The bromodomain containing proteins TRIM24 (Tripartite motif containing protein 24) and BRPF1 (bromodomain and PHD finger containing protein 1) are involved in the epigenetic regulation of gene expression and have been implicated in human cancer. Overexpression of TRIM24 correlates with poor patient prognosis and BRPF1 is a scaffolding protein required for the assembly of histone acetyltransferase complexes, where the gene of MOZ (monocytic leukemia zinc finger protein) was first identified as a recurrent fusion partner in leukemia patients (8p11 chromosomal rearrangements). IACS-9571 has low nanomolar affinities for TRIM24 and BRPF1 (ITC Kd = 31 nM and 14 nM, respectively). With its excellent cellular potency (EC50 = 50 nM) and favorable pharmacokinetic properties (F = 29%), IACS-9571 is a high-quality chemical probe for the evaluation of TRIM24 and/or BRPF1 bromodomain function in vitro and in vivo. (J Med Chem. 2015 Jun 10. [Epub ahead of print] )


PAPER
http://pubs.acs.org/doi/abs/10.1021/acs.jmedchem.5b00405

Structure-Guided Design of IACS-9571, a Selective High-Affinity Dual TRIM24-BRPF1 Bromodomain Inhibitor

Wylie S. Palmer^*†, Guillaume Poncet-Montange‡, Gang Liu†, Alessia Petrocchi†, Naphtali Reyna†, Govindan Subramanian†, Jay Theroff†, Anne Yau†, Maria Kost-Alimova†, Jennifer P. Bardenhagen†, Elisabetta Leo†, Hannah E. Shepard†, Trang N. Tieu†, Xi Shi†, Yanai Zhan†, Shuping Zhao†, Michelle C. Barton^§, Giulio Draetta†, Carlo Toniatti†, Philip Jones†, Mary Geck Do†, and Jannik N. Andersen†

^†Institute for Applied Cancer Science, and ^‡Core for Biomolecular Structure and Function, The University of Texas MD Anderson Cancer Center, 1881 East Road, Unit 1956, Houston, Texas 77054, United States

^§ Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center,

1515 Holcombe Boulevard

, Houston, Texas 77030, United States

J. Med. Chem., 2016, 59 (4), pp 1440–1454

DOI: 10.1021/acs.jmedchem.5b00405

Publication Date (Web): June 10, 2015

Copyright © 2015 American Chemical Society

*E-mail: wpalmer@mdanderson.org. Telephone: (001) 713-745-3022. Fax: (001) 713-745-8865.

The bromodomain containing proteins TRIM24 (tripartite motif containing protein 24) and BRPF1 (bromodomain and PHD finger containing protein 1) are involved in the epigenetic regulation of gene expression and have been implicated in human cancer. Overexpression of TRIM24 correlates with poor patient prognosis, and BRPF1 is a scaffolding protein required for the assembly of histone acetyltransferase complexes, where the gene of MOZ (monocytic leukemia zinc finger protein) was first identified as a recurrent fusion partner in leukemia patients (8p11 chromosomal rearrangements). Here, we present the structure guided development of a series of N,N-dimethylbenzimidazolone bromodomain inhibitors through the iterative use of X-ray cocrystal structures. A unique binding mode enabled the design of a potent and selective inhibitor 8i (IACS-9571) with low nanomolar affinities for TRIM24 and BRPF1 (ITC K_d = 31 nM and ITC K_d = 14 nM, respectively). With its excellent cellular potency (EC₅₀ = 50 nM) and favorable pharmacokinetic properties (F = 29%), 8i is a high-quality chemical probe for the evaluation of TRIM24 and/or BRPF1 bromodomain function in vitro and in vivo.

TFA salt of 8i (106 mg, 57%) as a white solid. 1H NMR (600 MHz, DMSO-d6) δ 9.46 (s, 1H), 9.30 (br-s, 1H), 7.19 (m, 2H), 7.07 (s, 1H), 6.90 (d, J = 9.0 Hz, 1H), 6.75 (s, 1H), 6.13 (t, J = 2.2 Hz, 1H), 5.71 (t, J = 2.0 Hz, 1H), 5.67 (t, J = 2.0 Hz, 1H), 3.84 (t, J = 5.9 Hz, 2H), 3.77 (m, 5H), 3.62 (s, 3H), 3.29 (s, 3H), 3.20 (s, 3H), 3.12–3.05 (m, 2H), 2.78 (d, J = 4.7 Hz, 6H), 1.77–1.63 (m, 6H), 0.95 (t, J = 7.3 Hz, 3H). 13C NMR (600 MHz, DMSO-d6) δ 160.3, 160.0, 159.3, 154.1, 152.0, 148.4, 143.9, 131.8, 128.2, 126.0, 121.9, 120.5, 110.4, 109.4, 106.4, 100.6, 95.9, 95.8, 95.2, 68.9, 66.7, 56.3, 55.6, 55.4, 42.1, 27.1, 27.0, 25.6, 21.9, 20.7, 10.4. MS (ESI) m/z 644 [M + H]+.
NMR

IACS -9571

 N-(6-(3-(4-(dimethylamino)butoxy)-5- propoxyphenoxy)-l,3-dimethyl-2-oxo-2,3-dihydro-lH-benzo[d]imidazol-5-yl)-3,4- dimethoxybenzenesulfonamide 2,2,2-trifluoroacetate

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.

ABSTRACT

251st ACS National Meeting & Exposition

13–17 March 2016

San Diego, United States

MEDI 5
Discovery and development of a potent dual TRIM24/BRPF1 bromodomain inhibitor, IACS -9571, using structure- based drug design Wylie S. Palmer 1 , wpalmer@mdanderson.org, Guillaume Poncet -Montagne 1 , Gang Liu 1 , Alessia Petrocchi 1 , N aphtali Reyna 1 , Govindan Subramanian 1 , Jay Theroff 1 , Maria Kost -Alimova 1 , Jennifer Bardenhagen 1 , Elisabetta Leo 1 , Hannah Sheppard 1 , Trang Tieu 1 , Shi Xi 1 , Yanai Zhan 1 , Shuping Zhao 1 , Michelle Barton 2 , Giulio Draetta 1 , Carlo Toniatti 1 , Philip Jones 1 , Mary Ge ck Do 1 , Jannik Andersen 1 . (1) Institute for Applied Cancer Science, The University of Texas, MD Anderson Cancer Center, Houston, Texas, United States (2) Department of Epigenetics and Molecular Carcinogenesis, The University of Texas, MD Anderson Cancer Center, Houston, Texas, United States
Bromodomains are an important class of chromatin remodeling proteins that recognize acetylated lysine residues on histone tails. As epigenetic targets they regulate gene transcription and offer a new way to treat diseas es, particularly in inflammation and oncology. The bromodomain and extra- terminal (BET) family has emerged as an important and druggable example of this class of proteins with the successful entry of small- molecule inhibitors into the clinic. Other families of bromodomains are only starting to be explored, such as the Tripartite Motif -containing 24 protein (TRIM24) and bromodomain- PHD finger protein 1 (BRPF1). Both proteins contain a dual PHD -bromo motif which have a role in recognizing specific histone mar ks. TRIM24 recognizes the dual histone marks of unmodified H3K4 and acetylated- H3K23 within the same histone tail. TRIM24 is a potent co- activator of ER -alpha and overexpression of TRIM24 has been linked to poor survival rates in breast cancer patients.
This presentation will describe the structure guided development of a series of N,N- dimethyl -benzimidazolones through the iterative use of X -ray cocrystal structures. A unique binding mode enabled the design of a potent and selective inhibitor (IACS -9571) with low nanomolar affinities for TRIM24 and BRPF1 (ITC Kd = 31 nM and ITC Kd = 14 nM, respectively). With its excellent cellular potency (EC 50 = 50 nM) and favorable pharmacokinetic properties, IACS -9571 is a high- quality chemical probe for the evaluation of TRIM24 and/or BRPF1 bromodomain function in vitro and in vivo

PATENT
WO-2016033416-A1

Synthesis of Intermediates:

N-(6-bromo-l ,3-dimethyl-2-oxo-2,3-dihydro-lH-benzo[d]imidazol-5-yl)-2,2,2- trifluoroacetamide (Intermediate 1):

Step 1 : 5-nitro-lH-benzo[d]imidazol-2(3H)-one:

To a 0 °C solution of 4-nitrobenzene- 1 ,2-diamine (44 g, 285 mmol) in 80 mL of DMF was added l, l'-carbonyldiimidazole (70 g, 428 mmol). The reaction mixture was stirred at RT for 4 h, then water (250 mL) was added. The resulting suspension was filtered, and the collected solids were washed with water (200 mL) and dried to give 5-nitro-lH- benzo[d]imidazol-2(3H)-one as a yellow solid (45 g, 88%). MS (ES+) C7H5N3O3 requires: 179, found: 180 [M+H]⁺.

