US010624968B2
(12 ) United States Patent (10 ) Patent No.: US 10,624,968 B2 Bennett et al. (45 ) Date of Patent : Apr. 21 , 2020
( 54 ) COMPOUNDS FOR TREATING CANCER WO 2005113554 12/2005 WO 2006078161 7/2006 WO 2006078846 7/2006 (71 ) Applicant: Bicycle Therapeutics Limited , WO 2006122806 11/2006 Cambridge (GB ) WO 2007016176 2/2007 WO 2007044729 4/2007 ( 72 ) Inventors : Gavin Bennett , Cambridge (GB ) ; WO 2007053452 5/2007 Daniel Paul Teufel , Cambridge (GB ) WO 2007070514 6/2007 WO 2007084786 7/2007 WO 2007129161 11/2007 ( 73 ) Assignee : BICYCLERD LIMITED , Cambridge WO 2008039218 4/2008 (GB ) WO 2008109943 9/2008 WO 2008118802 10/2008 ( * ) Notice : Subject to any disclaimer, the term of this WO 2009098450 8/2009 patent is extended or adjusted under 35 WO 2009114512 9/2009 WO 2010089115 8/2010 U.S.C. 154 ( b ) by 0 days. WO 2011090760 7/2011 WO 2013050615 4/2013 (21 ) Appl. No.: 15 /862,964 WO 2016067035 5/2016 WO 2017191460 11/2017 ( 22 ) Filed : Jan. 5 , 2018 WO 2018127699 7/2018 (65 ) Prior Publication Data OTHER PUBLICATIONS US 2018/0200378 A1 Jul. 19 , 2018 Paul Polakis . Antibody Drug Conjugates for Cancer Therapy. Pharmacol Rev. Jan 1 , 2016 ;68 ( 1 ): 3-19 . ( Year: 2016 ). * Related U.S. Application Data Heinis et al. Phage- encoded combinatorial chemical libraries based (60 ) Provisional application No. 62/ 443,508, filed on Jan. on bicyclic peptides . Nat Chem Biol, 2009 ; 5 (7 ): 502-07. ( Year: 2009) . * 6 , 2017 Eder et al. A phage display derived stabilised bicyclic peptide (51 ) Int. Ci. targeting MMP- 14 shows high imaging contrast in small animal A61K 47/64 ( 2017.01 ) PET imaging. Eur J Nucl Med Mol Imaging (2015 ) 42 (Suppl A61P 35/00 ( 2006.01 ) 1 ) :S140 - OP337 . ( Year : 2015 ). * A61K 38/08 Berge et al. , “ Pharmaceutical Salts , ” Journal of Pharmaceutical ( 2019.01 ) Sciences , vol. 66 , No. 1 , Jan. 1977 (pp . 1-19 ). CO7K 7764 ( 2006.01 ) ClinicalTrials.gov identified NCT02488759, “ An Investigational CO7K 7/50 ( 2006.01 ) Immuno -therapy Study to Investigate the Safety and Effectiveness (52 ) U.S. Cl. of Nivolumab , and Nivolumab Combination Therapy in Virus CPC A61K 47/64 (2017.08 ) ; A61K 38/08 associated Tumors (CheckMate358 ) ,” https://clinicaltrials.gov/ct2/ ( 2013.01 ) ; A61P 35/00 ( 2018.01 ); CO7K 7764 show / study/ NCT02488759 ( 7 pages ). ( 2013.01 ); CO7K 7/50 ( 2013.01 ) ClinicalTrials.gov identifier NCT02426892 , “ Nivolumab and HPV ( 58 ) Field of Classification Search 16 Vaccination in Patients with HPV - 16 Positive Incurable Solid CPC A61K 38/12 , A61K 47/64 ; A61K 38/00 ; Tumors , ” https://clinicaltrials.gov/ct2/show/study/NCT02426892 ( 8 C12N 2310/351 ; CO7K 16/2863 ; COOK pages ) 14/001 ; CO7K 7/64 ; CO7K 1/1072 ; COOK Okazaki et al. , “ A rheostat for immune responses : the unique 2319/70 properties of PD - 1 and their advantages for clinical application ,” See application file for complete search history. Nature Immunology, vol. 14 , No. 12, Dec. 2013 (pp . 1212-1218 ). Sounni et al ., “ MT1- MMP expression promotes tumor growth and ( 56 ) References Cited angiogenesis through an up -regulation of vascular endothelial growth factor expression , " FASEB Journal . vol. 16 , No. 6 , Apr. 2002 (pp . U.S. PATENT DOCUMENTS 555-564 ) . 6,552,065 B2 4/2003 Remiszewski et al . (Continued ) 7,390,799 B2 6/2008 Bruncko et al. 8,138,347 B2 3/2012 Adams et al. 8,906,682 B2 12/2014 June et al. Primary Examiner Marcela M Cordero Garcia 2013/0064791 A1 * 3/2013 Poelstra A61K 38/12 Assistant Examiner - Jia -Hai Lee 424 / 85.5 2013/0072598 Al * 3/2013 Yang COSH 1/06 (74 ) Attorney, Agent, or Firm Andrea L. C. Reid ; 524/10 Dechert LLP FOREIGN PATENT DOCUMENTS (57 ) ABSTRACT WO 2001042246 6/2001 WO 2002088112 11/2002 The present invention provides compounds, compositions WO 2003063794 8/2003 reof, and methods of using the same. WO 2004019973 3/2004 WO 2004077062 9/2004 WO 2004089925 10/2004 30 Claims, 17 Drawing Sheets WO 2004106328 12/2004 WO 2005007623 1/2005 Specification includes a Sequence Listing . US 10,624,968 B2 Page 2
( 56 ) References Cited
OTHER PUBLICATIONS Zou et al ., “ PD -L1 (B7 -H1 ) and PD - 1 pathway blockade for cancer therapy : Mechanisms, response biomarkers , and combinations, " Science Translational Medicine , vol. 8 , No. 328 , Mar. 2016 (34 pages ). Guangzhi et al. , “ The influence of the penetrating peptide iRGD on the effect of paclitaxel- loaded MT1 -AF7p -conjugated nanoparticles on glioma cells” , Biomaterials , vol. 34 , No. 21, 2013, pp . 5138 5148 International Search Report for PCT/GB2018 /050017 . * cited by examiner U.S. Patent Apr. 21 , 2020 Sheet 1 of 17 US 10,624,968 B2
BT17BDC - 23 in human plasma t = >> 24 hrs 1/2 cremaining 96 % remaining after 6 hrs BT17BDC - 23 in mouse plasma t = 6.4 hrs 1/2 57 % remaining after 6 hrs
2
Figure 1 U.S. Patent Apr. 21 , 2020 Sheet 2 of 17 US 10,624,968 B2
BT17BDC - 23 mouse plasma ( 4 UM + S ) with and without AESBF inhibitor
20
Figure 2 U.S. Patent Apr. 21 , 2020 Sheet 3 of 17 US 10,624,968 B2
BDC23 and MMAE metabolites
BDC23 , AEBSF
80 BDC23 , -AEBSF
%buluewal free MMAE, -AEBSE
20
free MMAE , & AEBSF
15 20 30 time (hrs ] Col1 vs 1293 > 1203 BD023 with AESSF
Figure 3 U.S. Patent Apr. 21 , 2020 Sheet 4 of 17 US 10,624,968 B2
2000 Vehicle the 1-1 0.1mg/ kg + 1-10.3mg/ kg + 1-1 1mg /kg 1500 1-1 3mg/ kg TumourVolume(mm”) 1000
500
0 2 6 8 10 12 14 A Days after start of dosing
Figure 4 U.S. Patent Apr. 21 , 2020 Sheet 5 of 17 US 10,624,968 B2
2000 Vehicle + 1-1 1mg/ kg on thin 1-1 1mg/ kg + N241 1500 TumourVolume(mm”) H 1000
500
0 0 2 4 6 8 10 12 14 Days after start of dosing
Figure 5 U.S. Patent Apr. 21 , 2020 Sheet 6 of 17 US 10,624,968 B2
3000 Vehicle , iv , biw E) . Docetaxel 20mpk , iv , qw 1-1 10mg/ kg 1-1 3mg/ kg 1-1 1mg/ kg 2000 TumourVolume(mm)
1000
0+ 0 20 40 60 A A A A ? ? ? ? ? Days after start of dosing
Figure 6 U.S. Patent Apr. 21 , 2020 Sheet 7 of 17 US 10,624,968 B2
1500 motor Vehicle E) . Docetaxel 20mpk , iv , qw |-1 3mg/ kg 1-1 1mg/ kg 1000 A mm)Volume(Tumour H 500 ---
0 0 10 20 30 ? Days after start of dosing
Figure 7 U.S. Patent Apr. 21, 2020 Sheet 8 of 17 US10,624,968 B2
26 ?? Vehicle , iV, biw 3. Docetaxel 20mpk, iv , qw 1-1 3mg/ kg 243 1-1 1mg/ kg weight(g)Body 22 1 20 .. Y 18 .....? ????? 16 0 10 20 30 A A ? A Days after start of dosing
Figure 8 U.S. Patent Apr. 21, 2020 Sheet 9 of 17 US 10,624,968 B2
? 1-3a , 10 mpk , biw 22 1-3a , 3 mpk, biw 1-3a , 1 mpk , biw 20 Vehicle
18 H
16 4 6 8 10 12 14 A A Days after start of dosing
B 26 1-7 , 10 mpk , biw 24 1-7,3 mpk , biw Bodyweight(g) 1-7 , 1 mpk , biw 22 Vehicle 20 18 16 2 6 8 10 12 14
< Days after start of dosing
? 1-4a , 10 mpk , biw 22 1-4a , 3 mpk , biw Bodyweight(g) 1-4a , 1 mpk , biw 20 Vehicle
18
16 2 4 8 10 12 14 n Days after start of dosing
D 26 1-8 , 10 mpk , biw 24 1-8 , 3 mpk , biw 1-8 , 1 mpk , biw Bodyweight(g) Vehicle 20 co 1 16 1 0 2 8 10 12 14
< A Days after start of dosing Figure 9 U.S. Patent Apr. 21 , 2020 Sheet 10 of 17 US 10,624,968 B2
A B
2500 , Vehicle 25009 Vehicle l - 3a , 1 mpk , biw 1-7 , 1 mpk , biw f- 3a , 3 mpk , biw 1-7,3 mpk , blu 20004 3-3a , 10 mpk , biw 2000 W 1-7, 10 mpk , biw
TumourVolume)(mm 1500 TumourVolume(mm) 1500
1000 1000
500 H
0 2 4 HH 6 12 14 4 6 8 12 14 Days after start of dosing Days after start ofdosing ?
2500 2500 Vehicle Vehicle 1-4a , 1 mpk , biw 4-8 , 1 mpk , biw 1-42, 3 mpk , biw 48 , 3 mpk , biw 2000 - 1-4a , 20 mpk , biw 2000 8-8 , 10 mpk , biw
TumourVolume(mm) 1500 TumourVolume(mm) 1500
1000 1000
500-1 H 5004 it 2 4 6 8 14 4 6 8 12 14 Days after start of dosing Days after start of dosing
Figure 10 U.S. Patent Apr. 21 , 2020 Sheet 11 of 17 US 10,624,968 B2
A 26 24 Bodyweight(g) 224 20 18 16 14- 0 2 4 6 8 10 12 14 Days after startA of dosing
B 1400 Vehicle 1200 1-12 , 1 mp /kg , qw TumourVolume(mm) 1000 met tona 1-12 , 3 mp/ kg , qw 800 H 1-12 , 10 mp /kg , qw 600 400 200 0 0 2 6 8 10 12 Days after start of dosing
Figure 11 U.S. Patent Apr. 21 , 2020 Sheet 12 of 17 US 10,624,968 B2
A
26 . 24
)Bodyweight(g 22
20
18
16+ 0 2 4 6 8 10 12 14 A Days after start of dosingA
B 1400 Vehicle 1200 within 1-16 , 1 mp/ kg , biw 1000 patronicPoornana 1-16 , 3 mp /kg , biw TumourVolumemm)( 800 1-16 , 10 mp/ kg , biw 600 400 200 0 0 2 4 6 8 10 12 14 A ? Days after start of dosing
Figure 12 U.S. Patent Apr. 21, 2020 Sheet 13 of 17 US 10,624,968 B2
OH Boc Boc HN . Boc OH Solid phase Weak acid (9 ) OH po C NH ?? NH 1 2 3 NH " NH2 " NH?
NO2 OH NO2 Boc N. , Boc N MMAE N NH 5 AN " NH2 H2N
OM
TFA K2CO3. ????? ( S ) (S ) NH2 6 HN H2N OH OH
OH
HN 7 HN OH ve HN 8 HN
NH2 H2N HO H?O L - NH NH NH NH HN (S ) NH H?C NH + ICH3 H2 NH C - S - C 1-12
?.? N OH P - 1 H2NH2C NH OH HN NH HN .
