THAT ARE NOT LOOK TWITTE

THAT ARE NOT LOOK TWITTE US009981971B2

( 12 ) United States Patent ( 10 ) Patent No . : US 9 , 981 , 971 B2 ( 45 ) Date of Patent : May 29 , 2018 Boersen

( 54 ) SOLID FORMS OF 1 - ETHYL - 7 - ( 2 - METHYL - 6 - 1H - 1 , 2 , 4 TRIAZOL - 3 - YL ) PYRIDIN - 3 - YL ) - 3 , 4 DIHYDROPYRAZINO [ 2 , 3 - BJPYRAZIN - 2 ( 1H ) ONE AS TOR KINASE INHIBITORS

( 71 ) Applicant : Signal Pharmaceuticals , LLC , San Diego , CA ( US )

( 72 ) Inventor : Nathan Boersen , Summit , NJ ( US ) ( 73 ) Assignee : Signal Pharmaceuticals , LLC , San

Diego , CA ( US )

7 , 476 , 665 B2 7 , 608 , 622 B2 7 , 700 , 594 B2 7 , 767 , 687 B2 7 , 902 , 187 B2 7 , 919 , 490 B2 7 , 968 , 556 B2 7 , 981 , 893 B2 8 , 034 , 948 B2 8 , 110 , 578 B2 8 , 268 , 809 B2 8 , 372 , 976 B2 8 , 383 , 634 B2 8 , 492 , 381 B2 8 , 569 , 494 B2 8 , 642 , 660 B2

2004 / 0063658 A1 2006 / 0004014 A1 2006 / 0142269 Al 2007 / 0036793 Al 2008 / 0194019 A1 2009 / 0181963 A1 2009 / 0281075 Al 2010 / 0144738 A1 2011 / 0257167 A1 2011 / 0318336 Al

1 / 2009 Burgey 10 / 2009 Liu et al . 4 / 2010 Chen et al . 8 / 2010 Oslob et al . 3 / 2011 Neagu et al . 4 / 2011 Neagu et al . 6 / 2011 Mortensen et al . 7 / 2011 Mortensen et al .

10 / 2011 Coquerel et al . 2 / 2012 Perin - Ninkovic et al . 9 / 2012 Kalman 2 / 2013 Mortensen et al . 2 / 2013 Mortensen et al . 7 / 2013 Perrin - Ninkovic et al .

10 / 2013 Harris et al . 2 / 2014 Goldfard 4 / 2004 Roberts et al . 1 / 2006 Hoffmann et al . 6 / 2006 Dykes 2 / 2007 Hardie et al . 8 / 2008 Cantley et al . 7 / 2009 Wyeth 11 / 2009 Roughton et al . 6 / 2010 Bommann et al .

10 / 2011 Chopra et al . 12 / 2011 Petricoin , III et al .

( Continued )

( * ) Notice : Subject to any disclaimer , the term of this patent is extended or adjusted under 35 U . S . C . 154 ( b ) by 0 days . days .

( 21 ) Appl . No . : 15 / 205 , 114 ( 22 ) Filed : Jul . 8 , 2016 ( 65 ) Prior Publication Data

US 2016 / 0318940 A1 Nov . 3 , 2016 FOREIGN PATENT DOCUMENTS

Related U . S . Application Data Continuation of application No . 14 / 686 , 879 , filed on Apr . 15 , 2015 , now Pat . No . 9 , 416 , 134 .

CA DE ( 63 )

2 458 699 3 / 2003 262 026 11 / 1988

( Continued )

OTHER PUBLICATIONS ( 60 ) Provisional application No . 62 / 003 , 173 , filed on May 27 , 2014 , provisional application No . 61 / 980 , 108 , filed on Apr . 16 , 2014 . Jordan , V . C . Nature Reviews : Drug Discovery , 2 , 2003 , 205 . *

( Continued ) Primary Examiner - Douglas M Willis ( 74 ) Attorney , Agent , or Firm — Jones Day

( 51 ) Int . Ci . C070 487 / 04 ( 2006 . 01 )

( 52 ) U . S . CI . CPC . . . . . . . . . . . . . . . CO7D 487 / 04 ( 2013 . 01 )

( 58 ) Field of Classification Search ??? . . . . . . . . . . C07D 487 / 04 USPC . . . . . . . . . . . . . . . . . . . . 544 / 350 ; 546 / 272 . 4 ; 548 / 266 . 4 See application file for complete search history .

( 57 ) ABSTRACT Provided herein are formulations , processes , solid forms and methods of use relating to the Compound for formula 1 :

( 56 ) References Cited

U . S . PATENT DOCUMENTS NN

Na

N

3 , 507 , 866 A 3 , 567 , 725 A 4 , 294 , 836 A 4 , 294 , 837 A 4 , 309 , 537 A 4 , 317 , 909 A 4 , 898 , 872 A 4 , 963 , 561 A 5 , 424 , 311 A 5 , 869 , 659 A 6 , 031 , 105 A 6 , 093 , 728 A 6 , 372 , 740 B1 6 , 566 , 367 B2 6 , 791 , 006 B2 6 , 800 , 436 B1 6 , 825 , 184 B2 6 , 855 , 723 B2 7 , 199 , 119 B2 7 , 247 , 621 B2 7 , 429 , 572 B2

4 / 1970 Jones et al . 3 / 1971 Grabowski et al .

10 / 1981 Lesher et al . 10 / 1981 Lesher et al .

1 / 1982 Lesher et al . 3 / 1982 Lesher et al . 2 / 1990 Campbell et al .

10 / 1990 Lesher et al . 6 / 1995 Billhardt - Troughton 2 / 1999 Stolle et al . 2 / 2000 Wright 7 / 2000 McMahon et al . 4 / 2002 Murata et al . 5 / 2003 Bakthayatchalam et al . 9 / 2004 Nezu et al .

10 / 2004 Jenne et al . 11 / 2004 Cirillo et al .

2 / 2005 McMahon et al . 4 / 2007 Burkitt et al . 7 / 2007 Hong et al . 9 / 2008 Clark

having the name 1 - ethyl - 7 - ( 2 - methyl - 6 - ( 1H - 1 , 2 , 4 - triazol - 3 yl ) pyridin - 3 - yl ) - 3 , 4 - dihydropyrazino [ 2 , 3 - b ] pyrazin - 2 ( 1H ) one and tautomers thereof .

6 Claims , 32 Drawing Sheets

US 9 , 981 , 971 B2

( 56 ) References Cited U . S . PATENT DOCUMENTS

2012 / 0028972 A1 2012 / 0046457 A1 2013 / 0102613 Al 2013 / 0142873 Al 2013 / 0158023 Al 2013 / 0225518 A1 2013 / 0245026 A1 2013 / 0245027 Al 2013 / 0245028 Al 2013 / 0245029 Al 2013 / 0245254 Al 2014 / 0046057 Al 2014 / 0113905 Al 2014 / 0314673 A1 2014 / 0314674 Al 2014 / 0314751 Al 2014 / 0314752 A1 2014 / 0314753 Al 2014 / 0315848 Al 2014 / 0315900 A1 2014 / 0315907 A1 2014 / 0315908 A1

2 / 2012 Wong et al . 2 / 2012 Kolla et al . 4 / 2013 Xu et al . 6 / 2013 Assaf et al . 6 / 2013 Ning et al . 8 / 2013 Xu et al . 9 / 2013 Xu et al . 9 / 2013 Xu et al . 9 / 2013 Xu et al . 9 / 2013 Xu et al . 9 / 2013 Harris et al . 2 / 2014 Cohen et al . 4 / 2014 Xu et al .

10 / 2014 Raymon et al . 10 / 2014 Raymon et al . 10 / 2014 Hege et al . 10 / 2014 Lopez - Girona et al . 10 / 2014 Hege et al . 10 / 2014 Raymon et al . 10 / 2014 Raymon et al . 10 / 2014 Raymon et al . 10 / 2014 Menon et al .

WO WO WO WO WO WO Wo wo wo WO WO WO WO WO WO WO WO WO WO WO WO WO wo WO WO WO WO WO Wo WO

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WO 2011 / 053518 Al WO 11 / 079114

WO 2011 / 097333

11 / 2007 11 / 2007 2 / 2008 2 / 2008 3 / 2008 3 / 2008 3 / 2008 3 / 2008 3 / 2008 3 / 2008 3 / 2008 3 / 2008 3 / 2008 3 / 2008 5 / 2008 5 / 2008 9 / 2008

11 / 2008 1 / 2009 1 / 2009 1 / 2009 4 / 2009 8 / 2009 1 / 2010 6 / 2010 6 / 2010 3 / 2011 5 / 2011 6 / 2011 8 / 2011

FOREIGN PATENT DOCUMENTS

OTHER PUBLICATIONS

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2001048882 2002100363 2002167387

WO 03 / 032989 WO 04 / 042002 WO 99 / 16438 WO 99 / 28320 WO 99 / 28459 WO 00 / 73306 WO 02 / 048152 WO 02 / 076954 WO 03 / 032989 WO 03 / 072557 WO 03 / 093290 WO 04 / 042002 WO 04 / 048365 WO 04 / 065378 WO 04 / 076454 WO 04 / 078754 WO 04 / 085409 WO 04 / 096797 WO 05 / 003147 WO 05 / 021519 WO 05 / 120511 WO 06 / 001266 WO 06 / 018182 WO 06 / 030031 WO 06 / 036883 WO 06 / 045828 WO 06 / 046031 WO 06 / 050076 WO 06 / 065703 WO 06 / 087530 WO 06 / 090167 WO 06 / 090169 WO 06 / 091737 WO 06 / 108103 WO 07 / 044698 WO 07 / 044729 WO 07 / 044813

WO 2007 / 047754 WO 07 / 060404 WO 07 / 066099 WO 07 / 066102 WO 07 / 080382 WO 07 / 125321 WO 07 / 129044 WO 07 / 129052

9 / 1990 5 / 1987 2 / 2001 4 / 2002 6 / 2002 4 / 1903 5 / 1904 4 / 1999 6 / 1999 6 / 1999 12 / 2000 6 / 2002

10 / 2002 4 / 2003 9 / 2003

11 / 2003 5 / 2004 6 / 2004 8 / 2004 9 / 2004 9 / 2004

10 / 2004 11 / 2004

1 / 2005 3 / 2005

12 / 2005 1 / 2006 2 / 2006 3 / 2006 4 / 2006 5 / 2006 5 / 2006 5 / 2006 6 / 2006 8 / 2006 8 / 2006 8 / 2006 8 / 2006

10 / 2006 4 / 2007 4 / 2007 4 / 2007 4 / 2007 5 / 2007 6 / 2007 6 / 2007 7 / 2007

11 / 2007 11 / 2007 11 / 2007

Hackam , et al . JAMA , 296 ( 14 ) , 2006 , 1731 - 1732 . * Barlin 1982 , “ Purine analogs as amplifiers of phleomycin . VII . Some 1H - inidazo [ 4 , 5 - b ] pyrazines and related compound , ” Austra lian Journal of Chemistry , vol . 35 : 2299 - 2306 . Beresnev et al . , 2000 , “ Interaction of 5 - methoxy - 1 , 2 , 4 - traizines with uras as a new route to 6 - azapurines , ” Medeleev Commu . , vol . 2 : 58 - 59 . Bergmann et al . , 1963 , “ 2 - Phenylpurines , their chemical and enzumological reactivity , " J . Chem Org . , pp . 3729 - 3735 . Booth et al . , 1992 , “ Synthesis of 9 - Hydroxyalkyl - substituted purines from the corresponding 4 - ( C Cyanoformimidoyl ) imidazole - 5 - amines , " J , Chem Society , Perkin Transactions 1 : Organic and Bio - Organic Chemstry , vol . 2119 - 26 . Booth et al . , 1995 , “ Synthesis of [ la , 26 , 3a - 2 , 3 bis ( benzyloxymethyl ) cyclobutlJimidazol - 5 - amines : important pre cursors to cyclobut - A derivatives , ” J . Chem Society , Perkin Tranac tions 1 : Organic and Bio - Organic Chemistry , vol . 6 , pp . 669 - 675 . Booth et al . , 2001 , “ The Reactions of Diaminomaleonitrile with Isocyanates and Either Aldehydes or Ketones Revisited , ” J . Org Chem , vol . 66 : 8436 - 8441 . Booth , et al . , 1994 , “ Synthesis of 4 - and 5 - Disubstituted 1 - Benzylimidazoles , Important Precursors of Purine Analogs , " J . of Heterocyclic of Chemistry , vol . 31 ( 2 ) : 345 - 50 . Caira et al . , 1998 , “ Crystalline Polymorphism of Organic Com pounds , ” Topics in Current Chemistry , Jan . 1 , 1998 , pp . 163 - 208 , vol . 198 , Springer , Berlin , DE . Carretero et al . 2010 , “ Integrative Genomic and Proteomic Analyses Indentity Targets for Lkb1 - Deficient Metastatic Lung Tumors , ” Cancer Cell , vol . 17 ( 6 ) : 547 - 559 . Chupakhin et al . , 2001 , “ A simple one pot synthesis of condensed 1 , 2 , 4 - triazines by using the tandem ApSnipso and Sn — Snipso reactions , " J . of Heterocyclic Chemistry , vol . 38 ( 4 ) : 901 - 907 . Cohen , 2005 , Protein Kinase Inhibitors for the Treatment of Dis ease : The Promise and the Problems , Handbook of Experimental Pharmacology , Springer Berlin Heidelberg , 167 : 1 - 7 . Cohen , P . 2001 , “ The role of protein phosphorylation in human health and disease , ” Eur . J . Biochem , vol . 268 : 5001 - 5010 . Cohen , P . 2002 , “ Protein kinases — the major drug targets of the twenty - first century ? ” Nature Reviews / Drug Discovery , vol . 1 : 309 315 . Coish , et al . , 2006 , “ Small molecule inhibitors of IKK kinase activity , ” Expert Opin . Ther . Patents , vol . 16 ( 1 ) : 1 - 12 .

WO WO WO WO WO WO WO WO WO WO WO WO wo WO WO WO WO WO WO WO WO wo

US 9 , 981 , 971 B2

( 56 ) References Cited OTHER PUBLICATIONS

Crofts et al . , 1997 “ Metabolism of 2 - amino - 1 - methyl - 6 phenylimidazo [ 4 , 5 - b ] pyridine ( PhIP ) by human cytochrome P4501B1 , " Carcinogenesis , vol . 18 ( 9 ) : 1793 - 1798 . Dang et al . , 1999 , “ Efficient synthesis of purines and purine nucelosides via an inverse electron demand diels — alder reaction , " J . Am Chem Soc . , vol . 121 ( 24 ) : 5833 - 5834 . Database Caplus Online Chemical Abstracts Service , Columbus , Ohio , Database Accession No . 1951 : 49974 ( XP - 002472261 ) ( 1951 ) Database Caplus Online Chemical Abstracts Service , Columbus , Ohio , Database Accession No . 1978 : 433195 ( XP - 002472262 ) ( 1978 ) . Database Caplus Online Chemical Abstracts Service , Columbus , Ohio , Database Accession No . 1966 : 26849 ( XP - 002472263 ) ( 1965 ) . Dornow et al . , 1957 , “ Synthese von2 - Oxy - imidazolo - ( 5 ' , 4 : 2 , 3 ) pyridinen ) , ” Arch Pharm . vol . 290 , pp . 20 - 31 ( w / English language abstract ) . Dzierba et al . , 2004 , “ Synthesis , structure - activity relationships , and in vivo properties of 3 , 4 - dihydro - 1H - pyrido [ 2 , 3 - b ] pyrazin - 2 - ones as corticotropin - releasing factor - 1 receptor antagonists , ” J of Medicinal Chemistry , vol . 47 , pp . 5783 - 5790 . Fabbro et al . , 2002 , “ Protein kinases as targets for anticancer agents : from inhibitors to useful drugs , ” Pharmacol Ther . , 93 ( 2 - 3 ) : 79 - 98 . Farhadi et al . , 2006 , “ The role of protein kinase C isoforms in modulating injury and repair of the intestinal barrier , " J . Pharm Exp . Ther . , vol . 316 ( 1 ) : 1 - 7 . Frandsen et al . , 1992 , “ Reaction of the N2 - acetoxy derivative of 2 - amino - 1 - methyl - 6 - phenylimidazo [ 4 , 5 , b ] pyridine . . , " Carcinogenesis , vol . 13 ( 4 ) : 629 - 635 . Furniss et al . , 1989 , “ Vogel ' s Textbook of Practical Organic Chem istry Fifth Edition , " 1989 , p . 132 - 136 , Longman . Gao et al . , 2010 , “ LKB1 inhibits lung cancer progression through lysyl oxidase and extracellular matrix remodeling , ” Proceedings of the National Academy of Sciences , vol . 107 ( 44 ) : 18892 - 18897 . Gao et al . : 2011 , “ LKB1 in lung cancerigenesis : a serine / threonine kinase as tumor suppressor , ” Protein & Cell , Gaodeng Jiaoyu Chubanshe , China , vol . 2 ( 2 ) : 99 - 107 . Georgakis and Younes , 2006 , “ From rapi nui to rapamycin : target ing PI3K / Akt / mTOR for cancer therapy , " Expert Rev . Anticancer Ther . , vol . 6 ( 1 ) : 131 - 140 . Gini et al . , 2013 , “ The mTOR Kinase Inhibitors , CC214 - 1 and CC214 - 2 , Preferentially Block the Growth of EGFRvIII - Activated Glioblastomas , " Clin Cancer Res 2013 ; 19 : 5722 - 5732 . Grimmiger et al . , 2010 , “ Targeting non - malignant disorders with tyrosine kinase inhibitors , ” Nat . Rev . Drug Disc . , 9 ( 12 ) : 956 - 970 . Hamad , 2001 , “ A new synthesis of 4 - cyano - 1 , 3 - dihydro - 2 - oxo - 2H imidazole - 5 - ( N ' - tosyl ) carboxamide : Reactive precursor for thiopurine analogues , ” J of Heterocyclic Chemistry , vol . 38 ( 4 ) : 939 944 . Hernan et al . , “ De novo germline mutation in the serine - threonine kinase STK11 / LKB1 gene associated with Peutz - Jeghers syn drome , " Clin Genet . , 66 ( 1 ) : 58 - 62 . Huang et al . , 2010 , " Genetic and epigenetic silencing of SCARAS may contribute to human hepatocellular carcinoma by activating FAK signaling , ” Journal of Clinical Investigation , American Society for Clinical investigation , vol . 120 ( 1 ) : 223 - 241 . Inge et al . , 2009 , “ Expression of LKB1 tumor suppressor in non - small cell lung cancer determines sensitivity to 2 - deoxyglucose , " Journal of Thoracic and Cardiovascular Surgery , vol . 137 ( 3 ) : 580 - 586 . Irie et al . , 2005 , “ Toward the development of new medicinal leads with selectivity for protein kinase C isozymes , ” The Chemical Record , vol . 5 : 185 - 195 . Itoh et al . , 2004 , “ A novel practical synthesis of C - 2 - arylpurines , " Advanced Synthesis & Catalysis , vol . 346 : 1859 - 1867 . Ji et al . , 2007 , “ LKB1 modulates lung cancer differentiation and metastasis , ” Nature , 448 ( 7155 ) : 807 - 810 .

Jones et al . , 1973 , " 6 - Substituted - 5 - chloro - 1 , 3 - dihydro - 2H imidazo ( 4 , 5 - b ) pyrazin - 2 - ones with hypotensive activity , " J . Med . Chem . , vol . 16 ( 5 ) : 537 - 542 . Kazaoka et al . , 2003 , “ Synthesis of 6 - substituted 9 - benzyl - 8 hydroxypurines with potential interferon - indcuing activity , ” Chemi cal & Pharmaceutical Bulletin , vol . 51 ( 5 ) : 608 - 611 . Killday et al . , 2001 , “ Microxine , a new cdc2 kinase inhibitor from the Australian marine sponge Microxina species , ” J . of Natural Products , vol . 64 ( 4 ) : 525 - 526 . Mahoney et al . , 2009 , “ LKB1 / KRAS mutant lung cancers constitute a genetic subset of NSCLC with increased sensitivity to MAPK and mTOR signalling inhibition , ” Br J Cancer , 100 ( 2 ) : 370 - 375 . Minehan et al . , 2000 , " Molecular recognition of DNA by Hoechst Benzimidazoles : Exploring beyond theopyrrole - imidazole hydroxypyrrole polyamide - pairing code , ” Helvitica Chima Acta , vol . 83 ( 9 ) : 2197 - 2213 . Nagashima et al . , 2004 , “ Solution - Phase parallel synthesis of an N - Alkylated dihydropteridinone library from fluorous amino acids , " J of Comb . Chemistry , vol . 6 ( 6 ) : 942 - 949 . Park et al . , 2000 , “ A novel mechanism of TRAF signaling revealed by structural and functional analyses of the TRADD - TRAF2 inter action , ” Cell , vol . 101 : 777 - 787 . Patani et al . , 1998 , “ Bioisosterim : A rational approach in dmg design , ” Chemical Reviews , vol . 96 : 3147 - 3176 . PCT Annex Communication Relating to the Results of the Partial International Search issued in connection with PCT / US2012 / 049281 , filed Aug . 2 , 2012 . PCT International Search Report issued in connection with PCT / US2012 / 049281 , filed Aug . 2 , 2012 . PCT Written Opinion of the International Searching Authority issued in connection with PCT / US2012 / 049281 , filed Aug . 2 , 2012 . Registry File Document for RN 863501 - 03 - 5 , 863502 - 39 - 0 and others ( Sep . 20 , 2005 ) . Seela et al . , 2004 , “ Product Class 17 : Purines , ” Science of Synthe sis , vol . 16 , pp . 945 - 1108 . Shaw et al . , 2004 , “ The LKB1 tumor suppressor negativiely regu lates mTOR signaling , " Cancer Cell , vol . 6 ( 1 ) : 91 - 99 . Shaw et al . 2009 , “ LKB1 and AMP — activated protein kinase control of mTOR signalling and growth , ” Acta . Physiol ( Oxf . ) 196 ( 1 ) : 65 - 80 . Shoji et al . 2012 , “ Genotype - dependent efficacy of a dual PI3K / mTOR inhibitor , NVP - BEZ235 , and an mTOR inhibitor , RAD001 , in endometrial carcinomas . " PloS one 7 . 5 , 2012 , e37431 . Singh et al . , 1994 , “ Novel CAMP PDE III Inhibitors : Imidazo ( 4 , 5 b ] pyridin - 2 ( 3H ) - ones and Thiazolo ( 4 , 5 - b ] pyridin - 2 ( 3h ) - ones and Their Analogs , " J . Med . Chem , vol . 37 ( 2 ) : 248 - 54 . Sridhar et al . , 2000 , “ Protein kinases as therapeutic targets , ” Pharm . Res . , 17 ( 11 ) : 1345 - 1353 . Wallace 2008 , “ Palladium - catalyzed synthesis of quinoxaline derivatives , ” Tetrahedron , vol . 64 : 9675 - 9684 . Wei et al . , 2009 , “ Chemopreventive efficacy of rapamycin on Peutz - Jeghers syndrome in a mouse model , ” Cancer Lett . , 277 ( 2 ) : 149 - 154 . Westover et al . , 1981 , “ Synthesis and antiviral activity of certain 9 - B - D - Riofuranoaylpurine - 6 - carboxamides , ” J . Med . Chem . , vol . 24 ( 8 ) : 941 - 46 . Wingo et al . , 2009 , “ Somatic LKB1 mutations promote cervical cancer progression , ” PloS One , 4 ( 4 ) : 1 - 8 . Yoneda et al . , 1976 , “ A transformationof 7 - azapteridines into 6 - azapurines ( Imidazo [ 4 , 5 - e ] - as - triazines ) , ” Heterocycles , vol . 4 ( 9 ) : 1503 - 1508 . Yoneda et al . , 1978 , “ Synthesis of imadazo [ 4 , 5 - e ] - as - triazine ( 6 - Azapurine ) Deriviatives , ” Chem & Pharm Bulletin , vol . 26 ( 10 ) : 3154 - 3160 . Yuan et al . , 2009 , “ Targeting tumorigenesis : development and use of mTOR inhibitors in cancer therapy , ” Journal of Hematology & Oncology , Biomed Central Ltd . , London UK , vol . 2 ( 1 ) : 45 . Zaki et al . , 2007 , “ The synthesis of imidazol [ 4 , 5 - d ] pyridines from a substituted imidazole and acyl or sulfonyl acetonitrile , " Tetrahe dron , vol . 63 ( 18 ) : 3745 - 3753 .