Step 2: l,3-dimethyl-5-nitro-lH-benzo[d]imidazol-2(3H)-one:

To a solution of 5-nitro-lH-benzo[d]imidazol-2(3H)-one (55 g, 309 mmol) in 150 mL of DMF was added K2CO3 (85 g, 618 mmol), the reaction mixture was cooled to 0 °C, then iodomethane (109 g, 772 mmol) was slowly added. The reaction mixture was stirred at RT overnight, then water was added to the reaction mixture. The resulting suspension was filtered and the collected solids were washed with water (200 mL) and dried to give 1,3- dimethyl-5-nitro-lH-benzo[d]imidazol-2(3H)-one as a yellow solid (55 g, 86%). MS (ES+) C9H9N3O3 requires: 207, found: 208 [M+H] ⁺.

Step 3: 5-amino-l,3-dimethyl-lH-benzo[d]imidazol-2(3H)-one:

 To a solution of l,3-dimethyl-5-nitro-lH-benzo[d]imidazol-2(3H)-one (50 g, 240 mmol) in 200 mL of EtOAc under an inert atmosphere was added 10% palladium on activated carbon (5 g, 24 mmol). The reaction mixture was then charged with hydrogen and stirred at RT under an ¾ atmosphere overnight. The reaction mixture was filtered through a pad of celite then concentrated to give 5-amino-l,3-dimethyl-lH-benzo[d]imidazol-2(3H)- one as a yellow solid (32 g, 68%). MS (ES+) C9H11N3O requires: 177, found: 178 [M+H]⁺.

Step 4: 5-amino-6-bromo-l ,3-dimethyl-lH-benzo[d]imidazol-2(3H)-one:

 To a 0 °C solution of 5-amino-l ,3-dimethyl-lH-benzo[d]imidazol-2(3H)-one (4 g, 22.6 mmol) in 25 mL of CHCI3 and 25 mL of AcOH was slowly added drop wise bromine (3.5 g, 22.6mmol). The mixture was stirred at RT for 30 min, then concentrated and purified by silica gel chromatography (1 : 1 EtOAc/ hexanes) to afford 5-amino-6-bromo-l ,3-dimethyl- lH-benzo[d]imidazol-2(3H)-one as a yellow solid (3.2 g, 69%). MS (ES+) C₉HioBrN₃0 requires: 256, found: 257 [M+H]⁺.

Step 5: N-(6-bromo-l ,3-dimethyl-2-oxo-2,3-dihydro-lH-benzo[d]imidazol-5-yl)-2,2,2- trifluoroacetamide:

To a 0 °C solution of 5-amino-6-bromo-l ,3-dimethyl-lH-benzo[d]imidazol- 2(3H)-one (1.50 g, 5.9 mmol) in DCM (45 ml) was added DMAP (72 mg, 0.59 mmol), triethylamine (1.63 ml, 11.7 mmol) and trifluoroacetic anhydride (0.91 ml, 6.4 mmol). The reaction mixture was stirred for 2 h and warmed to RT. The reaction mixture was then quenched with water and the organic phase was washed with brine, dried over sodium sulfate, filtered and concentrated to give N-(6-bromo-l,3-dimethyl-2-oxo-2,3-dihydro-lH- benzo[d]imidazol-5-yl)-2,2,2-trifluoroacetamide (Intermediate 1) as a yellow solid (2.20 g, 100%). MS (ES+) CiiH₉BrF₃N302 requires: 352, found 353 [M+H]⁺.

5-amino-6-(3-hydroxyphenoxy)-l,3-dimethyl-lH-benzo[d]imidazol-2(3H)-one (Intermediate 2, Route A):

To a mixture of 5-amino-6-(3-(benzyloxy)phenoxy)-l,3-dimethyl-lH- benzo[d]imidazol-2(3H)-one (400 mg, 1.07 mmol) in DCM (20 mL) at -78 °C was added tribromoborane (5.3 mL, 5.3 mmol). The mixture was warmed up to room temperature gradually, then quenched by methanol dropwise, concentrated, and purified by column chromatography (20-100% EtOAc/hexanes and then 0-40% methanol/EtOAc) to give 5- amino-6-(3-hydroxyphenoxy)-l,3-dimethyl-lH-benzo[d]imidazol-2(3H)-one as a solid (240 mg, 79%). MS (ES+) C15H15N3O3 requires: 285, found: 286 [M+H]⁺.

5-amino-6-(3-hydroxyphenoxy)-l,3-dimethyl-lH-benzo[d]imidazol-2(3H)-one (Intermediate 2, Route B):

Step 2

Step 1: 3-[(ieri-butyldimethylsilyl)oxy]phenol:

A mixture of lH-imidazole (2.25 g, 33.1 mmol), ieri-butylchlorodimethylsilane (3.83 g, 25.4 mmol) and resorcinol (5.6 g, 51 mmol) in THF (30 ml) was stirred at 80 °C for 5 h. The resulting suspension of the cooled reaction mixture was filtered and the collected filtrate was concentrated and purified by silica-gel chromatography (20:80 to 0:100, EtOAc/hexanes) to give 3-((ieri-butyldimethylsilyl)oxy)phenol (2.78 g, 49%). MS (ES+) C12H20O2S1 requires: 224, found 225 [M+H]⁺.

Step 2: 5-amino-6-(3-((ier^butyldimethylsilyl)oxy)phenoxy)-l ,3-dimethyl-lH- benzo[d]imidazol-2(3H)-one:

 A mixture of 3-((ieri-butyldimethylsilyl)oxy)phenol (1.39 g, 6.20 mmol), quinolin-8-ol (79 mg, 0.55 mmol), copper(I) chloride (20 mg, 0.21 mmol), potassium phosphate (526 mg, 2.48 mmol) and 5-amino-6-bromo-l ,3-dimethyl-lH-benzo[d]imidazol- 2(3H)-one (529 mg, 2.07 mmol) in diglyme (20 ml) in a 100 mL round-bottom flask was degassed under a nitrogen atmosphere and heated to 120 °C for 24 h. To the cooled reaction mixture was added silica gel, stirred for 2 min, then the mixture was filtered through a pad of silica gel. The collected filtrate was concentrated and purified by column chromatography (20:80 to 0: 100, EtOAc/hexanesthen 0: 100 to 40:60, MeOH/EtOAc) to give 5-amino-6-(3- ((ieri-butyldimethylsilyl)oxy)phenoxy)-l,3-dimethyl-lH-benzo[d]imidazol-2(3H)-one (521 mg, 63%). MS (ES+) C21H29N3O3S1 requires: 399, found 400 [M+H]⁺.