HO OH Figure 13 U.S. Patent Apr. 21 , 2020 Sheet 14 of 17 US 10,624,968 B2
AN HO ??? NH NB UN S ) NH Hac H20 -CH? NH 4-15
P.2 N CH? HaN tc NH OH HN
NA
HEN 1-16 HN HN CH HOC HOVOLCI HUN UN CHO CHO CN
P - 3
Cits SO CH : NH CHO CHO CH
Figure 13 (continued ) U.S. Patent Apr. 21 , 2020 Sheet 15 of 17 US 10,624,968 B2
234MMAF 2354Allyl- MMAF
Boca soron Boc . HN "NH? 4 - 3 ?? ??? - 23 ???????(S ) NH2
NH -0.05OH
H ( S ) "N { }s ( ( S ) *)s ( HN H?N Figure 14 U.S. Patent Apr. 21 , 2020 Sheet 16 of 17 US 10,624,968 B2
AN HN NH2 H2 NH OH HN “ NH CH3 -NH2 EN --NH HN H20 NH
Hac NH HN NH OH AN HO Hac NH? H? NH C- S H2 OHinte HN N8 CHA HAN
H2C NH HN
co HO P - 6
Figure 14 ( continued ) U.S. Patent Apr. 21 , 2020 Sheet 17 of 17 US 10,624,968 B2
( S
( R ) 11 ( R ) ) VS ) OV! ( R )
UN
HON NH HN r - NH HN
IS )
S ( S ) HN HN HN
HN NA
N -
UN NH2 NH2 NH NB H2 H2 OH HN "NH CH3 HNti UN N8 Cha NH HN H2C Mac
M2C NH H2C NM NE HN N NHO NH UN OH BN -NM
HO HO HO 9B IA
PdfpPh3 /4 , Ph?ih Pd (PPh3 / 4 , PhSiH 1-14 Figure 14 ( continued ) US 10,624,968 B2 2 COMPOUNDS FOR TREATING CANCER DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION CROSS - REFERENCE TO RELATED 1. Compound APPLICATION A proprietary phage display and cyclic peptide technology 5 (Bicycle technology ) was utilized to identify high affinity This application claims the benefit under 35 U.S.C. $ binding peptides to the membrane type 1- matrix metallo 119 ( e ) of U.S. Provisional Application No.62 /443,508 , filed proteinase (MT1 - MMP / MMP14 ) . MT1- MMP (MT1 ) is a Jan. 6 , 2017 , the content of which is incorporated herein in cell surface membrane protease normally involved in tissue its entirety by reference . remodeling which has been found to be over- expressed in 10 many solid tumors . Overexpression ofMT1 has been linked to cancer invasiveness and poor prognosis . While attempts BACKGROUND OF THE INVENTION to target the proteolytic activity of MT1 and other MMPs in MT1- MMP is a transmembrane metalloprotease that cancer were unsuccessful in clinical trials largely due to plays a major role in the extracellular matrix remodelling, 15 remainstoxicity ancaused attractive by insufficientcancer target selectivity for targeted , MT1 cytotoxic- MMP directly by degrading several of its components and indi delivery approaches. rectly by activating pro -MMP2 . MT1 -MMP is crucial for Diverse selection phage libraries containing 1011 to 1013 tumor angiogenesis (Sounni et al (2002 ) FASEB J. 16 ( 6 ), unique peptide sequences which are post- translationally 555-564 ) and is over - expressed on a variety of solid tumors . cyclized with thiol- reactive scaffolds were used to identify Accordingly , there remains a high unmet need in developing 20 small ( 1.5-2 kDa ) constrained bicyclic peptides binders inhibitors of MT1- MMP for the treatment of cancer (Bicycles ) to the hemopexin domain of MT1. Initial binders were subject to affinity maturation by directed screens and stabilization by chemical optimization . BRIEF DESCRIPTION OF THE DRAWINGS A bicyclic constrained peptide binder (Bicycle ) was iden FIG . 1 depicts the plasma stability of I- la in human and 25 apparenttified that Kd binds of toapproximately the hemopexin 2 nM domain . The ofBicycle MT1 withpeptide an mouse . ( N241 ) binds with similar affinity to the entire ectodomain of the protease but shows no binding to the catalytic domain . FIG . 2 depicts the plasma stability of l- la with and N241 also shows no binding toward any of the closely without AEBSF . related MMP family members tested (MMP15 , MMP16 , FIG . 3 depicts the plasma stability of l- la in mouse and 30 MMP24 , MMP1, Pro -MMP1 , MMP2) . Characterization of the formation of the MMAE metabolite . the pharmacologic effect of N241 on MT1 in vitro shows FIG . 4 depicts the efficacy of l -la in the HT1080 xeno that the peptide has no direct impact on the catalytic activity graft model. of the protease , nor related MMP catalytic activity (MMP1 , FIG . 5 depicts the displacement of I -la activity when MMP2ever , binding and MMP9 of fluorescently ) nor cell migration - tagged N241or invasion to MT1 . How on dosed at 1 mg/ kg in the HT1080 xenograft model by the 35 HT1080 fibrosarcoma cells results in the rapid internaliza binding peptide . tion and subsequent lysosomal localization of the com pound . In addition , 177Lu -loaded N241 demonstrates rapid FIG . 6 depicts the efficacy of l - la in the MT1 -MMP tumor localization when injected IV into mice bearing expressing non - small cell lung cancer (NSCLC ) patient MT1 -positive tumor xenografts , with levels as high as derived xenograft (PDX ) model. 15-20 % injected dose per gram of tumor in less than 60 FIG . 7 depicts the efficacy of I- la in the MT1 -MMP 40 minutes. In contrast, a non -binding Bicycle peptide shows low - expressing NSCLC PDX model . no tumor localization . These properties suggest that N241 may be a good delivery vehicle for cytotoxic payloads FIG . 8 depicts the tolerability of I - 1a in the MT1 -MMP targeting MT1- positive tumor cells . Bicycle drug conjugates low -expressing NSCLC PDX model. (BDCs ) with a variety of linkers and cytotoxic payloads FIG . 9 depicts body weight changes after administering 45 were prepared which retained binding to MT1. The anti 1-3a , 1-7 , 1-4a , and 1-8 to female Balb /c nude mice bearing tumor activity of select BDCs was demonstrated in MT1 HT1080 xenograft. Data points represent group mean body positive human tumor cell xenografts in mice . weight. Error bars represent standard error of the mean I - la is a Bicycle drug conjugate (BDC ) comprising a ( SEM ) . constrained bicyclic peptide that binds with high affinity and FIG . 10 depicts tumor volume traces after administering 50 specificity to membrane type 1 -matrix metalloprotease 1-3a , 1-7 , 1-4a , and 1-8 to female Balb / c nude mice bearing (MT1 - MMP ; MMP14 ) covalently linked via a Spacer - AA ? HT1080 xenograft. Data points represent group mean , error AA2- Linker to monomethyl auristatin E (MMAE ) , a potent bars represent standard error of the mean ( SEM ) . antimitotic agent .MT1 -MMP is naturally involved in tissue FIG . 11 depicts body weight changes ( A ) and tumor remodeling, however overexpression of the cell -surface volume (B ) trace after administering 1-12 to female BALB /c 55 proteasesiveness , hasas well been as tied poor to patient tumor prognosis aggressiveness for many and cancer inva nude mice bearing HT1080 xenograft. Data points represent indications. The Bicycle binder for I - la (N241 ) was identi group mean body weight. Error bars represent standard error fied using a proprietary phage display peptide technology of the mean (SEM ) . consisting of highly diverse phage libraries of linear amino FIG . 12 depicts body weight changes ( A ) and Tumor 60 acid sequences constrained into two loops by a central volume (B ) trace after administering 1-16 to female BALB / c chemical scaffold . While binding with similar affinity and nude mice bearing HT1080 xenograft. Data points represent specificity to that observed with monoclonal antibodies , the group mean body weight. Error bars represent standard error small size of a Bicycle peptide (1.5-2 kDa) aids in its rapid of the mean (SEM ). extravasation and tumor penetration making it an ideal FIG . 13 depicts the reaction schemeof I -12 , 1-15 , 1-16 and 65 format for the targeted delivery of cytotoxic payloads. 1-17 . One advantage of the AA -AAP linker is the specificity of FIG . 14 depicts the reaction scheme of 1-10 and 1-14 . toxin targeting and release . Non -specific release of toxin is US 10,624,968 B2 3 4 known and has been termed bystander activity . For example , In some embodiments , the present invention provides a antibody drug conjugates (ADCs ) have been shown to be Bicycle Drug Conjugate (“ BDC " ) of formula I: mostly activated through internalization and degradation of the antibody in the lysosome and release of activated metabolite that can kill tumor cells . Depending on the type 5 I of payload ( toxin ) and linker , activated metabolites can have bystander activity ( the ability to penetrate to neighboring Bicycle Spacer AA -AAP Linker Toxin cells ) and kill neighboring tumor cells . For example , the toxins DM1 and MMAE (the metabolite of Brentuximab 10 vedotin , trade name Adcetris ) have bystander activity while wherein : DM1- SMCC - lysine (the metabolite of Trastuzumab emta Bicycle is a polypeptide which is covalently bound to a sine, trade name Kadcyla ) and MMAF do not. Most of the molecular scaffold such that two or more peptide loops are BDC activity is not mediated by target -mediated internal 15 subtended between attachment points to the scaffold ; ization and degradation of BDC into active metabolites . Spacer is a bivalentmoiety that connects the Bicycle moiety Without being bound by any particular theory , it is believed with the AA -AA ? moiety ; that BDCs are mostly activated outside of the cells through AA -AAP is a bivalent moiety comprising at least one disulfide reduction for BDCs utilizing a disulfide linker and 20 citrulline moiety that connects the Spacer moiety with the protease cleavage by cathepsin B expressed on the tumor Linker moiety wherein each of AA and AA is an cell surface for BDCs utilizing a AA - AA ? linker such as independently selected natural or unnatural amino acid I - 1a , which is consistent with the observed half - life of moiety ; BDCs. Utilizing cleavage by cathepsin B has the potential Linker is a bivalent spacer moiety that connects the AA for a more selective release of toxin as cathepsin B activity 25 AA ? moiety with the Toxin moiety and has been found to be elevated in a variety of human tumors Toxin is a chemotherapeutic agent. and tumor cell lines. An alternative hypothesis is that BDCs deliver toxin via a non - targeted mechanism inside the cell In certain embodiments , the present invention provides a through pinocytosis and lysosome degradation . A further 30 compound of formula I - a or I- b : hypothesis is that BDCs deliver toxin via a combination of both internal and external activation . I - a A series of Bicycle - spacer -AA - AA ? -linker - Toxin BDCs were prepared , with varying spacer format to adjust the Bicycle Linker presentation of the Bicycle and evaluated for their anti 35 Spacer Val -Cit Toxin tumor activity in an MT1 -positive tumor xenograft model. The BDC selected for further assessment (1-1a ) was evalu I - b ated for efficacy in an array of tumor xenograft models . An AA ' -AA linker -MMAE construct (1-1a ) was among 40 Bicycle Spacer -Cit- Val Linker Toxin the most active constructs against MT1- positive EBC - 1 lung tumor xenografts . Dosing 1-1a on a 3x weekly schedule for two weeks, significant reduction in tumor growth was seen or a pharmaceutically acceptable salt thereof, wherein each regressionsat 1 mg/ kg , inwith this 3model mg/ kg . Theand therapeutic10 mg/ kg causingefficacy completeof I- la at 45 of Bicycle , Spacer, Linker and Toxin is as defined and 10 mg/kg is accompanied by brief body weight loss which described herein . is reversible upon discontinuation of treatment. In certain embodiments , the present invention provides a I -la , a Bicycle drug conjugate (BDC ), shows potent compound of formula I- c or 1- d : antitumor activity in human tumor xenograft models of lung 50 cancer . Without wishing to be bound by any particular theory, it is believed that the small size of the BDC may offer I - c a significant advantage to other targeted cytotoxic approaches such as antibody -drug conjugates due to rapid Bicycle Spacer -Trp -Cit Linker Toxin extravasation and improved tumor penetration . 55 In certain aspects , the present invention provides a I - d method of treating certain cancers in a subject, comprising administering to the subject an effective amount of a drug conjugate comprising a high affinity binder of MT1- MMP , Bicycle Spacer -Cit- Trp Linker Toxin such as I- 1 , or a pharmaceutically acceptable salt or com 60 position thereof. In certain aspects , the present invention provides a method of treating certain cancers in a subject , or a pharmaceutically acceptable salt thereof, wherein each comprising administering to the subject an effective amount of Bicycle , Spacer, Linker and Toxin is as defined and of a drug conjugate comprising a high affinity binder of 65 described herein . MT1- MMP , such as I- la , or a pharmaceutically acceptable In certain embodiments , the present invention provides a salt or composition thereof. compound of formula l - e or l - f : US 10,624,968 B2 5 6
I - e
Linker Toxin Bicycle Spacer
NH
NH2
I - f
Bicycle Spacer Linker Toxin
HN
H?N
or a pharmaceutically acceptable salt thereof, wherein each of Bicycle , Spacer, Linker and Toxin is as defined and described herein . In certain embodiments, the present invention provides a compound of formula 1- e ', I -e " , or l - e '" :
I- e '
Linker Toxin Bicycle Spacer
NH
NH2
I- e ”
Linker Toxin Bicycle Spacer
NH
NH2 US 10,624,968 B2 7 8 -continued I- e !!