US 9 , 981 , 971 B2

( 56 ) References Cited OTHER PUBLICATIONS

Zhong et al . , 2006 , “ LKB1 mutation in large cell carcinoma of the lung , ” Cancer Lung , vol . 53 ( 3 ) : 285 - 294 . * cited by examiner

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160 - - - - - - - - - - - - - - - - - - 80 - - - - -

25 50 - 1 . 80

75 100 225 250 275 300 125 150 175 200 Temperature ( °C


FIG . 10

TGMS _ S13029 _ SLPS 04 . 07 . 2013 12 : 49 : 26 Yrrrrrrrrrr

TOPS 31029 95 , 03 . 07 . 2013 17 : 57 : 57 TIME _ $ 13025 _ 9175 , 3 . 87000 ms - - - - - - - - - meeee - - - - - - - - - - - - - - -

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FIG . 11

Method Info : Method for Project $ 13029 , based an Fstgr03 with optimal wavelengt at 2547211

DAD1 C , Sig = 254 , 4 pz Fof ( 25JUN1300007 D nu 1200 1000 ????????????????????? 5

ETTETETT TETTE - - - - - - - - - - - - - - - - - - - - - - - - - - - -

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FIG . 15

TGMS S13029 ECP2 . 284 V1 25 . 06 . 2013 08 : 48 : 06 " TGMS 5 . 923 _ 6297 . 27413

4722 GM 913029 BP224 _ , ! . - 2007 - - - -

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Crystallics : TA - Lab 33

STAR SW 9 . 01


FIG . 16

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FIG . 20

TGMS S13029 AS14 . 458 V1 25 . 06 . 2013 08 : 28 : 39 my

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FIG . 21 Method Info atgro3 with optimal wavelengt ät : Method for Project S13029 , based on

2547

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FIG . 22

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FIG . 25 TGMS S13029 HF2 . 486 V7 04 . 07 . 2013 11 : 28 : 45

- - - - - - -

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FIG . 26 Method Info Method for Project $ 13029 , based on Fst gro3 with optimal navelanyt at

Arm wwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwww DADTC , Sig = 244 , 4 FFOA ( 26JUN1300003 . D ) TAU 14070 ?????????????????????????????????? 4 , 128 4897

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US 9 , 981 , 971 B2 SOLID FORMS OF ( see , e . g . , S . R . Byrn et al . , Solid State Chemistry of Drugs ,

1 - ETHYL - 7 - ( 2 - METHYL - 6 - ( 1H - 1 , 2 , 4 - TRIAZOL - 3 ( 1999 ) SSCI , West Lafayette ) . Moreover , multiple - compo YL ) PYRIDIN - 3 - YL ) - 3 , 4 - DIHYDROPYRAZINO [ 2 , nent crystal forms may potentially be susceptible to poly

3 - B ] PYRAZIN - 2 ( 1H ) - ONE AS TOR KINASE morphism , wherein a given multiple - component composi INHIBITORS 5 tion may exist in more than one three - dimensional This application is a continuation of U . S . application Ser . crystalline arrangement . The discovery of solid forms is of

No . 14 / 686 , 879 , filed Apr . 15 , 2015 , currently allowed , great importance in the development of a safe , effective , which claims the benefit of U . S . Provisional Application No . stable and marketable pharmaceutical compound . 61 / 980 , 108 , filed Apr . 16 , 2014 and the benefit of U . S . Notably , it is not possible to predict a priori if crystalline Provisional Application No . 62 / 003 , 173 , filed May 27 , 2014 , 10 forms of a compound even exist , let alone how to success the entire contents of each of which are incorporated herein fully prepare them ( see , e . g . , Braga and Grepioni , 2005 , by reference . “ Making crystals from crystals : a green route to crystal

engineering and polymorphism , ” Chem . Commun . 3635 1 . FIELD 3645 ( with respect to crystal engineering , if instructions are

Provided herein are solid forms of 1 - ethyl - 7 - ( 2 - methyl not very precise and / or if other external factors affect the 6 - ( 11 - 1 . 2 . 4 - triazol - 3 - ylpyridin - 3 - v1 ) - 3 , 4 - dihydropyrazino process , the result can be unpredictable ) ; Jones et al . , 2006 , [ 2 , 3 - b ] pyrazin - 2 ( 1H ) - one , compositions thereof , and meth Pharmaceutical Cocrystals : An Emerging Approach to ods of their use for the treatment of a disease , disorder , or Physical Property Enhancement , ” MRS Bulletin 31 : 875 - 879 condition . 20 ( At present it is not generally possible to computationally

predict the number of observable polymorphs of even the 2 . BACKGROUND simplest molecules ) ; Price , 2004 , “ The computational pre

diction of pharmaceutical crystal structures and polymor The identification and selection of a solid form of a phism , ” Advanced Drug Delivery Reviews 56 : 301 - 319

pharmaceutical compound is complex , given that a change 25 ( “ Price " ) ; and Bernstein , 2004 , “ Crystal Structure Prediction in solid form may affect a variety of physical and chemical and Polymorphism , ” ACA Transactions 39 : 14 - 23 ( a great properties , which may provide benefits or drawbacks in deal still needs to be learned and done before one can state processing , formulation , stability and bioavailability , among with any degree of confidence the ability to predict a crystal other important pharmaceutical characteristics . Potential structure , much less polymorphic forms ) . pharmaceutical solids include crystalline solids and amor 30 The compound chemically named 1 - ethyl - 7 - ( 2 - methyl - 6 phous solids . Amorphous solids are characterized by a lack _ 30 ( 1H - 1 , 2 , 4 - triazol - 3 - yl ) pyridin - 3 - yl ) - 3 , 4 - dihydropyrazino [ 2 , of long - range structural order , whereas crystalline solids are characterized by structural periodicity . The desired class of 3 - b ] pyrazin - 2 ( 1H ) - one and tautomers thereof ( collectively pharmaceutical solid depends upon the specific application ; referred to herein as “ Compound 1 ” ) was disclosed in U . S . amorphous solids are sometimes selected on the basis of , patent application Ser . No . 12 / 605 , 791 , filed Oct . 26 , 2009 , eg . an enhanced dissolution profile , while crystalline solids 35 and International Pub . No . WO 2010 / 062571 , the entireties may be desirable for properties such as , e . g . , physical or of each of which are incorporated by reference herein . We chemical stability ( see , e . g . , S . R . Vippagunta et al . , Adv . have discovered multiple solid forms of l - ethyl - 7 - ( 2 Drug . Deliv . Rev . , ( 2001 ) 48 : 3 - 26 ; L . Yu , Adv . Drug . Deliv . methyl - 6 - 1H - 1 , 2 , 4 - triazol - 3 - yl ) pyridin - 3 - yl ) - 3 , 4 - dihydro Rev . , ( 2001 ) 48 : 27 - 42 ) . pyrazino [ 2 , 3 - b ] pyrazin - 2 ( 1H ) - one .

Whether crystalline or amorphous , potential solid forms 40 Citation or identification of any reference in Section 2 of of a pharmaceutical compound include single - component this application is not to be construed as an admission that and multiple - component solids . Single - component solids the reference is prior art to the present application . consist essentially of the pharmaceutical compound in the absence of other compounds . Variety among single - compo 3 . SUMMARY nent crystalline materials may potentially arise from the 45 phenomenon of polymorphism , wherein multiple three - di Provided herein are solid forms of the Compound for mensional arrangements exist for a particular pharmaceuti formula 1 : cal compound ( see , e . g . , S . R . Byrn et al . , Solid State Chemistry of Drugs , ( 1999 ) SSCI , West Lafayette ) . The importance of discovering polymorphs was underscored by 50 the case of Ritonavir , an HIV protease inhibitor that was formulated as soft gelatin capsules . About two years after the product was launched , the unanticipated precipitation of a new , less soluble polymorph in the formulation necessitated the withdrawal of the product from the market until a more 55 consistent formulation could be developed ( see S . R . Chem burkar et al . , Org . Process Res . Dev . , ( 2000 ) 4 : 413 - 417 ) .

Additional diversity among the potential solid forms of a pharmaceutical compound may arise from the possibility of multiple - component solids . Crystalline solids comprising 60 two or more ionic species are termed salts ( see , e . g . , Hand having the name 1 - ethyl - 7 - ( 2 - methyl - 6 - ( 1H - 1 , 2 , 4 - triazol book of Pharmaceutical Salts : Properties , Selection and Use , 3 - yl ) pyridin - 3 - yl ) - 3 , 4 - dihydropyrazino [ 2 , 3 - b ] pyrazin - 2 P . H . Stahl and C . G . Wermuth , Eds . , ( 2002 ) , Wiley , Wein - ( 1H ) - one and tautomers thereof . heim ) . Additional types of multiple - component solids that Also provided herein are formulations of solid forms of may potentially offer other property improvements for a 65 the Compound of formula 1 and tautomers thereof . pharmaceutical compound or salt thereof include , e . g . , One crystal form provided herein has characteristic X - ray hydrates , solvates , co - crystals and clathrates , among others powder diffraction peaks at a two - theta angle of approxi

N

N

US 9 , 981 , 971 B2

mately 6 . 18 , 21 . 74 and 26 . 7 degrees . The X - ray powder differential thermal analysis thermogram comprising an diffraction pattern can further comprise characteristic X - ray endotherm between about 50° C . and about 140° C . with a powder diffraction peaks at a two - theta angle of approxi - maximum at approximately 80° C . when heated from about mately 12 . 34 , 22 . 5 and 23 . 42 degrees . The crystal form can 25° C . to about 300° C . The single differential thermal have a thermogravimetric analysis thermogram comprising 5 analysis thermogram can further comprise an exotherm a total mass loss of approximately 15 . 5 % of the total mass between about 160° C . and about 200° C . with a maximum of the crystal form when heated from about 25° C . to about at approximately 181° C . The single differential thermal 300° C . The crystal form can have a single differential analysis thermogram can further comprise an endotherm thermal analysis thermogram comprising an endotherm between about 225º C . and about 275° C . with a maximum between about 90° C . and about 185° C . with a maximum at 10 at approximately 251° C . The crystal form can be hydrated . approximately 140° C . when heated from about 25° C . to The crystal form can comprise 2 molar equivalents of water . about 300° C . The single differential thermal analysis ther The crystal form can substantially pure . mogram can further comprise an endotherm between about 240° C . and about 285° C . with a maximum at approxi Further provided herein is an amorphous form comprising mately 264° C . The crystal form can be 1 . 2 - ethanediol 15 Compound 1 , or a tautomer thereof . The amorphous form solvated . The crystal form can comprise 1 molar equivalent has a differential scanning calorimetry thermogram com of 1 , 2 - ethanediol . The crystal form can be substantially pure . prising an endotherm between about 160° C . and about 2009

A further crystal form provided herein has characteristic C . with a maximum at approximately 188 . 1° C . The amor X - ray powder diffraction peaks at a two - theta angle of phous form has a glass transition temperature at about 120° approximately 3 . 5 , 9 . 26 and 18 . 62 degrees . The X - ray 20 C . The amorphous form can be substantially pure . powder diffraction pattern can further comprise character The solid forms provided herein can be used as a medi istic X - ray powder diffraction peaks at a two - theta angle of cament . In certain embodiments , solid forms of Compound approximately 7 . 06 , 12 . 66 and 15 . 3 degrees . The crystal 1 and tautomers thereof are useful for treating or preventing form can have a thermogravimetric analysis thermogram cancer and conditions treatable or preventable by inhibition comprising a total mass loss of approximately 12 . 8 % of the 25 of a kinase pathway , for example , the mTOR / PI3K / Akt total mass of the crystal form when heated from about 25° pathway . The solid forms provided herein can be used in C . to about 300° C . The crystal form can have a single methods for treating or preventing cancer , an inflammatory differential thermal analysis thermogram comprising an condition , an immunological condition , a neurodegenerative endotherm between about 110° C . and about 175° C . with a disease , diabetes , obesity , a neurological disorder , an age maximum at approximately 160° C . when heated from about 30 related disease , a cardiovascular condition , or a conditions 25° C . to about 300° C . The single differential thermal treatable or preventable by inhibition of a kinase pathway . analysis thermogram can further comprise an endotherm The methods comprise administering an effective amount of between about 225° C . and about 275° C . with a maximum a crystal form provided herein to a subject in need thereof . at approximately 254° C . The crystal form can be 2 , 2 , 2 - The kinase pathway is the TOR kinase pathway . trifluoroethanol solvated . The crystal form can comprise 0 . 5 35 The solid forms of Compound 1 and tautomers thereof molar equivalents of 2 , 2 , 2 - trifluoroethanol . The crystal form can be used in methods for achieving a Response Evaluation can be substantially pure . Criteria in Solid Tumors ( RECIST 1 . 1 ) of complete

Another crystal form provided herein has characteristic response , partial response or stable disease in a subject . The X - ray powder diffraction peaks at a two - theta angle of methods comprise administering an effective amount of a approximately 10 . 66 , 21 . 94 and 26 . 26 degrees . The X - ray 40 crystal form provided herein to a subject having a solid powder diffraction pattern can further comprise character - tumor . istic X - ray powder diffraction peaks at a two - theta angle of The solid forms of Compound 1 and tautomers thereof approximately 10 . 14 , 18 . 1 and 22 . 66 degrees . The crystal can be used in methods for improving International Work form can have a thermogravimetric analysis thermogram shop Criteria ( IWC ) for NHL , International Uniform comprising a total mass loss of approximately 16 . 4 % of the 45 Response Criteria for Multiple Myeloma ( IURC ) , Eastern total mass of the crystal form when heated from about 25º Cooperative Oncology Group Performance Status ( ECOG ) C . to about 300° C . The crystal form can have a single or Response Assessment for Neuro - Oncology ( RANO ) differential thermal analysis thermogram comprising an Working Group for GBM . The methods comprise adminis endotherm between about 100° C . and about 175° C . with a tering an effective amount of a crystal form provided herein maximum at approximately 140° C . when heated from about 50 to a subject in need thereof . 25° C . to about 300° C . The single differential thermal The present embodiments can be understood more fully analysis thermogram can further comprise an endotherm by reference to the detailed description and examples , which between about 235° C . and about 275° C . with a maximum are intended to exemplify non - limiting embodiments . at approximately 258° C . The crystal form can be dimeth ylsulfoxide solvated . The crystal form can comprise 0 . 8 55 4 . BRIEF DESCRIPTION OF THE DRAWINGS molar equivalents of dimethylsulfoxide . The crystal form can be substantially pure . FIG . 1 depicts an X - ray powder diffractogram stack plot

Still another crystal form provided herein has character of Forms 1 , 2 , 3 , 4 and 5 of Compound 1 . istic X - ray powder diffraction peaks at a two - theta angle of FIG . 2 depicts an X - ray powder diffractogram plot of approximately 9 . 26 , 11 . 7 and 26 . 18 degrees . The X - ray 60 Form 1 of Compound 1 . powder diffraction pattern can further comprise character FIG . 3 depicts a digital image of Form 1 of Compound 1 . istic X - ray powder diffraction peaks at a two - theta angle of FIG . 4 depicts a thermogravimetrical analysis and single approximately 7 . 46 , 24 . 26 and 24 . 94 degrees . The crystal differential thermal analysis of Form 1 of Compound 1 . form can have a thermogravimetric analysis thermogram FIG . 5 depicts a thermogravimetric analysis coupled with comprising a total mass loss of approximately 9 . 4 % of the 65 mass spectroscopy of Form 1 of Compound 1 . total mass of the crystal form when heated from about 25° FIG . 6 depicts high performance liquid chromatography C . to about 300° C . The crystal form can have a single coupled with mass spectrometry of Form 1 of Compound 1 .

US 9 , 981 , 971 B2

FIG . 7 depicts an X - ray powder diffractogram stack plot 5 . DETAILED DESCRIPTION of Form 1 , Form 2 , and Form 2 after exposure to accelerated aging conditions ( AAC ) of Compound 1 . 5 . 1 Definitions

FIG . 8 depicts a digital image of Form 2 of Compound 1 ( A ) and a digital image of Form 2 of Compound 1 after 5 As used herein , the term “ pharmaceutically acceptable exposure to accelerated aging conditions ( B ) . salt ( s ) ” refers to a salt prepared from a pharmaceutically

FIG . 9 depicts a thermogravimetrical analysis and single acceptable non - toxic acid or base including an inorganic differential thermal analysis of Form 2 of Compound 1 . acid and base and an organic acid and base . Suitable

FIG . 10 depicts a thermogravimetric analysis coupled inted pharmaceutically acceptable base addition salts include , but 10 are not limited to metallic salts made from aluminum , with mass spectroscopy of Form 2 of Compound 1 .

FIG . 11 depicts high performance liquid chromatography calcium , lithium , magnesium , potassium , sodium and zinc or organic salts made from lysine , N , N - dibenzylethylene coupled with mass spectrometry of Form 2 of Compound 1 . diamine , chloroprocaine , choline , diethanolamine , ethylene FIG . 12 depicts an X - ray powder diffractogram stack plot diamine , meglumine ( N - methylglucamine ) and procaine .

of Form 1 , Form 3 , and Form 3 after exposure to accelerated 15 Suitable non - toxic acids include but are not limited to . aging conditions ( AAC ) of Compound 1 . inorganic and organic acids such as acetic , alginic , anthra FIG . 13 depicts a digital image of Form 3 of Compound nilic , benzenesulfonic , benzoic , camphorsulfonic , citric , 1 ( A ) and a digital image of Form 3 of Compound 1 after ethenesulfonic , formic , fumaric , furoic , galacturonic , glu exposure to accelerated aging conditions ( B ) . conic , glucuronic , glutamic , glycolic , hydrobromic , hydro

FIG . 14 depicts a thermogravimetrical analysis and single 20 chloric , isethionic , lactic , maleic , malic , mandelic , methane differential thermal analysis of Form 3 of Compound 1 . sulfonic , mucic , nitric , pamoic , pantothenic , phenylacetic ,

FIG . 15 depicts a thermogravimetric analysis coupled phosphoric , propionic , salicylic , stearic , succinic , sulfanilic , with mass spectroscopy of Form 3 of Compound 1 . sulfuric , tartaric acid , and p - toluenesulfonic acid . Specific

FIG . 16 depicts high performance liquid chromatography non - toxic acids include hydrochloric , hydrobromic , phos coupled with mass spectrometry of Form 3 of Compound 1 . 25 phoric , sulfuric , and methanesulfonic acids . Examples of

FIG . 17 depicts an X - ray powder diffractogram stack plot specific salts thus include hydrochloride and mesylate salts . of Form 1 , Form 4 as wet solid , Form 4 as dry solid , Others are well - known in the art , see for example , Reming amorphous form of Compound 1 and the mixture of Forms ton ' s Pharmaceutical Sciences , 18th eds . , Mack Publishing , 1 and 4 as dry solid after exposure to accelerated aging Easton Pa . ( 1990 ) or Remington : The Science and Practice

30 of Pharmacy , 19th eds . , Mack Publishing , Easton Pa . ( 1995 ) . conditions ( AAC ) of Compound 1 . Pharmaceutically acceptable salts of Compound 1 can be FIG . 18 depicts a digital image of Form 4 of Compound 1 as wet solid ( A ) and a digital image of Form 4 of formed by conventional and known techniques , such as by

reacting Compound 1 with a suitable acid as disclosed Compound 1 as dry solid ( B ) . above . Such salts are typically formed in high yields at FIG . 19 depicts a thermogravimetrical analysis and single 35 ysis and single 35 moderate temperatures , and often are prepared by merely differential thermal analysis of Form 4 of Compound 1 . isolating the compound from a suitable acidic wash in the FIG . 20 depicts a thermogravimetric analysis coupled final step of the synthesis . The salt - forming acid may

with mass spectroscopy of Form 4 of Compound 1 . dissolved in an appropriate organic solvent , or aqueous FIG . 21 depicts high performance liquid chromatography organic solvent , such as an alkanol , ketone or ester . On the

coupled with mass spectrometry of Form 4 of Compound 1 . 40 other hand , if Compound 1 is desired in the free base form , FIG . 22 depicts an X - ray powder diffractogram stack plot it may be isolated from a basic final wash step , according to

of Form 1 , Form 5 , and Form 5 after exposure to accelerated known techniques . For example , a typical technique for aging conditions ( AAC ) of Compound 1 . preparing hydrochloride salt is to dissolve the free base of

FIG . 23 depicts a digital image of Form 5 of Compound Compound 1 in a suitable solvent , and dry the solution 1 ( A ) and a digital image of Form 5 of Compound 1 after 45 thoroughly , as over molecular sieves , before bubbling exposure to accelerated aging conditions ( B ) . hydrogen chloride gas through it .

FIG . 24 depicts a thermogravimetrical analysis and single As used herein and unless otherwise indicated , the term differential thermal analysis of Form 5 of Compound 1 . " stereoisomer ” or “ stereomerically pure ” means one stereoi

FIG . 25 depicts a thermogravimetric analysis coupled somer of a compound that is substantially free of other with mass spectroscopy of Form 5 of Compound 1 . 50 stereoisomers of that compound . For example , a stereomeri

FIG . 26 depicts high performance liquid chromatography cally pure compound having one chiral center will be substantially free of the opposite enantiomer of the com coupled with mass spectrometry of Form 5 of Compound 1 . pound . A stereomerically pure compound having two chiral FIG . 27 depicts a differential scanning calorimetry ther centers will be substantially free of other diastereomers of mogram of amorphous Compound 1 . 55 the compound . A typical stereomerically pure compound FIG . 28 depicts an X - ray powder diffractogram of amor comprises greater than about 80 % by weight of one stereoi phous Compound 1 . somer of the compound and less than about 20 % by weight FIG . 29 depicts a Raman spectrum of amorphous Com of other stereoisomers of the compound , greater than about

pound 1 . 90 % by weight of one stereoisomer of the compound and FIG . 30 depicts a proton nuclear magnetic resonance 60 less than about 10 % by weight of the other stereoisomers of

spectrum of amorphous Compound 1 . the compound , greater than about 95 % by weight of one FIG . 31 depicts high performance liquid chromatography stereoisomer of the compound and less than about 5 % by

coupled with mass spectrometry of amorphous Compound weight of the other stereoisomers of the compound , or greater than about 97 % by weight of one stereoisomer of the

FIG . 32 depicts a differential scanning calorimetry ther - 65 compound and less than about 3 % by weight of the other mogram of amorphous Compound 1 for determination of its stereoisomers of the compound . Compounds can have chiral glass transition temperature . centers and can occur as racemates , individual enantiomers

US 9 , 981 , 971 B2

or diastereomers , and mixtures thereof . All such isomeric The term “ solid form ” refers to a physical form which is forms are included within the embodiments disclosed not predominantly in a liquid or a gaseous state . As used herein , including mixtures thereof . The use of stereomeri - herein and unless otherwise specified , the term “ solid form , ” cally pure forms of such compounds , as well as the use of when used herein to refer to Compound 1 , refers to a mixtures of those forms are encompassed by the embodi - 5 physical form comprising Compound 1 which is not pre ments disclosed herein . For example , mixtures comprising dominantly in a liquid or a gaseous state . A solid form may equal or unequal amounts of the enantiomers of a particular be a crystalline form , an amorphous form , or a mixture compound may be used in methods and compositions dis thereof . In certain embodiments , a solid form may be a closed herein . These isomers may be asymmetrically syn liquid crystal . In certain embodiments , the term “ solid forms thesized or resolved using standard techniques such as chiral 10 comprising Compound 1 ” includes crystal forms comprising

Compound 1 , amorphous forms comprising Compound 1 , columns or chiral resolving agents . See , e . g . , Jacques , J . , et and mixtures thereof . In certain embodiments , the solid form al . , Enantiomers , Racemates and Resolutions ( Wiley - Inter of Compound 1 is Form 1 , Form 2 , Form 3 , Form 4 , Form science , New York , 1981 ) ; Wilen , S . H . , et al . , Tetrahedron 5 , amorphous or a mixture thereof . 33 : 2725 ( 1977 ) ; Eliel , E . L . , Stereochemistry of Carbon 15 15 As used herein and unless otherwise specified , the term Compounds ( McGraw - Hill , N Y , 1962 ) ; and Wilen , S . H . , " crystalline ” when used to describe a compound , substance , Tables of Resolving Agents and Optical Resolutions p . 268 modification , material , component or product , unless other ( E . L . Eliel , Ed . , Univ . of Notre Dame Press , Notre Dame , wise specified , means that the compound , substance , modi Ind . , 1972 ) . fication , material , component or product is substantially

It should also be noted the compounds can include E and 20 crystalline as determined by X - ray diffraction . See , e . g . . Z isomers , or a mixture thereof , and cis and trans isomers or Remington : The Science and Practice of Pharmacy , 21st a mixture thereof . In certain embodiments , compounds are edition , Lippincott , Williams and Wilkins , Baltimore , Md . isolated as either the cis or trans isomer . In other embodi ( 2005 ) ; The United States Pharmacopeia , 23rd ed . , 1843 ments , compounds are a mixture of the cis and trans isomers . 1844 ( 1995 ) .

“ Tautomers ” refers to isomeric forms of a compound that 25 The term “ crystal form ” or “ crystalline form ” refers to a are in equilibrium with each other . The concentrations of the solid form that is crystalline . In certain embodiments , crystal isomeric forms will depend on the environment the com forms include salts . In certain embodiments , a crystal form pound is found in and may be different depending upon , for of a substance may be substantially free of amorphous forms example , whether the compound is a solid or is in an organic and / or other crystal forms . In certain embodiments , a crystal or aqueous solution . For example , in aqueous solution , 30 form of a substance may contain less than about 1 % , less pyrazoles may exhibit the following isomeric forms , which than about 2 % , less than about 3 % , less than about 4 % , less are referred to as tautomers of each other : than about 5 % , less than about 6 % , less than about 7 % , less

than about 8 % , less than about 9 % , less than about 10 % , less than about 15 % , less than about 20 % , less than about 25 % ,

35 less than about 30 % , less than about 35 % , less than about 40 % , less than about 45 % , or less than about 50 % by weight of one or more amorphous forms and / or other crystal forms . In certain embodiments , a crystal form of a substance may be physically and / or chemically pure . In certain embodi

As readily understood by one skilled in the art , a wide 40 ments , a crystal form of a substance may be about 99 % , variety of functional groups and other structures may exhibit about 98 % , about 97 % , about 96 % , about 95 % , about 94 % , tautomerism and all tautomers of Compound 1 are within the about 93 % , about 92 % , about 91 % , or about 90 % physically scope of the present invention . and / or chemically pure .