Step 3: 5-amino-6-(3-hydroxyphenoxy)-l,3-dimethyl-lH-benzo[d]imidazol-2(3H)-one:

To a 0 °C solution of 5-amino-6-(3-((ieri-butyldimethylsilyl)oxy)phenoxy)-l,3- dimethyl-lH-benzo[d]imidazol-2(3H)-one (623 mg, 1.56 mmol) in THF was added a solution of ieira-butylammonium fluoride (0.90 mL, 3.1 mmol) in THF, the reaction mixture was allowed to warm up to RT and then stirred for 1-2 h. The reaction mixture was quenched with 1 M hydrogen chloride (0.10 mL, 3.1 mmol) and then partitioned between EtOAc and water. The seperated organic layer was washed with water twice, then concentrated and purified by column chromatography (20-80% EtOAc/hexanes and 0-40% MeOH/DCM) to give 5-amino-6-(3-hydroxyphenoxy)-l ,3-dimethyl-lH-benzo[d]imidazol-2(3H)-one (120 mg, 27%) as a solid. MS (ES+) C15H15N3O3 requires: 285, found 286 [M+H]⁺.

EXAMPLE 10: N-(6-(3-(4-(dimethylamino)butoxy)-5-propoxyphenoxy)-l,3-dimethyl-2- oxo-2,3-dihydro-lH-benzo[d]imidazol-5-yl)-3,4-dimethoxybenzenesulfonamide 2,2,2-

To a solution of N-(6-(3-(4-aminobutoxy)-5-propoxyphenoxy)-l ,3-dimethyl-2- oxo-2,3-dihydro-lH-benzo[d]imidazol-5-yl)-3,4-dimethoxybenzenesulfonamide 2,2,2- trifluoroacetate (180 mg, 0.247 mmol) in methanol (3.0 ml) was added triethylamine (0.034 ml, 0.25 mmol), acetic acid (0.028 ml, 0.49 mmol), formaldehyde (0.054 ml, 2.0 mmol), and sodium triacetoxyborohydride (131 mg, 0.618 mmol). The reaction mixture was stirred at room temperature and checked by LCMS every 30 minutes. After 3 h the reaction was complete by LCMS. The reaction was quenched with a few drops of TFA and concentrated under reduced pressure. The residue was purified by prep-HPLC using a gradient of 20-60% ACN/water containing 0.1% TFA to afford N-(6-(3-(4-(dimethylamino)butoxy)-5- propoxyphenoxy)-l,3-dimethyl-2-oxo-2,3-dihydro-lH-benzo[d]imidazol-5-yl)-3,4- dimethoxybenzenesulfonamide 2,2,2-trifluoroacetate (106 mg, 57%) as a white solid. MS (ES+) C32H42N4O8S requires: 642, found 643 [M+H]⁺. ¾ NMR (600 MHz, DMSO-ifc) δ 9.46 (s, 1H), 9.30 (br-s, 1H), 7.19 (m, 2H), 7.07 (s, 1H), 6.90 (d, 7 = 9.0 Hz, 1H), 6.75 (s, 1H), 6.13 (t, 7 = 2.2 Hz, 1H), 5.71 (t, J = 2.0 Hz, 1H), 5.67 (t, J = 2.0 Hz, 1H), 3.84 (t, 7 = 5.9 Hz, 2H), 3.77 (m, 5H), 3.62 (s, 3H), 3.29 (s, 3H), 3.20 (s, 3H), 3.12-3.05 (m, 2H), 2.78 (d, 7 = 4.7 Hz, 6H), 1.77-1.63 (m, 6H), 0.95 (t, 7 = 7.3 Hz, 3H)

References

1: Palmer WS, Poncet-Montange G, Liu G, Petrocchi A, Reyna N, Subramanian G, Theroff J, Yau A, Kost-Alimova M, Bardenhagen JP, Leo E, Shepard HE, Tieu TN, Shi X, Zhan Y, Zhao S, Draetta G, Toniatti C, Jones P, Geck Do M, Andersen JN. Structure-Guided Design of IACS-9571, a Selective High-Affinity Dual TRIM24-BRPF1 Bromodomain Inhibitor. J Med Chem. 2015 Jun 10. [Epub ahead of print] PubMed PMID: 26061247.
US-20160060260-A1


Institute for Applied Cancer Science, The University of Texas, MD Anderson Cancer Center, Houston, Texas, United States

The University of Texas MD Anderson Cancer Center | University of Texas System


The new Institute for Applied Cancer Science will be located at the south campus of M.D.

Draetta arrived at MD Anderson in 2011 to direct the Institute for Applied Cancer Science. He oversees the moon shots platforms

Department of Epigenetics and Molecular Carcinogenesis, The University of Texas, MD Anderson Cancer Center, Houston, Texas, United States





///////IACS-9571, TRIM24, BRPF1 bromodomain inhibitor, IACS-9571,  IACS 9571,  IACS9571, BOARD OF REGENTS, UNIVERSITY OF TEXAS SYSTEM


CAS BASE 1800477-30-8
CAS OF 1:1 TRIFLUOROACETATE 1883598-69-3
c1(cc(cc(c1)OCCC)Oc3cc2N(C(N(c2cc3NS(=O)(=O)c4cc(c(cc4)OC)OC)C)=O)C)OCCCCN(C)C
CCCOC1=CC(=CC(=C1)OC2=C(C=C3C(=C2)N(C(=O)N3C)C)NS(=O)(=O)C4=CC(=C(C=C4)OC)OC)OCCCCN(C)C
TFA salt of 8i (106 mg, 57%) as a white solid. 1H NMR (600 MHz, DMSO-d6) δ 9.46 (s, 1H), 9.30 (br-s, 1H), 7.19 (m, 2H), 7.07 (s, 1H), 6.90 (d, J = 9.0 Hz, 1H), 6.75 (s, 1H), 6.13 (t, J = 2.2 Hz, 1H), 5.71 (t, J = 2.0 Hz, 1H), 5.67 (t, J = 2.0 Hz, 1H), 3.84 (t, J = 5.9 Hz, 2H), 3.77 (m, 5H), 3.62 (s, 3H), 3.29 (s, 3H), 3.20 (s, 3H), 3.12–3.05 (m, 2H), 2.78 (d, J = 4.7 Hz, 6H), 1.77–1.63 (m, 6H), 0.95 (t, J = 7.3 Hz, 3H). 13C NMR (600 MHz, DMSO-d6) δ 160.3, 160.0, 159.3, 154.1, 152.0, 148.4, 143.9, 131.8, 128.2, 126.0, 121.9, 120.5, 110.4, 109.4, 106.4, 100.6, 95.9, 95.8, 95.2, 68.9, 66.7, 56.3, 55.6, 55.4, 42.1, 27.1, 27.0, 25.6, 21.9, 20.7, 10.4. MS (ESI) m/z 644 [M + H]+.

Tripeptide Glycyl-L-Prolyl-L-Glutamate (Gly-Pro-Glu or GPE)

Gly-Pro-Glu

Synonym: GPE, Glycyl-prolyl-glutamic acid, (1-3)IGF-1

Pfizer (Originator)
Neuren Pharmaceuticals (Originator)

Glypromate; glycine-proline-glutamate (neuroprotectant), Neuren

• CAS Number 32302-76-4
• Empirical Formula C₁₂H₁₉N₃O₆
• Molecular Weight 301.30
• Psychiatric Disorders (Not Specified)
  Neurologic Drugs (Miscellaneous)
  Cognition Disorders, Treatment of
  Antiepileptic Drugs
  Antidepressants Biochem/physiol Actions

Gly-Pro-Glu is a neuroprotective compound and the N-terminal tripeptide of IGF-1. Gly-Pro-Glu is neuroprotective after central administration in animal models of neurodegenerative processes, such as Huntington’s, Parkinson’s, Alzheimer’s diseases, and varies acute brain injury animal models. The neuroprotective activity is not related to its affinity to glutamate receptor. Findings indicate that GPE mimics insulin-like growth factor I effects on the somatostatin system through a mechanism independent of β-amyloid clearance that involves modulation of calcium and glycogen synthase kinase 3β signaling.
GPE is a naturally occurring peptide fragment which had been in phase III clinical trials at Neuren Pharmaceuticals for use as prophylactic neuroprotection for patients undergoing coronary artery bypass graft (CABG) and valvuloplasty surgery. Although clinical evaluation in Australia continues, phase III trials evaluating the compound in the U.S. were discontinued based on negative results. The compound is found in normal brain tissue and, when injected intravenously, has been shown to act by multiple pathways to protect brain tissue from injury. The drug was originally developed by Pfizer, but rights were transferred to Neuren pursuant to a proprietary agreement between the companies.