Linker Toxin Bicycle Spacer NH 111||||
NH
NH2
15 or a pharmaceutically acceptable salt thereof , wherein each -continued of Bicycle , Spacer, Linker and Toxin is as defined and I- f described herein . In certain embodiments , the present invention provides a 20 compound of formula 1 - f' , I - f" , or I - f " : Bicycle Spacer Linker Toxin I- f
25 Bicycle Spacer HN Linker Toxin H?N
30 HN
H?N I - f" 35
Bicycle Spacer Linker Toxin 40 or a pharmaceutically acceptable salt thereof, wherein each of Bicycle , Spacer, Linker and Toxin is as defined and described herein . HN 45 H2N In certain embodiments , the present invention provides a compound of formula l- g or I- h :
l- g
HN
Linker Toxin Bicycle Spaceror
NH
NH2 US 10.624,968 B2 9 10 -continued
NH
Bicycle Spacer Linker Toxin
HN
H2N or a pharmaceutically acceptable salt thereof, wherein each 20 of Bicycle, Spacer , Linker and Toxin is as defined and described herein . In certain embodiments , the present invention provides a compound of formula 1 - g ', l - g " , or I - g '" :
l - g '
HN
Linker Toxin Bicycle Spacer
NH
NH2
l - g "
HN
Linker Toxin Bicycle Spacer
NH
NH2 US 10,624,968 B2 11 12 -continued I- g '"
HN
Linker Toxin Bicycle Spacer
NH
NH2
or a pharmaceutically acceptable salt thereof, wherein each of Bicycle , Spacer , Linker and Toxin is as defined and described herein . In certain embodiments , the present invention provides a compound of formula I - h ', I - h " , or I -ht " :
I- h '
NH
Bicycle Spacer Linker Toxin
HN
H2N
I - h "
NH
Bicycle Spacer Linker Toxin
HN
H2N US 10,624,968 B2 13 14 -continued I- h !"
NH
Bicycle Spacer Linker Toxin
HN
H?N
or a pharmaceutically acceptable salt thereof, wherein each ments , the polypeptide is BAla -Sar10 - A - CYNEFGCEDFY of Bicycle , Spacer, Linker and Toxin is as defined and DIC- (SEQ ID NO : 3 ) . In some embodiments , the polypep described herein . tide is -C ( D - Ala )NEFGCEDFYDIC- (SEQ ID NO : 4 ) . In some embodiments , the polypeptide is -C ( D - Ala )NE ( 1Nal ) The amino acid sequences described herein are written 25 (D - Ala )CEDFYD ( tBuGly )C- (SEQ ID NO : 5) . In some N - terminus to C - terminus, except where it is described embodiments , the polypeptide is -C - Y / M / F / V - U / O -UIZ - J differently . G - C - E - D - F - Y - Z - O - C- (SEQ ID NO : 6 ) . In some embodi I. Bicycle Moieties ments, the polypeptide is -C - Y /M / F / V -N / G - E / Q -F -G - C -E As described generally above, the present invention pro vides a BDC comprising a Bicycle moiety , a Spacer moiety , 30 polypeptideD - F - Y - D - I -C- is (-CSEQ - Y / MID / FNO - N /: G 7- ) E. /In Q - someF -G - C embodiments - E - D - F - Y -D - , I -theC a AA -AA ? moiety , a Linker moiety and a Toxin moiety . (SEQ ID NO : 8 ) . In some embodiments , the polypeptide is As used herein , the term Bicycle moiety refers to a -C - Y / M - N - E / Q - F - G - C - E - D - F - Y - D - I -C- (SEQ ID NO : 9 ). In bicyclic peptide covalently bound to a molecular scaffold . some embodiments , the polypeptide is -C - M - N - Q - F - G - C As defined above and described herein , a bicyclic peptide E - D - F - Y - D - I - C- (SEQ ID NO : 10 ) . In some embodiments , is a polypeptide which is covalently bound to a molecular 35 the polypeptide is -C - F - G - E - F - G - C - E - D - F - Y - D - I -C- (SEQ scaffold such that two or more peptide loops are subtended ID NO : 11 ) . In some embodiments, the polypeptide is between attachment points to the scaffold . -C - V - N - E -F -G -C - E - D -F - Y - D - I- C- (SEQ ID NO : 12 ). In In some embodiments , the polypeptide is a high affinity some embodiments , the polypeptide is -C - F - N - E - F -G - C - E binder of membrane type 1 metalloprotease (MT1 - MMP , D - F - Y -D - I- C- (SEQ ID NO : 13) . In some embodiments , the also known as MMP14 ) . In some embodiments , the poly- 40 polypeptide is -C - Y - N - E - Y -G - C - E - D - F - Y - D - I - C- (SEQ ID peptide is fully cross -reactive with murine, dog, cynomolgus NO : 14 ) . In some embodiments , the polypeptide is -C - Y - N and human MT1 -MMP . In some embodiments , the polypep E - W -G - C - E - D - F - Y - D - I- C- (SEQ ID NO : 15 ) . In some tide is selective forMT1 -MMP , but does not cross -react with embodiments , the polypeptide is -CKNRGFGCEDFYDIC MMP - 1 , MMP- 2 , MMP - 15 and MMP- 16 . In some embodi (SEQ ID NO : 16 ) . As described in WO 2016/067035 , the ments , the polypeptide is specific for human MT1 -MMP . In 45 content of which is incorporated herein by reference in its some embodiments , the polypeptide is specific for mouse entirety , Z represents a polar, negatively charged amino acid MT1 -MMP . In some embodiments, the polypeptide is spe residue selected from D or E , and J represents a non -polar cific for human and mouse MT1 -MMP . In some embodi aromatic amino acid residue selected from F , W and Y. As ments , the polypeptide is specific for human ,mouse and dog also described in WO 2016/067035 , D - Ala represents D -ala MT1- MMP . In some embodiments, the polypeptide is a high 50 nine; 1Nal represents 1 - naphthylalanine ; and tBuGly repre affinity binder ofMT1 - MMP Hemopexin domain (PEX ) . sents tert- butylglycine . In some embodiments , the polypeptide is a peptide In some embodiments , the polypeptide comprises L described in WO 2016/067035 , the content of which is amino acids. In some embodiments , the polypeptide com incorporated herein by reference in its entirety . prises Damino acids. In some embodiments , the polypeptide In some embodiments , the polypeptide is -C - X - U /O -X- 55 comprises a mixture of D and L amino acids . X - G - C -E -D - F - Y- X - X - C- (SEQ ID NO : 17) wherein X rep In some embodiments , the polypeptide is selected from resents any amino acid residue ; U represents a polar, those depicted in Table 1 , below . uncharged amino acid residue selected from N , C , Q , M , S In some embodiments , the polypeptide is selected from and T ; and O represents a non -polar aliphatic amino acid those depicted in Table la , below . residue selected from G , A , I , L , P and V. In some embodi- 60 In some embodiments , the polypeptide is selected from ments , the polypeptide is -C - X - U / O - X - X - G - E - D - F - Y - X - X those depicted in Table 1b , below . C- (SEQ ID NO : 1 ) wherein X represents any amino acid Molecular scaffolds are described in , for example , WO residue ; U represents a polar, uncharged amino acid residue 2009/098450 and references cited therein , particularly WO selected from N , C , Q , M , S and T ; and O represents a 2004/077062 and WO 2006/078161. non -polar aliphatic amino acid residue selected from G , A , 65 As noted in the foregoing documents , the molecular I, L , P and V. In some embodiments , the polypeptide is scaffold may be a small molecule , such as a small organic -CYNEFGCEDFYDIC- (SEQ ID NO : 2 ) . In some embodi molecule . US 10,624,968 B2 15 16 In one embodiment the molecular scaffold may be , or may be based on , natural monomers such as nucleosides, sugars, or steroids. For example the molecular scaffold may com prise a short polymer of such entities , such as a dimer or a trimer. In one embodiment the molecular scaffold is a 5 compound of known toxicity, for example of low toxicity . Examples of suitable compounds include cholesterols , nucleotides , steroids , or existing drugs such as tamazepam . In one embodiment the molecular scaffold may be a 10 macromolecule . In one embodiment the molecular scaffold is a macromolecule composed of amino acids, nucleotides or carbohydrates . or a derivative thereof, via bromide displacement. In one embodiment the molecular scaffold is formed via In one embodiment, the molecular scaffold reagent is a molecular scaffold reagent which comprises the molecular 15 2,4,6 - tris ( bromomethyl )mesitylene scaffold and reactive groups that are capable of reacting with functional group (s ) of the polypeptide to form covalent bonds. Br
The molecular scaffold reagent may comprise chemical 20 groups which form the linkage with a peptide, such as amines, thiols , alcohols , ketones, aldehydes, nitriles , car boxylic acids, esters , alkenes, alkynes , azides, anhydrides , Br succinimides , maleimides , alkyl halides and acyl halides . 25 In one embodiment, the molecular scaffold reagent may Br , comprise or may consist of wherein treatment with a polypeptide affords the molecular Br scaffold 30
Br 35 Br ,
40 minn
tris ( bromomethyl) benzene , especially 1,3,5 - tris (bromom via bromide displacement. This molecular scaffold reagent is ethyl) benzene ( TBMB ) or a derivative thereof. similar to 1,3,5 - tris (bromomethyl ) benzene but contains In one embodiment, the molecular scaffold may be 45 three additionalmethyl groups attached to the benzene ring . formed by treatment of the polypeptide with a molecular This has the advantage that the additional methyl groups scaffold reagent which comprise or may consist of tris may form further contacts with the polypeptide and hence (bromomethyl ) benzene, especially 1,3,5 - tris (bromomethyl ) add additional structural constraint. benzene ( TBMB ), 50 In one embodiment, the molecular scaffold reagent may comprise or may consist of N , N ', N " - (benzene - 1,3,5 - triyl) Br tris ( 2 - bromoacetamide ) ,
55 ( TBAB )
Br Br NH Br, 60
Br Br , IZ
65 or a derivative thereof, wherein treatment with a polypeptide wherein treatment with a polypeptide affords the molecular affords the molecular scaffold scaffold US 10,624,968 B2 17 18 Scaffold reactive groups that could be used in the molecu lar scaffold reagent to react with thiol groups of cysteines are alkyl halides (or also named halogenoalkanes or haloal NH kanes) . 5 Examples include bromomethylbenzene (the scaffold reactive group exemplified by TBMB ) or iodoacetamide . Other scaffold reactive groups that are used to selectively couple compounds to cysteines in proteins are maleimides. N Examples of maleimides which may be used as molecular ???? 10 scaffold reagents in the invention include : tris- ( 2 -maleimi doethyl) amine via bromide displacement. In one embodiment, the molecu- 15 lar scaffold may comprise or may consist of triacrylformal, N
( TATA ) 20 N.