It should also be noted that Compound 1 can contain The term " amorphous ” or “ amorphous form ” means that unnatural proportions of atomic isotopes at one or more of 45 the substance , component , or product in question is not the atoms . For example , Compound 1 may be radiolabeled substantially crystalline as determined by X - ray diffraction . with radioactive isotopes , such as for example tritium ( H ) , In particular , the term “ amorphous form ” describes a disor or carbon - 14 ( 14C ) , or may be isotopically enriched , such as dered solid form , i . e . , a solid form lacking long range with deuterium ( 2H ) , carbon - 13 ( 13C ) , or nitrogen - 15 ( 15N ) . crystalline order . In certain embodiments , an amorphous As used herein , an “ isotopologue ” is an isotopically 50 form of a substance may be substantially free of other enriched compound . The term “ isotopically enriched ” refers amorphous forms and / or crystal forms . In certain embodi to an atom having an isotopic composition other than the ments , an amorphous form of a substance may contain less natural isotopic composition of that atom . “ Isotopically than about 1 % , less than about 2 % , less than about 3 % , less enriched ” may also refer to a compound containing at least than about 4 % , less than about 5 % , less than about 10 % , less one atom having an isotopic composition other than the 55 than about 15 % , less than about 20 % , less than about 25 % , natural isotopic composition of that atom . The term “ isoto - less than about 30 % , less than about 35 % , less than about pic composition ” refers to the amount of each isotope 40 % , less than about 45 % , or less than about 50 % by weight present for a given atom . Radiolabeled and isotopically of one or more other amorphous forms and / or crystal forms enriched compounds are useful as therapeutic agents , e . g . , on a weight basis . In certain embodiments , an amorphous cancer and inflammation therapeutic agents , research 60 form of a substance may be physically and / or chemically reagents , e . g . , binding assay reagents , and diagnostic agents , pure . In certain embodiments , an amorphous form of a e . g . , in vivo imaging agents . All isotopic variations of substance be about 99 % , about 98 % , about 97 % , about 96 % , Compound 1 , whether radioactive or not , are intended to be about 95 % , about 94 % , about 93 % , about 92 % , about 91 % , encompassed within the scope of the embodiments provided or about 90 % physically and / or chemically pure . herein . In some embodiments , there are provided isotopo - 65 “ Treating " as used herein , means an alleviation , in whole logues of Compound 1 , for example , the isotopologues are or in part , of the disease or disorder , or symptoms associated deuterium , carbon - 13 , or nitrogen - 15 enriched Compound 1 . with the disease or disorder , or slowing , or halting of further

HN

US 9 , 981 , 971 B2

liver

CR

5

30

10 progression or worsening of the disease or disorder , or of secondary effects of disease , arrested tumor growth and symptoms associated with the disease or disorder . regression of tumors , among others . In the extreme , com

“ Preventing ” as used herein , means prevention of the plete inhibition , is referred to herein as prevention or che moprevention . In this context , the term " prevention ” onset , recurrence , or spread of the disease or disorder , or includes either preventing the onset of clinically evident

symptoms associated with the disorder or disease , in a 5 neoplasia altogether or preventing the onset of a preclini patient at risk for developing the disease or disorder . cally evident stage of neoplasia in individuals at risk . Also

The term " effective amount " in connection with a solid intended to be encompassed by this definition is the preven form of Compound 1 means , in one embodiment , an amount tion of transformation into malignant cells or to arrest or capable of alleviating , in whole or in part , symptoms asso reverse the progression of premalignant cells to malignant

10 cells . This includes prophylactic treatment of those at risk of ciated with a disorder or disease , or slowing or halting 10 developing the neoplasia . further progression or worsening of those symptoms , or , in In certain embodiments , the treatment of lymphoma may another embodiment , an amount capable of preventing or be assessed by the International Workshop Criteria ( IWC ) providing prophylaxis for the disease or disorder in a subject for non - Hodgkin lymphoma ( NHL ) ( see Cheson B D , Pfist at risk for developing the disease or disorder as disclosed 1 ner B , Juweid , M E , et . al . Revised Response Criteria for ner B . Juweid . ME . et . herein , such as cancer . In one embodiment an effective Malignant Lymphoma . J . Clin . Oncol : 2007 : ( 25 ) 579 - 586 ) , amount of Compound 1 is an amount that inhibits a kinase using the response and endpoint definitions shown below : in a cell , such as , for example , in vitro or in vivo . In one embodiment the kinase is mTOR , DNA - PK , PI3K or a Re Spleen , Bone Bone combination thereof . In some embodiments , the effective sponse Definition Nodal Masses Marrow amount of Compound 1 inhibits the kinase in a cell by 10 % , 20

( a ) FDG - avid or Disap Not 20 % , 30 % , 40 % , 50 % , 60 % , 70 % , 80 % , 90 % or 99 % , Infiltrate pearance PET positive prior palpa cleared on compared to the activity of the kinase in an untreated cell . of all to therapy ; mass of ble , repeat

The effective amount of Compound 1 , for example in a evidence any size permitted nodules biopsy ; if pharmaceutical composition , may be at a level that will of disease if PET negative disap indeterminate

( b ) Variably FDG - exercise the desired effect ; for example , about 0 . 005 mg / kg 23 peared by morphol avid or PET ogy , immuno of a subject ' s body weight to about 100 mg / kg of a patient ' s negative ; regression histochemis

body weight in unit dosage for both oral and parenteral to normal size on try should administration . As will be apparent to those skilled in the art , ?? be negative

PR Regres 250 % decrease in 250 % it is to be expected that the effective amount of Compound Irrelevant if sion of SPD of up to 6 decrease positive prior 1 disclosed herein may vary depending on the indication 30 measurable largest dominant in SPD of to therapy ;

being treated , e . g . , the effective amount of Compound 1 disease and masses ; no increase nodules cell type would likely be different for treating patients suffering from , no new sites in size of other ( for single should be or at risk for , inflammatory conditions relative to the effec nodes nodule in specified

( a ) FDG - avid or greatest tive amount of Compound 1 for treating patients suffering PET positive prior transverse from , or at risk of , a different disorder , e . g . , cancer or a to therapy ; one or diameter ) ; metabolic disorder . more PET positive

The term “ patient " includes an animal , including , but not at previously increase involved site in size of

limited to , an animal such as a cow , monkey , horse , sheep , ( b ) Variably FDG liver or pig , chicken , turkey , quail , cat , dog , mouse , rat , rabbit or avid or PET spleen guinea pig , in one embodiment a mammal , in another 4 negative ; regres

embodiment a human . sion on CT SD Failure to ( a ) FDG - avid or The term " cancer ” refers to any of various malignant attain CR / PR PET positive prior

neoplasms characterized by the proliferation of cells that can or PD to therapy ; PET invade surrounding tissue and metastasize to new body sites . positive at prior Both benign and malignant tumors are classified according sites of disease

and no new sites on to the type of tissue in which they are found . For example , CT or PET fibromas are neoplasms of fibrous connective tissue , and ( b ) Variably FDG melanomas are abnormal growths of pigment ( melanin ) avid or PET cells . Malignant tumors originating from epithelial tissue , negative ; no change

in size of previous e . g . , in skin , bronchi , and stomach , are termed carcinomas . 50 lesions on CT Malignancies of epithelial glandular tissue such as are found PD or Any new Appearance of a 250 % New or in the breast , prostate , and colon , are known as adenocar relapsed lesion or new lesion ( s ) 21 . 5 increase recurrent cinomas . Malignant growths of connective tissue , e . g . , disease increase cm in any axis , from nadir involvement

muscle , cartilage , lymph tissue , and bone , are called sarco by > 50 % of 50 % increase in the SPD mas . Lymphomas and leukemias are malignancies arising 55 previously in SPD of more of any

involved than one node , previous among white blood cells . Through the process of metastasis , sites from or 50 % increase in lesions tumor cell migration to other areas of the body establishes nadir longest diameter of neoplasms in areas away from the site of initial appearance . a previously Bone tissues are one of the most favored sites of metastases identifed node 21 of malignant tumors , occurring in about 30 % of all cancer cm in short axis cases . Among malignant tumors , cancers of the lung , breast , Lesions PET

positive if FDG - avid prostate or the like are particularly known to be likely to lymphoma or PET metastasize to bone . positive prior

In the context of neoplasm , cancer , tumor growth or tumor to therapy cell growth , inhibition may be assessed by delayed appear

Abbreviations : CR , complete remission ; FDG , [ F ] fluorodeoxyglucose ; PET , positron emission tomography ; CT , computed tomography ; PR , partial remission ; SPD , sum of the

of primary or secondary tumors , decreased occurrence of product of the diameters ; SD , stable disease ; PD , progressive disease . product of the diameters ; SD , stable primary or secondary tumors , slowed or decreased severity

?? no

SD

OS

US 9 , 981 , 971 B2 11 12

- continued End point Patients Definition Measured from

Parameter Parameter CR PR P D Primary

10 Neut

15

Overall All Death as a result of Entry onto 5 Hemoglobin > 11 . 0 g / dL > 11 g / dL or Decrease survival any cause study increase 50 % of > 2 g / dL Progression - free All Disease progression Entry onto over baseline from baseline survival or death as a result study secondary of any cause Secondary to CLL

Neutrophilst > 1500 / uL > 1500 / ul Event - free All Failure of treatment Entry onto or - 50 % survival or death as result of study improvement over

any cause baseline Time to All Time to progression or Entry onto progression death as a result study 15

of lymphoma Disease - free In CR Time to relapse or death Documentation Group A criteria define the tumor load ; Group B criteria survival as a result of lymphoma of response define the function of the hematopoietic system ( or marrow ) .

or acute toxicity of CR ( complete remission ) : all of the criteria have to be met , treatment 20 and patients have to lack disease - related constitutional

Response In CR Time to relapse or Documentation symptoms ; PR ( partial remission ) : at least two of the criteria duration or PR progression of response of group A plus one of the criteria of group B have to be met ; Lymphoma All Time to death as a result Entry onto SD is absence of progressive disease ( PD ) and failure to specific survival of lymphoma study Time to next All Time to new treatment End of primary achieve at least a PR ; PD : at least one of the above criteria

25 of group A or group B has to be met . Sum of the products treatment treatment treatment 25 01 80P AV 80P D of multiple lymph nodes ( as evaluated by CT scans in

Abbreviations : CR : complete remission ; PR : partial remission . clinical trials , or by physical examination in general prac tice ) . These parameters are irrelevant for some response

In one embodiment , the end point for lymphoma is categories . evidence of clinical benefit . Clinical benefit may reflect 30 In certain embodiments , the treatment of multiple improvement in quality of life , or reduction in patient myeloma may be assessed by the International Uniform symptoms , transfusion requirements , frequent infections , or Response Criteria for Multiple Myeloma ( IURC ) ( see Durie other parameters . Time to reappearance or progression of BGM , Harousseau J - L , Miguel J S , et al . International lymphoma - related symptoms can also be used in this end uniform response criteria for multiple myeloma . Leukemia , point . 35 2006 ; ( 10 ) 10 : 1 - 7 ) , using the response and endpoint defi

nitions shown below : In certain embodiments , the treatment of CLL may be assessed by the International Workshop Guidelines for CLL ( see Hallek M , Cheson B D , Catovsky D , et al . Guidelines Response Subcategory Response Criteria for the diagnosis and treatment of chronic lymphocytic SCR CR as defined below plus leukemia : a report from the International Workshop on Normal FLC ratio and

Absence of clonal cells in bone marrowb Chronic Lymphocytic Leukemia updating the National Can by immunohistochemistry or cer Institute - Working Group 1996 guidelines . Blood , 2008 ; immunofluorescence ( 111 ) 12 : 5446 - 5456 ) using the response and endpoint CR Negative immunofixation on the serum

and urine and Disappearance of any soft definitions shown therein and in particular : tissue plasmacytomas and < 5 % plasma cells in bone marrow

VGPR Serum and urine M - protein detectable by Parameter PR PR PD PD immunofixation but not on electrophoresis

or 90 % or greater reduction in serum Group A M - protein plus urine M - protein level < 100

mg per 24 h Lymphade - None > 1 . 5 cm Decrease 50 % Increase 50 % 250 % reduction of serum M - protein nopathy and reduction in 24 - h urinary M - protein Hepato None Decrease 250 % Increase 250 % by > 90 % or to < 200 mg per 24 h megaly If the serum and urine M - protein are Spleno Decrease 50 % Increase 50 % unmeasurable , d a 250 % decrease megaly in the difference between Blood < 4000 / uL Decrease 50 % Increase 250 % involved and uninvolved FLC levels lymphocytes from baseline over baseline is required in place of the M - protein Marrow Normocellular , < 30 % 50 % reduction in criteria

lymphocytes , no B - marrow infiltrate , If serum and urine M - protein are lymphoid nodules . or B - lymphoid unmeasurable , and serum free light Hypocellular marrow nodules assay is also unmeasurable , 250 % defines CRi ( 5 . 1 . 6 ) . reduction in plasma cells is required

Group B in place of M - protein , provided baseline bone marrow plasma cell percentage

Platelet > 100 000 / uL > 100 000 / uL or Decrease was 230 % increase > 50 % of 250 % In addition to the above listed criteria , over baseline from baseline if present at baseline , a 250 % reduction

secondary in the size of soft tissue plasmacytomas to CLL is also required

45

50 R

None 55

60

count

65

14 US 9 , 981 , 971 B2

13 - continued ( RANO ) Working Group regarding response criteria for

high - grade gliomas ( Wen P . , Macdonald , D R . , Reardon , D Response Subcategory Response Criteria A . , et al . Updated response assessment criteria for highgrade

gliomas : Response assessment in neuro - oncology working SD ( not recommended for use Not meeting criteria for CR , VGPR , as an indicator of response ; PR or progressive disease group . J Clin Oncol 2010 ; 28 : 1963 - 1972 ) . Primary modi stability of disease is best fications to the RANO criteria for Criteria for Time Point described by providing the Responses ( TPR ) can include the addition of operational time to progression estimates ) conventions for defining changes in glucocorticoid dose , and

the removal of subjects ' clinical deterioration component to Abbreviations : CR , complete response ; FLC , free light chain ; PR , partial response ; SD , stable disease ; SCR , stringent complete response ; VGPR , very good partial response ; Ssess " All response categories require two consecutive assessments made at anytime before the institution of any new therapy ; all categories also require no known evidence of MRI scan is defined as the assessment performed at the end progressive or new bone lesions if radiographic studies were performed . Radiographic studies are not required to satisfy these response requirements ; of the post - surgery rest period , prior to re - initiating com " Confirmation with repeat bone marrow biopsy not needed ; pound treatment . The baseline MM is used as the reference Presence / absence of clonal cells is based upon the Ka ratio . An abnormal k / ratio by immunohistochemistry and / or immunofluorescence requires a minimum of 100 plasma for assessing complete response ( CR ) and partial response cells for analysis . An abnormal ratio reflecting presence of an abnormal clone is k / of 4 : 1 e of an abnormal clone is k / k of > 4 : 1 15 ( PR ) . Whereas , the smallest SPD ( sum of the products of or < 1 : 2 . Measurable disease defined by at least one of the following measurements : Bone marrow perpendicular diameters ) obtained either at baseline or at plasma cells 230 % ; Serum M - protein 21 g / dl ( 210 gm / l ) [ 10 g / l ] ; Urine M - protein 2200 mg / 24 h ; Serum FLC assay : Involved FLC level 210 mg / dl ( 2100 mg / l ) ; provided serum subsequent assessments will be designated the nadir assess FLC ratio is abnormal . ment and utilized as the reference for determining progres

In certain embodiments , the treatment of a cancer may be sion . For the 5 days preceding any protocol - defined MM assessed by Response Evaluation Criteria in Solid Tumors 20 scan , subjects receive either no glucocorticoids or are on a ( RECIST 1 . 1 ) ( see Thereasse P . , et al . New Guidelines to stable dose of glucocorticoids . A stable dose is defined as the

same daily dose for the 5 consecutive days preceding the Evaluate the Response to Treatment in Solid Tumors . J . of the National Cancer Institute ; 2000 ; ( 92 ) 205 - 216 and Eisen MRI scan . If the prescribed glucocorticoid dose is changed

in the 5 days before the baseline scan , a new baseline scan hauer E . A . , Therasse P . , Bogaerts J . , et al . New response evaluation criteria in solid tumours : Revised RECIST guide - 25 25 is required with glucocorticoid use meeting the criteria line ( version 1 . 1 ) . European J . Cancer ; 2009 ; ( 45 ) 228 - 247 ) . described above . The following definitions will be used .

Measurable Lesions : Measurable lesions are contrast Overall responses for all possible combinations of tumor enhancing lesions that can be measured bidimensionally . A responses in target and non - target lesions with our without measurement is made of the maximal enhancing tumor the appearance of new lesions are as follows : 30 diameter ( also known as the longest diameter , LD ) . The greatest perpendicular diameter is measured on the same

Target Non - target New image . The cross hairs of bidimensional measurements Overall lesions lesions lesions response should cross and the product of these diameters will be

calculated . CR CR No Minimal Diameter : T1 - weighted image in which the Incomplete response / SD PR

Non - PD No sections are 5 mm with 1 mm skip . The minimal LD of a Non - PD No measurable lesion is set as 5 mm by 5 mm . Larger diameters Any Yes or no PD may be required for inclusion and / or designation as target

Yes or no lesions . After baseline , target lesions that become smaller Any Any Yes 40 than the minimum requirement for measurement or become

CR = complete response ; PR = partial response ; SD = stable disease ; and PD = progressive no longer amenable to bidimensional measurement will be disease . recorded at the default value of 5 mm for each diameter

With respect to the evaluation of target lesions , complete below 5 mm . Lesions that disappear will be recorded as 0 response ( CR ) is the disappearance of all target lesions , mm by 0 mm . partial response ( PR ) is at least a 30 % decrease in the sum 45 Multicentric Lesions : Lesions that are considered multi of the longest diameter of target lesions , taking as reference centric ( as opposed to continuous ) are lesions where there is the baseline sum longest diameter , progressive disease ( PD ) normal intervening brain tissue between the two ( or more ) is at least a 20 % increase in the sum of the longest diameter lesions . For multicentric lesions that are discrete foci of of target lesions , taking as reference the smallest sum enhancement , the approach is to separately measure each longest diameter recorded since the treatment started or the 50 enhancing lesion that meets the inclusion criteria . If there is appearance of one or more new lesions and stable disease no normal brain tissue between two ( or more ) lesions , they ( SD ) is neither sufficient shrinkage to qualify for partial will be considered the same lesion . response nor sufficient increase to qualify for progressive Nonmeasurable Lesions : All lesions that do not meet the disease , taking as reference the smallest sum longest diam - criteria for measurable disease as defined above will be eter since the treatment started . 55 considered non - measurable lesions , as well as all nonen

With respect to the evaluation of non - target lesions , hancing and other truly nonmeasurable lesions . Nonmeasur complete response ( CR ) is the disappearance of all non - able lesions include foci of enhancement that are less than target lesions and normalization of tumor marker level ; the specified smallest diameter ( i . e . , less than 5 mm by 5 incomplete response / stable disease ( SD ) is the persistence of mm ) , nonenhancing lesions ( e . g . , as seen on T1 - weighted one or more non - target lesion ( s ) and / or the maintenance of 60 post - contrast , T2 - weighted , or fluid - attenuated inversion tumor marker level above the normal limits , and progressive recovery ( FLAIR ) images ) , hemorrhagic or predominantly disease ( PD ) is the appearance of one or more new lesions cystic or necrotic lesions , and leptomeningeal tumor . Hem and / or unequivocal progression of existing non - target orrhagic lesions often have intrinsic T1 - weighted hyperin lesions . tensity that could be misinterpreted as enhancing tumor , and

The procedures , conventions , and definitions described 65 for this reason , the pre - contrast T1 - weighted image may be below provide guidance for implementing the recommen examined to exclude baseline or interval sub - acute hemor dations from the Response Assessment for Neuro - Oncology rhage .

CR 3 5 No

PR CR PR SD PD Any

SD

PD PD PD

15 US 9 , 981 , 971 B2

16 At baseline , lesions will be classified as follows : Target As used herein , and unless otherwise specified , the terms

lesions : Up to 5 measurable lesions can be selected as target about ” and “ approximately . ” when used in connection with lesions with each measuring at least 10 mm by 5 mm , doses , amounts , or weight percent of ingredients of a com representative of the subject ' s disease ; Non - target lesions : All other lesions , including all nonmeasurable lesions ( in - 5 position or a dosage form , mean a dose , amount , or weight cluding mass effects and T2 / FLAIR findings ) and any mea percent that is recognized by those of ordinary skill in the art surable lesion not selected as a target lesion . At baseline , to provide a pharmacological effect equivalent to that target lesions are to be measured as described in the defi nition for measurable lesions and the SPD of all target obtained from the specified dose , amount , or weight percent . lesions is to be determined . The presence of all other lesions 10 Specifically , the terms “ about ” and “ approximately , " when is to be documented . At all post - treatment evaluations , the used in this context , contemplate a dose , amount , or weight baseline classification of lesions as target and non - target percent within 15 % , more specifically within 10 % , more lesions will be maintained and lesions will be documented and described in a consistent fashion over time ( e . g . , specifically within 5 % , of the specified dose , amount , or recorded in the same order on source documents and 15 weight percent . eCRFs ) . All measurable and nonmeasurable lesions must be As used herein , and unless otherwise specified , the terms assessed using the same technique as at baseline ( e . g . , subjects should be imaged on the same MM scanner or at “ about ” and “ approximately , ” when used in connection with least with the same magnet strength ) for the duration of the a numeric value or range of values which is provided to study to reduce difficulties in interpreting changes . At each 20 characterize a particular solid form , e . g . , a specific tempera evaluation , target lesions will be measured and the SPD calculated . Non - target lesions will be assessed qualitatively ture or temperature range , such as , for example , that describ and new lesions , if any , will be documented separately . At ing a melting , dehydration , desolvation or glass transition each evaluation , a time point response will be determined for temperature ; a mass change , such as , for example , a mass target lesions , non - target lesions , and new lesion . Tumor 25 progression can be established even if only a subset of change as a function of temperature or humidity ; a solvent lesions is assessed . However , unless progression is or water content , in terms of , for example , mass or a observed , objective status ( stable disease , PR or CR ) can percentage ; or a peak position , such as , for example , in only be determined when all lesions are assessed .

Confirmation assessments for overall time point 30 analysis by IR or Raman spectroscopy or XRPD ; indicate responses of CR and PR will be performed at the next that the value or range of values may deviate to an extent scheduled assessment , but confirmation may not occur if scans have an interval of < 28 days . Best response , incorpo deemed reasonable to one of ordinary skill in the art while rating confirmation requirements , will be derived from the still describing the particular solid form . Specifically , the series of time points . 35 terms “ about ” and “ approximately , " when used in this In certain embodiments , treatment of a cancer may be assessed by the inhibition of phosphorylation of SORP , context , indicate that the numeric value or range of values 4E - BP1 , AKT and / or DNA - PK in circulating blood and / or may vary , in particular embodiments , within 20 % , 10 % , 9 % , tumor cells , and / or skin biopsies or tumor biopsies / aspirates , 8 % , 7 % , 6 % , 5 % , 4 % , 3 % , 2 % , 1 . 5 % , 1 % , 0 . 5 % , 0 . 25 % of before , during and / or after treatment with a TOR kinase 40 the recited value or range of values . For example , in some inhibitor . For example , the inhibition of phosphorylation of SORP . 4E - BP1 , AKT and / or DNA - PK is assessed in B - cells , embodiments , the value of an XRPD peak position may vary T - cells and / or monocytes . In other embodiments , treatment by up to 20 . 2 degrees two theta while still describing the of a cancer may be assessed by the inhibition of DNA particular XRPD peak . dependent protein kinase ( DNA - PK ) activity in skin samples 45 and / or tumor biopsies / aspirates , such as by assessment of the amount of pDNA - PK S2056 as a biomarker for DNA 5 . 2 Compound 1 damage pathways , before , during , and / or after TOR kinase inhibitor treatment . In one embodiment , the skin sample is The solid forms , formulations and methods of use pro irradiated by UV light . 50 vided herein relate to Compound 1 : In the extreme , complete inhibition , is referred to herein as prevention or chemoprevention . In this context , the term " prevention ” includes either preventing the onset of clini cally evident cancer altogether or preventing the onset of a preclinically evident stage of a cancer . Also intended to be 55 encompassed by this definition is the prevention of trans formation into malignant cells or to arrest or reverse the progression of premalignant cells to malignant cells . This includes prophylactic treatment of those at risk of develop ing a cancer .

As used herein and unless otherwise indicated , the term " substantially pure ” when used to describe a polymorph of a compound , i . e . a crystal form or an amorphous form of a compound , means a crystal form or an amorphous form of the compound that comprises that crystal form or amorphous 65 having the name 1 - ethyl - 7 - ( 2 - methyl - 6 - 1H - 1 , 2 , 4 - triazol form and is substantially free of other polymorphs of the 3 - yl ) pyridin - 3 - yl ) - 3 , 4 - dihydropyrazino [ 2 , 3 - b ] pyrazin - 2 compound ( 1H ) - one , and tautomers .

CZ

N

US 9 , 981 , 971 B2 17

Tautomers of Compound 1 include the following : 18

HN

IZ

N

Compound 1 can be prepared using reagents and methods 15 with single differential thermal analysis ( TGA - SDTA ) , and known in the art , including the methods provided in U . S . thermogravimetric analysis coupled with mass spectroscopy Pat . No . 8 , 110 , 578 , filed on Oct . 26 , 2009 ; US Patent ( TGA - MS ) . The particle size and size distribution of the Publication Application No . 2011 / 0137028 , filed on Oct . 25 , solid form provided herein may be determined by conven 2010 ; and U . S . Provisional Patent Application No . 61 / 813 , tional methods , such as laser light scattering technique . 064 , filed on Apr . 17 , 2013 , the entire contents of each of 20 The purity of the solid forms provided herein may be which are incorporated herein by reference . determined by standard analytical methods , such as thin

It should be noted that if there is a discrepancy between layer chromatography ( TLC ) , gel electrophoresis , gas chro a depicted structure and a name given that structure , the matography , high performance liquid chromatography depicted structure is to be accorded more weight . In addi - ( HPLC ) , and mass spectrometry ( MS ) . tion , if the stereochemistry of a structure or a portion of a 25 It should be understood that the numerical values of the structure is not indicated with , for example , bold or dashed peaks of an X - ray powder diffraction pattern may vary lines , the structure or portion of the structure is to be slightly from one machine to another or from one sample to interpreted as encompassing all stereoisomers of it . another , and so the values quoted are not to be construed as

absolute , but with an allowable variability , such as 10 . 2 5 . 3 Solid Forms of Compound 1 30 degrees two theta ( 20 ) ( see United State Pharmacopoeia ,

page 2228 ( 2003 ) ) . In certain embodiments , provided herein are solid forms 5 . 3 . 1 Form 1 of Compound 1

of Compound 1 or tautomers thereof . In certain embodi - In certain embodiments , provided herein is Form 1 of ments , the solid form is crystalline . In certain embodiments , Compound 1 . the solid form is a single - component solid form . In certain 35 In one embodiment , Form 1 is an anhydrous form of embodiments , the solid form is a solvate . Compound 1 . In another embodiment , Form 1 of Compound

While not intending to be bound by any particular theory , 1 is crystalline . certain solid forms are characterized by physical properties , In certain embodiments , a solid form provided herein , e . g . , stability , solubility and dissolution rate , appropriate for e . g . , Form 1 , is substantially crystalline , as indicated by , e . g . , pharmaceutical and therapeutic dosage forms . Moreover , 40 X - ray powder diffraction measurements . In one embodi while not wishing to be bound by any particular theory , ment , Form 1 of Compound 1 has an X - ray powder diffrac certain solid forms are characterized by physical properties tion pattern substantially as shown in FIG . 2 . In one embodi ( e . g . , density , compressibility , hardness , morphology , cleav - ment , Form 1 of Compound 1 has one or more characteristic age , stickiness , solubility , water uptake , electrical properties , X - ray powder diffraction peaks at a two - theta angle of thermal behavior , solid - state reactivity , physical stability , 45 approximately 7 . 94 , 9 . 74 , 11 . 94 , 15 . 86 , 17 . 3 , 17 . 86 , 19 . 46 , and chemical stability ) affecting particular processes ( e . g . , 25 . 14 , 26 . 42 , 27 . 06 , 27 . 98 or 29 . 38 degrees as depicted in yield , filtration , washing , drying , milling , mixing , tableting , FIG . 2 . In a specific embodiment , Form 1 of Compound 1 flowability , dissolution , formulation , and lyophilization ) has one , two , three , four , five , six , seven or eight character which make certain solid forms suitable for the manufacture istic X - ray powder diffraction peaks at a two - theta angle of of a solid dosage form . Such properties can be determined 50 approximately 9 . 74 , 11 . 94 , 15 . 86 , 17 . 3 , 25 . 14 , 26 . 42 , 27 . 06 using particular analytical chemical techniques , including or 27 . 98 degrees . In another embodiment , Form 1 of Com solid - state analytical techniques ( e . g . , X - ray diffraction , pound 1 has one , two , three or four characteristic X - ray microscopy , spectroscopy and thermal analysis ) , as powder diffraction peaks at a two - theta angle of approxi described herein and known in the art . mately 9 . 74 , 15 . 86 , 25 . 14 or 27 . 06 degrees . In another

The solid forms provided herein ( e . g . , Form 1 , Form 2 , 55 embodiment , Form 1 of Compound 1 has one , two , three , Form 3 , Form 4 , Form 5 and amorphous of Compound 1 ) four , five , six , seven , eight , nine , ten , eleven or twelve may be characterized using a number of methods known to characteristic X - ray powder diffraction peaks as set forth in a person having ordinary skill in the art , including , but not Table 25 . limited to , single crystal X - ray diffraction , X - ray powder In one embodiment , Form 1 of Compound 1 has a digital diffraction ( XRPD ) , microscopy ( e . g . , scanning electron 60 image substantially as shown in FIG . 3 . microscopy ( SEM ) ) , thermal analysis ( e . g . , differential scan - In one embodiment , provided herein is a crystalline form ning calorimetry ( DSC ) , thermal gravimetric analysis of Compound 1 having a single differential thermal analysis ( TGA ) , and hot - stage microscopy ) , spectroscopy ( e . g . , infra - ( SDTA ) thermogram as depicted in FIG . 4 comprising an red , Raman , and solid - state nuclear magnetic resonance ) , endothermic event between about 240° C . and about 285° C . single differential thermal analysis ( SDTA ) , high perfor - 65 with a maximum at about 268 . 9° C . when heated from mance liquid chromatography coupled with mass spectros - approximately 25° C . to approximately 300° C . ( see Table copy ( HPLC - MS ) , thermogravimetrical analysis coupled 24 ) .