When amino acids join together (forming short groups called polypeptides, or much longer chains called proteins) the amine group of one amino acid joins with the carboxyl group of the next, making a peptide bond. These bonds don’t ionise at different pHs, but can be hydrolised — broken — reforming the amino acids. GPE is formed from the amino acids glycine, proline and glutamic acid:

This tripeptide has 3 pH-sensitive groups, each with its own pK_a. What the university chemists needed to do was work out what form GPE is in when it is active in the brain, what parts of the molecule are critical to its effectiveness, and how to ‘tweak’ the molecule (by changing the side chains) so that it will remain in the brain for longer than the naturally-occurring substance.   They also needed to make sure the final compound passes through the blood-brain barrier (that prevents most substances in the blood from entering and affecting the brain). If possible, they also wanted a compound that could be taken in pill form without being broken down in the stomach. It was also essential that the compound was safe for people to take!
Neuren Pharmaceuticals

After initial work on GPE at the university, the research was passed to a spin-off research group called Neuren Pharmaceuticals Ltd, which takes compounds discovered by the University of Auckland and develops them into medicines. Neuren developed GPE intoGlypromate® and are working with researchers in the US (including the US Military, who have a keen interest in a medicine that will reduce brain damage after head injuries) to test the compound on patients. There is considerable interest in Glypromate® world-wide, because at present there is nothing that reduces cell death after brain injuries. The chances of winning a race are pretty high when you’re the only competitor!Glypromate® is being tested on heart-bypass patients because up to 70% of bypass patients are affected mentally after their surgery. It’s thought that tiny clots form after the heart is restarted, and that these travel to the brain and cause mini-strokes. Unlike naturally-occurring strokes, or the brain damage caused by accident or war, the bypass surgery is planned, so before and after tests can be done on the patients to see exactly what effect the treatment has. Early results look very promising.
Glypromate is just one of the compounds Neuren is working on. Others may develop into treatments for Multiple Sclerosis, Parkinson’s Disease or Alzheimer’s Disease as well as various kinds of cancer. The company’s links with overseas research groups mean that compounds developed in New Zealand are able to be tested in the US and gain the FDA approval which will allow them to be used in most countries in the world.

The tripeptide Glycyl-L-Prolyl-L-Glutamate (Gly-Pro-Glu or GPE) is a naturally occurring peptide, which is proteolytically cleaved from insulin-like growth factor-1 (IGF-1). IGF-1 is a potent neurotrophic factor produced endogenously in damaged regions of the brain. It has been postulated that some of the neuroprotective actions of IGF-1 are mediated by GPE although the precise mechanism of action remains unclear. GPE has a different mode of action to IGF-1 as GPE does not bind to the IGF-1 receptor. Rather, GPE has been shown to bind with low affinity to the N-methyl-D-aspartate (NMDA) receptor and also elicit a biological response via other mechanisms. GPE facilitates the release of dopamine through interaction with the NMDA receptor but GPE stimulated acetylcholine release is via an unknown, non-NMDA pathway.
It has been demonstrated that GPE can act as a neuronal rescue agent following brain injury or disease, including hypoxic-ischemic brain injury, NMDA challenge, chemical toxins and in animal models of Parkinson's and Alzheimer's disease. Analogs of GPE are thus of interest in the development of novel pharmaceutical agents for the treatment of central nervous system (CNS) injuries and neurodegenerative disorders among others.
CURRENT STATUS

Neuren Pharmaceuticals was developing Glypromate (glycine-proline glutamate), a naturally occurring small-molecule neuroprotectant derived from IGF-1 which inhibits caspase III dependent apoptosis, for the potential treatment of neurodegenerative diseases by iv infusion. By June 2008, a phase III trial had begun . However, in December 2008, the company discontinued further development of the drug after it failed to show an observable effect [972907]. In November 2005, the company was seeking to outlicense the drug [771417].
Neuren is also investigating the Glypromate analog, NNZ-2566 for similar indications.

In August 2006, Neuren expected Glypromate to be eligible for Orphan Drug status for neurodegenerative diseases and planned to apply for Fast Track status for the drug.

SYDNEY, Australia, Sept. 4 /PRNewswire-FirstCall/ -- Neuren Pharmaceuticals today announced that physicians from Madigan Army Medical Center (Madigan) in Tacoma, Washington, will conduct an investigator- initiated Phase 2 trial to determine the safety and efficacy of Glypromate(R) in reducing brain injury caused by out of hospital cardiac arrest. The trial will start in mid-2007 and will be managed by The Henry M. Jackson Foundation for the Advancement of Military Medicine (Jackson Foundation) in consultation with the clinical investigators at Madigan.
The proposed study will be an investigator-initiated study which means that the Investigational New Drug (IND) application will be submitted to the FDA by the Army investigators rather than by Neuren. Neuren will provide the drug product as well as access to preclinical, clinical and regulatory documents related to Glypromate(R). The Company's only financial commitment will be compensation to the Jackson Foundation for administrative costs incurred in coordinating the study. Neuren will retain all commercial rights to Glypromate(R) in these indications.
Cardiac arrest involves the sudden, complete cessation of heart function and circulation leading rapidly to neurological and other organ system damage. Among patients who survive, the consequences of neurological damage resulting from lack of blood flow and oxygen to the brain represent the primary adverse outcomes. This occurs in up to 80% of survivors and causes cognitive impairment such as occurs in patients undergoing major cardiac surgery, the focus for Neuren's upcoming Phase 3 study with Glypromate(R). However recovery without residual neurological damage after cardiac arrest is rare.
There are no drugs approved to reduce the neurological damage caused by cardiac arrest. Neuren believes that Glypromate(R) for this indication will be eligible for Orphan Drug designation. Orphan Drug designation provides for a period of market exclusivity following approval as well as possible access to US government grants. In addition, because of the serious nature of neurological impairment resulting from cardiac arrest and the lack of available drug therapy, Neuren intends to apply for Fast Track designation which provides for accelerated clinical development and review.
While the Army's investigator-initiated trial will focus on out of hospital cardiac arrest, if this trial is successful, Neuren, the Jackson Foundation and the Army investigators are considering additional trials of Glypromate(R) to reduce brain damage resulting from related conditions including in-hospital cardiac arrest and treatment of patients with ventricular fibrillation, the heart rhythm disturbance associated with more than 75% of cardiac arrests.
Under the agreement, the Jackson Foundation will provide support to the Army investigators in clinical trial preparations, protocol development, obtaining human subjects clearance, coordination of patient enrolment, data management and analysis, and preparation of study reports.
Mr David Clarke, CEO of Neuren said: "This is a very important development for Neuren in that it reflects a growing appreciation of the potential for Glypromate(R) to reduce neurological damage. It also, of course, reinforces the value and strength of Neuren's relationship with the US Army physicians and scientists. Cardiac arrest is a devastating clinical event and one for which a drug to reduce the neurological consequences is clearly needed. The addition of this trial will now give Neuren a very strong and cost effective portfolio of clinical trials in 2007 -- a Phase 3 and a Phase 2 for Glypromate(R) and the two Phase 2 trials with NNZ-2566."
Approximately 300,000 deaths result from cardiac arrest in the US each year, making cardiac arrest one of the leading causes of death. According to the American Heart Association, each year approximately 160,000 people in the US experience sudden cardiac arrest outside of a hospital or in a hospital emergency department.
Neuren estimates that the number of patients in the US that could be treated for out of hospital cardiac arrest and related indications is approximately 400,000 which could represent a potential market of US$800 million.
About Madigan Army Medical Center
Madigan Army Medical Center, located in Tacoma, Washington, is one of the major US Army medical centers, providing clinical care to over 120,000 active, reserve and retired military personnel and dependents. The hospital has a medical staff of more than 1,000 with 200 physicians and nurses in training. Madigan's Department of Clinical Investigations, which is dedicated to writing, performing, and regulating clinical research, is conducting approximately 200 clinical trials across a wide spectrum of indications from Phase I to IV.
About the Jackson Foundation
The Jackson Foundation is a private, not-for-profit organisation that supports the US military in conducting medical research and clinical trials and has established relationships with more than 160 military medical organisations worldwide. It was founded in 1983, in part, to foster cooperative relationships between the military medical community and the private sector, including pharmaceutical sponsors. The Jackson Foundation manages Phase I - IV clinical trials utilizing an established network of military medical centers across the United States.
About Glypromate(R)
Glypromate(R) is a peptide fragment of IGF-1 and is being developed by Neuren as a potential therapeutic candidate for diseases caused by some forms of chronic or acute brain injury. Glypromate(R) has been shown to act by multiple pathways to protect brain tissue from injury. Neuren has successfully completed a Phase I safety study and a Phase IIa safety and pharmacokinetics study and plans to initiate a Phase III study in late 2006.
About Neuren Pharmaceuticals
Neuren Pharmaceuticals is a biotechnology company developing novel therapeutics in the fields of brain injury and diseases and metabolic disorders. The Neuren portfolio consists of six product families, targeting markets with large unmet needs and limited competition. Neuren has three lead candidates, Glypromate(R) andNNZ-2566, presently in the clinic in development to treat a range of acute neurological conditions, and NNZ-2591, in preclinical development for Parkinson's and other chronic conditions. Neuren has commercial and development partnerships with the US ArmyWalter Reed Army Institute of Research, Metabolic Pharmaceuticals,UCLA Medical Center and the National Trauma Research Institute in Melbourne.
For more information, please visit Neuren's website at http://www.neurenpharma.com
Company David Clarke CEO of Neuren T: 1800 259 181 (Australia) T: +64 9 3 367 7167 ext 82308 (New Zealand) M: +64 21 988 052 Media and investor relations Rebecca Piercy Buchan Consulting T: +61 9827 2800 M: +61 422 916 422
CONTACT: David Clarke, CEO of Neuren, 1-800-259-181(Australia), or
+64-9-3-367-7167 ext 82308 (New Zealand), or +64-21-988-052 (mobile); or
Media and investor relations - Rebecca Piercy of Buchan Consulting,
+61-9827-2800, +61-422-916-422 (mobile)
Web site: http://www.neurenpharma.com/
REFERENCES
1 EP 0366638
2 WO 2005042000
3 WO 2008153929
4 WO 2009033805
5 WO 2009033806
Synthesis off isotopically labelled glycyl-L-prolyl-L-glutamic acid (Glypromate(R)) and derivatives
J Label Compd Radiopharm 2006, 49(6): 571
An efficient fmoc solid-phase synthesis of an amphiphile of the neuroprotective agent glycyl-prolyl-glutamic acid
Synlett (Stuttgart) 2014, 25(15): 2221
Intracellular pathways activated by Insulin-like growth factor 1 and its derivates
40th Annu Meet Soc Neurosci (November 13-17, San Diego) 2010, Abst 167.13