N ' N
25 wherein treatment with a polypeptide affords the molecular scaffold
30 wherein treatment with a polypeptide affords the molecular scaffold 35
40 N N
45 via Michael addition ; tris- ( 2 -maleimidoethyl ) benzene
50 via Michael addition . In some embodiments , the molecular scaffold is selected from those depicted in Table 1 , below . In some embodiments , the molecular scaffold is selected 55 from those depicted in Table 1a , below . In some embodiments , the molecular scaffold is selected from those depicted in Table 1b , below . The molecular scaffold reagent of the invention contains chemical groups that allow functional groups of the poly 60 peptide of the invention to form covalent links with the molecular scaffold . Said chemical groups are selected from a wide range of functionalities including amines, thiols , alcohols , ketones, aldehydes, nitriles, carboxylic acids, 65 esters , alkenes , alkynes , anhydrides, succinimides , maleim wherein treatment with a polypeptide affords the molecular ides , azides , alkyl halides and acyl halides . scaffold US 10,624,968 B2 19 20 In some embodiments , a Bicycle is of formula II:
II wan 5
10 Ring A
15 wherein : each of L ' , LP , and L ’ is independently a C1-6 bivalent straight or branched saturated or unsaturated hydrocarbon chain wherein 1-2 methylene units of the chain are 20 independently and optionally replaced with S , -N( R ) - , C ( O ) , CONC ( O ) N (( RR2 ) , —N (R ) C ( 0 ) ; via Michael addition , and tris - maleimido )benzene each R is independently hydrogen or C14 alkyl; Ring A is a 6 -membered saturated, partially unsaturated , or 25 aromatic ring having 0-3 heteroatoms independently selected from nitrogen , oxygen , or sulfur ; each “ loop ” comprises a peptide targeting MT1 -MMP ; and \ indicates the site of attachment to the Spacer. In some embodiments , a Bicycle is of formula II' : 30
II '
35 wherein treatment with a polypeptide affords the molecular 40 Ring A scaffold uni *L3
45 wherein : each of L ', L ’ , and L is independently a C - bivalent straight or branched saturated or unsaturated hydrocarbon chain wherein 1-2 methylene units of the chain are independently and optionally replaced with S 50 -N ( R ) , C ( O ) , C ( O ) N ( R ) — , or - N ( R ) C ( O ) each R is independently hydrogen or C1-4 alkyl; Ring A is a 6 -membered saturated, partially unsaturated , or aromatic ring having 0-3 heteroatoms independently selected from nitrogen , oxygen , and sulfur ; sotros 55
60 via Michael addition . Selenocysteine is also a natural amino acid which has a similar reactivity to cysteine and can be used for the same reactions . Thus, wherever cysteine is 65 mentioned , it is typically acceptable to substitute selenocys teine unless the context suggests otherwise . US 10,624,968 B2 21 22 comprises a polypeptide targeting MT1 -MMP , and is selected from those depicted in Table la, below . In some embodiments , L2 is selected from those depicted in Table 1b , below . In some embodiments , L is CH , — . In some embodi ments, L ' is - C (O )CH , CH , — . In some embodiments, L 3 moltom 5 is CH SCH , — . In some embodiments , L is CH NHCH2 In some embodiments , L is CH_N indicates the site of attachment to the Spacer . (CH3 ) CH2 In some embodiments , L ’ is CH SCH_C As defined above and described herein , each of L ', L , (O )NH— . In some embodiments , L is CH NHCH , C (O ) and L ’ is independently a C - bivalent straight or branched NH— . In some embodiments , Lºis - CH N (CH2 ) CH CO ) saturated or unsaturated hydrocarbon chain wherein 1-2 10 NH— In some embodiments , ? is CH SCH2CH2C methylene units of the chain are independently and option (0 ) — . In some embodiments , L is CH NHCH2CH2C ally replaced with S- , -N ( R ? ) — , C ( O ) - , -C ( O )N (0 ) — In some embodiments , L * is CHAN ( CH3 ) ( R ) — , -N ( R ) C ( 0 ) CH2CH2C ( O ) — . In some embodiments, L is selected from In some embodiments , each of L ' , L², and L ’ is indepen- 15 those depicted in Table 1 , below . In some embodiments , L3 dently a C1-6 bivalent straight or branched saturated or is selected from those depicted in Table 1a, below . In some unsaturated hydrocarbon chain wherein 1-2 methylene units embodiments , L ’ is selected from those depicted in Table 1b , of the chain are independently and optionally replaced with below . S , N ( R ) , or C ( O ) . In some embodiments , As defined above and described herein , each R is inde each of L ' , L², and L ’ is independently a C1-6 bivalent 20 pendently hydrogen or C1-4 alkyl. straight or branched saturated or unsaturated hydrocarbon In some embodiments , R is hydrogen . In some embodi chain wherein 1 methylene unit of the chain is independently ments , R is C1-4 alkyl. In some embodiments, R is methyl . and optionally replaced with s- , -N ( R ) , or In some embodiments , R is ethyl. C ( O ) - . In some embodiments , each of L ', L², and L ’ is In some embodiments , R is selected from those depicted orindependently unsaturated hydrocarbona C1-6 bivalent chain straight wherein or branched 1 methylene saturated unit 25 in Table 1 , below . In some embodiments, R is selected from of the chain is optionally replaced with S— In some those depicted in Table la, below . In some embodiments , R embodiments , each of L ’ , L², and L ’ is independently a C1-6 is selected from those depicted in Table 1b , below . bivalent straight or branched saturated or unsaturated hydro As defined above and described herein , Ring A is a carbon chain wherein 1 methylene unit of the chain is 30 6 -membered saturated , partially unsaturated , or aromatic optionally replaced withN( R ) — . In some embodiments , ring having 0-3 heteroatoms independently selected from each of L ' , L² , and L is independently a C1-6 bivalent nitrogen , oxygen , or sulfur. straight or branched saturated or unsaturated hydrocarbon In some embodiments , Ring A is chain wherein 1 methylene unit of the chain is optionally replaced with C ( O ) - , 35 In some embodiments, each of L ' , L², and L is CH2 In some embodiments , each of L ’, L², and L ’ is -C (O ) CH2CH2– In some embodiments , each of L ' , L ?, and L3 is CH SCH , — 40 In some embodiments , L ' is CH2 In some embodi ments , L ' is C (O )CH2CH , — . In some embodiments , L ' is CH SCH2 In some embodiments , L is CH NHCH2 In some embodiments , Ll is CH N ( CH3) CH2— . In some embodiments , L ' is CH SCH_C45 In some embodiments , Ring A is ( O )NH— . In some embodiments , L ' is CH_NHCH C ( O ) NH— . In some embodiments , L ' is — CH N (CH3 ) CH_C ( O ) NH— . In some embodiments , L ' is CH SCH2CH2C ( 0 ) — . In some embodiments , L ' is CH , NHCH , CHÚC ( 0 ) In some embodiments , Ll is CHÚN ( CH?) 50 CH2CH2C (O ) . In some embodiments , L '1 is selected from N. those depicted in Table 1 , below . In some embodiments , L is selected from those depicted in Table 1a , below . In some embodiments , L ' is selected from those depicted in Table 1b , below . 55 In some embodiments , L2 is CH2 In some embodi ments , L² is -C (O )CH2CH2 In some embodiments , L2 In some embodiments , Ring A is is CH SCH , In some embodiments , L2 is CH NHCH , - In some embodiments , L2 is _CH_N (CH3 ) CH , — . In some embodiments , L2 is CH SCH , C 60 ( O )NH— . In some embodiments , L² is CH NHCH , C (O ) NH— . In some embodiments , L² is CH_N (CH3 ) CH C ( O ) NH— In some embodiments , L2 is -CH SCH2CH2C (0 ) In some embodiments , L2 CH , NHCH , CH?C (0 ) In some embodiments , L2 is CH , N ( CH?) 65 ? CH2CH2C (O ) . In some embodiments , L2 is selected from those depicted in Table 1 , below . In some embodiments , L2 US 10.624,968 B2 23 24 In some embodiments , Ring A is In some embodiments , 5 m is a polypeptide selected from those depicted in Table 1 , KA 10 below . In some embodiments, In some embodiments , Ring A is 15 m is a polypeptide selected from those depicted in Table la, 20 below . In some embodiments ,
In some embodiments , Ring A is 25 you nhun 30 is a polypeptide selected from those depicted in Table 1b , below . mihu In some embodiments , a Bicycle is : In some embodiments , Ring A is selected from those 35 depicted in Table 1 , below . In some embodiments , Ring A is selected from those depicted in Table la, below . In some HO embodiments , Ring A is selected from those depicted in H2N Table 1b , below . H3C NH NH As defined above and described herein , 40 NH NH whe NH NH 45 H2C 1
H2C -CH3 H2 H2 NH 50 C - s ? OH comprises a polypeptide targeting MT1 -MMP . H2C In some embodiments , H2C 55 H2N NH OH . HN
NH 60 HN -??
?? . 65 OH comprises a polypeptide selected from SEQ ID NOS . 1-17 as described herein . US 10.624,968 B2 25 26 In some embodiments , a Bicycle is :
HO H2N 5 H3C NH NH
NH NH 10 mbre NH H?ç NH ho1 15
"CH3 HC 20 NH H2 H2 -C -S - C
N HC CH3 25 s H2C H2N NH OH . HN 30
HN NH 35 HO OH
40
45 In some embodiments , a Bicycle is :
H2N . NH H2N . NH HN HN OH CH3 CH3 H3C , CH3 H3C H3C HO ( R) , CH3 CH3 NH2. US 10,624,968 B2 27 28 In some embodiments , a Bicycle is :
NH2 HN NH S CH3 H3C S H3C , CH3 CH3 H3C OH H3C (S ) H3C CH3 CH3 CH3 CH3 NH) .
In some embodiments , a Bicycle is selected from those 20 domain is a 9 membered bivalent polysarcosine moiety . In depicted in Table 1 , below . In some embodiments , a Bicycle some embodiments , the polysarcosine domain is a 10 mem is selected from those depicted in Table 1a , below . In some bered bivalent polysarcosine moiety . In some embodiments , embodiments , a Bicycle is selected from those depicted in Table 1b , below . the polysarcosine domain is a 11 membered bivalent poly II. Spacer Moieties 25 sarcosine moiety . In some embodiments , the polysarcosine As used herein Spacer refers to a bivalent moiety that domain is a 12 membered bivalent polysarcosine moiety . In connects the Bicycle moiety with the AA - AA ? moiety . some embodiments , the polysarcosine domain is a 13 mem One of ordinary skill in the art will appreciate that a bered bivalent polysarcosine moiety . In some embodiments , variety of Spacer moieties are amenable to achieve connec tion of the Bicycle with the AA - AA ? moiety . 30 the polysarcosine domain is a 14 membered bivalent poly In certain embodiments , the Spacer moiety is a bivalent sarcosine moiety . In some embodiments , the polysarcosine moiety comprising an alanine , a polysarcosine domain , a domain is a 15 membered bivalent polysarcosine moiety . beta - alanine and a glutaryl moiety . In some embodiments , In some embodiments , the Spacer moiety is the polysarcosine domain is a 5-15 membered bivalent 35 polysarcosine moiety . In some embodiments , the polysar cosine domain is a 5 membered bivalent polysarcosine moiety . In some embodiments , the polysarcosine domain is rythboys a 6 membered bivalent polysarcosine moiety . In some embodiments , the polysarcosine domain is a 7 membered 40 bivalent polysarcosine moiety . In some embodiments , the In certain embodiments , the Spacer moiety is a bivalent polysarcosine domain is a 8 membered bivalent polysar thiopropanoyl maleimido caproylmoiety . In other embodi cosine moiety . In some embodiments , the polysarcosine ments , the Spacer moiety is selected from :
10 thereby US 10,624,968 B2 29 30 or
5 IZ
In some embodiments , the Spacer moiety is 10
thighhin 15 In some embodiments , the Spacer moiety is 20
0
25 NH NN
In some some embodiments , the Spacer moiety is
NH
HN
55 In some embodiments , the Spacer moiety is
CH3 CH3 CH3 - N N N N N the CH3 CH3 CH3 US 10,624,968 B2 31 32 -continued CH3 CH3 CH3 H3C CH3 N N HN CH3 CH3
10 In some embodiments , the Spacer moiety is
NH2 HN = NH CH3 H3C OH CH3 O CH3 0 CH3 CH3 O CH3 - | CH3 w N HZ CH3 CH3 CH3 O CH3 CH3
In some embodiments , the Spacer moiety is selected from In some embodiments, AA - AAP is those depicted in Table 1 , below . 25 In some embodiments , the Spacer moiety is selected from those depicted in Table la , below . In some embodiments , the Spacer moiety is selected from those depicted in Table 1b , below . 30 III . AA - AA ? Moieties nhu As used herein AA -AA2 is a bivalent moiety at least one citrulline moiety that connects the Spacer moiety with the Linker moiety , wherein AA and AA ? represent amino acids 35 HN and the bond between them can be selectively cleaved by enzymes expressed on tumor cells . In some embodiments , H2N AA - AA is a bivalent moiety at least one citrulline moiety and is selectively cleaved by cathepsin . 40 In some embodiments , each of AA and AA² is an L amino acid . In some embodiments , each of AA and AA is a D amino acid . In some embodiments , one of AA and AA ? is an L amino acid , and the other one is a D amino acid . In some embodiments , AA - AAP is - Val- Cit- . In some 45 embodiments , AA -AA ? is -Cit- Val- . In some embodiments , AAL- AA is In some embodiments , AA -AAP is 50 55 Kry AY 60
NH NH
NH2. 65 NH2 US 10,624,968 B2 33 34 In some embodiments , AA -AA ’ is In some embodiments , AA - AAP is
5 my HV nhu 10
NH HN
NH2. H2N 15 In some embodiments , AA - AA ? is
20 In some embodiments , AA - AAP is - Trp -Cit- . In some embodiments , AA -AA ’ is -Cit - Trp-. In some embodiments , AA "-AAP is tyy 25
HN *NH 30 NH2.
In some embodiments , AA -AA is 35
NH NH2 mku 40
HN 45 H2N In some embodiments , AA -AAP is
50
In some embodiments, AA -AAP is NH .
55 Hoy mahu HM nhu 60
HN HN H2N H2N 65 US 10,624,968 B2 35 36 In some embodiments , AA - AAP is In some embodiments , AA - AAP is
5 HN HN
10 NH mine
15 HN NH NH2. H?N 20 In some embodiments , AA -AA ? is In some embodiments, AA -AA2 is
25
NH . HN 30 mobra mku 35
HN NH H?N 40 NH2. In some embodiments , AA -AAP is
45 In some embodiments , AA - AA ? is NH .
50 HN nhu 55 mku HN 60 H2N
NH In some embodiments , the AAP- AA ? moiety is selected from those depicted in Table 1 , below . In some embodiments , the AA '- AA moiety is selected NH2 65 from those depicted in Table la , below . In some embodiments , the AA -AA ? moiety is selected from those depicted in Table 1b , below . US 10,624,968 B2 37 38 IV . Linker Moieties In some embodiments, the Toxin is monomethyl auristatin F As used herein Linker refers to a bivalent spacer moiety (MMAF ) : that connects AA -AA ? moiety with the Toxin moiety . In some embodiments , a Linker is a self -immolative linker . One of ordinary skill in the art will appreciate that a 5 variety of Linker moieties are amenable to achieve connec OH tion of the AA - AA ? moiety with the Toxin moiety . In some embodiments , the Linker is a para -aminobenzyl NH moiety . In some embodiments , the Linker is 10 N pro Arrow mahu 15 In some embodiments , the Toxin is DM1: In some embodiments , the Linker is a bivalent self immolative spacer moiety . In some embodiments , the Linker is para -aminobenzyl carbamate , 20
HN
H mbro 25 OH
( PABC ) . SH . In some embodiments , the Linker moiety is selected from those depicted in Table 1 , below . 30 In some embodiments , the Linker moiety is selected from those depicted in Table 1a , below . In some embodiments , the Linker moiety is selected from CI those depicted in Table 1b , below . V. Toxin Moieties 35 As used herein Toxin refers to a chemotherapeutic agent . One of ordinary skill in the art will appreciate that a variety of Toxin moieties are amenable to achieve the chemotherapeutic effects of the present invention . In some embodiments , the Toxin can be connected at any 40 available position . In some embodiments , the Toxin can be connected at any available OH , C ( O ) OH , SH , In some embodiments , the Toxin is DM4: -NH2, or- NHCHZ. In some embodiments , the Toxin is monomethyl auristatin H E (MMAE ) : 45 OH H
OH H H 50 HH
O
. 11 55
CI H N 60 NH
NH 65 SH . US 10.624,968 B2 39 40 In some embodiments , the Toxin is SN38 : In some embodiments , the Toxin is monomethyl auristatin E (MMAE ) :
5 ?? .