US 9 , 981 , 971 B2 19 20

In one embodiment , provided herein is a crystalline form stirring the slurry during steps 1 - 5 ; 6 ) repeating steps 2 - 58 of Compound 1 having a thermogravimetric ( TGA ) thermo - times ; and 7 ) filtering the slurry to yield a solid . graph corresponding substantially to the representative TGA In one embodiment , Form 2 is a 1 , 2 - ethanediol solvated thermogram as depicted in FIG . 5 . In certain embodiments , form of Compound 1 . In one embodiment , Form 2 is a the crystalline form exhibits a TGA thermogram comprising 5 1 , 2 - ethanediol mono - solvated form of Compound 1 . In a total mass loss of approximately 0 . 44 % of the total mass another embodiment , Form 2 of Compound 1 is crystalline . of the sample between approximately 30° C . and approxi - In certain embodiments , a solid form provided herein , mately 250° C . when heated from approximately 20° C . to e . g . , Form 2 , is substantially crystalline , as indicated by , e . g . , approximately 300° C . Thus , in certain embodiments , the X - ray powder diffraction measurements . In one embodi crystalline form loses about 0 . 44 % of its total mass when 10 ment , Form 2 of Compound 1 has an X - ray powder diffrac heated from about ambient temperature to about 300° C . tion pattern substantially as shown in FIG . 7 ( middle pat

In still another embodiment , Form 1 of Compound 1 is tern ) . In one embodiment , Form 2 of Compound 1 has one substantially pure . In certain embodiments , the substantially or more characteristic X - ray powder diffraction peaks at a pure Form 1 of Compound 1 is substantially free of other two - theta angle of approximately 6 . 18 , 10 . 02 , 11 . 54 , 12 . 34 , solid forms , e . g . , amorphous form . In certain embodiments , 15 13 . 86 , 18 . 54 , 21 . 74 , 22 . 5 , 23 . 42 , 24 . 54 , 25 . 5 , 26 . 02 , 26 . 7 , the purity of the substantially pure Form 1 of Compound 1 27 . 82 , 28 . 34 or 34 . 14 degrees as depicted in FIG . 7 . In a is no less than about 95 % pure , no less than about 96 % pure , specific embodiment , Form 2 of Compound 1 has one , two , no less than about 97 % pure , no less than about 98 % pure , three , four , five , six , seven or eight characteristic X - ray no less than about 98 . 5 % pure , no less than about 99 % pure , powder diffraction peaks at a two - theta angle of approxi no less than about 99 . 5 % pure , or no less than about 99 . 8 % 20 mately 6 . 18 , 12 . 34 , 18 . 54 , 21 . 74 , 22 . 5 , 23 . 42 , 26 . 7 or 28 . 34 pure . degrees . In another embodiment , Form 2 of Compound 1 has

5 . 3 . 2 Form 2 of Compound 1 one , two , three or four characteristic X - ray powder diffrac In certain embodiments , provided herein is Form 2 of tion peaks at a two - theta angle of approximately 6 . 18 , 12 . 34 ,

Compound 1 . 21 . 74 or 26 . 7 degrees . In another embodiment , Form 2 of In certain embodiments , Form 2 is obtained by crystalli - 25 Compound 1 has one , two , three , four , five , six , seven , eight ,

zation from certain solvent systems , for example , solvent n ine , ten , eleven , twelve , thirteen , fourteen , fifteen or sixteen systems comprising one or more of the following solvents or characteristic X - ray powder diffraction peaks as set forth in solvent combinations : 1 , 2 - ethanediol and THF . In certain Table 26 . embodiments , Form 2 provided herein is obtained by slurry In one embodiment , Form 2 of Compound 1 has a digital crystallization , evaporation crystallization or thermocycling 30 image substantially as shown in FIG . 8A . crystallization ( see Table 23 ) . In one embodiment , provided herein is a crystalline form

In certain embodiments , provided herein are methods for of Compound 1 having a thermogravimetric ( TGA ) thermo making Form 2 of Compound 1 , comprising obtaining a graph corresponding substantially to the representative TGA slurry of Form 1 of Compound 1 in a solvent , stirring the thermogram as depicted in FIG . 10 . In certain embodiments , slurry , collecting solid from the slurry by filtration ( e . g . , 35 the crystalline form exhibits a TGA thermogram comprising centrifuge filtration ) and optionally washing ( e . g . , washing a total mass loss of approximately 15 . 5 % of the total mass with the solvent ) and drying . In certain embodiments , pro - of the sample between approximately 95° C . and approxi vided herein are methods for making Form 2 of Compound mately 175° C . when heated from approximately 25° C . to 1 , comprising obtaining a slurry of Compound 1 in 1 , 2 - approximately 300° C . Thus , in certain embodiments , the ethanediol , stirring the slurry , collecting solid from the 40 crystalline form loses about 15 . 5 % of its total mass when slurry by centrifuge filtration and optionally washing with heated from about ambient temperature to about 300° C . In 1 , 2 - ethanediol and drying . certain embodiments , the crystalline form contains 1 molar

In certain embodiments , provided herein are methods for equivalent of solvent in the crystal lattice corresponding to making Form 2 of Compound 1 , comprising dissolving approximately 1 mole of 1 , 2 - ethanediol per mole of Com Form 1 of Compound 1 in a solvent to yield a solution , 45 pound 1 . The theoretical 1 , 2 - ethanediol content of a 1 , 2 filtering the solution if Form 1 does not dissolve completely , ethanediol mono - solvate of Compound 1 is 15 . 6 % by and evaporating the solution under certain air pressure to weight , matching the TGA weight loss observed . In certain yield a solid . In certain embodiments , provided herein are embodiments , the crystalline form is a 1 , 2 - ethanediol mono methods for making Form 2 of Compound 1 , comprising solvate of Compound 1 . dissolving Form 1 of Compound 1 in 1 , 2 - ethanediol / THF 50 In one embodiment , provided herein is a crystalline form ( 50 / 50 ) to yield a solution , filtering the solution if Form 1 of Compound 1 having a single differential thermal analysis does not dissolve completely , and evaporating the solution ( SDTA ) thermogram as depicted in FIG . 9 comprising an under 200 mbar air pressure to yield a solid . endothermic event between about 95° C . and about 176° C .

In certain embodiments , provided herein are methods for with a maximum at about 137° C . when heated from making Form 2 of Compound 1 , comprising 1 ) obtaining a 55 approximately 25° C . to approximately 300° C . ( see Table slurry of Form 1 of Compound 1 in a solvent ; 2 ) heating the 24 ) . slurry until a first temperature ( e . g . , about 30° C . to about In one embodiment , provided herein is a crystalline form 50° C . ) ; 3 ) cooling the slurry to a second temperature ( e . g . , of Compound 1 having a single differential thermal analysis about - 5° C . to about 15° C . ) ; 4 ) keeping the slurry at the ( SDTA ) thermogram as depicted in FIG . 9 comprising an second temperature for a period of time ; 5 ) stirring the slurry 60 endothermic event between about 240° C . and about 285° C . during steps 1 - 5 ; 6 ) repeating steps 2 - 5 ( e . g . , from 6 to 10 with a maximum at about 264° C . when heated from times ) ; and 7 ) filtering the slurry to yield a solid . In certain approximately 25° C . to approximately 300° C . ( see Table embodiments , provided herein are methods for making 24 ) . Form 2 of Compound 1 , comprising 1 ) obtaining a slurry of In still another embodiment , Form 2 of Compound 1 is Form 1 of Compound 1 in 1 , 2 - ethanediol ; 2 ) heating the 65 substantially pure . In certain embodiments , the substantially slurry to about 40° C . ; 3 ) cooling the slurry to about 5° C . ; pure Form 2 of Compound 1 is substantially free of other 4 ) keeping the slurry at about 5° C . for about 30 minutes ; 5 ) solid forms , e . g . , amorphous form . In certain embodiments ,

21 US 9 , 981 , 971 B2

22 the purity of the substantially pure Form 2 of Compound 1 Compound 1 for about two hours , diffusing chloroform on is no less than about 95 % pure , no less than about 96 % pure , to the amorphous form of Compound 1 for about two weeks , no less than about 97 % pure , no less than about 98 % pure , and collecting the solid . no less than about 98 . 5 % pure , no less than about 99 % pure , In one embodiment , Form 3 is a 2 , 2 , 2 - trifluoroethanol no less than about 99 . 5 % pure , or no less than about 99 . 8 % 5 solvated form of Compound 1 . In one embodiment , Form 3 pure . is a 2 , 2 , 2 - trifluoroethanol hemi - solvated form of Compound

5 . 3 . 3 Form 3 of Compound 1 1 . In another embodiment , Form 3 of Compound 1 is In certain embodiments , provided herein is Form 3 of crystalline .

Compound 1 . In one embodiment , Form 3 is a chloroform solvated form In certain embodiments , Form 3 is obtained by crystalli - " of Compound 1 . In one embodiment , Form 3 is a chloroform

zation from certain solvent systems , for example , solvent hemi - solvated form of Compound 1 . systems comprising one or more of the following solvents or In one embodiment , Form 3 is an acetone solvated form solvent combinations : 2 , 2 , 2 - trifluoroethanol ( TFE ) com - of Compound 1 . In one embodiment , Form 3 is an acetone bined with either water or cyclohexane , chloroform and the 15 hemi - solvated form of Compound 1 . solvent mixture of isopropanol and acetone . In certain In one embodiment , Form 3 is an isopropanol solvated embodiments , Form 3 is obtained by evaporative crystalli - form of Compound 1 . In one embodiment , Form 3 is an zation , hot - filtration crystallization , vapor diffusion into isopropanol hemi - solvated form of Compound 1 . liquid crystallization or vapor diffusion onto solid crystalli - In certain embodiments , a solid form provided herein zation ( see Table 23 ) . 20 ( e . g . , Form 3 ) is substantially crystalline , as indicated by ,

In certain embodiments , provided herein are methods for e . g . , X - ray powder diffraction measurements . In one making Form 3 of Compound 1 , comprising mixing Form 1 embodiment , Form 3 of Compound 1 has an X - ray powder of Compound 1 with a solvent or solvent mixture , filtering diffraction pattern substantially as shown in FIG . 12 ( middle the mixture to yield a solution if Form 1 does not dissolve pattern ) . In one embodiment , Form 3 of Compound 1 has completely , and evaporating the solution under certain air 25 one or more characteristic X - ray powder diffraction peaks at pressure to yield a solid . In certain embodiments , provided a two - theta angle of approximately 3 . 5 , 7 . 06 , 9 . 26 , 10 . 5 . herein are methods for making Form 3 of Compound 1 , 12 . 66 , 15 . 3 or 18 . 62 degrees as depicted in FIG . 12 . In comprising mixing Form 1 of Compound 1 with a 1 : 1 another embodiment , Form 3 of Compound 1 has one , two , solution of TFE and water , filtering the mixture to yield a three or four characteristic X - ray powder diffraction peaks at

solution if Form 1 does not dissolve completely , and evapo 30 a two - theta angle of approximately 3 . 5 , 9 . 26 , 15 . 3 or 18 . 62 rating the solution of TFE and water under 200 mbar air degrees . In another embodiment , Form 3 of Compound 1 has

one , two , three , four , five , six or seven characteristic X - ray pressure to yield a solid . powder diffraction peaks as set forth in Table 27 . In certain embodiments , provided herein are methods for In one embodiment , Form 3 of Compound 1 has a digital making Form 3 of Compound 1 , comprising obtaining a 35 image substantially as shown in FIG . 13A . slurry of Form 1 of Compound 1 in a solvent , heating the In one embodiment provided herein is a crystalline form slurry to a first temperature ( e . g . , about 30° C . to about 70 % of Compound 1 having a thermogravimetric ( TGA ) thermo C . ) , filtering the slurry to yield a solution , cooling down the graph corresponding substantially to the representative TGA solution to a second temperature ( e . g . , about 15° C . to about thermogram as depicted in FIG . 15 . In certain embodiments . 35° C . ) to yield solid precipitation , and collecting the solid . 40 the crystalline form exhibits a TGA thermogram comprising In certain embodiments , provided herein are methods for a total mass loss of approximately 12 . 8 % of the total mass making Form 3 of Compound 1 , comprising obtaining a of the sample between approximately 35º C . and approxi slurry of Form 1 of Compound 1 in a 1 : 1 solution of acetone mately 190° C . when heated from approximately 25° C . to and isopropanol , heating the slurry to about 60° C . , filtering approximately 300° C . Thus , in certain embodiments , the the slurry to yield a solution , cooling down the solution to 45 crystalline form loses about 12 . 8 % of its total mass when about 25° C . to yield solid precipitation , and collecting the heated from about ambient temperature to about 300° C . In solid . certain embodiments , the crystalline form contains 0 . 5 molar

In certain embodiments , provided herein are methods for equivalents of solvent in the crystal lattice corresponding to making Form 3 of Compound 1 , comprising obtaining a approximately 0 . 5 mole of 2 , 2 , 2 - trifluoroethanol per mole of saturated solution of Form 1 of Compound 1 in a solvent , 50 Compound 1 . The theoretical 2 , 2 , 2 - trifluoroethanol content diffusing an anti - solvent into the saturated solution , collect - of a 2 , 2 , 2 - trifluoroethanol hemi - solvate of Compound 1 is ing precipitated solid if there is precipitation , and evaporat 11 . 5 % by weight , matching the TGA weight loss observed . ing the solvent to collect solid if there is no precipitation . In In certain embodiments , the crystalline form is a 2 , 2 , 2 certain embodiments , provided herein are methods for mak - trifluoroethanol hemi - solvate of Compound 1 . ing Form 3 of Compound 1 , comprising obtaining a satu - 55 In one embodiment , provided herein is a crystalline form rated solution of Form 1 of Compound 1 in TFE , diffusing of Compound 1 having a single differential thermal analysis cyclohexane into the saturated solution , collecting precipi - ( SDTA ) thermogram as depicted in FIG . 14 comprising an tated solid if there is precipitation , and evaporating the endothermic event between about 110° C . and about 175° C . solvent to collect solid if there is no precipitation . with a maximum at about 149° C . when heated from

In certain embodiments , provided herein are methods for 60 approximately 25° C . to approximately 300° C . ( see Table making Form 3 of Compound 1 , comprising obtaining 24 ) . amorphous form of Compound 1 , diffusing a solvent on to In one embodiment , provided herein is a crystalline form the amorphous form of Compound 1 for a period of time of Compound 1 having a SDTA thermogram comprising an ( e . g . , about 1 week to about 1 month ) , and collecting the endothermic event as depicted in FIG . 14 between about solid . In certain embodiments , provided herein are methods 65 225º C . and about 275° C . with a maximum at about 254° for making Form 3 of Compound 1 , comprising obtaining C . when heated from approximately 25° C . to approximately amorphous form of Compound 1 by grinding Form 1 of 300° C . ( see Table 24 ) .

23 US 9 , 981 , 971 B2

24 In still another embodiment , Form 3 of Compound 1 is 18 . 1 , 20 . 62 , 21 . 94 , 22 . 66 , 24 . 34 or 26 . 26 degrees . In another

substantially pure . In certain embodiments , the substantially embodiment , Form 4 of Compound 1 has one , two , three or pure Form 3 of Compound 1 is substantially free of other four characteristic X - ray powder diffraction peaks at a solid forms , e . g . , amorphous form . In certain embodiments , two - theta angle of approximately 10 . 14 , 10 . 66 , 21 . 94 or the purity of the substantially pure Form 3 of Compound 1 5 26 . 26 degrees . In another embodiment , Form 4 of Com is no less than about 95 % pure , no less than about 96 % pure , pound 1 has one , two , three , four , five , six , seven , eight , no less than about 97 % pure , no less than about 98 % pure , nine , ten , eleven or twelve characteristic X - ray powder no less than about 98 . 5 % pure , no less than about 99 % pure , diffraction peaks as set forth in Table 28 . no less than about 99 . 5 % pure , or no less than about 99 . 8 % In one embodiment , Form 4 of Compound 1 as wet solid pure . 10 has a digital image substantially as shown in FIG . 18A . In

5 . 3 . 4 Form 4 of Compound 1 one embodiment , Form 4 of Compound 1 as dry solid has a In certain embodiments , provided herein is Form 4 of digital image substantially as shown in FIG . 18B .

Compound 1 . In one embodiment , provided herein is a crystalline form In certain embodiments , Form 4 is obtained by crystalli - of Compound 1 having a thermogravimetric ( TGA ) thermo

zation from certain solvent systems , for example , solvent 15 graph corresponding substantially to the representative TGA systems comprising one or more of the following solvents or thermogram as depicted in FIG . 20 . In certain embodiments , solvent combinations : dimethylsulfoxide , water and toluene . the crystalline form exhibits a TGA thermogram comprising In certain embodiments , Form 4 is obtained by anti - solvent a total mass loss of approximately 16 . 4 % of the total mass crystallization and vapor diffusion into liquid crystallization of the sample between approximately 35° C . and approxi

In certain embodiments , provided herein are methods for 20 mately 180° C . when heated from approximately 25° C . to making Form 4 of Compound 1 , comprising dissolving approximately 300° C . Thus , in certain embodiments , the Form 1 of Compound 1 in a solvent , adding an anti - solvent , crystalline form loses about 16 . 4 % of its total mass when collecting solid from the solution by filtration , and option heated from about ambient temperature to about 300° C . In ally washing ( e . g . , washing with the mixture of solvent and certain embodiments , the crystalline form contains 0 . 8 molar anti - solvent at the same ratio of the solution ) and drying . In 25 equivalents of solvent in the crystal lattice corresponding to certain embodiments , provided herein are methods for mak - approximately 0 . 8 mole of dimethylsulfoxide per mole of ing Form 4 of Compound 1 , comprising dissolving Form 1 Compound 1 . The theoretical dimethylsulfoxide content of a of Compound 1 in dimethylsulfoxide , adding water , collect - 0 . 8 molar equivalent dimethylsulfoxide solvate of Com ing solid from the solution by filtration , and optionally pound 1 is 18 . 9 % by weight , matching the TGA weight loss washing with the mixture of dimethylsulfoxide and water at 30 observed . In certain embodiments , the crystalline form is a the same ratio of the solution and drying . In certain embodi - 0 . 8 molar equivalent dimethylsulfoxide solvate of Com ments , provided herein are methods for making Form 4 of pound 1 . Compound 1 , comprising dissolving Form 1 of Compound In one embodiment , provided herein is a crystalline form 1 in dimethylsulfoxide , adding toluene , collecting solid from of Compound 1 having a SDTA thermogram as depicted in the solution by filtration , and optionally washing with the 35 FIG . 19 comprising an endothermic event between about mixture of dimethylsulfoxide and toluene at the same ratio 100° C . and about 175° C . with a maximum at about 139° of the solution and drying . C . when heated from approximately 25° C . to approximately

In certain embodiments , provided herein are methods for 300° C . ( see Table 24 ) . making Form 4 of Compound 1 , comprising obtaining a In one embodiment , provided herein is a crystalline form saturated solution of Form 1 of Compound 1 in a solvent , 40 of Compound 1 having a SDTA thermogram as depicted in diffusing an anti - solvent into the saturated solution , collect - FIG . 19 comprising an endothermic event between about ing precipitated solid if there is precipitation , and evaporat - 235° C . and about 275º C . with a maximum at about 258° ing the solvent to collect solid if there is no precipitation . In C . when heated from approximately 25° C . to approximately certain embodiments , provided herein are methods for mak - 300° C . ( see Table 24 ) . ing Form 4 of Compound 1 , comprising obtaining a satu - 45 In still another embodiment , Form 4 of Compound 1 is rated solution of Form 1 of Compound 1 in DMSO , diffusing substantially pure . In certain embodiments , the substantially water into the saturated solution , collecting precipitated pure Form 4 of Compound 1 is substantially free of other solid if there is precipitation , and evaporating the solvent to solid forms , e . g . , amorphous form . In certain embodiments , collect solid if there is no precipitation . the purity of the substantially pure Form 4 of Compound 1

In one embodiment , Form 4 is a dimethylsulfoxide sol - 50 is no less than about 95 % pure , no less than about 96 % pure , vated form of Compound 1 . In one embodiment , Form 4 is no less than about 97 % pure , no less than about 98 % pure , a 0 . 8 molar equivalent dimethylsulfoxide solvated form of no less than about 98 . 5 % pure , no less than about 99 % pure , Compound 1 . In another embodiment , Form 4 of Compound no less than about 99 . 5 % pure , or no less than about 99 . 8 % 1 is crystalline . pure .

In certain embodiments , a solid form provided herein , 55 5 . 3 . 5 Form 5 of Compound 1 e . g . , Form 4 , is substantially crystalline , as indicated by , e . g . , In certain embodiments , provided herein is Form 5 of X - ray powder diffraction measurements . In one embodi - Compound 1 . ment , Form 4 of Compound 1 has an X - ray powder diffrac In certain embodiments , Form 5 is obtained by crystalli tion pattern substantially as shown in FIG . 17 ( middle zation from certain solvent systems , for example , solvent pattern ) . In one embodiment , Form 4 of Compound 1 has 60 systems comprising one or more of the following solvents or one or more characteristic X - ray powder diffraction peaks at solvent combinations : THF , water , 1 , 4 - dioxane , methanol a two - theta angle of approximately 8 . 22 , 10 . 14 , 10 . 66 , and ethanol . In certain embodiments , Form 5 is obtained by 14 . 02 , 18 . 1 , 20 . 62 , 21 . 94 , 22 . 66 , 23 . 78 , 24 . 34 , 25 . 42 or hot - filtration crystallization , anti - solvent crystallization or 26 . 26 degrees as depicted in FIG . 17 . In a specific embodi - evaporative crystallization . ment , Form 4 of Compound 1 has one , two , three , four , five , 65 In certain embodiments , provided herein are methods for six , seven or eight characteristic X - ray powder diffraction making Form 5 of Compound 1 , comprising obtaining a peaks at a two - theta angle of approximately 10 . 14 , 10 . 66 , slurry of Form 1 of Compound 1 in a solvent , heating the

US 9 , 981 , 971 B2 25 26

slurry to a temperature ( e . g . , about 50° C . to about 70° C . ) X - ray powder diffraction measurements . In one embodi for a period of time ( e . g . , about 10 minutes to about 2 hours ) , ment , Form 5 of Compound 1 has an X - ray powder diffrac filtering the slurry to yield a solution , cooling down the tion pattern substantially as shown in FIG . 22 ( middle solution to a temperature ( e . g . , about 10° C . to about 35° C . ) , pattern ) . In one embodiment , Form 5 of Compound 1 has collecting solid from the solution by filtration , and option - 5 one or more characteristic X - ray powder diffraction peaks at ally washing ( e . g . , washing with the solvent ) and drying . In a two - theta angle of approximately 6 . 02 , 7 . 46 , 9 . 26 , 11 . 7 , certain embodiments , provided herein are methods for mak - 12 . 18 , 19 . 78 , 22 . 02 , 23 . 74 , 24 . 26 , 24 . 94 , 26 . 18 , 27 . 06 or ing Form 5 of Compound 1 , comprising obtaining a slurry 29 . 86 degrees as depicted in FIG . 22 . In a specific embodi of Form 1 of Compound 1 in a solvent mixture of THF and ment , Form 5 of Compound 1 has one , two , three , four , five , water ( 50 / 50 ) , heating the slurry at about 60° C . for about 10 six , seven or eight characteristic X - ray powder diffraction one hour , filtering the slurry to yield a solution , cooling peaks at a two - theta angle of approximately 7 . 46 , 9 . 26 , 11 . 7 , down the solution to about 25° C . , collecting solid from the 22 . 02 , 23 . 74 , 24 . 26 , 24 . 94 or 26 . 18 degrees . In another solution by filtration , and optionally washing with the sol - embodiment , Form 5 of Compound 1 has one , two , three or vent mixture of THF and water ( 50 / 50 ) and drying . In certain four characteristic X - ray powder diffraction peaks at a embodiments , provided herein are methods for making 15 two - theta angle of approximately 9 . 26 , 11 . 7 , 24 . 94 or 26 . 18 Form 5 of Compound 1 , comprising obtaining a slurry of degrees . In another embodiment , Form 5 of Compound 1 has Form 1 of Compound 1 in a solvent mixture of methanol and one , two , three , four , five , six , seven , eight , nine , ten , eleven , water ( 50 / 50 ) , heating the slurry at about 60° C . for about twelve or thirteen characteristic X - ray powder diffraction one hour , filtering the slurry to yield a solution , cooling peaks as set forth in Table 29 . down the solution to about 25° C . , collecting solid from the 20 In one embodiment , Form 5 of Compound 1 has a digital solution by filtration , and optionally washing with the sol image substantially as shown in FIG . 23A . vent mixture of methanol and water ( 50 / 50 ) and drying . In In one embodiment , provided herein is a crystalline form certain embodiments , provided herein are methods for mak of Compound 1 having a thermogravimetric ( TGA ) thermo ing Form 5 of Compound 1 , comprising obtaining a slurry graph corresponding substantially to the representative TGA of Form 1 of Compound 1 in a solvent mixture of 1 , 4 - 25 thermogram as depicted in FIG . 25 . In certain embodiments , dioxane and water ( 50 / 50 ) , heating the slurry at about 60° C . the crystalline form exhibits a TGA thermogram comprising for about one hour , filtering the slurry to yield a solution , a total mass loss of approximately 9 . 4 % of the total mass of cooling down the solution to about 25° C . , collecting solid the sample between approximately 35° C . and approxi from the solution by filtration , and optionally washing with mately 240° C . when heated from approximately 25° C . to the solvent mixture of 1 , 4 - dioxane and water ( 50 / 50 ) and 30 approximately 300° C . Thus , in certain embodiments , the drying . In certain embodiments , provided herein are meth - crystalline form loses about 9 . 4 % of its total mass when ods for making Form 5 of Compound 1 , comprising obtain - heated from about ambient temperature to about 300° C . In ing a slurry of Form 1 of Compound 1 in a solvent mixture certain embodiments , the crystalline form contains 2 molar of ethanol and water ( 50 / 50 ) , heating the slurry at about 60° equivalents of solvent in the crystal lattice corresponding to C . for about one hour , filtering the slurry to yield a solution , 35 approximately 2 moles of water per mole of Compound 1 . cooling down the solution to about 25° C . , collecting solid The theoretical water content of a dihydrate of Compound 1 from the solution by filtration , and optionally washing with is 10 . 2 % by weight , matching the TGA weight loss solvent mixture of ethanol and water ( 50 / 50 ) and drying . observed . In certain embodiments , the crystalline form is a