EP2667715A1 *	Jan 27, 2012	Dec 4, 2013	Neuren Pharmaceuticals Limited	Treatment of autism spectrum disorderes using glycyl-l-2-methylprolyl-l-glutamic acid
EP2667715A4 *	Jan 27, 2012	Jul 23, 2014	Neuren Pharmaceuticals Ltd	Treatment of autism spectrum disorderes using glycyl-l-2-methylprolyl-l-glutamic acid
US8940732	Jan 15, 2010	Jan 27, 2015	Massachusetts Institute Of Technology	Diagnosis of autism spectrum disorders and its treatment with an antagonist or inhibitor of the 5-HT2c receptor signaling pathway
US9212204	Jan 26, 2015	Dec 15, 2015	Neuren Pharmaceuticals Limited

WO2005042000A1 *	22 Oct 2004	12 May 2005	David Charles Batchelor	Neuroprotective effects of gly-pro-glu following intravenous infusion
WO2005097161A2 *	30 Mar 2005	20 Oct 2005	Peter D Gluckman	Gpe and g-2mepe, caffeine and alkanol for treatment of cns injury
WO2006127702A2 *	23 May 2006	30 Nov 2006	Neuren Pharmaceuticals Ltd	Analogs of glycyl-prolyl-glutamate
EP0366638A2 *	24 Oct 1989	2 May 1990	KabiGen AB	Neuromodulatory peptide
US20020151522 *	13 Mar 2002	17 Oct 2002	Tajrena Alexi	Regulation of weight

 

Reference
1	*	ALONSO DE DIEGO, SERGIO A. ET AL: "New Gly-Pro-Glu (GPE) analogues: Expedite solid-phase synthesis and biological activity" BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, vol. 16, no. 5, 2006, - 1392 page 1396, XP002527092
2	*	SARA V R ET AL: "IDENTIFICATION OF GLY-PRO-GLU (GPE), THE AMINOTERMINAL TRIPEPTIDE OF INSULIN-LIKE GROWTH FACTOR 1 WHICH IS TRUNCATED IN BRAIN, AS A NOVEL NEUROACTIVE PEPTIDE" BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, ACADEMIC PRESS INC. ORLANDO, FL, US, vol. 165, no. 2, 15 December 1989 (1989-12-15), pages 766-771, XP000992688 ISSN: 0006-291X

//////Gly-Pro-Glu, GPE, Glycyl-prolyl-glutamic acid,  32302-76-4, Tripeptide,  Glycyl-L-Prolyl-L-Glutamate, Glypromate®, (1-3)IGF-1 , PHASE 3, Glypromate,  glycine-proline-glutamate, neuroprotectant, Neuren

Neuren’s NNZ-2566 shows clinical benefit in Rett syndrome trial

Promising results in Phase 2 clinical trial

by Michael Tranfaglia, MD
FRAXA Medical Director

This isn’t a Fragile X trial, but the Neuren compound, NNZ-2566, that is in trials now for Fragile X has shown significant positive effects in a Phase 2 trial for Rett syndrome.
The results of the trial are interesting, in that improvement was seen a Rett syndrome-specific rating scale compared to placebo, and there was also improvement noted on the CGI-I (Clinical Global Impression of Improvement) and Caregiver Top 3 Concerns. However, there was no effect seen on ABC scores (Aberrant Behavior Checklist) compared to placebo. Many in the Fragile X field have noted the inadequacies of the ABC; indeed, it was never designed or intended to be an outcome measure for clinical trials. In this case, a Rett-specific rating scale called the Motor-Behavior Assessment (MBA) showed a statistically significant and clinically meaningful treatment effect at the highest dose of the Neuren compound compared to placebo.
This is great news for those of us in the Fragile X community for several reasons:

• It shows that this compound really does something—it seems to have useful properties in actual patients, and that’s not trivial.
• It demonstrates that disease-specific symptoms can improve significantly on the drug, and that improvement can be measured in a relatively short clinical trial.
• It shows that a drug can have beneficial effects on core features of a genetically based developmental disorder, even if the more general rating scales (like the ABC) show no change.

–
This last point is strongly reminiscent of the experience of many families and clinicians in recent Fragile X clinical trials, where the drugs showed no advantage compared to placebo based on rating scales, but genuine improvement was noted in many subjects, with significant deterioration upon discontinuation of the drugs. Thus the calls for improved rating scales which can “capture” these core, disease-specific therapeutic effects. The NeurenFragile X trial is using some Fragile X-specific outcome measures which will hopefully lead to similar positive results.
The fact that this result is good news for Neuren also means that the company should remain financially viable for longer, so that they can continue the development of this compound for a number of indications—more “shots on goal”.
Of course, the usual caveats apply: this was a small study, and these results need to be replicated in a larger Phase 3 trial. Still, there’s a realistic possibility that we may see a similar result in Fragile X!