10
HO 15 OH some embodiments , the Toxin is doxorubicin : HN
OH 20 OH
*** PRIOH Olun. 25
OH NH2. NH
"HOH 30 NH
In some embodiments , the Toxin is a duocarmycin analog : 35
40 In some embodiments , the toxin moiety is selected from NH2. those depicted in Table 1 , below . In some embodiments , the toxin moiety is selected from those depicted in Table la , below . 45 In some embodiments , the toxin moiety is selected from those depicted in Table 1b , below . Exemplary compounds of the invention are set forth in Table 1 , below . Table 1. Exemplary Compounds US 10,624,968 B2 41 42
I-1
HO HO -CH3 NH NH NH NH HO. ??. NH H2 NH -CS HN
HN H2 H2N H3C NH .HN HO O med ?? H2C NH H2CO NH
HACH2N 0
H10
NH H2N
OH US 10,624,968 B2 43 44
I-2
HO. HO.
NH CH2 NAMAN NH NH NH ~?? NH N ??. H2 NH S-C NH
H2N NH H2
NH HO HN NH HO. H?CH2N 0 -continued onlineenishet
NH US 10.624.968 B2 45 46
I-3
HO CH3 HO. NH NH NH NH HO ??. View NH, H2 S-C NH
HN N H2N H2
H3C NH NH ??. S HC H2C s HN H2C HN H2CH2N NH
10
N
-continued N H O =
N NH H2N
H Me
HO ? OMe Me
Me 'N CI OMe US 10.624.968 B2 47 48
1-4
HO. CH3 OH
NH . NH OH NH NH NH HO HN H2 -S-C HN
NH H2N H2 gi Ö H3C NH NH HO S H2C H2C S H2C NH NH H2CH2N
N' H10
-continued O
H 'N NH H2N
CH3 H Me N OH H OMe Me
Me N. Me CI OMe US 10,624,968 B2 49 50
I-5
HO HO CH3 NH HN NH NH NH N HO JOH ??, H2 -S-C NH
H3C1NH ??. QH HN HN NH HCH2N to
-continued onventionateursNH US 10,624,968 B2 51 52
9-1 OH -CH3 HO NH NH NH NH HO ??. via H2 NH S-C -??
HN
NH HO. H2C NHY NH H2CH2N 0
-continued lagingobtentaNH
HO US 10.624,968 B2 53 54 In some embodiments , the present invention provides a compound set forth in Table 1, above, or a pharmaceutically acceptable salt thereof. Exemplary compounds of the invention are also set forth in Table la , below . 5 Table 1a . Exemplary Compounds US 10,624,968 B2 55 56
I-la
HO HO NH -CH3 NH OH NH NH NH HO HN H2 S-C NH
H2 H2N H3C|_NH NH NH ??. S H2C HC HN NH 0H2C HÓCH2N
10 2
N. HN H2N
No.
HO US 10.624.968 B2 57 58
1-3a
HO CH3 HO. NH NH NH NH HO NH N ??. NH, H2 S-C NH
HN N H2N H2
H3C NH NH ??. S H2C H2C s HN H2C H2C HN
NH H2N
10 yeN
-continued N H O =
i
N
N NH H2N
S H Me H OH H OMe Me
Me N. Me CI OMe US 10.624.968 B2 59 09
I-4a
HO HO. CH3 NH NH HO NH NH NH HO HN H2 -S-C HN
HN H2N H2
Ö NH HO. H3C HN H2C H2C s NH H2C NH HDCH2N
10
-continued O
H 'N NH H2N
CH3 H Me HO ? OMe Me
'N Me Me CI OMe US 10.624.968 B2 61 79
I-5a
HO -CH3 HO NH NH HO NH NH N NH HO NH H2 -S-C NH
N H?N HBC_NH H2 NH HO. HN S HC H2C NH ÖH2C HN
H?CH2N 0
10
-continued
H NH OYNH towayinor Nh. -
HO US 10,624,968 B2 63 64
I-6a -OH -CH3 HO NH NH HO NH NH N NH HO 'NH, H2 C-S- NH
N H.N' NH H2
H3C NH NH HO. - H,C H,C S NH HN OHC H.CH2N
NEN -continued
HN MaranathanagarH.N HO US 10.624,968 B2 65 66 In some embodiments , the present invention provides a compound set forth in Table la , above, or a pharmaceuti cally acceptable salt thereof. Exemplary compounds of the invention are also set forth in Table 1b , below . 5 Table 1b . Exemplary Compounds US 10,624,968 B2 L9 89
I-7
HO CH3 NH CH3 NH NH NH OH NH N HO HN H2 -S-C NH
HN H2N H2
NH ??. OH HN S H2C H2C HC 's HN HCHN HN
10
N
N H
N H NH H2N
H Me HO ? OMe Me
N. Me Me CI oMe US 10,624,968 B2 69 70
8-1
HO. CH3 NH CH3 NH NH T NH HO N ??. H2 NH -S-C NH
HN HN H2N H2
NH ??. H3C NH H2C H2C s NH H2C NH H?cHN
N H ti 10
-continued N H
?????????N H HN H2N
CH3 H Me N OH H OMe Me
Me N. Me OMe US 10,624,968 B2 71 72
I-9 HO
O -CH3 NH CH3 NH NH NH OH NHO N HO NH H2 -S-C -??
H2N HBC_NH. H2 NH NH ?? / O H2C / ??, HC HN H2CO HÓCH2N ??10 -continued
NHto H2N
HO US 10,624,968 B2 73 74
1-10 HO CH3 HO. 0 NH NH HO. NH NH N NH HO H2 NH S-C NH
H2N H?CNH H2 NH HO ,CH H,C
,CHO NH C,HH.N HN Bal-continued ??????????
HO. US 10,624,968 B2 75 9L
I-11
OH CH3 HO. NH NH HO NH NH N NH HO. ??. H2 S-C HN
NH H2N H2
NH ??. H3C HN O S H2C H2C S HN H2C0 HN H2CH2N
N
N=1
N -continued NH
HN H2N
HO US 10.624.968 B2 LL 78
I-12
HO CH3 HO NH NH HN NH HO. NH N HO NH. H, -S-C -HN
NH. H2N H?
HC NH HO. HN S PH H2C 7 H2C HN NH H?CH2N
NH HN -continued
H2N'
'???'…'' Nn
HO US 10.624.968 B2 79 08
13I-
HO OH CH3 NH NH NH OH NH NH N HO H2 NH S-C NH
H2N HBC_NH H2 NH NH OH HC H2C NELYNH 0H2C HN H2CH2N O
NH NH
-continued NH
ZE
N NH H2N w
HO US 10.624,968 B2 81 8 (
-14
HO CH3 HO NH NH HO NH NH NH HO H, NH -S-C NH•
H2N' -HN: H? NH HC HN OH S ?°H NH. H2C H2C NH H2CH2N
N.
NH HN -continued N
HN' ??????? H2N"
HO US 10,624,968 B2 83 84
I-15 1-16 OH CH3 CH3 ? NH ??3 NH H3O NH OH NH NH N HO H2 CH3 NH S-C NH ??30 NH H2N H2 CH3O
NH ??. CH3O H3C HN ?, H2C / HN CH3Ö NH ÖH2C H?CH2N CH3O CH3 CH3O
NH HN CH3Ö -continued CH3 HN H3C H?N HN IntoH2N
CH3 CH; CH3 IN CH3CH3H3C ??CH3 H3C 'N CH3
CH3 O-CH CH,6 OH OH US 10,624,968 B2 85 86
1-17 NH2 CH3 = O DZ 0CH3
CH3 CH30
NH HO. OCH3 H2N HN CH3 CH3O
CH30
OCH3
CH3O CH3 CH3O H3C CH3 CH3 H3C O -continued CH3 N
CH3 O OH H3C
NH 0 ZT HN H2N HN H2N CH3 ??3 CH; H3C CH3 CH3H3C 1.X S CH3 H3C "CH3
CH; CH3 CH3Ö CH3 OH TamilH3C US 10,624,968 B2 L8 88
NH2
CH3 I H3C CH3
NH2 NH HN OH H3C
CH3 Z H3C 0 CH3 -continued TIZ
CH3 H3C CH3 CH3 r
H3C. TZ
ZI
S NH2 NH HN OH
CH3 H3C uhrn US 10,624,968 B2 89 90 In some embodiments , the present invention provides a protamine sulfate , disodium hydrogen phosphate , potassium compound set forth in Table 1b , above , or a pharmaceuti hydrogen phosphate , sodium chloride , zinc salts , colloidal cally acceptable salt thereof. silica , magnesium trisilicate , polyvinyl pyrrolidone, cellu 2. Pharmaceutically Acceptable Compositions lose -based substances, polyethylene glycol, sodium car According to another embodiment, the invention provides 5 boxymethylcellulose , polyacrylates, waxes , polyethylene a composition comprising a compound of the present inven polyoxypropylene- block polymers , polyethylene glycol and tion , or a pharmaceutically acceptable salt thereof, and a wool fat. pharmaceutically acceptable carrier, adjuvant, or vehicle . Compositions of the present invention may be adminis As used herein , the term “ pharmaceutically acceptable tered orally , parenterally , by inhalation spray , topically , salt” refers to those salts which are , within the scope of 10 rectally , nasally , buccally , vaginally or via an implanted sound medical judgment, suitable for use in contact with the reservoir . The term “ parenteral” as used herein includes tissues of humans and lower animals without undue toxicity , subcutaneous , intravenous , intramuscular, intra -articular , irritation , allergic response and the like, and are commen intra -synovial , intrasternal, intrathecal, intrahepatic , intral surate with a reasonable benefit /risk ratio . Pharmaceutically esional and intracranial injection or infusion techniques . acceptable salts are well known in the art . For example , S. 15 Preferably , the compositions are administered orally , intra M. Berge et al. , describe pharmaceutically acceptable salts peritoneally or intravenously. Sterile injectable forms of the in detail in J. Pharmaceutical Sciences, 1977 , 66 , 1-19 , compositions of this invention may be aqueous or oleagi incorporated herein by reference. Pharmaceutically accept nous suspension . These suspensions may be formulated able salts of the compounds of this invention include those according to techniques known in the art using suitable derived from suitable inorganic and organic acids and bases. 20 dispersing or wetting agents and suspending agents . The Examples of pharmaceutically acceptable , nontoxic acid sterile injectable preparation may also be a sterile injectable addition salts are salts of an amino group formed with solution or suspension in a non - toxic parenterally acceptable inorganic acids such as hydrochloric acid , hydrobromic acid , diluent or solvent, for example as a solution in 1,3 -butane phosphoric acid , sulfuric acid and perchloric acid or with diol. Among the acceptable vehicles and solvents that may organic acids such as acetic acid , oxalic acid , maleic acid , 25 be employed are water, Ringer's solution and isotonic tartaric acid , citric acid , succinic acid or malonic acid or by sodium chloride solution . In addition , sterile , fixed oils are using other methods used in the art such as ion exchange . conventionally employed as a solvent or suspending Other pharmaceutically acceptable salts include adipate , medium . alginate , ascorbate , aspartate , benzenesulfonate , benzoate , For this purpose , any bland fixed oil may be employed bisulfate , borate , butyrate , camphorate , camphorsulfonate , 30 including synthetic mono - or di- glycerides . Fatty acids, such citrate , cyclopentanepropionate , digluconate , dodecylsul as oleic acid and its glyceride derivatives are useful in the fate , ethanesulfonate , formate , fumarate , glucoheptonate, preparation of injectables , as are natural pharmaceutically glycerophosphate , gluconate , hemisulfate , heptanoate , acceptable such as olive oil or castor oil, especially in hexanoate , hydroiodide, 2 -hydroxy - ethanesulfonate , lacto their polyoxyethylated versions. These oil solutions or sus bionate , lactate , laurate , lauryl sulfate , malate , maleate , 35 pensions may also contain a long -chain alcohol diluent or malonate , methanesulfonate , 2 -naphthalenesulfonate , nico dispersant, such as carboxymethyl cellulose or similar dis tinate, nitrate , oleate, oxalate , palmitate , pamoate , pectinate , persing agents that are commonly used in the formulation of persulfate , 3 -phenylpropionate , phosphate , pivalate , propi pharmaceutically acceptable dosage forms including emul onate , stearate , succinate, sulfate , tartrate, thiocyanate , sions and suspensions. Other commonly used surfactants , p -toluenesulfonate , undecanoate , valerate salts , and the like . 40 such as Tweens, Spans and other emulsifying agents or Salts derived from appropriate bases include alkali metal , bioavailability enhancers which are commonly used in the alkaline earth metal , ammonium and N + (C1-4alkyl ) 4 salts . manufacture of pharmaceutically acceptable solid , liquid , or Representative alkali or alkaline earth metal salts include other dosage forms may also be used for the purposes of sodium , lithium , potassium , calcium , magnesium , and the formulation . like. Further pharmaceutically acceptable salts include , 45 Pharmaceutically acceptable compositions of this inven when appropriate , nontoxic ammonium , quaternary ammo tion may be orally administered in any orally acceptable nium , and amine cations formed using counterions such as dosage form including , but not limited to , capsules , tablets , halide , hydroxide , carboxylate , sulfate , phosphate , nitrate , aqueous suspensions or solutions. In the case of tablets for loweralkyl sulfonate and aryl sulfonate . oral use, carriers commonly used include lactose and corn The term “ subject, ” as used herein , is used interchange- 50 starch . Lubricating agents , such as magnesium stearate , are ably with the term “ patient" and means an animal, preferably also typically added . For oral administration in a capsule a mammal. In some embodiments , a subject or patient is a form , useful diluents include lactose and dried cornstarch . human . In other embodiments , a subject (or patient) is a When aqueous suspensions are required for oral use , the veterinary subject (or patient) . In some embodiments , a active ingredient is combined with emulsifying and suspend veterinary subject ( or patient) is a canine , a feline, or an 55 ing agents . If desired , certain sweetening , flavoring or col equine subject. oring agents may also be added . The term “ pharmaceutically acceptable carrier , adjuvant, Alternatively , pharmaceutically acceptable compositions or vehicle ” refers to a non - toxic carrier , adjuvant, or vehicle of this invention may be administered in the form of that does not destroy the pharmacological activity of the suppositories for rectal administration . These can be pre compound with which it is formulated . Pharmaceutically 60 pared by mixing the agent with a suitable non -irritating acceptable carriers , adjuvants or vehicles that may be used excipient that is solid at room temperature but liquid at rectal in the compositions of this invention include , but are not temperature and therefore will melt in the rectum to release limited to , ion exchangers , alumina , aluminum stearate , the drug . Such materials include cocoa butter , beeswax and lecithin , serum proteins, such as human serum albumin , polyethylene glycols. buffer substances such as phosphates, glycine , sorbic acid , 65 Pharmaceutically acceptable compositions of this inven potassium sorbate , partial glyceride mixtures of saturated tion may also be administered topically , especially when the vegetable fatty acids, water , salts or electrolytes , such as target of treatment includes areas or organs readily acces US 10,624,968 B2 91 92 sible by topical application , including diseases of the eye , oils ( in particular, cottonseed , groundnut, corn , germ , olive , the skin , or the lower intestinal tract. Suitable topical for castor, and sesame oils ), glycerol, tetrahydrofurfuryl alco mulations are readily prepared for each of these areas or hol, polyethylene glycols and fatty acid esters of sorbitan , organs . and mixtures thereof. Besides inert diluents , the oral com Topical application for the lower intestinal tract can be 5 positions can also include adjuvants such as wetting agents , effected in a rectal suppository formulation ( see above ) or in emulsifying and suspending agents , sweetening, flavoring , a suitable enema formulation . Topically -transdermal patches and perfuming agents . may also be used . Injectable preparations, for example , sterile injectable For topical applications , provided pharmaceutically aqueous or oleaginous suspensions may be formulated acceptable compositions may be formulated in a suitable 10 according to the known art using suitable dispersing or ointment containing the active component suspended or wetting agents and suspending agents . The sterile injectable dissolved in one or more carriers. Carriers for topical preparation may also be a sterile injectable solution , sus administration of compounds of this invention include , but pension or emulsion in a nontoxic parenterally acceptable are not limited to , mineral oil , liquid petrolatum , white diluent or solvent, for example , as a solution in 1,3 -butane petrolatum , propylene glycol, polyoxyethylene , polyoxypro- 15 diol. Among the acceptable vehicles and solvents that may pylene compound , emulsifying wax and water. Alterna be employed are water, Ringer's solution , U.S.P. and iso tively , provided pharmaceutically acceptable compositions tonic sodium chloride solution . In addition , sterile , fixed oils can be formulated in a suitable lotion or cream containing are conventionally employed as a solvent or suspending the active components suspended or dissolved in one or medium . For this purpose any bland fixed oil can be more pharmaceutically acceptable carriers . Suitable carriers 20 employed including synthetic mono- or diglycerides . In include, but are not limited to , mineral oil , sorbitan monos addition , fatty acids such as oleic acid are used in the tearate , polysorbate 60 , cetyl esters wax , cetearyl alcohol, preparation of injectables . 