In certain embodiments , provided herein are methods for dihydrated form of Compound 1 . making Form 5 of Compound 1 , comprising obtaining a 40 In one embodiment , provided herein is a crystalline form saturated solution of Form 1 of Compound 1 in a solvent of Compound 1 having a SDTA thermogram as depicted in diffusing an anti - solvent into the saturated solution , collect - FIG . 24 comprising an endothermic event between about ing precipitated solid if there is precipitation , and evaporat - 50° C . and about 140° C . with a maximum at about 80° C . ing the solvent to collect solid if there is no precipitation . In when heated from approximately 25° C . to approximately certain embodiments , provided herein are methods for mak - 45 300° C . ( see Table 24 ) . ing Form 5 of Compound 1 , comprising obtaining a satu - In one embodiment , provided herein is a crystalline form rated solution of Form 1 of Compound 1 in THF , diffusing of Compound 1 having a SDTA thermogram as depicted in water into the saturated solution , collecting precipitated FIG . 24 comprising an exothermic event between about solid if there is precipitation , and evaporating the solvent to 160° C . and about 200° C . with a maximum at about 181° collect solid if there is no precipitation . 50 C . when heated from approximately 25° C . to approximately

In certain embodiments , provided herein are methods for 300° C . ( see Table 24 ) . making Form 5 of Compound 1 , comprising mixing Form 5 In one embodiment , provided herein is a crystalline form of Compound 1 with a solvent , filtering the mixture to yield of Compound 1 having a SDTA thermogram as depicted in a solution if Form 1 does not dissolve completely , and FIG . 24 comprising an endothermic event between about evaporating the solution under certain air pressure to yield 55 225° C . and about 275° C . with a maximum at about 251° solid . In certain embodiments , provided herein are methods C . when heated from approximately 25° C . to approximately for making Form 5 of Compound 1 , comprising mixing 300° C . ( see Table 24 ) . Form 1 of Compound 1 with THF / water ( 50 : 50 ) , filtering the In still another embodiment , Form 5 of Compound 1 is mixture to yield a solution if Form 1 does not dissolve substantially pure . In certain embodiments , the substantially completely , and evaporating the solution under 200 mbar air 60 pure Form 5 of Compound 1 is substantially free of other pressure to yield solid . solid forms , e . g . , amorphous form . In certain embodiments ,

In one embodiment , Form 5 is a hydrated form of Com - the purity of the substantially pure Form 5 of Compound 1 pound 1 . In one embodiment , Form 5 is a dihydrated form is no less than about 95 % pure , no less than about 96 % pure , of Compound 1 . In another embodiment , Form 5 of Com - no less than about 97 % pure , no less than about 98 % pure , pound 1 is crystalline . 65 no less than about 98 . 5 % pure , no less than about 99 % pure ,

In certain embodiments , a solid form provided herein , no less than about 99 . 5 % pure , or no less than about 99 . 8 % e . g . , Form 5 , is substantially crystalline , as indicated by , e . g . , pure .

27 US 9 , 981 , 971 B2

28 5 . 3 . 6 Amorphous Compound 1 The solid forms provided herein are for use as a medica

ment . In certain embodiments , provided herein is amorphous Provided herein are methods for treating or preventing a Compound 1 . cancer , comprising administering a solid form of Compound In certain embodiments , provided herein are methods for 5 1 provided herein or a pharmaceutical composition thereof

making amorphous Compound 1 , comprising 1 ) equilibrat - to a patient having a cancer . ing the temperature of a sample of one of the solid forms of The solid forms provided herein are for use in a method Compound 1 provided herein at room temperature ; 2 ) heat for treating or preventing cancer , an inflammatory condition , ing the sample to a first temperature at a first rate ; 3 ) holding an immunological condition , a neurodegenerative disease , the sample isothermally for a period of time ; 4 ) cooling the 10 diabetes , obesity , a neurological disorder , an age - related

disease , a cardiovascular condition , or a conditions treatable sample to a second temperature at a second rate ; 5 ) heating or preventable by inhibition of a kinase pathway . The the sample to a third temperature at about a third rate ; and method comprises administering an effective amount of a 6 ) collecting remaining solids . In one embodiment , the crystal form to a subject in need thereof . In one embodiment , sample is Form 1 of Compound 1 . In one embodiment , the the kinase pathway is the TOR kinase pathway . first temperature is higher than the melting point of one of 15 In some embodiments , the cancer is an advanced unre the solid forms of Compound 1 provided herein . In one spectable solid tumor , or a hematologic malignancy . For embodiment , the second temperature is lower than room example , the hematologic malignancy is CLL , NHL , or temperature . In another embodiment , the third temperature MM . In some such embodiments , the cancer has progressed is higher than the glass transition temperature of the amor on standard anti - cancer therapy , or the patient is not able to

20 tolerate standard anti - cancer therapy . In yet others , the phous solid form of Compound 1 provided herein . In another cancer is a cancer for which no approved therapy exists . In embodiment , the first and third rates are about 10° C . / min some embodiments , the cancer is resistant to standard and the second rate is about 30° C . / min , independently from therapy . In another , the patient has relapsed after standard each other . In one embodiment , the period of time at which therapy . In one embodiment , the cancer is a neoplasm the sample is held isothermally is about 5 minutes . 25 metastasis .

In certain embodiments , provided herein are methods for In certain embodiments , the cancer is a blood borne tumor . making amorphous Compound 1 , comprising 1 ) equilibrat In certain embodiments , the cancer is a lymphoma , a ing the temperature of a sample of Form 1 at about 25° C . ; leukemia or a multiple myeloma . 2 ) heating the sample to about 275º C . at a rate of about 10° In certain embodiments , the cancer is non - Hodgkin ' s

C . / min ; 3 ) holding the sample isothermally for about 5 30 lymphoma . In certain embodiments , the non - Hodgkin ' s minutes ; 4 ) cooling the sample to about - 10° C . at a rate of lymphoma is diffuse large B - cell lymphoma ( DLBCL ) , about 30° C . / min ; 5 ) heating the sample to about 150° C . at follicular lymphoma ( FL ) , acute myeloid leukemia ( AML ) , about 10° C . at a rate of about 10° C . / min ; and 6 ) collecting mantle cell lymphoma ( MCL ) , or ALK + anaplastic large cell remaining solids . lymphoma . In one embodiment , the non - Hodgkin ' s lym

35 phoma is advanced solid non - Hodgkin ' s lymphoma . In one In one embodiment , amorphous Compound 1 has a glass embodiment , the non - Hodgkin ' s lymphoma is diffuse large

transition temperature ( Tg ) at about 120° C . B - cell lymphoma ( DLBCL ) . In one embodiment , amorphous Compound 1 has an In certain embodiments , the cancer is a B - cell lymphoma .

X - ray powder diffraction pattern substantially as shown in In certain embodiments , the B - cell lymphoma is a B - cell FIG . 28 . 40 non - Hodgkin ' s lymphoma selected from diffuse large B - cell

lymphoma , Burkitt ' s lymphoma / leukemia , mantle cell lym In one embodiment , provided herein is an amorphous phoma , mediastinal ( thymic ) large B - cell lymphoma , folli solid form of Compound 1 having a DSC thermogram as cular lymphoma , marginal zone lymphoma ( including depicted in FIG . 32 comprising an endothermic event extranodal marginal zone B - cell lymphoma and nodal mar between about 160° C . and about 200° C . with a maximum 45 ginal zone B - cell lymphoma ) , lymphoplamacytic lym at about 188 . 1° C . when heated from approximately 25° C . phoma / Waldenstrom macroglobulinemia . In some embodi to approximately 300° C . ( see FIG . 32 ) . ments , the B - cell lymphoma is chronic lymphocytic

In still another embodiment , amorphous Compound 1 is leukemia / small lymphocytic lymphoma ( CLL / SLL ) . In one substantially pure . In certain embodiments , the substantially embodiment , the B - cell lymphoma is Waldenstrom macro pure amorphous Compound 1 is substantially free of other 50 globulinemia . solid forms , e . g . , Form 1 . Form 2 , Form 3 , Form 4 or Form In one embodiment , the B - cell non - Hodgkin ' s lymphoma 5 . In certain embodiments , the purity of the substantially is refractory B - cell non - Hodgkin ' s lymphoma . In one pure amorphous Compound 1 is no less than about 95 % pure amorphous Compound 1 is no less than about 95 % embodiment , the B - cell non - Hodgkin ' s lymphoma is pure , no less than about 96 % pure , no less than about 97 % relapsed B - cell non - Hodgkin ' s lymphoma . pure , no less than about 98 % pure , no less than about 98 . 5 % 55 In certain embodiments , the cancer is a T - cell lymphoma . pure , no less than about 99 % pure , no less than about 99 . 5 % The B - cell disorders chronic lymphocytic leukemia / small pure , or no less than about 99 . 8 % pure . lymphocytic lymphoma ( CLL / SLL ) represent 2 ends of a

spectrum of the same disease process differing in the degree of blood / marrow involvement ( CLL ) versus lymph node

5 . 4 Methods of Use 60 involvement ( SLL ) . In another embodiment , the cancer is CLL characterized

The solid forms provided herein or combinations of the by deletion of chromosome 11922 , loss of ATM expression , various solid forms provided herein can be used in the mutation of IgVH , wild type IgVH , wild type p53 / ATM , methods provided herein . The solid forms provided herein or mutation of p53 or dysfunctional p53 . combinations of the various solid forms provided herein can 65 In other embodiments , the cancer is a multiple myeloma . be used in the treatment of all diseases , disorders or condi In certain embodiments , the cancer is a cancer of the head , tions provided herein . neck , eye , mouth , throat , esophagus , bronchus , larynx , phar

US 9 , 981 , 971 B2 29 30

ynx , chest , bone , lung , colon , rectum , stomach , prostate , In another embodiment , the cancer is E - twenty six ( ETS ) urinary bladder , uterine , cervix , breast , ovaries , testicles or overexpressing Ewings sarcoma . other reproductive organs , skin , thyroid , blood , lymph In another embodiment , the cancer is head and neck nodes , kidney , liver , pancreas , and brain or central nervous squamous cell carcinoma ( HNSCC ) characterized by dele system . 5 tion of chromosome 11922 or loss of ataxia telangiectasia

In other embodiments , the cancer is a solid tumor . In mutated ( ATM ) expression . certain embodiments , the solid tumor is a relapsed or refrac In another embodiment , the cancer is glioblastoma mul tory solid tumor . tiforme ( GBM ) characterized by 06 - methylguanine - DNA

In other embodiments , the solid tumor can be an advanced methyltransferase ( MGMT ) methylation . * 10 In other embodiments , the cancer is a cancer associated solid tumor . with the pathways involving mTOR , PI3K , or Akt kinases In other embodiments , the solid tumor can be a neuroen and mutants or isoforms thereof . Other cancers within the docrine tumor , glioblastoma multiforme ( GBM ) , hepatocel scope of the methods provided herein include those associ

lular carcinoma ( HCC ) , breast cancer , colorectal cancer ated with the pathways of the following kinases : P13Ka . ( CRC ) , salivary cancer , pancreatic cancer , adenocystic can 15 PI3KB , P1318 , KDR , GSK30 , GSK3B , ATM , ATX , ATR , cer , adrenal cancer , esophageal cancer , renal cancer , leio CFMS , and / or DNA - PK kinases and mutants or isoforms myosarcoma , paraganglioma , head and neck squamous cell thereof . In some embodiments , the cancers associated with carcinoma , E - twenty six ( ETS ) overexpressing castration mTOR / PI3K / Akt pathways include solid and blood - borne resistant prostate cancer or E - twenty six ( ETS ) overexpress - tumors , for example , multiple myeloma , mantle cell lym ing Ewings sarcoma . 20 phoma , diffused large B - cell lymphoma , acute myeloid

In one embodiment , the solid tumor is a neuroendocrine lymphoma , follicular lymphoma , chronic lymphocytic leu tumor . In certain embodiments , the neuroendocrine tumor is kemia ; and solid tumors , for example , breast , lung , endo a neuroendocrine tumor of gut origin . In certain embodi - metrial , ovarian , gastric , cervical , and prostate cancer ; glio ments , the neuroendocrine tumor is of non - pancreatic origin . blastoma ; renal carcinoma ; hepatocellular carcinoma ; colon In certain embodiments , the neuroendocrine tumor is non - 25 carcinoma ; neuroendocrine tumors ; head and neck tumors ; pancreatic of gut origin . In certain embodiments , the neu - and sarcomas , such as Ewing ' s sarcoma . roendocrine tumor is of unknown primary origin . In certain In certain embodiments , provided herein are methods for embodiments , the neuroendocrine tumor is a symptomatic achieving a Response Evaluation Criteria in Solid Tumors endocrine producing tumor or a nonfunctional tumor . In ( for example , RECIST 1 . 1 ) of complete response , partial certain embodiments , the neuroendocrine tumor is locally 30 response or stable disease in a patient having a solid tumor , unrespectable , metastatic moderate , well differentiated , low comprising administering a solid form of Compound 1 ( grade 1 ) or intermediate ( grade 2 ) . In certain embodiments , provided herein or a pharmaceutical composition thereof to the neuroendocrine tumor is of non - gut origin . In one said patient . In certain embodiments , provided herein are embodiment , the neuroendocrine tumor of non - gut origin , is methods for achieving a National Cancer Institute - Spon rapamycin resistant . In one embodiment , the neuroendocrine 35 sored Working Group on Chronic Lymphocytic Leukemia tumor of non - gut origin is a bronchial neuroendocrine ( NCI - WG CLL ) of complete response , partial response or tumor , or a neuroendocrine tumor with origin in an organ stable disease in a patient having leukemia , comprising above the diaphragm , for example , a laryngeal neuroendo administering a solid form of Compound 1 provided herein crine tumor , a pharyngeal neuroendocrine tumor , or a thy - or a pharmaceutical composition thereof to said patient . In roid neuroendocrine tumor . In one embodiment , the neu - 40 certain embodiments , provided herein are methods for roendocrine tumor of non - gut origin is a symptomatic achieving a Prostate Cancer Working Group 2 ( PCWG2 ) endocrine producing tumor or a nonfunctional tumor . In one Criteria of complete response , partial response or stable embodiment , the neuroendocrine tumor of non - gut origin is disease in a patient having prostate cancer , comprising locally unrespectable , metastatic moderate , well differenti administering a solid form of Compound 1 provided herein ated , low ( grade 1 ) or intermediate ( grade 2 ) . 45 or a pharmaceutical composition thereof to said patient . In

In one embodiment , the solid tumor is non - small cell lung certain embodiments , provided herein are methods for cancer ( NSCLC ) . achieving an International Workshop Criteria ( IWC ) for

In another embodiments the solid tumor is glioblastoma non - Hodgkin ' s lymphoma of complete response , partial multiforme ( GBM ) . response or stable disease in a patient having non - Hodgkin ' s

In another embodiment , the solid tumor is hepatocellular 50 lymphoma , comprising administering a solid form of Com carcinoma ( HCC ) . pound 1 provided herein or a pharmaceutical composition

In another embodiment , the solid tumor is breast cancer . thereof to said patient . In certain embodiments , provided In one embodiment , the breast cancer is hormone receptor herein are methods for achieving an International Uniform positive . In one embodiment , the breast cancer is estrogen Response Criteria ( IURC ) for multiple myeloma of com receptor positive ( ER + , ER + / Her2 or ER + / Her2 + ) . In one 55 plete response , partial response or stable disease in a patient embodiment , the breast cancer is estrogen receptor negative having multiple myeloma , comprising administering a solid ( ER - / Her2 + ) . In one embodiment , the breast cancer is triple form of Compound 1 provided herein or a pharmaceutical negative ( TN ) ( breast cancer that does not express the genes composition thereof to said patient . In certain embodiments , and / or protein corresponding to the estrogen receptor ( ER ) , provided herein are methods for achieving a Responses progesterone receptor ( PR ) , and that does not overexpress 60 Assessment for Neuro - Oncology ( RANO ) Working Group the Her2 / neu protein ) . for glioblastoma multiforme of complete response , partial

In one embodiment , the solid tumor is an advanced solid response or stable disease in a patient having glioblastoma tumor . multiforme , comprising administering a solid form of Com

In another embodiment , the cancer is head and neck pound 1 provided herein or a pharmaceutical composition squamous cell carcinoma . 65 thereof to said patient .

In another embodiment , the cancer is E - twenty six ( ETS ) In certain embodiments , the solid forms provided herein overexpressing castration - resistant prostate cancer . are for use in a method for achieving a Response Evaluation

da

US 9 , 981 , 971 B2 32

Criteria in Solid Tumors ( RECIST 1 . 1 ) of complete Form 5 and amorphous form of Compound 1 and one or response , partial response or stable disease in a subject . The more pharmaceutically acceptable excipients or carriers . methods comprise administering an effective amount of a In one embodiment , the pharmaceutical compositions solid form to a subject having a solid tumor . provided herein comprise tautomers of one or more solid

In certain embodiments , the solid forms provided herein 5 forms of Compound 1 and one or more pharmaceutically are for use in a method for improving International Work - acceptable excipients or carriers . shop Criteria ( IWC ) for NHL , International Uniform In one embodiment , the pharmaceutically acceptable Response Criteria for Multiple Myeloma ( IURC ) , Eastern excipients and carriers are selected from binders , diluents , Cooperative Oncology Group Performance Status ( ECOG ) disintegrants and lubricants . In another embodiment , the or Response Assessment for Neuro - Oncology ( RANO ) 10 pharmaceutically acceptable excipients and carriers further Working Group for GBM . The method comprises adminis include one or more antioxidants ( e . g . EDTA or BHT ) . tering an effective amount of a solid form to a subject in need In certain embodiments , the binders include , but are not thereof . limited to , cellulose ( e . g . , microcrystalline cellulose , such as In certain embodiments , provided herein are methods for AVICEL® PH 101 , AVICEL® PH112 , and AVICEL® PH increasing survival without disease progression of a patient 15 having a cancer , comprising administering a solid form of 102 ) and starch ( e . g . , pregelatinized starch ( STARCH Compound 1 provided herein or a pharmaceutical compo 1500® ) ) . In one embodiment , the binder is cellulose . In sition thereof to said patient . another embodiment , the binder is microcrystalline cellu

In certain embodiments , provided herein are methods for lose . In yet another embodiment , the binder is AVICEL® PH treating a cancer , the methods comprising administering a 20 101 . In yet another embodiment , the binder is AVICEL® PH solid form of Compound 1 provided herein or a pharma 102 . In yet another embodiment , the binder is starch . In yet ceutical composition thereof to a patient having a cancer , another embodiment , the binder is pregelatinized starch . In wherein the treatment results in prevention or retarding of still another embodiment , the binder is STARCH 1500? . clinical progression , such as cancer - related cachexia or In certain embodiments , the diluents include , but are not increased pain . 25 limited to , lactose ( e . g . , lactose monohydrate ( FAST FLOR

In some embodiments , provided herein are methods for 316 ) and lactose anhydrous ) , cellulose ( e . g . , microcrystal treating a cancer , the methods comprising administering a line cellulose , such as AVICEL® PH 101 and AVICEL® PH solid form of Compound 1 provided herein or a pharma - 102 ) , and mannitol . In one embodiment , the diluent is ceutical composition thereof to a patient having a cancer , lactose . In another embodiment , the diluent is lactose mono wherein the treatment results in one or more of inhibition of 30 hydrate . In yet another embodiment , the diluent is FAST disease progression , increased Time To Progression ( TTP ) , FLO? 316 . In yet another embodiment , the diluent is lactose increased Progression Free Survival ( PFS ) , and / or increased anhydrous . In yet another embodiment , the diluent is cellu Overall Survival ( OS ) , among others . lose . In yet another embodiment , the diluent is microcrys

talline cellulose . In yet another embodiment , the diluent is 5 . 5 Pharmaceutical Compositions 35 AVICEL® PH 101 . In still another embodiment , the diluent

is AVICEL® PH 102 ) . Solid forms of Compound 1 provided herein are useful for In certain embodiments , the disintegrants include , but are

the preparation of pharmaceutical compositions , comprising not limited to , starch ( e . g . , corn starch ) and carboxymethyl an effective amount of a solid form of Compound 1 and a cellulose ( e . g . , croscarmellose sodium , such as AC - DI pharmaceutically acceptable carrier or vehicle . In some 40 SOL® ) , and sodium starch glycolate . In one embodiment , embodiments , the pharmaceutical compositions described the disintegrant is starch . In another embodiment , the dis herein are suitable for oral , parenteral , mucosal , transdermal integrant is corn starch . In yet another embodiment , the or topical administration . disintegrant is carboxymethyl cellulose . In yet another

In one embodiment , the pharmaceutical compositions embodiment , the disintegrant is croscarmellose sodium . In provided herein comprise a solid form of Compound 1 and 45 still another embodiment , the disintegrant is AC - DI - SOL® . one or more pharmaceutically acceptable excipients or car - In certain embodiments , the lubricants include , but are not riers . In one embodiment , the pharmaceutical compositions limited to , starch ( e . g . , corn starch ) , magnesium stearate , and provided herein comprise Form 1 of Compound 1 and one stearic acid . In one embodiment , the lubricant is starch . In or more pharmaceutically acceptable excipients or carriers . another embodiment , the lubricant is corn starch . In yet In one embodiment , the pharmaceutical compositions pro - 50 another embodiment , the lubricant is magnesium stearate . In vided herein comprise Form 2 of Compound 1 and one or still another embodiment , the lubricant is stearic acid . more pharmaceutically acceptable excipients or carriers . In In another embodiment , the pharmaceutical compositions one embodiment , the pharmaceutical compositions provided provided herein comprise a solid form of Compound 1 and herein comprise Form 3 of Compound 1 and one or more one or more pharmaceutically acceptable excipients or car pharmaceutically acceptable excipients or carriers . In one 55 riers , each independently selected from carboxymethyl cel embodiment , the pharmaceutical compositions provided lulose , cellulose , lactose , magnesium stearate , starch , and herein comprise Form 4 of Compound 1 and one or more stearic acid . pharmaceutically acceptable excipients or carriers . In one In one embodiment , the pharmaceutical compositions embodiment , the pharmaceutical compositions provided provided herein comprise about 2 . 5 - 10 % by weight of a herein comprise Form 5 of Compound 1 and one or more 60 solid form of Compound 1 , about 70 - 90 % by weight of pharmaceutically acceptable excipients or carriers . In one diluent ( s ) / binder ( s ) , about 1 - 5 % by weight of embodiment , the pharmaceutical compositions provided disintegrant ( s ) , and about 0 . 1 - 2 % by weight of lubricant ( s ) . herein comprise amorphous Compound 1 and one or more In another embodiment , the pharmaceutical compositions pharmaceutically acceptable excipients or carriers . In one provided herein comprise about 10 % by weight of a solid embodiment , the pharmaceutical compositions provided 65 form of Compound 1 , about 59 . 85 % by weight of mannitol , herein comprise one or more of the following solid forms or about 25 % by weight of microcrystalline cellulose , about solid form combinations : Form 1 , Form 2 , Form 3 , Form 4 , 3 % by weight of sodium starch glycolate , about 1 % by

US 9 , 981 , 971 B2 33 34

weight of silicon dioxide , about 0 . 5 % by weight of stearic about 250 mg , about 280 mg , about 300 mg , about 350 mg , acid , and about 0 . 65 % by weight of magnesium stearate . about 400 mg , about 500 mg , about 560 mg , about 600 mg ,

In another embodiment , the pharmaceutical compositions about 700 mg , about 750 mg , about 800 mg , about 1000 mg provided herein comprise about 10 % by weight of a solid or about 1400 mg of a solid form of Compound 1 . In a form of Compound 1 , about 59 . 45 % by weight of mannitol , 5 particular embodiment , provided herein are unit dosage about 25 % by weight of microcrystalline cellulose , about formulations that comprise about 2 . 5 mg , about 5 mg , about 3 % by weight of sodium starch glycolate , about 1 % by 7 . 5 mg , about 8 mg , about 10 mg , about 15 mg , about 20 mg , weight of silicon dioxide , about 0 . 5 % by weight of stearic about 25 mg , about 30 mg , about 40 mg , about 45 mg , about acid , about 0 . 4 % BHT , and about 0 . 65 % by weight of 50 mg , about 60 mg or about 100 mg of a solid form of magnesium stearate . 10 Compound 1 or a tautomer thereof . In a particular embodi In another embodiment , the pharmaceutical compositions ment , provided herein are unit dosage formulations that provided herein comprise about 10 % by weight of a solid form of Compound 1 , about 59 . 35 % by weight of mannitol , comprise about 1 mg , about 2 mg , about 5 mg , about 7 . 5 mg about 25 % by weight of microcrystalline cellulose , about and about 10 mg . 3 % by weight of sodium starch glycolate , about 1 % by 15 5 In some embodiments , a unit dosage form comprising weight of silicon dioxide , about 0 . 5 % by weight of stearic Compound 1 , or a tautomer thereof can be administered acid , about 0 . 5 % disodium EDTA , and about 0 . 65 % by once daily ( CD ) , twice daily ( BID ) , three times daily , four weight of magnesium stearate . times daily or more often .