BRIVARACETAM

BRIVARACETAM, UCB-34714

(2S)-2-[(4R)-2-oxo-4-propylpyrrolidin-1-yl]butanamide

(2S)-2-[(4R)-2-Oxo-4-propyl-1-pyrrolidinyl]butanamide

1-Pyrrolidineacetamide, α-ethyl-2-oxo-4-propyl-, (αS,4R)-

 CAS 357336-20-0

Molecular Formula:	C11H20N2O2
Molecular Weight:	212.2887 g/mol

UNII-U863JGG2IA

UCB; For the treatment of partial onset seizures related to epilepsy, Approved February 2016

Brivaracetam, the 4-n-propyl analog of levetiracetam, is a racetam derivative with anticonvulsant properties.[1][2] Brivaracetam is believed to act by binding to the ubiquitous synaptic vesicle glycoprotein 2A (SV2A).[3] Phase II clinical trials in adult patients with refractory partial seizures were promising. Positive preliminary results from stage III trials have been recorded,[4][5] along with evidence that it is around 10 times more potent[6] for the prevention of certain types of seizure in mouse models than levetiracetam, of which it is an analogue.

On 14 January 2016, the European Commission,[7] and on 18 February 2016, the USFDA[8] approved brivaracetam under the trade nameBriviact (by UCB). The launch of this anti-epileptic is scheduled for the first quarter of that year. Currently, brivaracetam is still not approved in other countries like Australia, Canada and Switzerland.

Brivaracetam was approved by European Medicine Agency (EMA) on Jan 14, 2016 and approved by the U.S. Food and Drug Administration (FDA) on Feb 18, 2016. It was developed and marketed as Briviact® by UCB in EU/US.

Brivaracetam is a selective high-affinity synaptic vesicle protein 2A ligand, as an adjunctive therapy in the treatment of partial-onset seizures with or without secondary generalization in adult and adolescent patients from 16 years of age with epilepsy.

Briviact® is available in three formulations, including film-coated tablets, oral solution and solution for injection/infusion. And it will be available as 10 mg, 25 mg, 50 mg, 75 mg and 100 mg film-coated tablets, a 10 mg/ml oral solution, and a 10 mg/ml solution for injection/infusion. The recommended starting dose is either 25 mg twice a day or 50 mg twice a day, depending on the patient’s condition. The dose can then be adjusted according to the patient’s needs up to a maximum of 100 mg twice a day. Briviact can be given by injection or by infusion (drip) into a vein if it cannot be given by mouth.

European Patent No. 0 162 036 Bl discloses compound (S)-α-ethyl-2-oxo-l- pyrrolidine acetamide, which is known under the International Non-proprietary Name of Levetiracetam.

Levetiracetam

Levetiracetam is disclosed as a protective agent for the treatment and prevention of hypoxic and ischemic type aggressions of the central nervous system in European patent EP 0 162 036 Bl. This compound is also effective in the treatment of epilepsy.

The preparation of Levetiracetam has been disclosed in European Patent No. 0 162 036 and in British Patent No. 2 225 322.

International patent application having publication number WO 01/62726 discloses 2-oxo-l -pyrrolidine derivatives and methods for their preparation. It particularly discloses compound (2S)-2-[(4R)-2-oxo-4-propyl-pyrrolidin-l-yl] butanamide known under the international non propriety name of brivaracetam.

Brivaracetam

International patent application having publication number WO 2005/121082 describes a process of preparation of 2-oxo-l -pyrrolidine derivatives and particularly discloses a process of preparation of (2S)-2-[(4S)-4-(2,2-difluorovinyl)-2-oxo-pyrrolidin-l- yl]butanamide known under the international non propriety name of seletracetam.

Seletracetam

Kenda et al., in J. Med. Chem. 2004, 47, 530-549, describe processes of preparation of 2-oxo-l -pyrrolidine derivatives and particularly discloses compound 1-((1S)-I- carbamoyl-propyl)-2-oxo-pyrrolidone-3-carboxylic acid as a synthetic intermediate.

WO2005028435

CLIPS

Find better ways to make old and new epilepsy drugs. J. Surtees and co-inventors disclose alternative processes for making active pharmaceutical ingredients (APIs) that are used to treat epilepsy and seizures. One compound that can be prepared by their processes is the established drug levetiracetam (1, Figure 1), marketed under the trade name Keppra. Because 1 is now off-patent, there is obvious interest in new drugs.

The inventors also claim that seletracetam (2) and brivaracetam (3) (Figure 2) can be prepared by their processes. These drugs are apparently much more active than 1.

All of the drugs are used as single isomers, so a stereoselective synthesis is desirable. The inventors describe two routes for preparing the molecules; the first, shown in Figure 1, is the synthesis of 1 by the reaction between pyrrolidone (4) and chiral bromo amide 5 in the presence of a base. GC analysis showed that the conversion is 40.3% and that the product contains 51% of the (S)-enantiomer and 49% of the (R)-isomer. No details of their separation are given, although the use of chiral HPLC is discussed.

The same reaction is used to prepare derivative 6 of 1. Compound 7 is prepared from the corresponding hydroxy ester and then condensed with 4 to give 6. Chiral HPLC showed that the product is a mixture of 89.3% (S)-enantiomer 6 and 10.7% of its (R)-isomer.

The inventors do not describe the detailed preparation of 2, but they report that acid 8 is prepared in 41% yield from pyrrolidone 9 and acid10 in the presence of NaH (Figure 2). Ammonolysis of 8 produces 2; no reaction details are provided.

In a reaction similar to the preparation of 8, acid 11 is prepared from 10 and pyrrolidone 12. The product is isolated in 77% yield and can be converted to 3 by ammonolysis. Again, no details are provided for this reaction.

The second route for preparing the substituted pyrrolidones does not start with simple pyrrolidones and is the subject of additional claims. The route involves a cyclization reaction, shown in Figure 3. The preparation of enantiomer 13 begins with the reaction of racemic salt 14and optically pure bromo ester 15. This step produces intermediate 16, isolated as a yellow oil. The crude material is treated with 2-hydroxypyridine (2-HP) to cyclize it to 17. This ester is hydrolyzed to give acid 18. Conversion to 13 is carried out by adding ClCO2Et, followed by reaction with liquid NH3 in the presence of K2CO3. The overall yield of 13 is 32%.

This route is also used to prepare levetiracetam (1) by treating 5 with the HCl salt of amino ester 19 to give 20, recovered as its HCl salt in 49% yield. The salt is basified with Et3N and treated with 2-HP to cyclize it to 1, initially isolated as an oil. GC analysis showed 100% conversion, and chiral HPLC showed that the product contains 98.6% (S)-isomer and 1.4% (R)-isomer.

The inventors also prepared 1 and its (R)-enantiomer 21 by using a similar reaction scheme with alternative substrates to 5. Figure 4 outlines the route, which starts from protected hydroxy amide 22 and amino ester 23. When the reaction is carried out in the presence of Cs2CO3, the product is (R)-enantiomer24, which is used without purification to prepare 21 by treating it with 2-HP. Chiral HPLC showed that the product is 94% (R) and 6% (S).

When the reaction between 22 and 23 is run with K2CO3, the product is (S)-enantiomer 25. This is used to prepare 1, but the product contains only 79% (S)-isomer.

The inventors do not comment on the apparent stereoselectivity of the carbonate salts in the reaction of 22 with 23. This is an intriguing finding and worthy of investigation. (UCB S.A. [Brussels]. US Patent 8,338,621

SYNTHESIS

PATENT

WO2005028435

Example 1: Synthesis of (2S)-2-((4R)-2-oxo-4-n-propyl-l-pyrrolidinyl)butanamide 1.1 Synthesis of (2S)-2-aminobutyramide free base

1800 ml of isopropanol are introduced in a 5L reactor. 1800 g of (2S)-2- aminobutyramide tartrate are added under stirring at room temperature. 700 ml of a 25% aqueous solution of ammonium hydroxide are slowly added while maintaining the temperature below 25°C. The mixture is stirred for an additional 3 hours and then the reaction is allowed to complete at 18°C for 1 hour. The ammonium tartrate is filtered. Yield : 86%.