In another embodiment , the pharmaceutical compositions In certain embodiments , provided herein are methods for provided herein comprise about 10 % by weight of a solid 20 preparing a composition provided herein , comprising : ( i ) form of Compound 1 , about 58 . 95 % by weight of mannitol , weighing out the desired amount of a solid form of Com about 25 % by weight of microcrystalline cellulose , about pound 1 ( e . g . , Form 1 , Form 2 , Form 3 , Form 4 , Form 5 or 3 % by weight of sodium starch glycolate , about 1 % by amorphous ) and the desired amount of excipients ( such as weight of silicon dioxide , about 0 . 5 % by weight of stearic lactose monohydrate , croscarmellose sodium and / or micro acid , about 0 . 5 % disodium EDTA , about 0 . 4 % BHT , and 25 crystalline cellulose ) ; ( ii ) mixing or blending the solid form about 0 . 65 % by weight of magnesium stearate . of Compound 1 and the excipients ; ( iii ) passing the mixture

In certain embodiments , provided herein are pharmaceu of the solid form of Compound 1 and excipients through a tical compositions comprising an opaque coating . Without screen ( such as a 25 mesh screen ) ; ( iv ) mixing or blending being limited by theory , it was found that a more opaque the solid form of Compound 1 and the excipients after coating protected the drug product from degradation . In 30 passage through the screen ; ( v ) weighing out the desired some embodiments , the pharmaceutical composition is for - amount of lubricating agents ( such as stearic acid and mulated as a tablet . In some such embodiments , the tablet is magnesium stearate ) ; ( vi ) passing the lubricating agents film coated . In some embodiments , the tablet is film coated through a screen ( such as a 35 mesh screen ) ; ( vii ) mixing or to a weight gain of 1 - 8 % . In others , the film coating is about blending the solid form of Compound 1 , the excipients and 5 % by weight of the tablet . 35 the lubricating agents ; ( viii ) compressing the mixture of the

In certain embodiments , provided herein are pharmaceu - solid form of Compound 1 , the excipients and the lubricating tical compositions , wherein the amounts of the recited agents ( such as into a tablet form ) ; and optionally ( ix ) components can independently be varied by 1 % , 2 % , 3 % , coating the compressed mixture of the solid form of Com 4 % , 5 % , 6 % , 7 % , 8 % , 9 % , 10 % , 15 % , 20 % or 25 % . pound 1 thereof , the excipients and the lubricating agents

The pharmaceutical compositions provided herein can be 40 with a coating agent ( such as Opadry pink , yellow or beige ) . provided in a unit - dosage form or multiple - dosage form . A In certain embodiments , the methods for preparing a com unit - dosage form , as used herein , refers to physically dis - position provided herein are carried out in the dark , under crete unit suitable for administration to a human and animal yellow light or in the absence of UV light . subject , and packaged individually as is known in the art . In certain embodiments , the pharmaceutical compositions Each unit - dose contains a predetermined quantity of an 45 provided herein comprise Form 1 of Compound 1 , including active ingredient ( s ) sufficient to produce the desired thera - substantially pure Form 1 . peutic effect , in association with the required pharmaceutical In certain embodiments , the pharmaceutical compositions carriers or excipients . Examples of a unit - dosage form provided herein comprise Form 2 of Compound 1 , including include an individually packaged tablet or capsule . A unit - substantially pure Form 2 . dosage form may be administered in fractions or multiples 50 In certain embodiments , the pharmaceutical compositions thereof . A multiple - dosage form is a plurality of identical provided herein comprise Form 3 of Compound 1 , including unit - dosage forms packaged in a single container to be substantially pure Form 3 . administered in segregated unit - dosage form . In certain embodiments , the pharmaceutical compositions

In another embodiment , provided herein are unit dosage provided herein comprise Form 4 of Compound 1 , including formulations that comprise between about 0 . 1 mg and about 55 substantially pure Form 4 . 2000 mg , about 1 mg and 200 mg , about 35 mg and about In certain embodiments , the pharmaceutical compositions 1400 mg , about 125 mg and about 1000 mg , about 250 mg provided herein comprise Form 5 of Compound 1 , including and about 1000 mg , or about 500 mg and about 1000 mg substantially pure Form 5 . solid form of Compound 1 , or a solid form thereof . In certain embodiments , the pharmaceutical compositions

In a particular embodiment , provided herein are unit 60 provided herein comprise amorphous Compound 1 , includ dosage formulation comprising about 0 . 1 mg , about 0 . 25 ing substantially pure amorphous Compound 1 . mg , about 0 . 5 mg , about 1 mg , about 2 mg , about 2 . 5 mg , about 5 mg , about 7 . 5 mg , about 8 mg , about 10 mg , about 6 . EXAMPLES 15 mg , about 20 mg , about 25 mg , about 30 mg , about 35 mg , about 40 mg , about 45 mg , about 50 mg , about 60 mg , 65 The following Examples are presented by way of illus about 70 mg , about 75 mg , about 100 mg , about 125 mg , tration , not limitation . The following abbreviations are used about 140 mg , about 150 mg , about 175 mg , about 200 mg , in descriptions and examples :

US 9 , 981 , 971 B2 35 36

2MXETOH : 2 - Methoxyethanol ( ACN ) , n - butanol ( n - BuOH ) , absolute ethanol ( EtOH ) , etha AAC : Accelerated aging conditions ( 48 hours at 40° C . nol / water ( 1 : 1 ) , methanol ( MeOH ) , 2 - propanol ( IPA ) , ethyl

and 75 % RH ) acetate ( EtOAc ) , methylene chloride ( DCM ) , methyl ethyl ACN : Acetonitril ketone ( MEK ) , methyl t - butyl ether ( MTBE ) , heptane , tolu Am : Amorphous ene , tetrahydrofuran ( THF ) , dimethyl sulfoxide ( DMSO ) , AmPhos : p - Dimethylamino phenylditbutylphosphine N - methylpyrrolidone ( NMP ) , N , N - dimethylformamide API : Active Pharmaceutical Ingredient ( DMF ) and water . AS : ID for anti - solvent crystallization experiment Boc : tert - Butoxycarbonyl All of the solid samples generated in the polymorph dba : Dibenzylidene acetone 10 screen were analyzed by XRPD . XRPD analysis was con DCM : Dichloromethane ducted on a Crystallics T2 high - throughput X - ray powder DIPEA : N , N - Diisopropylethylamine diffractometer using Cu Ka radiation at 1 . 54 Å . The instru DMF : N , N - Dimethylformide ment was equipped with a fine focus X - ray tube . The voltage DMSO : Dimethylsulfoxide and amperage of the X - ray generator were set at 45 kV and DSC : Differential Scanning calorimetry 15 40 mA , respectively . The divergence slits were set at 4 mm ECP : ID for evaporative experiment and 2 mm and the measuring slits were set at 0 . 5 mm and 0 . 2 EDTA : Ethylenediamine tetraacetate mm . Diffracted radiation was measured using a Peltier ESI : Electrospray ionization cooled Si ( Li ) solid - state detector . A theta - two theta con EtOH : Ethanol tinuous scan at 2 . 40° / minutes ( 0 . 5 sec / 0 . 02° step ) from 1 . 5° FTIR : Fourier Transform Infra Red Spectroscopy 20 to 41 . 50 20 was used . A sintered alumina standard was used GRP : Grinding experiment to check the peak positions . HF : ID for hot - filtration crystallization experiment DSC analyses were performed on a DSC822e instrument HPLC : High performance liquid chromatography IPA : 2 - Propanol ( Mettler - Toledo GmbH , Switzerland ) . Indium was used as LCMS : Liquid Chromatography with Mass Spectroscopy 25 v 25 the calibration standard . Approximately 2 - 5 mg of sample MeOH : Methanol was placed into a DSC pan . The sample was heated under mp : Melting point nitrogen at a rate of 10° C . / min , up to a final temperature of MS : Mass spectrometry 300° C . Melting points were reported as the extrapolated Ms : Mesylate or methanesulfonyl onset temperatures . MTBE : tert - Butyl methyl ether 30 TGA analyses were performed on a TA instrument 25000 MTBE : methyl tert - butyl ether Thermogravimetric Analyzer . Calcium oxalate was used for NBS : N - Bromosuccinimide a performance check . Approximately 5 - 20 mg of accurately NMP : N - Methyl - 2 - pyrrolidone weighed sample was placed on a pan and loaded into the NMP : N - methylpyrrolidinone TGA furnace . The sample was heated under nitrogen at a NMR : Nuclear magnetic resonance rate of 10° C . / min , up to a final temperature of 300° C . PSU : ID for cooling - evaporative crystallization experi ment TGA / SDTA analyses were performed on a TGA

QSA : ID for Phase 1 experiments SDTA851e instrument ( Mettler - Toledo GmbH , Switzer RH : Relative Humidity land ) . The TGA / SDTA851e instrument was calibrated for RT : Room Temperature 40 temperature with indium and aluminium . Samples were S : Solvent weighed into 100 ul aluminium crucibles and sealed . The SDTA : Single Differential Thermal Analysis seals were pin - holed and the crucibles heated in the TGA SLP : ID for slurry experiment from 25 to 300° C . at a heating rate of 10° C . / min . Dry N , SM : Starting material gas was used for purging . TA : Thermal Analysis Morphology analysis of the samples was carried out on an TCP : ID for thermocycling and reflux experiment Tf : triflate or trifluoromethanesulfonyl Olympus microscope . Small amounts of samples were dis TFA : Trifluoroacetic acid persed in mineral oil on a glass slide with cover slips and TFE : 2 , 2 , 2 - Trifluoroethanol viewed with 20x or 50x magnification . TGA : Thermogravimetric Analysis 50 Hygroscopicity was determined on a Surface Measure TGA - MS / TG - MS : Thermogravimetric Analysis coupled ment Systems DVS . Typically a sample size of 2 - 10 mg was with Mass Spectroscopy loaded into the DVS instrument sample pan and the sample THF : Tetrahydrofuran

TLC : Thin layer chromatography was analyzed on a DVS automated sorption analyzer at VDL : ID for vapor diffusion into solutions experiment 55 room temperature . The relative humidity was increased from VDS : ID for vapor diffusion onto solids experiment 0 % to 90 % RH at 10 % RH step then 95 % RH . The relative XRPD : X - Ray Powder Diffraction humidity was then decreased in a similar manner to accom

6 . 1 Solid Forms plish a full adsorption / desorption cycle . For selected 60 hydrated forms , the analysis started at 50 % RH and

6 . 1 . 1 Polymorph Screen increased to 90 % RH at 10 % RH step . The relative humidity A polymorph screen of Compound 1 was performed to was then decreased in a similar manner to 0 % RH followed investigate whether different solid forms could be generated

under various conditions , such as different solvents , tem by increasing to 50 % RH . perature and humidity changes . 65 High Performance Liquid Chromatography ( HPLC ) was

The solvents used in the polymorph screen were either performed according to the conditions in Table 1 and gra HPLC or reagent grade , including acetone , acetonitrile dient program in Table 2 .

35

45

US 9 , 981 , 971 B2 38 37

TABLE 1 TABLE 3

High Performance Liquid Chromatography Operating Parameters of Crystal16 ® multiple - reactor system ( HPLC ) experiemental conditions Temperature range - 15° C . to 150° C .

5 Manufacturer Heating / cooling Agilent Individually programmable per reactor block

HPLC Temperature profile HP1200sl Unlimited heating / cooling / hold steps UV - detector per run programmable HP DAD Temperature control accuracy 0 . 1° C . MS - detector HP1100 API - ES MSD VL - type Heating / cooling ramps Programmable between 0° C . and 20° Column Waters Sunfire C18 ( 100 x 4 . 6 mm ; 3 . 5 um ) 16 C . / min Column Temperature 35° C . Stirrer speed ( magnetic stirrer bars ) Programmable from 0 - 1250 rpm Mobile Phase A 10 mM ammonium acetate Turbidity measurement Per individual reactor in transmission Mobile Phase B Acetonitrile 100 % Flow Rate 1 . 0 ml / min Post time 1 min 6 . 1 . 2 Experiments and Methods UV - Detector DAD 15 6 . 1 . 2 . 1 Solubility Experiment : Range 200 - 400 nm Wavelength 254 nm In order to select the screening solvents and to determine Slit width 4 nm the concentration range to be used in the screen , a quanti Time 0 - 20 min tative solubility assessment was performed on the starting MS - Detector MSD material , Form 1 of Compound 1 . A set of 15 solvents was Scan positive 20 analyzed . For each solvent , a standard 1 . 8 ml screw cap vial Mass Range 70 - 1000 amu was loaded with about 30 mg of the starting material , Form Fragmentator 70 1 of Compound 1 , 400 uL of solvent and a magnetic stirring Time 0 - 12 min bar . The vials were then closed and equilibrated at 25° C . for Autosampler :

24 h while stirring . The resulting mixtures ( slurries ) were Temperature Not controlled 25 filtered ( 0 . 5 micron ) and the isolated mother liquors diluted Injection mode loop to two dilutions selected according to the calibration curve . Injection volume 5 uL Quantities of Compound 1 in the diluted solutions were Needle wash 2 / 3 ; ACN / H2O ( v / v ) determined via HPLC analysis . The calibration curve was Dilution solvent 0 . 1 % TFA water / acetonitrile ( v / v = 50 / 50 ) obtained from two independently prepared stock solutions of

30 Compound 1 in 0 . 1 % TFA in Water / Acetonitrile ( 50 : 50 ) . Subsequent to the solubility determination , the wet solids

TABLE 2 were harvested and analyzed by XRPD . Moreover , the residual solvent was evaporated from each vial ( slurry )

High Performance Liquid Chromatography under vacuum at ambient temperature . All of the resulting ( HPLC ) experiemental gradient program 35 residues were analyzed by XRPD to check for new ( crys

talline ) forms . Time ( mins ) % A % B In addition to the solubility determination , 15 slurry

90 10 experiments of Form 1 of Compound 1 were performed with 10 at 50° C . for 24 hours in 15 solvents ( same 15 solvents ) . 10 40 Table 4 summarizes the experimental conditions . At the end

of the slurry time , the solids were separated from the solutions by centrifugation , harvested wet and dried and

The compound integrity is expressed as a peak - area analyzed by XRPD and digital imaging . percentage , calculated from the area of each peak in the chromatogram , except the ‘ injection peak ’ , and the total 43 TABLE 4 peak - area , as follows : Experimental conditions for 30 slurry conversion experiments ,

combined with solubility determination

90 90

90

peak - area peak – area % = ! - * 100 %

total – area

Sample Mass 50

Solvent Volume

( UL ) Temperature

( °C . ) Solvent ( mo Dissolved No No No

55 No No No No No

1 , 2 - Ethanediol The peak - area percentage of the compound of interest is 1 , 4 - Dioxane

employed as an indication of the purity of the component in Diethyl Ether Chloroform the sample . 2 - Methoxyethanol

Crystal16® multiple - reactor system ( Avantium Technolo Cyclohexane gies ) holds 16 ( 4x4 ) standard HPLC glass vials ( 11 . 5 mm p - Xylene

Cumene diameter , flat bottomed , 1 . 8 mL volume ) . A unit consists of Isopropyl Acetate four independently heated aluminum reactor blocks encased Anisole

Ethyl formate in a robust bench top setup . These blocks are electrically 1 - Propanol heated and cooled by a combination of Peltier elements and 1 , 2 - Dimethoxyethane a cryostat . In order to prevent condensation of water on the 2 - Butanone reactor blocks and electronics during runs at temperatures Acetonitrile

1 , 2 - Ethanediol below 10° C . , the Crystal16® system provides an inlet for a 65 14 1 , 4 - Dioxane dry purge gas ( typically nitrogen ) . Operating Parameters are Diethyl Ether provided in Table 3 .

No

32 . 9 33 . 0 33 . 5 31 . 1 29 . 3 30 . 3 27 . 5 29 . 7 29 . 2 30 . 8 32 . 7 29 . 8 30 . 4 28 . 6 30 . 8 46 . 5 48 . 4 51 . 0

400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400

UNNNNNNNNNNNNNNN Ou üüüüüüüüüüüüü No No No No No No No

50 No No 50

US 9 , 981 , 971 B2 39 40

TABLE 4 - continued determine the change in mass ( see Table 7 and Table 8 ) . The materials used in desiccators to reach the defined relative

Experimental conditions for 30 slurry conversion experiments , humidity are presented in Table 6 . combined with solubility determination

5 TABLE 6 Solvent Volume ( UL )

Mass ( mg ) Solvent Dissolved

Temperature ( ° C . ) Preparation of the different humidity ranges

No

10 No No No

50

50 No 8 .

50 15 No No 50

20

?

35 ?

? ?? ??

Chloroform 53 . 0 400 50 Relative Humidity at RT Method 2 - Methoxyethanol 50 . 0 400 No 50 cyclohexane 51 . 9 400 0 % P205 ( powder ) p - Xylene 41 . 7 400 50 % MgNO3 ( saturated solution ) Cumene 47 . 4 400 50 75 % NaCl ( saturated solution ) Isopropyl Acetate 48 . 1 400 No 100 % Climate chamber with water vapors Anisole 51 . 3 400 Ethyl formate 50 . 7 400 No 1 - Propanol 48 . 2 400 No 15 1 , 2 - Dimethoxyethane 51 . 5 400 No TABLE 7 2 - Butanone 46 . 5 400 50 Acetonitrile 55 . 9 400 Initial experimental conditions for the four

vials used to determine the water uptake

6 . 1 . 2 . 2 Feasibility Study Empty vial Starting The experimental conditions of the feasibility study with Relative Empty vial weight + Starting material

Compound 1 are summarized in Table 5 . The freeze drying Humidity weight ( mg ) material ( mg ) weight ( mg ) experiments were performed in 1 . 8 ml vials . Approximately 0 % 2297 . 5 2318 . 2 20 . 7 20 mg of starting material were weight in a HPLC vial and 50 % 2314 . 1 2334 . 5 20 . 4 dissolved in five different solvent mixtures . The starting of 75 % 2285 . 5 2306 . 3 20 . 8

100 % 2333 . 3 2354 . 2 20 . 9 material , Form 1 , did not dissolve in THF / water ( 90 / 10 or 50 / 50 ) and ethanol / water ( 90 / 10 ) ; therefore these experi mental samples were not freeze - dried . Form 1 dissolved in TFE and TFE / water ( 90 / 10 ) . These two experimental TABLE 8 samples were freeze - dried in liquid nitrogen , followed by placing the vials in a freeze - dryer for 24 hours . The obtained Experimental conditions for the 20 stability tests solid was then harvested and analyzed by XRPD and digital Relative humidity Time Starting material weight ( mg ) imaging Grinding experiments were performed in stainless steel 0 % 3 days

1 weeks grinding vials , containing 2 stainless steel grinding balls and 2 weeks a frequency of 30 Hz . Following the experiments , XRPD 3 weeks analysis was performed to assess the crystallinity of the 4 weeks materials . 50 % 3 days

1 weeks 5 . 1 2 weeks 4 . 6

TABLE 5 3 weeks 5 . 2 4 weeks

Conditions applied for the feasibility study on Form 1 75 % 3 days 1 weeks 4 . 4

Sample Solvent Solu 2 weeks Mass Volume bility Dis 3 weeks ( mg ) Solvent ( UL ) ( mg / mL ) solved Comments 4 weeks

100 % 3 days 24 . 8 THF / water < 25 No Freeze drying 1 weeks

( 90 / 10 ) 2 weeks 24 . 2 Ethanol / water 400 < 60 Freeze drying 3 weeks

( 90 / 10 ) 4 weeks 6 . 3 25 . 1 THF / Water 1000 Freeze drying

( 50 / 50 ) 20 . 7 TFE / water 1000 Freeze drying 6 . 1 . 2 . 4 Experimental Methods of the Polymorph Screen

( 90 / 10 ) 21 . 9 TFE ing : 1000 100022 Yes Yes Freeze drying 20 . 0 None Grinding 1 The screening experiments for Compound 1 comprised 96

hour experiments at microliter ( UL ) scale and 125 experiments at None Grinding 2 55 milliliter ( mL ) scale . The following ten crystallization pro

hours cedures were applied : cooling - evaporation , evaporative , cooling crystallization with hot filtration , crash crystalliza

6 . 1 . 2 . 3 Physical Stability Study at Room Temperature and tion with anti - solvent addition , slurry conversion , vapor Different RH diffusion into solutions , vapor diffusion onto solid , thermo

Physical stability studies over a prolonged period of time 60 cycling , reflux and grinding . ( e . g . , 4 weeks ) were conducted in desiccators at defined Cooling - Evaporative Crystallization Experiments at ul relative humidity ( 0 % , 50 % , 75 % and 100 % ) . At regular Scale : intervals ( e . g . , 3 days , 1 week , 2 weeks , 3 weeks and 4 The 96 cooling - evaporative experiments at uL scale were weeks ) , the materials were analyzed by XRPD . To determine performed in well plates , employing 12 different solvents if the material absorb water molecules under different rela - 65 and 12 mixtures of solvents in Table 9 and four temperature tive humidity levels , four more additional vials were placed profiles in Table 9 . About 4 mg solid dose of Compound 1 in the desiccators to weight them back periodically and was in each well of the microliter well plate . Subsequently ,

40 5 . 5 4 . 6

5 . 4 ?

?

_ _ 45 45 ?

1000 wo No ?

JIO 22

100001 No 50

Yes

20 . 0

US 9 , 981 , 971 B2 42

TABLE 10

Temperature profiles employed for the 96 cooling - evaporative experiments

80 uL of the screening solvent was added into the well to reach a concentration of 50 mg / ml .

The plates , with each well individually sealed , were placed in a Crystal Breeder to undergo a temperature profile as described in Table 10 . The plates were placed under vacuum after completion of the temperature profile . The solvents were evaporated for several days at 200 mbar or 5 mbar and analyzed by XRPD and digital imaging . Follow ing , the solid samples were exposed to accelerated aging conditions ( 2 days at 40° C . / 75 % RH ) and re - analyzed by 10 XRPD and digital imaging .

TABLE 9

Heating Hold Cooling Tstart rate Tmax time rate Tend ( °C . ) ( ° C . / min ) ( ° C . ) ( min ) ( ° C . / h ) ( °C . )

Temperature profile #

Age time ( h )

ex

NP 20 20

10 . 0 10 . 0 10 . 0

60 60

60 60

1 . 0 20 . 0 20 . 0

5 5

48 3 20

Experimental conditions for the 96 ul cooling - evaporation experiments

Solvent Temperature Conditions

1 , 2 - Ethanediol

Anisole

2 - Methoxyethanol

Acetonitrile / Anisole ( 50 / 50 )

1 , 2 - Dimethoxyethane / 1 Pentanol ( 50 / 50 ) Isobutanol

Isopropyl Acetate / 2 Methoxyethanol ( 50 / 50 ) Water

1 , 4 - Dioxane / Water ( 50 / 50 )

1 , 4 - Dioxane

Water / Ethanol ( 50 / 50 )

Isopropyl Acetate

Water / Methanol ( 50 / 50 )

Temperature Temperature Temperature Temperature Profile # 1 Profile # 2 Profile # 3 Profile # 4 Temperature Temperature Temperature Temperature Profile # 1 Profile # 2 Profile # 3 Profile # 4 Temperature Temperature Temperature Temperature Profile # 1 Profile # 2 Profile # 3 Profile # 4 Temperature Temperature Temperature Temperature Profile # 1 Profile # 2 Profile # 3 Profile # 4 Temperature Temperature Temperature Temperature Profile # 1 Profile # 2 Profile # 3 Profile # 4 Temperature Temperature Temperature Temperature Profile # 1 Profile # 2 Profile # 3 Profile # 4 Temperature Temperature Temperature Temperature Profile # 1 Profile # 2 Profile # 3 Profile # 4 Temperature Temperature Temperature Temperature Profile # 1 Profile # 2 Profile # 3 Profile # 4 Temperature Temperature Temperature Temperature Profile # 1 Profile # 2 Profile # 3 Profile # 4 Temperature Temperature Temperature Temperature Profile # 1 Profile # 2 Profile # 3 Profile # 4 Temperature Temperature Temperature Temperature Profile # 1 Profile # 2 Profile # 3 Profile # 4 Temperature Temperature Temperature Temperature Profile # 1 Profile # 2 Profile # 3 Profile # 4 Temperature Temperature Temperature Temperature Profile # 1 Profile # 2 Profile # 3 Profile # 4 Temperature Temperature Temperature Temperature

Profile # 1 Profile # 2 Profile # 3 Profile # 4 Temperature Temperature Temperature Temperature Profile # 1 Profile # 2 Profile # 3 Profile # 4 Temperature Temperature Temperature Temperature Profile # 1 Profile # 2 Profile # 3 Profile # 4 Temperature Temperature Temperature Temperature Profile # 1 Profile # 2 Profile # 3 Profile # 4 Temperature Temperature Temperature Temperature Profile # 1 Profile # 2 Profile # 3 Profile # 4 Temperature Temperature Temperature Temperature Profile # 1 Profile # 2 Profile # 3 Profile # 4 Temperature Temperature Temperature Temperature Profile # 1 Profile # 2 Profile # 3 Profile # 4 Temperature Temperature Temperature Temperature Profile # 1 Profile # 2 Profile # 3 Profile # 4 Temperature Temperature Temperature Temperature Profile # 1 Profile # 2 Profile # 3 Profile # 4 Temperature Temperature Temperature Temperature Profile # 1 Profile # 2 Profile # 3 Profile # 4 Temperature Temperature Temperature Temperature Profile # 1 Profile # 2 Profile # 3 Profile # 4

Acetonitrile

isopropanol / Tetrahydrofuran ( 50 / 50 ) Methanol / Acetonitrile

( 50 / 50 ) Tetrahydrofuran / Ethanol ( 50 / 50 ) Tetrahydrofuran

Methanol

Tetrahydrofuran / Chloroform ( 50 / 50 ) Methanol / Chloroform

( 50 / 50 ) Chloroform

Acetonitrile / Dichloromethane ( 50 / 50 ) Ethyl Formate

US 9 , 981 , 971 B2 44 43

TABLE 10 - continued TABLE 12 - continued

Experimental conditions and results for the hot filtration experiments Temperature profiles employed for the 96 cooling - evaporative experiments 5

Starting Heating Hold Cooling Age

Exp . Temperature profile #

Tstart rate Tmax time rate Tend time ( °C . ) ( °C . / min ) ( °C . ) ( min ) ( °C . / h ) ( °C . ) ( h )

Solvent material Solid after volume weight Temperature ( UL ) ( mg ) profile 10 No . Stock solvent description

m Yes No +

2000 No 15

atu

20 10 . 0 60 60 1 . 0 20 48 13 Tetrahydrofuran / Acetonitrile 5000 30 . 0 20 1 0 . 0 60 60 20 . 0 20 3 Anisole / Chloroform 5000 5000 30 . 0 30 . 0 No

15 Butanone , 2 - / N - Methyl - 2 30 . 0

pyrrolidone 6 . 1 . 2 . 5 Cooling Crystallization with Hot Filtration : The crystallization method with hot filtration comprised

15 solvent mixtures . Supersaturated solutions were prepared 6 . 1 . 2 . 6 Anti - Solvent Crystallization : by stirring slurries ( see Table 12 ) at 60° C . for one hour and 20 For the crash - crystallization experiments with anti - sol then filtering the slurries . All the solutions were then placed vent addition , 15 different crystallization conditions were in a Crystal16® system to undergo a cooling profile and applied , using the selected solvents and eleven different aged for 62 h ( see Table 11 ) . If solids precipitated after the anti - solvents ( see Table 14 ) . Stock solutions were prepared temperature profile , they were harvested wet and dried and in each solvent ( see Table 13 ) . These solutions were sa analyzed by XRPD and digital imaging . The experiments 25 rated with Form 1 of Compound 1 and equilibrated for 24 h with no solid after the temperature profile were left to before filtering . The stock solutions were then liquid dosed evaporate under vacuum . The obtained dry solid samples into the experimental vials , followed by the anti - solvent were analyzed by XRPD and digital imaging . All the solid addition . The anti - solvent was added to each solvent vial samples were exposed to accelerated aging conditions ( 2 with a solvent to anti - solvent ratio of 1 : 0 . 25 . In the case of days at 40° C . / 75 % RH ) and re - analyzed by XRPD and 30 no precipitation occurred , this ratio was increased to 1 : 1 or digital imaging . 1 : 4 with a waiting time of 60 minutes between the additions .

After the last addition the samples were left stirring at TABLE 11 ambient temperature for 24 hours . The precipitated solids 35 were isolated from the mother liquor and analyzed wet and

Cooling profile employed for the hot filtration experiments dried by XRPD and digital imaging . The samples , in which no precipitation occurred , were placed under vacuum and the dried solids were analyzed by XPRD and digital imag Cooling rate

Tinitial ( C . ) Hold ( min ) ( °C . / h ) Tfinal ( °C . ) Hold ( hrs ) ing . All the solids were exposed to accelerated aging con 40 ditions ( 2 days at 40° C . / 75 % RH ) and re - analyzed by XRPD and digital imaging .

TABLE 13

45 Stock solution for the anti - solvent addition experiments TABLE 12 Experimental conditions and results for the hot filtration experiments

Exp . Starting

material weight ( mg )

Solvent Solution

volume concentration ( UL ) ( mg / mL ) No . Solvent ( s ) Starting

Solvent material Solid after volume weight Temperature ( UL ) ( mg ) profile

50 Exp . No . Stock solvent description

30 No 30 Yes ??? .

5000 2000 5000 3000

30 . 0 30 . 0 30 . 0 30 . 0

120 Yes No 55

60

30 30 30 va u AWNA

6

5000 5000 5000 500

5000 5000 1000 1000 500

5000 500

30 30

120 3000 5000 5000 2000

Acetonitrile / Ethyl Formate Tetrahydrofuran / Water Water / Methanol N , N - Dimethylformamide / Cumene Water / 1 , 4 - Dioxane Isopropanol / Acetone Ethanol / Water Ethanol / N - Methyl - 2 pyrrolidone Tetrahydrofuran / 1 , 2 Dimethoxyethane Dimethyl Sulfoxide / Water Isopropyl Acetate / Diethyl Ether 2 - Methoxyethanol / Chloroform

30 . 0 30 . 0 30 . 0 30 . 0

1 Tetrahydrofuran 22 - Methoxyethanol 3 Tetrahydrofuran 4 N - Methyl - 2 - pyrrolidone 5 1 , 4 - Dioxane 6 N , N - Dimethylformamide 7 N - Methyl - 2 - pyrrolidone 8 1 , 4 - Dioxane 9 N - Methyl - 2 - pyrrolidone

10 Tetrahydrofuran 11 2 - Methoxyethanol 12 N , N - Dimethylformamide 13 Tetrahydrofuran 14 Dimethyl Sulfoxide 15 Dimethyl Sulfoxide

Yes Yes Yes No

30 6 30 0

60 30 5000 6 9 4000 28 . 0 Yes 30 a

O 30 O

10 30 . 0 No 5000 5000

30 30 30 30 30

5000 1000 1000 5000 5000 500 500 500

11 30 . 0 No 8 a 60

12 2000 32 . 0 Yes 65 30

US 9 , 981 , 971 B2 45 46 TABLE 14

Results and experimental conditions for the anti - solvent addition experiments

Exp No . Solvent

Solvent volume

( UL ) Anti - solvent

Starting Material wt ( mg ) A * B * C *

AS : S ratio

Yes No

0 . 25 4

30 . 0 30 . 0 30 . 0 60 . 0

No No No

No Yes No No Yes 4

1 THF 2 2MXETOH 3 THF 4 N - Methyl

2 - pyrrolidone 5 1 , 4 - Dioxane 6 DMF 7 NMP 8 1 , 4 - Dioxane 9 NMP

10 THF

No No No Yes NO Yes

5000 Heptane 5000 Cumene 5000 Cyclohexane 500 Ethyl

formate 5000 p - Xylene 1000 Isopropylether 500 Cyclohexane

5000 Heptane 500 TBME 5000 2 , 2 , 4 - Trimethyl

pentane 5000 Ethyl acetate 1000 Water 5000 Water 500 Water 500 Toluene

30 . 0 30 . 0 60 . 0 30 . 0 30 . 0 30 . 0

No Yes No Yes 0 . 25

No No 11 2MXETOH 12 DMF 13 THF 14 DMSO 15 DMSO

30 . 0 30 . 0 30 . 0 30 . 0 30 . 0 30 . 0

No Yes No

Yes No Yes

No - No

No - Yes

0 . 25 4 0 . 25 4

* A = whether or not any precipitation after addition to 0 . 25 : 1 ( AS : S ) ; B = whether or not any precipitation after addition to 1 : 1 ( AS : S ) ; C = whether or not any precipitation after addition to 4 : 1 ( AS : S ) .

No Yes

62 Yes

No NO No No

Yes Yes

6 . 1 . 2 . 7 Slurry Conversion Experiment : 25 TABLE 15 - continued Experiments were carried out by adding about 30 mg of Form 1 of Compound 1 to 500 uL of a test solvent . The Experimental conditions of the slurry experiments

resulting mixture was agitated for at least 24 hours at 25° C . Dissolved Solids Upon reaching equilibrium , the saturated supernatant solu Solvent Starting Concen - at initial after tion was removed . The solid resulting from the equilibration 30 Exp Fyn volume Material tration temper - two was filtered and air - dried before analysis . No . Solvent ( UL ) wt ( mg ) ( mg / mL ) ature ature weeks

A total of ten slurry experiments were performed with Form 1 of Compound 1 with ten solvents at ambient 4 Acetonitrile 500 30 . 4 60 . 8 No Yes

5 1 , 2 500 30 . 5 61 temperature for two weeks ( see Table 15 ) . After the slurry Ethanediol time , the solids were separated from the solutions by cen 6 6 Isopropyl Isopropyl 500 500 31 . 0 31 . 0 62 No Yes trifugation , harvested wet and analyzed by XRPD and 35 Acetate digital imaging . The solids were then exposed to accelerated 7 p - Xylene 500 30 . 3 60 . 6 Yes aging conditions ( 2 days at 40° C . / 75 % RH ) , followed by 8 2 - Butanone 500 29 . 8 59 . 6 Yes XRPD re - analysis . 9 Cumene 500 29 . 8 59 . 6

10 Anisole 500 30 . 1 60 . 2 TABLE 15

Experimental conditions of the slurry experiments 6 . 1 . 2 . 8 Evaporative Experiments The 15 evaporative experiments were done by dissolving

Dissolved Solids Form 1 of Compound 1 in 15 different solvent mixtures in Solvent Starting Concen - at initial after Table 16 . The starting material , Form 1 of Compound 1 was volume Material tration temper - two No . Solvent ( ul ) wt ( mg ) ( mg / mL ) ature weeks 45 added into solvent and if the starting material did not

dissolve in the solvent completely , the mixtures were filtered 1 Water 500 29 . 7 59 . 4 No Yes and then the clear solutions were evaporated . The solvents 2 Methanol / 500 500 30 . 0 30 . 0 60 No Yes Yes

Water were slowly evaporated under vacuum ( 200 mbar or 5 mbar ) ( 50 / 50 ) until dryness to yield solid . The solid was analyzed by

3 Ethanol / 500 30 . 0 60 No Yes 50 XRPD and digital imaging . The solid was then exposed to Water ( 50 / 50 ) accelerated aging conditions ( 2 days at 40° C . / 75 % RH ) ,

followed by XRPD re - analysis and digital imaging . TABLE 16

40

Exp

60 No

Experimental conditions of the evaporative experiments

Starting Material wt

( mg )

Solvent volume Concentration ( UL ) ( mg / mL ) Exp No . Solvent D *

30 . 2 31 . 5

5000 5000

60 . 4 63

Yes No

w Na

Ethanol / Chloroform ( 50 / 50 ) 2 , 2 , 2 - trifluoroethanol / Water ( 50 / 50 ) 1 , 4 - Dioxane / Ethyl formate ( 50 / 50 ) Methanol / Acetonitrile ( 50 / 50 )

29 . 8 5000 59 . 6 No

A 28 . 7 5000 57 . 4 No

US 9 , 981 , 971 B2 47 48

TABLE 16 - continued Experimental conditions of the evaporative experiments

Starting Material wt

Exp No . ( mg )

Solvent volume

( UL ) Solvent Concentration

( mg / mL ) D *

26 . 5 5000 53 No

30 . 1 60 . 2 5000 5000

Yes No 31 62

29 . 5 28 . 9

5000 5000

59 57 . 8

Yes No

Acetonitrile / Chloroform ( 50 / 50 ) Water / Tetrahydrofuran ( 50 / 50 ) Isopropanol / 2 - Butanone ( 50 / 50 ) Methanol / 1 , 4 - Dioxane ( 50 / 50 ) 2 - Methoxyethanol / Isopropyl Acetate ( 50 / 50 ) Ethanol / Water ( 50 / 50 ) Water / NMP ( 50 / 50 ) THF / TBME ( 50 / 50 ) 1 , 4 - Dioxane / Water ( 50 / 50 ) 1 , 2 - Ethanediol / THF ( 50 / 50 ) Acetone / isopropanol ( 50 / 50 )

No No

30 . 4 29 . 4 29 . 8 30 . 6 28 . 8 30 . 1

5000 5000 5000 5000 5000 5000

60 . 8 58 . 8 59 . 6 61 . 2 57 . 6 60 . 2

No No

14 Yes No 15

* D = whether or not all the starting material dissolved at initial temperature .

Exp ( mg ) S *

Yes

40 Yes Yes Yes OOO vau AWNIO Yes

6 . 1 . 2 . 9 Vapor Diffusion into Solutions : transferred into 1 . 8 ml vials , which were left open and For the 15 vapor diffusion into solution experiments , placed in closed 40 ml vials containing 2 ml of solvent ( see

saturated solutions of Form 1 of Compound 1 were exposed Table 18 ) . The material was exposed to solvent vapors at to solvent vapors at room temperature for two weeks . Stock 25 room temperature for two weeks . After that time , the experi solutions were prepared in each solvent . These solutions ments were harvested and analyzed by XRPD and digital were saturated with Form 1 of Compound 1 and equilibrated imaging . Following , all the solids were exposed to acceler for 24 h before filtering into a set of 8 ml vials . These vials ated aging conditions ( 40° C . and 75 % RH ) for two days , were left open and placed in closed 40 ml vials containing followed by XRPD re - analysis and digital imaging . 2 ml of anti - solvent ( see Table 17 ) . After two weeks , the 30 samples were checked on solid formation . When solid was TABLE 18 formed the solid samples were analyzed wet by XRPD and Experimental conditions of the vapor diffusion onto solids experiments digital imaging . If no precipitation occurred , the samples were placed under vacuum and the resulted solid samples 25 Starting Solvent were analyzed by XRPD and digital imaging . Subsequently , Material wt volume

Solvent ( UL ) all the solid samples were exposed to accelerated aging conditions ( 2 days at 40° C . / 75 % RH ) , followed by XRPD 30 . 3 tert - Butyl methyl ether 2000

30 . 2 2000 re - analysis and digital imaging . 1 , 2 - Ethanediol 29 . 8 Chloroform 2000 30 . 3 Methanol 2000

TABLE 17 30 . 0 Ethyl Formate 2000 29 . 8 Cyclohexane 2000

Experimental conditions of the vapor diffusion into solution 30 . 6 Acetonitrile 2000 Yes experiments 30 . 3 Heptane 2000 Yes

29 . 7 isopropyl ether 2000 Yes Starting 29 . 8 Pentane , n 2000 Yes

Material Solvent 30 . 3 Toluene 2000 Yes Exp wt volume 29 . 7 Isobutyl acetate 2000 Yes No . ( mg ) Solvent ( UL ) Anti - solvent 30 . 5 2 - Ethoxyethanol 2000

14 30 . 0 Water 2000 Yes 29 . 8 2 - Methoxyethanol 5000 Anisole No 29 . 7 Acetone 2000 Yes 30 . 5 DMF 1000 Acetonitrile 30 . 1 Tetrahydrofuran 5000 Diethyl ether * S = whether or not there was any solid left after two weeks . 29 . 9 Dimethyl Sulfoxide 600 Water Yes 29 . 6 1 , 4 - Dioxane 5000 Cyclohexane 6 . 1 . 2 . 11 Thermocycling Experiments 29 . 9 DMF 1000 n - Pentane 29 . 7 NMP 600 Ethanol A total of 15 slurries of Compound 1 in solvents were 29 . 8 2 , 2 , 2 1000 Cyclohexane No 55 prepared at room temperature ( see Table 19 ) . The mixtures trifluoroethanol 30 . 1 NMP 600 Heptane were placed in the Crystal Breeder to undergo the tempera No 29 . 6 Dimethyl Sulfoxide 500 Isopropyl ether No ture profile as follows : a ) heated with a heating rate of 5° 30 . 0 2 - Methoxyethanol 5000 Acetone No C . / h until reaching 40° C . ; b ) cooled with a cooling rate of

12 29 . 9 Tetrahydrofuran 5000 Chloroform 5° C . / h until reaching 5° C . ; c ) held at 5° C . for 30 min ; d ) 13 30 . 4 Dimethyl Sulfoxide 500 Ethyl acetate No 14 30 . 5 Tetrahydrofuran 5000 n - Pentane Yes 60 repeated the cycle 8 times ; and e ) being stirred at 300 rpm 60 15 29 . 9 1 , 4 - Dioxane 5000 Dichloromethane No during the temperature profile .

After the completion of the cycling program , the solids * S = whether or not there is any solid formed after two weeks . were separated from the liquids and analyzed wet and dried

6 . 1 . 2 . 10 Vapor Diffusion onto Solids by XRPD and digital imaging . All the solids were then For the 15 vapor diffusion onto solids experiments , amor - 65 exposed to accelerated aging conditions ( 2 days at 40°

phous Compound 1 was prepared by grinding crystalline C . / 75 % RH ) , followed by XRPD re - analysis and digital Compound 1 for two hours . The amorphous material was imaging .

S * Yes

200 15 Yes Yes 50

vau AWNA No No No

10 11 O

No

US 9 , 981 , 971 B2 49 50

TABLE 21 - continued TABLE 19

Experimental conditions of the thermocycling experiments Experimental conditions for the reflux experiments

Yes No

No

Yes ? ??

No No

15 13 Yes OOONOU 30 Yes

Dissolved Dissolved Starting Solvent Concen - at initial Exp Material volumetration temper Starting Solvent Concen - at initial No . wt ( mg ) Solvent ( UL ) ( mg / mL ) ature S * Exp Material volume tration temper

No . wt ( mg ) Solvent ( LL ) ( mg / mL ) ature ature S * 1 29 . 7 tert - Butyl 1000 29 . 7 No

methyl ether 9 29 . 8 Ethyl acetate 1000 29 . 8 Yes 30 . 8 Chloroform 1000 30 . 8 No Yes 10 10 31 . 3 2 - Methyl 1000 31 . 3 No Yes 29 . 5 Methanol 1000 29 . 5 No Yes 29 . 5 1 , 2 - Di 1000 29 . 5 Yes tetrahydro

methoxy furan ethane 11 29 . 5 29 . 5 Ethanol Ethanol 1000 1000 29 . 5 29 . 5 No Yes

29 . 8 p - Xylene 1000 29 . 8 No Yes 30 . 6 2 - Butanone 1000 30 . 6 No Yes 29 . 7 Acetonitrile 1000 29 . 7 No Yes 29 . 5 Cyclohexane 1000 29 . 5 No Yes 30 . 0 Water 1000 30 No 14 1000 29 . 5 29 . 5 Acetonitrile No Yes 1000 29 . 7 No Acetone 29 . 7 Yes 30 . 0 1 , 4 - Dioxane 1000 No 15 29 . 5 Isopropanol 1000 29 . 5 No Yes 30 . 1 1 , 2 - Ethanediol 1000 30 . 1 No Yes 30 . 5 Ethyl Formate 1000 30 . 5 No Yes * S = whether or not there was any solid left after Tprofile in Table 20 .

12 30 . 6 2 - Butanone 1000 30 . 6 No Yes 30 . 0 Isopropanol 1000 No 30 20 Yes 6 . 1 . 2 . 13 Grinding Experiments 30 . 3 Tetrahydro 1000 30 . 3 No Yes

furan In ten grinding experiments ( see Table 22 ) , about 30 mg 30 . 1 Cumene 1000 1000 30 . 1 30 . 1 No No Yes Yes Form 1 of Compound 1 was ground in metal grinding vials

* S = whether or not there was any solid left after the eight cycles . charged with two metal grinding balls . Then 10 ul of solvent 25 was added . The samples were ground for 1 hour with a

6 . 1 . 2 . 12 Reflux Experiments frequency of 30 Hz . In the 15 reflux experiments ( see Table 21 ) , the starting material , Form 1 of Compound 1 , was mixed with selected The ground solids were harvested and analyzed by XRPD solvents in 1 . 8 mL vials to give slurries . The slurries were and digital imaging . Then the solids were exposed to accel then kept at a constant temperature ( slightly below the 30 the 30 erated aging conditions ( 40° C . / 75 % RH ) for two days , corresponding boiling point of the chosen solvent ) for one followed by XRPD re - analysis and digital imaging . week and afterwards at 5° C . for two days ( see Table 20 ) . TABLE 22

11

13 14

15

EXP I start

25

?? M ?? ?? uuuu 10 10 48 23 au Ova AWNA ooooooooo 45

10

TABLE 20 Experimental conditions for the grinding experiments 35

Temperature profile ( Trofile ) applied to the reflux experiments Starting Exp Material wt Solvent volume Concentration Heating Hold Cooling No . Solvent ( UL ) ( mg / mL ) Exp Tetor rate Tmax time rate Tend Age time

No . No . ( °C . ) ( °C . / min ) ( °C . ) ( h ) ( °C . / h ) ( °C . ) ( h ) 29 . 9 Ethanol 10 2990 40 30 . 3 1 , 2 - Ethanediol 3030

50 168 10 48 30 . 7 Acetonitrile 3070 25 168 10 5 48 30 . 0 Isobutanol 3000

70 168 29 . 6 Toluene 2960 80 168 10 48 29 . 7 Isopropyl Acetate 2970

30 . 6 Anisole 3060 29 . 8 Water 2980 After the temperature profile the solids were analyzed wet 43 29 . 9 Acetone 2990

by XRPD and digital imaging . Then all the solids were 30 . 1 Cumene 3010

exposed to accelerated aging conditions ( 2 days at 40° C . / 75 % RH ) , followed by XRPD re - analysis and digital imaging . Provided herein are five crystalline forms identified by the

50 polymer screen . Form 1 was found to be a stable anhydrous TABLE 21 crystalline form that melts at approximated 268 . 9° C . Form

2 , a 1 , 2 - ethanediol mono - solvated form of Compound 1 , Experimental conditions for the reflux experiments was prepared at least when 1 , 2 - ethanediol was used as

solvent in a slurry conversion experiment . Form 3 , a 2 , 2 , 2 Dissolved Dissolved » 55 trift , trifluorotoluene hemi - solvated form of Compound 1 , was Starting Solvent Concen - at initial Exp Material volumetration temper prepared from at least one evaporative experiment in TFE / No . wt ( mg ) Solvent ( UL ) ( mg / mL ) ature S * water ( 50 : 50 ) . Form 4 , a 0 . 8 molar equivalent DMSO

29 . 5 Ethyl formate 1000 29 . 5 No Yes solvated form of Compound 1 , was prepared at least from 2 29 . 7 tert - Butyl 1000 29 . 7 No Yes anti - solvent crystallization by using DMSO as solvent and

methyl ether water as anti - solvent . Form 5 , a dihydrated form of Com 30 . 1 Acetone 1000 30 . 1 No 30 . 0 Methyl acetate 1000 30 No Yes pound 1 , was prepared in hot - filtration experiments at least 29 . 5 Chloroform 1000 29 . 5 Yes when water was used as part of the crystallization solvent . 30 . 1 Methanol 1000 30 . 1 No A summary of the experimental conditions which the new 29 . 8 Tetrahydro 1000 29 . 8 No Yes 65 solid forms were produced is presented in Table 23 . A furan 30 . 1 Isopropyl ether 1000 30 . 1 No Yes summary of physical data of solid forms is presented in

Table 24 .

Yes

o vauw NA No Yes

US 9 , 981 , 971 B2 51

TABLE 23 52

TABLE 25 - continued Summary of experimental conditions of the solid forms X - Ray Diffraction Peaks for Form 1 of Compound 1

Form Crystallization Method Solvent Two - theta angle o d Space ( 8 )

Relative Intensity ( % )

1 , 2 - ethanediol Evaporative Slurry Thermocycling Evaporative Hot - filtration Vapor diffusion into liquids

27 . 06 27 . 98 29 . 38

3 . 29 3 . 19 3 . 04

44 . 83 26 . 77 10 . 14 w

10

2 , 2 , 2 - trifuoroethanol Isopropanol / acetone ( 50 : 5 ) 2 , 2 , 2 - trifuoroethanol ( s ) , cyclohexane ( AS ) Chloroform DMSO ( S ) , water ( AS ) DMSO ( S ) , toluene ( AS ) DMSO ( S ) , water ( AS ) THF / water ( 50 : 50 ) Water / methanol ( 50 : 50 ) Water / 1 , 4 - dioxane ( 50 : 50 ) Ethanol / water ( 50 : 50 ) THF ( S ) , water ( AS ) Water / THF ( 50 : 50 )

Vapor diffusion onto solids Anti - solvent Anti - solvent Vapor diffusion into liquids Hot - filtration Hot - filtration Hot - filtration Hot - filtration Anti - solvent Evaporative

u

TABLE 24

Physical Characterization of Solid Forms of Compound 1

Endo - therms ( ° C . )

Purity % by

HPLC )

Physical stability ( existing forms

after 48 h 40° C . / 75 % RH )

FIG . 3 is a digital image of Form 1 of Compound 1 . FIGS . 4 and 5 provide TGA / SDTA signal and TGA - MS

data , respectively , of Form 1 . The TGA thermogram of Form 1 in FIG . 4 does not shows

15 any significant mass loss when heated from 25° C . to 300° C . The SDTA data of Form 1 in FIG . 4 shows a melting event at 268 . 9° C . , corresponding to the melting point of Form 1 of Compound 1 .

The TGA thermogram of Form 1 in FIG . 5 comprises a 20 total mass loss of approximately 0 . 44 % of the total mass of

the sample between approximately 30° C . and approxi mately 250° C . when heated from approximately 25° C . to approximately 300° C . Thus , Form 1 loses about 0 . 44 % of its total mass when heated from about ambient temperature to about 300° C . These observations suggest that Form 1 is anhydrous crystalline material .

FIG . 6 provides HPLC and MS data of Form 1 . The peak retention time is 4 . 9 minutes and indicates the sample purity is 99 . 90 % ( area % ) .

6 . 1 . 2 . 15 Form 2 The XRPD pattern , crystal habit , TGA , SDTA , TGA - MS ,

HPLC and MS of Form 2 of Compound 1 are shown in FIGS . 7 - 11 . Form 2 was prepared in slurry conversion

35 experiments when 1 , 2 - ethanediol was used as solvent . Form 2 appears stable under accelerated aging conditions ( 2 days at 40° C . / 75 % RH ) .

FIG . 7 provides an overlay of XRPD patterns ( from bottom to top ) of : starting material ( Form 1 of Compound 1 ) ,

40 Form 2 as obtained from slurry conversion experiment in 1 , 2 ethanediol and Form 2 after exposure to accelerated aging conditions ( AAC ) . A list of X - Ray Diffraction Peaks for Form 2 of Compound 1 is provided below in Table 26 .

Form Form Nature

1 11 11 % 99 . 9 100

Stable Form 2

268 . 9 95 - 176 ( broad ) ,

264

?? 91 . 7 Form 3

Anhydrate Solvate ( 15 . 5 % of 1 , 2 ethanediol 1 molecule of 1 , 2 - ethanediol per molecule of API ) Solvate ( 12 . 8 % of TFE - 0 . 5 molecule of TFE per molecule of API ) Solvate ( 16 . 4 % of DMSO - 0 . 8 molecule of DMSO per molecule of API ) Hydrate ( 9 . 4 % of water - 1 . 9 molecules of water per molecule of API )

149 ( broad ) ,

254

4 93 . 6 Forms 1 + 4 139 ( broad ) , 258

5 90 . 1 Form 5 80 ( broad ) , 181 ( exo ) , 251

TABLE 26 X - Ray Diffraction Peaks for Form 2 of Compound 1

6 . 1 . 2 . 14 Form 1 The XRPD pattern , crystal habit , TGA , SDTA , TGA - MS ,

HPLC and MS of Form 1 of Compound 1 are shown in FIGS . 2 - 6 .

FIG . 2 provides an XRPD pattern of Form 1 of Compound 50 — 1 . A list of X - Ray Diffraction Peaks for Form 2 of Com pound 1 is provided below in Table 25 .

Relative Intensity ( % ) Two - theta angle o d Space ( 8 )

TABLE 25 55 55

X - Ray Diffraction Peaks for Form 1 of Compound 1

6 . 18 10 . 02 11 . 54 12 . 34 13 . 86 18 . 54 21 . 74 22 . 5 23 . 42 24 . 54 25 . 5 26 . 02 26 . 7 27 . 82 28 . 34 34 . 14

14 . 28 8 . 82 7 . 66 7 . 16 6 . 38 4 . 78 4 . 08 3 . 95 3 . 79 3 . 62 3 . 49 3 . 42 3 . 33 3 . 2 3 . 15 2 . 62

86 . 62 17 . 74 28 . 21 49 . 02 19 . 58 32 . 73 71 . 24 35 . 65 47 . 77 30 . 05 12 . 63 20 . 22 81 . 52 15 . 25 34 . 21 16 . 39

Two - theta angle o d Space ( 8 ) Relative

Intensity ( % )

11 . 12 9 . 07 60

7 . 4 .