1.2 Synthesis of 5-hydroxy-4-n-propyl-furan-2-one

Heptane (394 ml) and morpholine (127.5 ml) are introduced in a reactor. The mixture is cooled to 0°C and glyoxylic acid (195 g, 150 ml, 50w% in water) is added. The mixture is heated at 20°C during 1 hour, and then valeraldehyde (148.8 ml) is added . The reaction mixture is heated at 43°C during 20 hours. After cooling down to 20CC, a 37 % aqueous solution of HCl (196.9 ml) is slowly added to the mixture, which is then stirred during 2 hours.

After removal of the heptane phase, the aqueous phase is washed three times with heptane. Diisopropyl ether is added to the aqueous phase. The organic phase is removed, and the aqueous phase further extracted with diisopropyl ether (2x). The diisopropyl ether phases are combined, washed with brine and then dried by azeotropic distillation. After filtration and evaporation of the solvent, 170g of 5- hydroxy-4-n-propyl-furan-2-one are obtained as a brown oil. Yield: 90.8 %

1.3 Synthesis of (2S)-2-((4R)-2-oxo-4-n-propyl-l-pyrrolidinyl)butanamide and (2S)-2-((4S)-2-oxo-4-n-propyl-l-pyrrolidinyl)butanamide

(S, R) (S, S) The (2S)-2-aιninobutyrarnide solution in isopropanol containing 250 g obtained as described here above is dried by azeotropic distillation under vacuum. To the dried (2S)-2-am obutyraιnide solution is added 5-hydroxy-4-n-propyl-furan-2-one (290 g) between 15°C and 25 °C; the mixture is heated to 30 °C and kept for at least 2 hours at that temperature. Acetic acid (1, 18 eq.), Pd/C catalyst (5 w/w%; Johnson Matthey 5% Pd on carbon – type 87L) are then added and hydrogen introduced into the system under pressure. The temperature is kept at 40 °C maximum and the H2 pressure maintained between 0,2 bar and 0,5 bar followed by stirring for at least 20 hours following the initial reaction. The solution is then cooled to between 15 °C and 25 °C and filtered to remove the catalyst. The solution of product in isopropanol is solvent switched to a solution of product in isopropyl acetate by azeotropic distillation with isopropyl acetate. The organic solution is washed with aqueous sodium bicarbonate followed by a brine wash and then filtered. After recristallisation, 349 g of (2S)-2-((4R)-2- oxo-4-n-propyl-l-pyrrolidinyl)butanamide and (2S)-2-((4S)-2-oxo-4-n-propyl-l- pyιτolidinyl)butanamide are obtained (Yield: 82.5%).

1.4 Preparation of (2S)-2-((4R)-2-oxo-4-n-propyl-l-pyrrolidinyl)butanamide The chromatographic separation of the two diastereoisomers obtained in 1.3 is performed using of (CHIRALPAK AD 20 um) chiral stationary phase and a 45/55 (volume /volume) mixture of n-heptane and ethanol as eluent at a temperature of 25 + 2°C. The crude (2S)-2-((4R)-2-oxo-4-n-propyl-l-pyrrolidinyl)butanamide thus obtained is recristallised in isopropylacetate, yielding pure (2S)-2-((4R)-2-oxo-4-n-propyl-l- pyrrolidinyl)butanamide (Overall yield: 80%) .

Example 2: Synthesis of (2S)-2-((4R)-2-oxo-4-n-propyl-l-pyrrolidinyl)butanamide

Example 1 is repeated except that in step 1.1 a solution of (2S)-2- aminoburyramide.HCl in isopropanol is used (27.72 g, 1.2 equivalent), which is neutralised with a NHs/isopropanol solution (3,4-3,7 mol/L). The resulting ainmonium chloride is removed from this solution by filtration and the solution is directly used for reaction -with 5-hydroxy-4-n-propyl-furan-2-one (23.62 g, 1.0 equivalent) without intermediate drying of the (2S)-2-aminobutyramide solution. Yield after separation of the two diastereoisomers and recristallisation: approximately 84%.

Ref ROUTE1

1. WO0162726A2.

2. WO2005028435A1 / US2007100150A1.

3. J. Med. Chem. 1988, 31, 893-897.

4. J. Org. Chem. 1981, 46, 4889-4894.

PATENT

https://www.google.com/patents/WO2007031263A1?cl=en

Example 3-Synthesis of brivaracetam (I)

3.a. Synthesis of (S) and (R) 2-((R)-2-oxo-4-propyl-pyrrolidin-l-yl)-butyric acid methyl ester fVIaa*) and (Wlab)

(VIaa) (VIab) A slurry of 60% sodium hydride suspension in mineral oil (0.94g, 23.4 mmol) in tetrahydrofuran (30 mL) is cooled at 0°C under a nitrogen atmosphere. A solution of substantially optically pure (R)-4-propyl-pyrrolidin-2-one (Ilia) (2g, 15.7 mmol) in tetrahydrofuran (2 mL) is added over a 15 minutes period. The reaction mixture is stirred 10 min at 0°C then a solution of methyl-2-bromo-butyric acid methyl ester (V) (3.69g, 20.4 mmol) in tetrahydrofuran (2mL) was added over a 20 minutes period. The reaction mixture is stirred at O0C until maximum conversion of starting material and the reaction mixture is then allowed to warm to room temperature and diluted with water (20 mL). Tetrahydrofuran is removed by evaporation and the residue is extracted with isopropyl acetate (20 ml + 10 mL). The combined organic layers are dried on anhydrous magnesium sulfate and evaporated to afford 3g (13.2 mmol, 86 %) of a mixture of epimers of compound (Via), as a mixture respectively of epimer (VIaa) and epimer (VIab). 1H NMR(400 MHz, CDCI3) of the mixture of epimers (VIaa) and (VIab) : δ = 4.68

(dd, J= 10.8, J= 5.1, 2×1 H) ; 3.71 (s, 2x3H); 3.60 (t app, J= 8.2, IH); 3.42 (t app, J= 8.7, IH); 313 (dd, J= 9.2, J = 6.8, IH); 2.95 (dd, J= 9.2, J= 6.8, IH); 2.56 (dd, J= 16.6, J = 8.7, 2xlH); 2.37 (dm, 2xlH); 2.10 (m, 2xlH); 2.00 (m, 2xlH); 1.68 (m, 2xlH); 1.46 (m, 2x2H); 1.36 (m, 2x2H); 0.92 (m, 2x6H).

13C NMR (400 MHz, CDCl3) of the mixture of epimers (VIaa) and (VIab) : δ =

175.9; 175.2; 171.9; 55.3; 52.4; 49.8; 49.5; 38.0; 37.8; 37.3; 36.9; 32.5; 32.2; 22.6; 22.4; 21.0; 14.4; 11.2; 11.1

HPLC (GRAD 90/10) of the mixture of epimers (VIaa) and (VIab): retention time= 9.84 minutes (100 %)

GC of the mixture of epimers (VIaa) and (VIab): retention time = 13.33 minutes (98.9 %)

MS of the mixture of epimers (VIaa) and (VIab) (ESI) : 228 MH+

3.b. Ammonolysis of compound of the mixture of (VIaa) and (VIab)

(VIaa) (VIab) (I) (VII)

A solution of (VIaa) and (VIab) obtained in previous reaction step (1.46g, 6.4 mmol) in aqueous ammonia 50 % w/w (18 mL) at 00C is stirred at room temperature for 5.5hours. A white precipitate that appears during the reaction, is filtered off, is washed with water and is dried to give 0.77g (3.6 mmol, yield = 56 %) of white solid which is a mixture of brivaracetam (I) and of compound (VII) in a 1 :1 ratio.

1H NMR of the mixture (I) and (VII) (400 MHz, CDCI3) : δ = 6.36 (s, broad, IH); 5.66 (s, broad, IH); 4.45 (m, IH); 3.53 (ddd, J= 28.8, J= 9.7, J= 8.1, IH); 3.02 (m, IH); 2.55 (m, IH); 2.35 (m, IH); 2.11 (m, IH); 1.96 (m, IH); 1.68 (m, IH); 1.38 (dm, 4H); 0.92 (m, 6H). 13c NMR of the mixture (I) and (VII) (400 MHz, CDCl3) : δ = 176.0; 175.9; 172.8;

172.5; 56.4; 56.3; 50.0; 49.9; 38.3; 38.1; 37.3; 37.0; 32.3; 32.2; 21.4; 21.3; 21.0; 20.9; 14.4; 10.9; 10.8

HPLC (GRAD 90/10) of the mixture of (I) and (VII) retention time= 7.67 minutes (100 %)

Melting point of the mixture of (I) and (VII) = 104.90C (heat from 400C to 1200C at 10°C/min)

Compounds (I) and (VII) are separated according to conventional techniques known to the skilled person in the art. A typical preparative separation is performed on a 11.7g scale of a 1 :1 mixture of compounds (I) and (VII) : DAICEL CHIRALPAK® AD 20 μm, 100*500 mm column at 300C with a 300 mL/minutes debit, 50 % EtOH – 50 % Heptane. The separation affords 5.28g (45 %) of compound (VII), retention time = 14 minutes and 5.2Og (44 %) of compounds (I), retention time = 23 minutes.

1H NMR of compound (I) (400 MHz, CDCl3): δ = 6.17 (s, broad, IH); 5.32 (s, broad, IH); 4.43 (dd, J= 8.6, J= 7.1, IH); 3.49 (dd, J= 9.8, J= 8.1, IH); 3.01 (dd, J= 9.8, J= 7.1, IH); 2.59 (dd, J= 16.8, J= 8.7, IH); 2.34 (m, IH); 2.08 (dd, J= 16.8, J= 7.9, IH); 1.95 (m, IH); 1.70 (m, IH); 1.47-1.28 (m, 4H); 0.91 (dt, J= 7.2, J= 2.1, 6H)

HPLC (GRAD 90/10) of compound (I) : retention time = 7.78 minutes

1H NMR of compound (VII) (400 MHz, CDCl3): δ = 6.14 (s, broad, IH); 5.27 (s, broad, IH); 4.43 (t app, J = 8.1, IH); 3.53 (t app, J = 9.1, IH); 3.01 (t app, J = 7.8, IH); 2.53 (dd, J = 16.5, J = 8.8, IH); 2.36 (m, IH); 2.14 (dd, J = 16.5, J = 8.1, IH); 1.97 (m, IH); 1.68 (m, IH); 1.43 (m, 2H); 1.34 (m, 2H); 0.92 (m, 6H)

3c. Epimerisation of compound of (2RV2-((R)-2-oxo-4-propyl-pyπOlidin-l-ylV butyramide (VID

Compound (VII) (200 mg, 0.94 mmol) is added to a solution of sodium tert- butoxide (20 mg, 10 % w/w) in isopropanol (2 mL) at room temperature. The reaction mixture is stirred at room temperature for 18h. The solvent is evaporated to afford 200 mg

(0.94 mmol, 100 %) of a white solid. Said white solid is a mixture of brivaracetam (I) and of (VII) in a ratio 49.3 / 50.7.

HPLC (ISO80): retention time= 7.45 min (49.3%) brivaracetam (I); retention time= 8.02 minutes (50.7%) compound (VII).

Route 2

Reference：ROUTE 2

1. WO2007031263A1 / US2009318708A1.

PATENT

http://www.google.com/patents/WO2007065634A1?cl=en

(scheme 3).

Scheme 3

scheme 4.

5h. Synthesis of brivaracetam and (V) A suspension of (Id) and (Ie) (0.6 g, 2.3 mmol) in MIBK (10 mL) is heated at

120°C for 6 hours. The resulting solution is concentrated and separated on chromatography column (Silicagel 600.068-0.200 mm, cyclohexane/EtOAc : 10/90) to give 0.13 g of brivaracetam (0.6 mmol, 26 %, ee = 94 %) and (V).

1H NMR (400 MHz, CDCl3): δ = 6.17 (s, broad, IH); 5.32 (s, broad, IH); 4.43 (dd, J= 8.6, J= 7.1, IH); 3.49 (dd, J= 9.8, J= 8.1, IH); 3.01 (dd, J= 9.8, J= 7.1, IH); 2.59 (dd, J= 16.8, J= 8.7, IH); 2.34 (m, IH); 2.08 (dd, J= 16.8, J= 7.9, IH); 1.95 (m, IH); 1.70 (m, IH); 1.47-1.28 (m, 4H); 0.91 (dt, J= 7.2,J= 2.1, 6H).

HPLC (method 90/10) : Retention time = 7.78 minutes Chiral HPLC : Retention time = 9.66 minutes (97%) MS (ESI): 213 MH+

Route 3

Reference：1. WO2007065634A1 / US2009012313A1.

References

1.  von Rosenstiel P (Jan 2007). “Brivaracetam (UCB 34714)”. Neurotherapeutics 4 (1): 84–7.doi:10.1016/j.nurt.2006.11.004.PMID 17199019.
2.  Malawska B, Kulig K (Jul 2005). “Brivaracetam UCB”. Current Opinion in Investigational Drugs 6 (7): 740–746. PMID 16044671.
3.  Rogawski MA, Bazil CW (Jul 2008). “New molecular targets for antiepileptic drugs: alpha(2)delta, SV2A, and K(v)7/KCNQ/M potassium channels”. Current Neurology and Neuroscience Reports 8 (4): 345–352. doi:10.1007/s11910-008-0053-7.PMC 2587091.PMID 18590620.
4.  Clinical trial number NCT00464269 for “Double-blind, Randomized Study Evaluating the Efficacy and Safety of Brivaracetam in Adults With Partial Onset Seizures” at ClinicalTrials.gov
5.  Rogawski MA (Aug 2008). “Brivaracetam: a rational drug discovery success story”. British Journal of Pharmacology 154 (8): 1555–7.doi:10.1038/bjp.2008.221. PMC 2518467. PMID 18552880.
6.  Matagne A, Margineanu DG, Kenda B, Michel P, Klitgaard H (Aug 2008). “Anti-convulsive and anti-epileptic properties of brivaracetam (ucb 34714), a high-affinity ligand for the synaptic vesicle protein, SV2A”. British Journal of Pharmacology 154 (8): 1662.doi:10.1038/bjp.2008.198. PMID 18500360.
7.  http://www.ema.europa.eu/ema/index.jsp?curl=pages/medicines/human/medicines/003898/human_med_001945.jsp&mid=WC0b01ac058001d124
8.  http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm486827.htm

Brivaracetam

Names
IUPAC name (2S)-2-[(4R)-2-oxo- 4-propylpyrrolidin-1-yl] butanamide
Identifiers
CAS Number	357336-20-0
ChEMBL	ChEMBL607400
ChemSpider	8012964
Jmol interactive 3D	Image
PubChem	9837243
UNII	U863JGG2IA
InChI[show]
SMILES[show]
Properties
Chemical formula	C11H20N2O2
Molar mass	212.15 g/mol
Pharmacology
ATC code	N03AX23
Legal status	Investigational
Routes of administration	Oral
Pharmacokinetics:
Bioavailability	Nearly 100%
Protein binding	<20%
Metabolism	Hydrolysis, CYP2C8-mediated hydroxylation
Biological half-life	8 hrs
Excretion	>75% renal
Except where otherwise noted, data are given for materials in their standard state (at 25 °C [77 °F], 100 kPa).

//////BRIVARACETAM, UCB, 2016 FDA, UCB-34714

CCCC1CC(=O)N(C1)C(CC)C(=O)N