7 . 94 9 . 74

11 . 94 15 . 86 17 . 3 17 . 86 19 . 46 25 . 14 26 . 42

5 . 58 5 . 12 4 . 96 4 . 56 3 . 54 3 . 37

11 . 54 87 . 52 33 . 02 37 . 83 26 . 24 20 . 51 11 . 69 79 . 73 25 . 15

65 FIG . 8A is a digital image of Form 2 of Compound 1 . FIG . 8B is a digital image of Form 2 of Compound 1 after exposure to accelerated aging conditions .

54 US 9 , 981 , 971 B2

53 FIGS . 9 and 10 provide TGA / SDTA signal and TGA - MS The TGA thermogram of Form 3 in FIG . 14 shows a mass

data , respectively , of Form 2 as obtained from a slurry loss corresponding to a broad endothermic event observed in conversion experiment in 1 , 2 - ethanediol . the SDTA signal between 110° C . and 175° C . with a

The TGA thermogram of Form 2 in FIG . 9 shows a mass maximum at about 149° C . , which may be the desolvation of loss corresponding to a broad endothermic event observed in 5 Form 3 . After the desolvation , the SDTA data of Form 3 in the SDTA signal between 95 and 176° C . with a maximum FIG . 14 shows a melting event at 254° C . , corresponding to at about 137° C . , which may be the desolvation of Form 2 the melting point of the starting material , Form 1 of Com of Compound 1 . After the desolvation , the SDTA data of pound 1 . The temperature difference of the melting of the Form 2 in FIG . 9 shows a melting event at 264° C . , anhydrous Form 1 ( Tpeak 264° C . ) and after desolvation of corresponding to the melting point of Form 1 of Compound Form 3 ( Tpeak 254° C . ) can be attributed to the partial

degradation observed in the HPLC analysis . The chemical The TGA thermogram of Form 2 in FIG . 10 comprises a purity of Form 3 sample was determined by HPLC in FIG .

total mass loss of approximately 15 . 5 % of the total mass of 16 to be 91 . 8 % . the sample between approximately 95° C . and approxi The TGA thermogram of Form 3 in FIG . 15 comprises a mately 175° C . when heated from approximately 25° C . to total mass loss of approximately 12 . 8 % of the total mass of approximately 300° C . Thus , Form 2 loses about 15 . 5 % of the sample between approximately 40° C . and approxi its total mass when heated from about ambient temperature mately 190° C . when heated from approximately 25° C . to

to about 300° C . The thermal data indicates that Form 2 approximately 300° C . Thus , Form 3 loses about 12 . 8 % of its total mass when heated from about ambient temperature contains 1 molar equivalent of solvent in the crystal lattice 20 to about 300° C . The thermal data indicates that Form 3 corresponding to approximately 1 mole of 1 , 2 - ethanediol contains 0 . 5 molar equivalents of solvent in the crystal per mole of Compound 1 . The theoretical 1 , 2 - ethanediol lattice corresponding to approximately 0 . 5 mole of 2 , 2 , 2

content of a 1 , 2 - ethanediol mono - solvate of Compound 1 is trifluoroethanol per mole of Compound 1 . The theoretical 15 . 6 % by weight , matching the TGA weight loss observed . 2 , 2 , 2 - trifluoroethanol content of a 2 , 2 , 2 - trifluoroethanol These observations suggest that Form 2 is a 1 , 2 - ethanediol 25 hemi - solvate of Compound 1 is 11 . 5 % by weight , matching mono - solvate of Compound 1 . the TGA weight loss observed . These observations suggest

FIG . 11 provides HPLC and MS data of Form 2 as that Form 3 is a 2 , 2 , 2 - trifluoroethanol hemi - solvate of obtained from the slurry conversion experiment in 1 , 2 Compound 1 . ethanediol . The peak retention time is 4 . 8 minutes and FIG . 16 provides HPLC and MS data of Form 3 as indicates the sample purity is 100 % ( area % ) . obtained from an evaporative experiment in TFE / water

6 . 1 . 2 . 16 Form 3 ( 50 : 50 ) . The peak retention time is 4 . 8 minutes with a The XRPD pattern , crystal habit , TGA , SDTA , TGA - MS , sample purity of 91 . 8 % ( area % ) .

HPLC and MS of Form 3 of Compound 1 are shown in 6 . 1 . 2 . 17 Form 4 FIGS . 12 - 16 . Form 3 was produced in a variety of crystal - The XRPD pattern , crystal habit , TGA , SDTA , TGA - MS , lization solvents , including : 2 , 2 , 2 - trifluoroethanol ( TFE ) 3 HPLC and MS of Form 4 of Compound 1 are shown in combined with either water or cyclohexane , chloroform and FIGS . 17 - 21 . Form 4 was prepared by anti - solvent crystal the solvent mixture of isopropanol and acetone . Most of the lization with DMSO as solvent and water as anti - solvent . Form 3 samples showed a yellowish color . The samples used Form 4 is physically unstable and converses to Form 1 or for further analyses were prepared in the evaporative experi - mixtures of Forms 1 and 4 upon exposure to accelerated ment in TFE / water ( 50 : 50 ) . 40 aging conditions . Most likely after long term stability con

FIG . 12 provides an overlay of XRPD patterns ( from ditions , full conversion to Form 1 may occur . bottom to top ) of : starting material ( Form 1 of Compound 1 ) , FIG . 17 provides an overlay of XRPD patterns ( from Form 3 as obtained from evaporative experiment in TFE / bottom to top ) of : starting material , Form 1 of Compound 1 ; water ( 50 : 50 ) and Form 3 after exposure to accelerated Form 4 as wet solid obtained from an anti - solvent experi aging conditions ( AAC : 2 days at 40° C . / 75 % RH ) . A list of 45 ment using DMSO as sols ment using DMSO as solvent and water as anti - solvent ; X - Ray Diffraction Peaks for Form 3 of Compound 1 is Form 4 as dried solid obtained from an anti - solvent experi provided below in Table 27 . ment using DMSO as solvent and water as anti - solvent ;

Amorphous Form of Compound 1 as wet solid from an TABLE 27 anti - solvent experiment using DMSO as solvent and water

50 as anti - solvent after exposure to accelerated aging condi X - Ray Diffraction Peaks for Form 3 of Compound 1 _ tions ( AAC : 2 days at 40° C . / 75 % RH ) ; mixture of Forms 1 Relative and 4 as dried solid obtained after exposure to accelerated

Two - theta angle o d Space ( Å ) Intensity ( % ) aging conditions ( AAC ) . A list of X - Ray Diffraction Peaks for Form 4 of Compound 1 is provided below in Table 28 . 3 . 5 25 . 21 29 . 74

7 . 06 16 . 87 9 . 26 9 . 54 79 . 97 TABLE 28

10 . 5 11 . 23 12 . 66 6 . 98 13 . 38 X - Ray Diffraction Peaks for Form 4 of Compound 1

5 . 78 19 . 31 18 . 62 4 . 76 Relative

Two - theta angle o d Space ( 8 ) Intensity ( % ) FIG . 13A is a digital image of Form 3 of Compound 1 . 8 . 22 10 . 74 12 . 38

FIG . 13B is a digital image of Form 3 of Compound 1 after 10 . 14 8 . 71 28 . 85 10 . 66 8 . 29 42 . 92 exposure to accelerated aging conditions . 14 . 02 6 . 31 19 . 57

FIGS . 14 and 15 provide TGA / SDTA signal and TGA - MS 65 18 . 1 4 . 9 25 . 78 data , respectively , of Form 3 as obtained from an evapora 20 . 62 4 . 3 24 . 43

tive experiment in TFE / water ( 50 : 50 ) .

12 . 51

8 . 42

15 . 3 20 . 63

60

US 9 , 981 , 971 B2 55 56

TABLE 29 TABLE 28 - continued X - Ray Diffraction Peaks for Form 4 of Compound 1 X - Ray Diffraction Peaks for Form 5 of Compound 1

Relative Intensity ( % ) Two - theta angle o d Space ( 8 ) Two - theta angle o d Space ( 8 )

Relative Intensity ( % )

13

10

3 . 4

21 . 94 4 . 05 84 . 94 6 . 02 14 . 66 22 . 66 3 . 92 27 . 92 7 . 46 11 . 84 31 . 65 23 . 78 3 . 74 19 . 31 9 . 26 9 . 54 76 . 44 24 . 34 3 . 65 24 . 73 11 . 7 7 . 55 79 . 47 25 . 42 3 . 5 18 . 72 12 . 18 7 . 26 19 . 72 26 . 26 3 . 39 29 . 32 19 . 78 4 . 48 8 . 74

22 . 02 4 . 03 24 . 68 23 . 74 3 . 74 26 . 68

FIG . 18A is a digital image of Form 4 of Compound 1 as 24 . 26 3 . 66 28 . 87 24 . 94 3 . 57 wet solid obtained from an anti - solvent experiment using 32 . 55 26 . 18 55 . 24 DMSO as solvent and water as anti - solvent . FIG . 18B is a 15 27 . 06 3 . 29 16 . 87

digital image of Form 4 of Compound 1 as dry solid 29 . 86 2 . 99 16 . 03

obtained from an anti - solvent experiment using DMSO as solvent and water as anti - solvent . FIG . 23A is a digital image of Form 5 of Compound 1 .

FIGS . 19 and 20 provide TGA / SDTA signal and TGA - MS 20 FIG . 23B is a digital image of Form 5 of Compound 1 after data , respectively , of Form 4 as obtained from an anti exposure to accelerated aging conditions . solvent experiment using DMSO as solvent and water as FIGS . 24 and 25 provide TGA / SDTA signal and TGA - MS anti - solvent . data , respectively , of Form 5 obtained from a hot - filtration The TGA thermogram of Form 4 in FIG . 19 shows a mass experiment in THF / water ( 50 : 50 ) . loss corresponding to a broad endothermic event observed in 25 The TGA thermogram of Form 5 in FIG . 24 shows a mass the SDTA signal between 100 and 175° C . with a maximum loss corresponding to a broad endothermic event observed in at about 140° C . , which may be the desolvation of Form 4 . the SDTA signal at T 80° C . which is likely related to the After desolvation , the SDTA shows a melting event at 258° dehydration process , followed by re - crystallization at 181° C . , corresponding to the melting point of Form 1 of Com - C . and melting of Form 1 at 251° C . The large difference of pound 1 . The temperature difference between the melting of 30 the melting temperature of Form 1 here compared to that of the anhydrous Form 1 ( Tpeak 264° C . ) and the melting after the starting material Form 1 ( 264° C . ) can be attributed to desolvation of Form 4 ( Tpeak 258° C . ) can be attributed to the the different history of the two solids . Note that Form 5 was partial degradation observed in the HPLC analysis . The produced only when water was used in mixture with other chemical purity of Form 4 sample was determined by HPLC solvents , e . g . , THF , 1 , 4 - dioxane , methanol and ethanol . The in FIG . 21 to be 93 . 6 % . 35 slurry experiment in water for two weeks at room tempera

The TGA thermogram of Form 4 in FIG . 20 comprises a ture produced the anhydrous starting material , Form 1 . This total mass loss of approximately 16 . 4 % of the total mass of observation might be explained by the fact that Form 1 of the sample between approximately 35º C . and approxi Compound 1 is practically insoluble in water . Some disso mately 180° C . when heated from approximately 25° C . to lution of the starting material is needed to produce the approximately 300° C . Thus , Form 4 loses about 16 . 4 % of tu F 40 dihydrated Form 5 , which is provided by the organic solvent

( THF , 1 , 4 - dioxane , methanol or ethanol ) , followed by pre its total mass when heated from about ambient temperature cipitation of Form 5 . The chemical purity of Form 5 sample to about 300° C . The thermal data indicate that Form 4 was determined by HPLC in FIG . 26 to be 90 . 1 % . contains 0 . 8 molar equivalents of solvent in the crystal The TGA thermogram of Form 5 in FIG . 25 comprises a lattice corresponding to approximately 0 . 8 mole of dimeth - 45 total mass loss of approximately 9 . 4 % of the total mass of ylsulfoxide per mole of Compound 1 . The theoretical dim the sample between approximately 35° C . and approxi ethylsulfoxide content of a 0 . 8 molar equivalent dimethyl mately 240° C . when heated from approximately 25° C . to sulfoxide solvate of Compound 1 is 18 . 9 % by weight , approximately 300° C . Thus , Form 5 loses about 9 . 4 % of its matching the TGA weight loss observed . These observations total mass when heated from about ambient temperature to suggest that Form 4 is a dimethylsulfoxide solvate of 50 about 300° C . The thermal data indicates that Form 5 Compound 1 . contains 2 molar equivalents of solvent in the crystal lattice

FIG . 21 provides HPLC and MS data of Form 4 as corresponding to approximately 2 moles of water per mole obtained from an anti - solvent experiment using DMSO as o f Compound 1 . The theoretical water content of a dihydrate solvent and water as anti - solvent . The peak retention time is of Compound 1 is 10 . 2 % by weight , matching the TGA 4 . 8 minutes with a sample purity of 93 . 6 % ( area % ) . 55 weight loss observed . These observations suggest that Form

6 . 1 . 2 . 18 Form 5 5 is a dihydrated form of Compound 1 . The XRPD pattern , crystal habit , TGA , SDTA , TGA - MS , FIG . 26 provides HPLC and MS data of Form 5 as solid

HPLC and MS of Form 5 of Compound 1 are shown in obtained from a hot - filtration experiment in THF / water FIGS . 22 - 26 . Form 5 was prepared in hot - filtration experi - ( 50 : 50 ) . The peak retention time is 4 . 8 minutes with a ments in THF / water ( 50 : 50 ) . Form 5 appears stable for at 60 sample purity of 89 . 9 % ( area % ) . least two days under accelerated aging conditions . 6 . 1 . 2 . 19 Amorphous Form

FIG . 22 provides an overlay of XRPD patterns ( from The DSC , XRPD pattern , Raman spectrum , NMR , HPLC bottom to top ) of : starting material , Form 1 of Compound 1 ; and MS of amorphous Compound 1 are shown in FIGS . Form 5 of Compound 1 ; and Form 5 of Compound 1 after 27 - 32 . exposure to accelerated aging conditions ( AAC : 2 days at 65 Amorphous Compound 1 was prepared by 1 ) equilibrat 40° C . / 75 % RH ) . A list of X - Ray Diffraction Peaks for Form i ng the temperature of a sample of Form 1 at 25° C . ; 2 ) 5 of Compound 1 is provided below in Table 29 . heating up the sample to 275º C . at a rate of 10° C . / min ; 3 )

US 9 , 981 , 971 B2 57 58

pound 1 . 10

5

10 10

30

+

1

104 104 104

holding the sample isothermally for 5 minutes ; 4 ) cooling Study 1 : the sample to - 10° C . at a rate of 30° C . / min ; 5 ) heating the A 23 - 1 study evaluates the effect of diluents , disintegrant sample to 150° C . at a rate of 10° C . / min ; and 6 ) collecting and drug loading on tablet physical properties and chemical remaining solids . stability . Examples of formulation compositions are shown The differential scanning calorimetry thermal analysis of 5 in T in Table 30 . Initial tablet development is carried out in amorphous Compound 1 in FIG . 27 shows that the glass normal room UV light . transition temperature ( Tg ) of amorphous Compound 1 is at 120° C . TABLE 30 FIG . 28 provides an XRPD pattern of amorphous Com

Exemplary Formulation Composition FIG . 30 provides a proton nuclear magnetic resonance Of Various Tablet Formulations

spectrum of amorphous Compound 1 . FIG . 31 provides HPLC and MS data of amorphous Solid Form of Compound 1 ( mg ) 0 . 5 0 . 5 0 . 5 0 . 5 5

Compound 1 . Microcrystalline Cellulose ( mg ) 63 . 75 83 . 75 59 . 25 79 . 25 The DSC thermogram of amorphous Compound 1 in FIG . 15 Partially pregelatinized corn starch

32 shows a broad endothermic event between 160 and 200° ( mg ) C . with a maximum at about 188 . 1° C . Lactose monohydrate , spray dried 30

( mg ) 6 . 2 Biological Examples Crospovidone ( mg )

20 . Croscarmellose Na ( mg ) 4 6 . 2 . 1 Biochemical Assays Silicon dioxide ( mg )

0 . 75 Magnesium Stearate ( mg ) 0 . 75 0 . 75 TOR HTR - FRET Assay . The following is an example of 0 . 75 an assay that can be used to determine the TOR kinase total uncoated tablet ( mg ) 100 100 100 100 inhibitory activity of solid forms of Compound 1 . A solid Opadry II coating ( mg ) form of Compound 1 is dissolved in DMSO and prepared as 25 10 mM stocks and diluted appropriately for the experiments . total coated tablet ( mg ) 104 104 104 104 104 Reagents are prepared as follows :

“ Simple TOR buffer ” ( used to dilute high glycerol TOR fraction ) : 10 mM Tris pH 7 . 4 , 100 mM NaCl , 0 . 1 % Tween Study 2 : 20 , 1 mM DTT . Invitrogen recombinant TOR enzyme ( cat # 30 PV4753 ) is diluted in this buffer to an assay concentration of A study is conducted to evaluate the effect of antioxidant 0 . 200 ug / mL . ( e . g . , butylated hydroxyl toluene , BHT ) and chelating agent

ATP / Substrate solution : 0 . 075 mM ATP , 12 . 5 mM MnCl2 , ( e . g . , disodium edentate , Na , - EDTA ) on the stability of solid 50 mM Henes , pH 7 . 4 . 50 mM B - GOP 250 nM Microcystin forms of Compound 1 in formulated product . The impact of LR , 0 . 25 mM EDTA , 5 mM DTT , and 3 . 5 ug / mL GST - 35 dosage form ( tablet vs capsule ) on the stability of solid p70S6 . forms of Compound 1 is evaluated .

Detection reagent solution : 50 mM HEPES , pH 7 . 4 , Examples of formulation compositions are shown in 0 . 01 % Triton X - 100 , 0 . 01 % BSA , 0 . 1 mM EDTA , 12 . 7 Table 31 . All of the processes are carried out in dark . ug / mL Cy5 - aGST Amersham ( Cat # PA92002V ) , 9 ng / mL

- phospho p70S6 ( Thr389 ) ( Cell Signaling Mouse Mono - 40 TABLE 31 clonal # 9206L ) , 627 ng / mL a - mouse Lance Eu ( Perkin Elmer Cat # AD0077 ) . Exemplary Formulation Composition

To 20 uL of the Simple TOR buffer is added 0 . 5 uL of test % w / w solid form in DMSO . To initiate the reaction 5 uL of ATP / Substrate solution is added to 20 uL of the Simple TOR 45 Cap - Cap - Cap - Cap - Tab - Cap buffer solution ( control ) and to the compound solution Ingredients sule sule sule sule let sule prepared above . The assay is stopped after 60 minutes by Solid Form of 0 . 5 0 . 5 0 . 5 0 . 5 0 . 5 0 . 5 adding 5 uL of a 60 mM EDTA solution ; 10 uL of detection Compound 1 reagent solution is then added and the mixture is allowed to Mannitol 84 94 . 1 - 93 . 6 83 . 6 - sit for at least 2 hours before reading on a Perkin - Elmer 50 ( Mannogem EZ )

MCC PH112 94 . 1 Envision Microplate Reader set to detect LANCE Eu TR Lactose 93 . 6 FRET ( excitation at 320 nm and emission at 495 / 520 nm ) . Sodium starch DNA - PK Assay . glycolate DNA - PK assay is performed using the procedures sup stearic acid

70 plied in the Promega DNA - PK assay kit ( catalog # V7870 ) . 55 55 Butylated hydroxy 0 . 4 toluene DNA - PK enzyme can be purchased from Promega ( Pro Naz - EDTA 0 . 5

mega cat # V5811 ) . Mg stearate

6 . 3 Formulation Examples Total 100 100 100 100 100 100

0 . 5 0 . 5

5

? ? 93 . 6 w w w

1 1 1 - - -

- o

05 0 . 5

- 1

60 Certain formulations comprising solid forms of Com Study 3 : pound 1 are prepared and tested for a number of physical and

chemical properties . Modifications are made and subsequent Further study can be conducted to study the influence of formulations are also tested , until formulations possessing coating and desiccant on the stability of Compound 1 tablets . desirable physical and chemical properties are found . The 65 All processes can be carried out under yellow light to following example describes these formulations and their prevent any UV light exposure to the Compound 1 formu testing . lations .

US 9 , 981 , 971 B2 59

An exemplary formulation composition is provided in Table 32 .

60 TABLE 34 - continued

Exemplary Low Strength Tablet Formulation # 1 TABLE 32 Exemplary Formulation Composition Of Tablet Amount

( weight % ) Ingredients lients % w / w % w / w Ingredient Source

0 . 5 83 . 6 Mannitol

10 Roquette FMC Biopolymer

57 . 2 3 . 0 1 sodium 3 . 0

Solid form of Compound 1 Mannitol ( Mannogem EZ ) MCC PH112 Sodium starch glycolate stearic acid Butylated hydroxy toluene Naz - EDTA Mg stearate

?? ?? ? carboxymethylcellulose magnesium stearate Nitika Chemicals 1 . 0 1 . 0 0 . 4

0 . 5

15 Total

TABLE 35 Exemplary Low Strength Tablet Formulation # 2

TABLE 33 20 Ingredient Amount

( weight % ) Source Exemplary Tablet Formulations

% w / w ( mg ) Batch #

Solid form of Compound 1 microcrystalline cellulose pregelatinized starch sodium carboxymethylcellulose magnesium stearate

FMC Biopolymer Colorcon FMC Biopolymer

0 . 7 75 . 3 20 . 0

3 . 0 Ingredients 1 2 3 4 25 1 . Nitika Chemicals 1 . 0

9

s w Can Gunm de TABLE 36

Solid form of Compound 1 ( active ingredient ) Mannitol ( Mannogem EZ ) Microcrystalline Cellulose ( PH 112 ) Sodium Starch Glycolate Silicon dioxide Stearic acid Disodium EDTA BHT Magnesium Stearate

30

m Exemplary Low Strength Tablet Formulation # 3 0 . 5 0 . 5 0 . 5 0 . 5

0 . 5 0 . 5 0 . 5 0 . 5 0 . 4 0 . 65

Ingredient Amount

( weight % ) Source 0 . 4 0 . 65 0 . 65 0 . 65 - 35 0 . 7

Total Color

100 Yellow

100 Yellow

100 Yellow

100 Yellow

Solid form of Compound 1 microcrystalline cellulose Lactose monohydrate sodium carboxymethylcellulose magnesium stearate

FMC Biopolymer Meggle Pharma FMC Biopolymer

38 . 1 57 . 2

3 . 0

Nitika Chemicals 1 . 0

pared 40 TABLE 37

Exemplary High Strength Tablet Formulation # 1 45

Amount ( weight % ) Ingredient Source

Preparation of Tablets : The blends according to Table 34 to Table 39 are prepared

as follows . Microcrystalline cellulose is weighed and added to an amber colored straight sided glass jar . The lid is closed and the jar is shook in order to coat the inside of the jar . Active ingredient ( solid form of Compound 1 ) is added and blended for 10 minutes at 46 rpm using a Turbula mixer . The blend is passed through a 25 mesh screen and blended again for 10 minutes at 46 rpm using a Turbula mixer . The resulting blend is passed through a 35 mesh screen . Remain ing excipients are added , except for lubricant ( magnesium stearate ) . The resulting mixture is blended for 10 minutes at 46 rpm using a Turbula mixer . 6 grams of the resulting blend is added an amber glass jar . Lubricant is added and blended for 1 minute and 35 seconds at 46 rpm using a Turbula mixer . For low strength tablet formulations , 140 mg tablets se are prepared using a 7 . 14 mm punch and die . For high strength tablet formulations , 400 mg tablets are prepared using a 10 . 3 mm punch and die .

Solid form of Compound 1 microcrystalline cellulose Mannitol sodium carboxymethylcellulose magnesium stearate

FMC Biopolymer Roquette FMC Biopolymer

25 . 0 28 . 4 42 . 6

3 . 0 50

Nitika Chemicals 1 . 0

TABLE 38 Exemplary High Strength Tablet Formulation # 2

TABLE 34 Amount

( weight % ) Ingredient Source

Exemplary Low Strength Tablet Formulation # 1 Solid form of Compound 1 microcrystalline cellulose pregelatinized starch sodium carboxymethylcellulose magnesium stearate

25 . 0 51 . 0 20 . 0

3 . 0

FMC Biopolymer Colorcon FMC Biopolymer

Amount ( weight % ) Ingredient Source

65 Nitika Chemicals 1 . 0 Solid form of Compound 1 microcrystalline cellulose

0 . 7 38 . 1 FMC Biopolymer

61 TABLE 39

US 9 , 981 , 971 B2 62

What is claimed is : 1 . An amorphous form of the compound of formula ( I ) :

Exemplary High Strength Tablet Formulation # 3

Amount Ingredient Source ( weight % )

10 10 28 . 4 2

1 . 0 15

Solid form of Compound 1 25 . 0 microcrystalline cellulose FMC Biopolymer 28 . 4 Lactose monohydrate Meggle Pharma 42 . 6

sodium FMC Biopolymer 3 . 0

carboxymethylcellulose magnesium stearate Nitika Chemicals 1 . 0 15

or a tautomer thereof , wherein the amorphous form has an X - ray powder dif

The above formulations are subjected to a 6 week stability fraction pattern as shown in FIG . 28 . study . 2 . The amorphous form of claim 1 , which has a differ

20 ential scanning calorimetry thermogram comprising an The embodiments disclosed herein are not to be limited in endotherm between 160° C . + 5° C . and 200° C . + 5° C . with

scope by the specific embodiments disclosed in the a maximum at 188 . 1° C . - 5° C . 3 . The amorphous form of claim 1 , which has a glass examples which are intended as illustrations of a few aspects w aspects transition temperature at 120° C . - 5° C .

of the disclosed embodiments and any embodiments that are 25 4 . The amorphous form of claim 1 , which is pure . 5 . A method for modulating target of rapamycin kinase functionally equivalent are encompassed by the present activity in a subject , comprising administering to a subject

disclosure . Indeed , various modifications of the embodi - in need thereof an effective amount of the amorphous form ments disclosed herein are in addition to those shown and of claim 1 .

6 . The method of claim 5 , wherein the subject suffers from described herein will become apparent to those skilled in the 30 a condition , disorder or disease selected from the group art and are intended to fall within the scope of the appended consisting of an inflammatory condition , an immunological

condition , a cardiovascular condition , a neurological disor claims . der , a neurodegenerative disease , an age - related disease , Anumber of references have been cited , the disclosures of diabetes and obesity .

which are incorporated herein by reference in their entirety . * * * * *

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