2 -octyldodecanol , benzyl alcohol and water . Injectable formulations can be sterilized , for example , by For ophthalmic use , provided pharmaceutically accept filtration through a bacterial- retaining filter, or by incorpo able compositions may be formulated as micronized sus- 25 rating sterilizing agents in the form of sterile solid compo pensions in isotonic , pH adjusted sterile saline, or, prefer sitions which can be dissolved or dispersed in sterile water ably , as solutions in isotonic , pH adjusted sterile saline, or other sterile injectable medium prior to use . either with or without a preservative such as benzylalkonium In order to prolong the effect of a compound of the present chloride. Alternatively , for ophthalmic uses , the pharmaceu invention , it may be desirable to slow the absorption of the tically acceptable compositions may be formulated in an 30 compound from subcutaneous or intramuscular injection . ointment such as petrolatum . This may be accomplished by the use of a liquid suspension Pharmaceutically acceptable compositions of this inven of crystalline or amorphous material with poor water solu tion may also be administered by nasal aerosol or inhalation . bility . The rate of absorption of the compound then depends Such compositions are prepared according to techniques upon its rate of dissolution that, in turn , may depend upon well- known in the art of pharmaceutical formulation and 35 crystal size and crystalline form . Alternatively , delayed may be prepared as solutions in saline, employing benzyl absorption of a parenterally administered compound form is alcohol or other suitable preservatives , absorption promoters accomplished by dissolving or suspending the compound in to enhance bioavailability , fluorocarbons, and /or other con an oil vehicle . Injectable depot forms are made by forming ventional solubilizing or dispersing agents . microencapsule matrices of the compound in biodegradable In certain embodiments , pharmaceutically acceptable 40 polymers such as polylactide -polyglycolide . Depending compositions of this invention are formulated for oral upon the ratio of compound to polymer and the nature of the administration . Such formulationsmay be administered with particular polymer employed , the rate of compound release or without food . In some embodiments , pharmaceutically can be controlled . Examples of other biodegradable poly acceptable compositions of this invention are administered mers include poly (orthoesters ) and poly ( anhydrides ). Depot without food . In other embodiments , pharmaceutically 45 injectable formulations are also prepared by entrapping the acceptable compositions of this invention are administered compound in liposomes or microemulsions that are compat with food . ible with body tissues . Pharmaceutically acceptable compositions of this inven Compositions for rectal or vaginal administration are tion can be administered to humans and other animals orally , preferably suppositories which can be prepared by mixing rectally , parenterally , intracisternally , intravaginally , intrap- 50 the compounds of this invention with suitable non - irritating eritoneally , topically ( as by powders , ointments , or drops ), excipients or carriers such as cocoa butter, polyethylene bucally , as an oral or nasal spray , or the like , depending on glycol or a suppository wax which are solid at ambient the severity of the infection being treated . In certain embodi temperature but liquid at body temperature and therefore ments , the compounds of the invention may be administered melt in the rectum or vaginal cavity and release the active orally or parenterally at dosage levels of about 0.01 mg/kg 55 compound . to about 50 mg/ kg and preferably from about 1 mg/kg to Solid dosage forms for oral administration include cap about 25 mg/ kg , of subject body weight per day, one or more sules , tablets , pills , powders, and granules . In such solid times a day, to obtain the desired therapeutic effect . dosage forms, the active compound is mixed with at least Liquid dosage forms for oral administration include, but one inert, pharmaceutically acceptable excipient or carrier are not limited to , pharmaceutically acceptable emulsions, 60 such as sodium citrate or dicalcium phosphate and / or a ) microemulsions , solutions, suspensions , syrups and elixirs . fillers or extenders such as starches, lactose , sucrose , glu In addition to the active compounds , the liquid dosage forms cose , mannitol, and silicic acid , b ) binders such as , for may contain inert diluents commonly used in the art such as , example , carboxymethylcellulose , alginates, gelatin , poly for example , water or other solvents, solubilizing agents and vinylpyrrolidinone , sucrose , and acacia , c ) humectants such emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl 65 as glycerol, d ) disintegrating agents such as agar- agar, carbonate , ethyl acetate , benzyl alcohol, benzyl benzoate , calcium carbonate , potato or tapioca starch , alginic acid , propylene glycol, 1,3 -butylene glycol, dimethylformamide , certain silicates, and sodium carbonate , e ) solution retarding US 10,624,968 B2 93 94 agents such as paraffin , f ) absorption accelerators such as As used herein , the terms “ treatment, ” “ treat, ” and “ treat quaternary ammonium compounds, g ) wetting agents such ing” refer to reversing, alleviating , delaying the onset of, or as, for example , cetyl alcohol and glycerolmonostearate , h ) inhibiting the progress of a disease or disorder, or one or absorbents such as kaolin and bentonite clay, and i) lubri more symptoms thereof, as described herein . In some cants such as talc , calcium stearate , magnesium stearate , 5 embodiments , treatment may be administered after one or solid polyethylene glycols , sodium lauryl sulfate , and mix more symptoms have developed . In other embodiments , treatmentmay be administered in the absence of symptoms. tures thereof. In the case of capsules , tablets and pills , the For example , treatment may be administered to a susceptible dosage form may also comprise buffering agents . individual prior to the onset of symptoms ( e.g., in light of a Solid compositions of a similar type may also be 10 history of symptoms and / or in light of genetic or other employed as fillers in soft and hard - filled gelatin capsules susceptibility factors ). Treatment may also be continued using such excipients as lactose or milk sugar as well as high after symptoms have resolved , for example to prevent or molecular weight polyethylene glycols and the like. The delay their recurrence . solid dosage forms of tablets , dragees, capsules, pills , and In some embodiments , the present invention provides a granules can be prepared with coatings and shells such as 15 method for treating cancer as described herein . enteric coatings and other coatings well known in the Cancer pharmaceutical formulating art. They may optionally con Cancer includes, in one embodiment, without limitation , tain opacifying agents and can also be of a composition that leukemias ( e.g., acute leukemia , acute lymphocytic leuke they release the active ingredient( s ) only , or preferentially , in mia , acute myelocytic leukemia , acute myeloblastic leuke a certain part of the intestinal tract , optionally, in a delayed 20 mia , acute promyelocytic leukemia , acute myelomonocytic manner . Examples of embedding compositions that can be leukemia , acute monocytic leukemia , acute erythroleuke used include polymeric substances and waxes . Solid com mia , chronic leukemia , chronic myelocytic leukemia , positions of a similar type may also be employed as fillers chronic lymphocytic leukemia ) , polycythemia vera , lym in soft and hard - filled gelatin capsules using such excipients phoma (e.g. , Hodgkin's disease or non -Hodgkin's disease ) , as lactose or milk sugar as well as high molecular weight 25 Waldenstrom’s macroglobulinemia , multiple myeloma, polethylene glycols and the like . heavy chain disease , and solid tumors such as sarcomas and A compound of the present invention , or a pharmaceuti carcinomas ( e.g. , fibrosarcoma, myxosarcoma, liposarcoma, cally acceptable salt thereof, can also be in micro -encapsu chondrosarcoma, osteogenic sarcoma , chordoma , angiosar lated form with one or more excipients as noted above . The coma, endotheliosarcoma , lymphangiosarcoma, lymphan solid dosage forms of tablets , dragees, capsules, pills , and 30 gioendotheliosarcoma, synovioma , mesothelioma, Ewing's granules can be prepared with coatings and shells such as tumor, leiomyosarcoma, rhabdomyosarcoma, colon carci enteric coatings , release controlling coatings and other coat noma, pancreatic cancer , breast cancer, ovarian cancer , ings well known in the pharmaceutical formulating art . In prostate cancer, squamous cell carcinoma , basal cell carci such solid dosage forms the active compound may be noma, adenocarcinoma, sweat gland carcinoma, sebaceous admixed with at least one inert diluent such as sucrose , 35 gland carcinoma , papillary carcinoma, papillary adenocar lactose or starch . Such dosage forms may also comprise , as cinomas, cystadenocarcinoma, medullary carcinoma, bron is normal practice , additional substances other than inert chogenic carcinoma, renal cell carcinoma, hepatoma, bile diluents , e.g., tableting lubricants and other tableting aids duct carcinoma, choriocarcinoma, seminoma , embryonal such a magnesium stearate and microcrystalline cellulose . In carcinoma, Wilm's tumor, cervical cancer, uterine cancer, the case of capsules , tablets and pills , the dosage forms may 40 testicular cancer , lung carcinoma, small cell lung carcinoma , also comprise buffering agents . They may optionally contain bladder carcinoma, epithelial carcinoma, glioma, astrocy opacifying agents and can also be of a composition that they toma , glioblastoma multiforme (GBM , also known as glio release the active ingredient( s ) only , or preferentially , in a blastoma) , medulloblastoma , craniopharyngioma, certain part of the intestinal tract, optionally, in a delayed ependymoma, pinealoma, hemangioblastoma , acoustic neu manner. Examples of embedding compositions that can be 45 roma, oligodendroglioma , schwannoma, neurofibrosarcoma, used include polymeric substances and waxes. meningioma, melanoma , neuroblastoma, and retinoblas Dosage forms for topical or transdermal administration of toma ) . a compound of this invention include ointments , pastes, In some embodiments , the cancer is glioma, astrocytoma, creams, lotions , gels , powders , solutions, sprays , inhalants glioblastoma multiforme (GBM , also known as glioblas or patches . The active component is admixed under sterile 50 toma) , medulloblastoma, craniopharyngioma, ependymoma, conditions with a pharmaceutically acceptable carrier and pinealoma, hemangioblastoma, acoustic neuroma, oligoden any needed preservatives or buffers as may be required . droglioma, schwannoma, neurofibrosarcoma, meningioma, Ophthalmic formulation , ear drops, and eye drops are also melanoma, neuroblastoma, or retinoblastoma. contemplated as being within the scope of this invention . In some embodiments , the cancer is acoustic neuroma, Additionally , the present invention contemplates the use of 55 astrocytoma ( e.g. Grade IPilocytic Astrocytoma, Grade transdermal patches, which have the added advantage of II — Low - grade Astrocytoma, Grade III — Anaplastic Astro providing controlled delivery of a compound to the body. cytoma, or Grade IV – Glioblastoma (GBM ) ), chordoma, Such dosage forms can be made by dissolving or dispensing CNS lymphoma, craniopharyngioma, brain stem glioma, the compound in the proper medium . Absorption enhancers ependymoma, mixed glioma, optic nerve glioma, sub can also be used to increase the flux of the compound across 60 ependymoma , medulloblastoma, meningioma, metastatic the skin . The rate can be controlled by either providing a rate brain tumor , oligodendroglioma , pituitary tumors, primitive controlling membrane or by dispersing the compound in a neuroectodermal (PNET ) tumor, or schwannoma. In some polymer matrix or gel. embodiments , the cancer is a type found more commonly in 3. Uses of Compounds and Pharmaceutically Acceptable children than adults , such as brain stem glioma, craniophar Compositions 65 yngioma, ependymoma, juvenile pilocytic astrocytoma Compounds and compositions described herein are gen (JPA ), medulloblastoma, optic nerve glioma, pineal tumor, erally useful for treatment of cancer . primitive neuroectodermal tumors (PNET ), or rhabdoid US 10,624,968 B2 95 96 tumor. In some embodiments , the patient is an adult human . cystadenocarcinoma or uterine papillary serous carcinoma In some embodiments , the patient is a child or pediatric (UPSC ) ; prostate cancer ; testicular cancer; gallbladder can patient. cer ; hepatocholangiocarcinoma; soft tissue and bone syn Cancer includes, in another embodiment, without limita ovial sarcoma ; rhabdomyosarcoma; osteosarcoma; chondro tion , mesothelioma, hepatobilliary (hepatic and billiary 5 sarcoma; Ewing sarcoma; anaplastic thyroid cancer ; duct ) , bone cancer , pancreatic cancer , skin cancer, cancer of adrenocortical carcinoma; pancreatic cancer ; pancreatic the head or neck , cutaneous or intraocular melanoma, ovar ductal carcinoma or pancreatic adenocarcinoma; gastroin ian cancer , colon cancer, rectal cancer , cancer of the anal testinal /stomach (GIST ) cancer ; lymphoma; squamous cell region , stomach cancer, gastrointestinal (gastric , colorectal, carcinoma of the head and neck ( SCCHN ) ; salivary gland and duodenal) , uterine cancer, carcinoma of the fallopian 10 cancer ; glioma, or brain cancer ; neurofibromatosis - 1 asso tubes, carcinoma of the endometrium , carcinoma of the ciated malignant peripheral nerve sheath tumors (MPNST ) ; cervix , carcinoma of the vagina , carcinoma of the vulva , Waldenstrom's macroglobulinemia ; or medulloblastoma. Hodgkin's Disease , cancer of the esophagus, cancer of the In some embodiments , the cancer is selected from renal small intestine , cancer of the endocrine system , cancer of the cell carcinoma, hepatocellular carcinoma (HCC ) , hepato thyroid gland , cancer of the parathyroid gland , cancer of the 15 blastoma, colorectal carcinoma , colorectal cancer , colon adrenal gland , sarcoma of soft tissue, cancer of the urethra , cancer, rectal cancer , anal cancer, ovarian cancer, ovarian cancer of the penis , prostate cancer, testicular cancer, epithelial cancer, ovarian carcinoma, fallopian tube cancer, chronic or acute leukemia , chronic myeloid leukemia , lym papillary serous cystadenocarcinoma , uterine papillary phocytic lymphomas , cancer of the bladder, cancer of the serous carcinoma (UPSC ) , hepatocholangiocarcinoma, soft kidney or ureter , renal cell carcinoma, carcinoma of the renal 20 tissue and bone synovial sarcoma, rhabdomyosarcoma, pelvis , non -Hodgkins's lymphoma , spinal axis tumors , brain osteosarcoma, chondrosarcoma, anaplastic thyroid cancer , stem glioma, pituitary adenoma, adrenocortical cancer, gall adrenocortical carcinoma, pancreatic cancer, pancreatic duc bladder cancer, multiple myeloma , cholangiocarcinoma , tal carcinoma, pancreatic adenocarcinoma, glioma, brain fibrosarcoma, neuroblastoma, retinoblastoma, or a combi cancer , neurofibromatosis - 1 associated malignant peripheral nation of one or more of the foregoing cancers . 25 nerve sheath tumors (MPNST ) , Waldenstrom's macroglobu In some embodiments , the cancer is selected from hepato linemia , or medulloblastoma. cellular carcinoma, ovarian cancer , ovarian epithelial cancer , In some embodiments , the cancer is selected from hepato or fallopian tube cancer; papillary serous cystadenocarci cellular carcinoma (HCC ), hepatoblastoma, colon cancer, noma or uterine papillary serous carcinoma (UPSC ) ; pros rectal cancer, ovarian cancer , ovarian epithelial cancer , ovar tate cancer ; testicular cancer; gallbladder cancer ; hepatocho- 30 ian carcinoma , fallopian tube cancer , papillary serous cys langiocarcinoma ; soft tissue and bone synovial sarcoma; tadenocarcinoma, uterine papillary serous carcinoma rhabdomyosarcoma; osteosarcoma ; chondrosarcoma; Ewing (UPSC ), hepatocholangiocarcinoma, soft tissue and bone sarcoma; anaplastic thyroid cancer, adrenocortical adenoma; synovial sarcoma, rhabdomyosarcoma, osteosarcoma, ana pancreatic cancer ; pancreatic ductal carcinoma or pancreatic plastic thyroid cancer , adrenocortical carcinoma, pancreatic adenocarcinoma; gastrointestinal/ stomach (GIST ) cancer; 35 cancer , pancreatic ductal carcinoma, pancreatic adenocarci lymphoma; squamous cell carcinoma of the head and neck noma , glioma, neurofibromatosis - 1 associated malignant (SCCHN ) ; salivary gland cancer ; glioma , or brain cancer; peripheral nerve sheath tumors (MPNST ), Waldenstrom's neurofibromatosis -1 associated malignant peripheral nerve macroglobulinemia , or medulloblastoma. sheath tumors (MPNST ) ; Waldenstrom’s macroglobuline In some embodiments , the cancer is hepatocellular carci mia ; or medulloblastoma . 40 noma (HCC ). In some embodiments , the cancer is hepato In some embodiments , the cancer is selected from hepato blastoma . In some embodiments , the cancer is colon cancer . cellular carcinoma (HCC ), hepatoblastoma, colon cancer, In some embodiments , the cancer is rectal cancer. In some rectal cancer , ovarian cancer, ovarian epithelial cancer , fal embodiments , the cancer is ovarian cancer, or ovarian car lopian tube cancer , papillary serous cystadenocarcinoma, cinoma. In some embodiments , the cancer is ovarian epi uterine papillary serous carcinoma (UPSC ), hepatocholan- 45 thelial cancer. In some embodiments , the cancer is fallopian giocarcinoma, soft tissue and bone synovial sarcoma, rhab tube cancer. In some embodiments , the cancer is papillary domyosarcoma, osteosarcoma , anaplastic thyroid cancer, serous cystadenocarcinoma. In some embodiments , the can adrenocortical adenoma, pancreatic cancer, pancreatic duc cer is uterine papillary serous carcinoma (UPSC ) . In some tal carcinoma, pancreatic adenocarcinoma, glioma, neurofi embodiments , the cancer is hepatocholangiocarcinoma. In bromatosis - 1 associated malignant peripheral nerve sheath 50 some embodiments , the cancer is soft tissue and bone tumors (MPNST ) , Waldenstrom’s macroglobulinemia , or synovial sarcoma. In some embodiments , the cancer is medulloblastoma. rhabdomyosarcoma . In some embodiments , the cancer is In some embodiments , the present invention provides a osteosarcoma. In some embodiments , the cancer is anaplas method for treating a cancer that presents as a solid tumor, tic thyroid cancer . In some embodiments , the cancer is such as a sarcoma, carcinoma, or lymphoma, comprising the 55 adrenocortical carcinoma. In some embodiments , the cancer step of administering a disclosed compound , or a pharma is pancreatic cancer , or pancreatic ductal carcinoma. In some ceutically acceptable salt thereof, to a patient in need embodiments , the cancer is pancreatic adenocarcinoma. In thereof. Solid tumors generally comprise an abnormal mass some embodiments , the cancer is glioma. In some embodi of tissue that typically does not include cysts or liquid areas . ments , the cancer is malignant peripheral nerve sheath In some embodiments , the cancer is selected from renal cell 60 tumors (MPNST ) . In some embodiments , the cancer is carcinoma, or kidney cancer , hepatocellular carcinoma neurofibromatosis - 1 associated MPNST. In some embodi (HCC ) or hepatoblastoma , or liver cancer; melanoma ; breast ments , the cancer is Waldenstrom's macroglobulinemia . In cancer ; colorectal carcinoma, or colorectal cancer ; colon some embodiments , the cancer is medulloblastoma. cancer ; rectal cancer; anal cancer; lung cancer , such as The present invention further features methods and com non - small cell lung cancer (NSCLC ) or small cell lung 65 positions for the diagnosis , prognosis and treatment of cancer (SCLC ) ; ovarian cancer , ovarian epithelial cancer, viral- associated cancers , including human immunodefi ovarian carcinoma, or fallopian tube cancer; papillary serous ciency virus (HIV ) associated solid tumors , human papil US 10,624,968 B2 97 98 loma virus (HPV )-16 positive incurable solid tumors , and present invention , or a composition comprising said com adult T - cell leukemia , which is caused by human T -cell pound . In some embodiments , a cancer is as described in leukemia virus type I (HTLV - I) and is a highly aggressive detail herein . form of CD4 + T- cell leukemia characterized by clonal In some embodiments , the invention relates to a method integration of HTLV - I in leukemic cells (See https: // clini- 5 of treating cancer in a patient comprising the step of admin caltrials.gov/ct2/show/study/NCT02631746 ) ; as well as istering to said patient a compound of the present invention , virus -associated tumors in gastric cancer, nasopharyngeal or a composition comprising said compound , wherein the cancer is selected from colorectal cancer , non -small cell carcinoma, cervical cancer, vaginal cancer, vulvar cancer , lung cancer, breast cancer, gastric cancer, sarcoma, squamous cell carcinoma of the head and neck , and Merkel 10 myeloma, nasalpharyngeal/ laryngeal / oesophageal cancer , cell carcinoma. (See https://clinicaltrials.gov/ct2/show/ ovarian cancer , epithelial cancer, melanoma , glioma , astro study /NCT02488759 ; see also https://clinicaltrials.gov/ct2/ cytoma, glioblastoma, neuroblastoma, mesothlioma , bladder show / study /NCT0240886 ; https://clinicaltrials.gov/ct2/ cancer , hepatocellular carcinoma, and prostate cancer . In show /NCT02426892 ) some embodiments , the cancer is colorectal cancer. In some In some embodiments , the present invention provides a 15 embodiments , the cancer is non -small cell lung cancer. In method for treating a tumor in a patient in need thereof, some embodiments , the cancer is breast cancer . In some comprising administering to the patient any of the com embodiments , the cancer is gastric cancer. In some embodi pounds , salts or pharmaceutical compositions described ments , the cancer is sarcoma. In some embodiments , the herein . In some embodiments , the tumor comprises any of cancer is myeloma. In some embodiments , the cancer is the cancers described herein . In some embodiments , the 20 nasalpharyngeal /laryngeal / oesophageal cancer. In some tumor comprises melanoma cancer . In some embodiments , embodiments , the cancer is ovarian cancer. In some embodi the tumor comprises breast cancer . In some embodiments , ments , the cancer is epithelial cancer. In some embodiments , the tumor comprises cancer. In some embodiments the the cancer is melanoma. In some embodiments, the cancer is tumor comprises small cell lung cancer (SCLC ) . In some glioma. In some embodiments , the cancer is astrocytoma. In embodiments the the tumor comprises non - small cell lung 25 some embodiments , the cancer is glioblastoma. In some cancer (NSCLC ) . embodiments , the cancer is neuroblastoma. In some embodi In some embodiments , the tumor is treated by arresting ments , the cancer is mesothelioma. In some embodiments , the cancer is bladder cancer. In some embodiments , the further growth of the tumor. In some embodiments , the cancer is hepatocellular carcinoma. In some embodiments , tumor is treated by reducing the size ( e.g. , volume or mass ) 30 the cancer is prostate cancer. of the tumor by at least 5 % , 10 % , 25 % , 50 % , 75 % , 90 % or In some embodiments , the cancer is triple negative breast 99 % relative to the size of the tumor prior to treatment. In cancer. In some embodiments , the cancer is Her2 positive some embodiments , tumors are treated by reducing the breast cancer. In some embodiments , the cancer is lung quantity of the tumors in the patient by at least 5 % , 10 % , adenocarcinoma. In some embodiments , the cancer is lung 25 % , 50 % , 75 % , 90 % or 99 % relative to the quantity of 35 squamous cell carcinoma. In some embodiments , the cancer tumors prior to treatment . is fibrosarcoma. In some embodiments , the cancer is leio The compounds and compositions, according to the myosarcoma. In some embodiments , the cancer is rhab method of the present invention , may be administered using domyosarcoma. In some embodiments , the cancer is Osteo any amount and any route of administration effective for sarcoma. In some embodiments , the cancer is esophageal treating or lessening the severity of cancer. The exact 40 squamous cell carcinoma . In some embodiments , the cancer amount required will vary from subject to subject , depend is intestinal adenocarcinoma. In some embodiments , the ing on the species , age, and general condition of the subject , cancer is hepatocellular carcinoma. In some embodiments , the severity of the disease or condition, the particular agent, the cancer is uterine squamous cell carcinoma. its mode of administration , and the like . Compounds of the Co - Administration of Additional Therapeutic Agents invention are preferably formulated in dosage unit form for 45 Depending upon the particular condition , or disease , to be ease of administration and uniformity of dosage. The expres treated , additional therapeutic agents that are normally sion “ dosage unit form ” as used herein refers to a physically administered to treat that condition , may also be present in discrete unit of agent appropriate for the patient to be the compositions of this invention . As used herein , addi treated . It will be understood , however , that the total daily tional therapeutic agents that are normally administered to usage of the compounds and compositions of the present 50 treat a particular disease , or condition , are known as “ appro invention will be decided by the attending physician within priate for the disease , or condition , being treated . ” the scope of sound medical judgment. The specific effective In some embodiments , the present invention provides a dose level for any particular patient or organism will depend method of treating a disclosed disease or condition com upon a variety of factors including the disorder being treated prising administering to a patient in need thereof an effective and the severity of the disorder ; the activity of the specific 55 amount of a compound disclosed herein or a pharmaceuti compound employed ; the specific composition employed ; cally acceptable salt thereof and co -administering simulta the age, body weight , general health , sex and diet of the neously or sequentially an effective amount of one or more patient; the time of administration , route of administration , additional therapeutic agents , such as those described herein . and rate of excretion of the specific compound employed ; In some embodiments , the method includes co -administer the duration of the treatment; drugs used in combination or 60 ing one additional therapeutic agent. In some embodiments , coincidental with the specific compound employed , and like the method includes co -administering two additional thera factors well known in the medical arts . The term “ patient” , peutic agents . In some embodiments , the combination of the as used herein , means an animal , preferably a mammal, and disclosed compound and the additional therapeutic agent or most preferably a human . agents acts synergistically . In some embodiments , the invention relates to a method 65 In some embodiments , the additional therapeutic agent is of treating MT1- positive cancer in a patient comprising the selected from an immunostimulatory therapeutic compound . step of administering to said patient a compound of the In some embodiments , the immunostimulatory therapeutic US 10,624,968 B2 99 100 compound is selected from elotuzumab , mifamurtide, an ( NCT02520791 ) ; GSK3359609 (Merck ) , an agonistic anti agonist or activator of a toll- like receptor, or an activator of ICOS antibody, in Phase 1 (NCT02723955 ) ; JTX -2011 RORyt . ( Jounce Therapeutics ), an agonistic anti- ICOS antibody , in In some embodiments , the method further comprises Phase 1 (NCT02904226 ). administering to said patient a third therapeutic agent, such 5 Other checkpoint inhibitors that may be used in the as an immune checkpoint inhibitor. In some embodiments , present invention include killer IgG - like receptor (KIR ) the method comprises administering to the patient in need inhibitors. KIR inhibitors that are being studied in clinical thereof three therapeutic agents selected from a compound trials include lirilumab (IPH2102 /BMS - 986015 , Innate disclosed herein or a pharmaceutically acceptable salt Pharma /Bristol -Myers Squibb ), an anti- KIR antibody , in thereof, an immunostimulatory therapeutic compound , and 10 leukemias (NCT01687387 , NCT02399917 , NCT02481297 , an immune checkpoint inhibitor. NCT02599649) , multiple myeloma (NCT02252263 ) , and Other checkpoint inhibitors that may be used in the lymphoma (NCT01592370 ); IPH2101 (1-7F9 , Innate present invention include OX40 agonists . OX40 agonists Pharma) in myeloma (NCT01222286 and NCT01217203 ); that are being studied in clinical trials include PF -04518600 / and IPH4102 ( Innate Pharma) , an anti -KIR antibody that PF - 8600 (Pfizer ), an agonistic anti -OX40 antibody, in meta- 15 binds to three domains of the long cytoplasmic tail static kidney cancer (NCT03092856 ) and advanced cancers (KIR3DL2 ), in lymphoma (NCT02593045 ) . and neoplasms (NCT02554812 ; NCT05082566 ) ; Other checkpoint inhibitors that may be used in the GSK3174998 (Merck ) , an agonistic anti- OX40 antibody, in present invention include CD47 inhibitors of interaction Phase 1 cancer trials ( NCT02528357 ) ; MEDI0562 (Medim between CD47 and signal regulatory protein alpha (SIRPa ) . mune/ AstraZeneca ), an agonistic anti - OX40 antibody, in 20 CD47 /SIRPa inhibitors that are being studied in clinical advanced solid tumors (NCT02318394 and NCT02705482 ) ; trials include ALX -148 (Alexo Therapeutics ), an antagonis MEDI6469 , an agonistic anti -OX40 antibody (Medimmune / tic variant of (SIRPa ) that binds to CD47 and prevents AstraZeneca ) , in patients with colorectal cancer CD47 /SIRPa -mediated signaling , in phase 1 (NCT02559024 ) , breast cancer (NCT01862900 ) , head and (NCT03013218 ) ; TTI -621 (SIRPa -Fc , Trillium Therapeu neck cancer (NCT02274155 ) and metastatic prostate cancer 25 tics ), a soluble recombinant fusion protein created by linking (NCTO1303705 ); and BMS- 986178 (Bristol - Myers Squibb ) the N - terminal CD47 -binding domain of SIRPa with the Fc an agonistic anti -OX40 antibody, in advanced cancers domain of human IgG1, acts by binding human CD47 , and (NCT02737475 ) . preventing it from delivering its “ do not eat ” signal to Other checkpoint inhibitors that may be used in the macrophages, is in clinical trials in Phase 1 (NCT02890368 present invention include CD137 (also called 4 - IBB ) ago- 30 and NCT02663518 ); CC - 90002 (Celgene ), an anti -CD47 nists . CD137 agonists that are being studied in clinical trials antibody, in leukemias (NCT02641002 ) ; and Hu5F9 -G4 include utomilumab ( PF -05082566 , Pfizer ) an agonistic (Forty Seven , Inc. ), in colorectal neoplasms and solid anti- CD137 antibody , in diffuse large B -cell lymphoma tumors ( NCT02953782 ), acute myeloid leukemia ( NCT02951156 ) and in advanced cancers and neoplasms (NCT02678338 ) and lymphoma (NCT02953509 ) . (NCT02554812 and NCT05082566 ) ; urelumab (BMS- 35 Other checkpoint inhibitors that may be used in the 663513, Bristol -Myers Squibb ) , an agonistic anti- CD137 present invention include CD73 inhibitors . CD73 inhibitors antibody, in melanoma and skin cancer (NCT02652455 ) and that are being studied in clinical trials include MEDI9447 glioblastoma and gliosarcoma (NCT02658981 ). (Medimmune ), an anti- CD73 antibody , in solid tumors Other checkpoint inhibitors that may be used in the (NCT02503774 ) ; and BMS - 986179 ( Bristol -Myers Squibb ) , present invention include CD27 agonists . CD27 agonists 40 an anti -CD73 antibody , in solid tumors (NCT02754141 ). that are being studied in clinical trials include varlilumab Other checkpoint inhibitors that may be used in the ( CDX - 1127 , Celldex Therapeutics ) an agonistic anti- CD27 present invention include agonists of stimulator of interferon antibody , in squamous cell head and neck cancer, ovarian genes protein (STING , also known as transmembrane pro carcinoma, colorectal cancer, renal cell cancer, and glioblas tein 173 , or TMEM173 ). Agonists of STING that are being toma (NCT02335918 ); lymphomas (NCT01460134 ); and 45 studied in clinical trials include MK - 1454 (Merck ) , an glioma and astrocytoma ( NCT02924038 ). agonistic synthetic cyclic dinucleotide, in lymphoma Other checkpoint inhibitors that may be used in the (NCT03010176 ); and ADU - S100 (MIW815 , Aduro Bio present invention include glucocorticoid - induced tumor tech /Novartis ) , an agonistic synthetic cyclic dinucleotide, in necrosis factor receptor (GITR ) agonists . GITR agonists that Phase 1 (NCT02675439 and NCT03172936 ) . are being studied in clinical trials include TRX518 (Leap 50 Other checkpoint inhibitors that may be used in the Therapeutics ), an agonistic anti -GITR antibody, in malig present invention include CSFIR inhibitors . CSF1R inhibi nant melanoma and other malignant solid tumors tors that are being studied in clinical trials include pexidar ( NCT01239134 and NCT02628574 ) ; GWN323 (Novartis ) , tinib (PLX3397 , Plexxikon ) , a CSFIR small molecule an agonistic anti -GITR antibody, in solid tumors and lym inhibitor , in colorectal cancer, pancreatic cancer , metastatic phoma (NCT 02740270 ); INCAGN01876 ( Incyte /Agenus ) , 55 and advanced cancers (NCT02777710 ) and melanoma, non an agonistic anti- GITR antibody , in advanced cancers small cell lung cancer, squamous cell head and neck cancer , (NCT02697591 and NCT03126110 ) ; MK -4166 (Merck ), an gastrointestinal stromal tumor (GIST ) and ovarian cancer agonistic anti -GITR antibody , in solid tumors (NCT02452424 ) ; and 1MC - CS4 (LY3022855 , Lilly ), an (NCT02132754 ) and MEDI1873 (Medimmune / AstraZen anti- CSF - 1R antibody , in pancreatic cancer eca ) , an agonistic hexameric GITR - ligand molecule with a 60 (NCT03153410 ) , melanoma (NCT03101254 ) , and solid human IgG1 Fc domain , in advanced solid tumors tumors (NCT02718911 ) ; and BLZ945 (4- [ 2 (( 1R ,2R ) -2 -hy (NCT02583165 ) . droxycyclohexylamino )-benzothiazol - 6 - yloxyl] -pyridine -2 Other checkpoint inhibitors that may be used in the carboxylic acid methylamide , Novartis ) , an orally available present invention include inducible T - cell co -stimulator inhibitor of CSFIR , in advanced solid tumors ( ICOS, also known as CD278 ) agonists . ICOS agonists that 65 (NCT02829723 ) . are being studied in clinical trials include MEDI- 570 (Med Other checkpoint inhibitors that may be used in the immune ) , an agonistic anti - ICOS antibody, in lymphomas present invention include NKG2A receptor inhibitors . US 10,624,968 B2 101 102 NKG2A receptor inhibitors that are being studied in clinical In another aspect , the present invention provides a method trials include monalizumab (IPH2201 , Innate Pharma) , an of treating cancer in a patient in need thereof, wherein said anti- NKG2A antibody, in head and neck neoplasms method comprises administering to said patient a compound (NCT02643550 ) and chronic lymphocytic leukemia disclosed herein or a pharmaceutically acceptable salt (NCT02557516 ) . 5 thereof in combination with one or more additional thera peutic agents selected from a platinum -based therapeutic , a In some embodiments , the immune checkpoint inhibitor is taxane, a nucleoside inhibitor , or a therapeutic agent that selected from nivolumab , pembrolizumab , ipilimumab , ave interferes with normal DNA synthesis , protein synthesis, cell lumab , durvalumab , atezolizumab , or pidilizumab . replication , or will otherwise inhibit rapidly proliferating In another aspect, the present invention provides a method 10 cells . of treating cancer in a patient in need thereof, wherein said In some embodiments , the platinum - based therapeutic is method comprises administering to said patient a compound selected from cisplatin , carboplatin , oxaliplatin , nedaplatin , disclosed herein or a pharmaceutically acceptable salt picoplatin , or satraplatin . thereof in combination with one or more additional thera peutic agents selected from an indoleamine ( 2,3 )-dioxy- 15 taxelIn , some docetaxel embodiments, albumin , -thebound taxane paclitaxel is selected, cabazitaxel from pacli, or genase (IDO ) inhibitor, a Poly ADP ribose polymerase SID530 . (PARP ) inhibitor, a histone deacetylase (HDAC ) inhibitor , a In some embodiments , the therapeutic agent that inter CDK4 /CDK6 inhibitor, or a phosphatidylinositol 3 kinase feres with normal DNA synthesis , protein synthesis , cell (PI3K ) inhibitor. replication , or will otherwise interfere with the replication of In some embodiments , the IDO inhibitor is selected from 20 rapidly proliferating cells is selected from trabectedin , epacadostat , indoximod , capmanitib , GDC -0919 , mechlorethamine, vincristine, temozolomide , cytarabine , PF - 06840003 , BMS: F001287 , Phy906 /KD108 , or an lomustine , azacitidine, omacetaxine mepesuccinate , aspara enzyme that breaks down kynurenine . ginase Erwinia chrysanthemi, eribulin mesylate , capace In some embodiments , the PARP inhibitor is selected trine, bendamustine , ixabepilone, nelarabine , clorafabine , from olaparib , rucaparib , or niraparib . 25 trifluridine , or tipiracil . In some embodiments , the HDAC inhibitor is selected In some embodiments , the method further comprises from vorinostat, romidepsin , panobinostat, belinostat, enti administering to said patient a third therapeutic agent, such nostat, or chidamide . as an immune checkpoint inhibitor. In some embodiments , In some embodiments , the CDK 4/6 inhibitor is selected the method comprises administering to the patient in need from palbociclib , ribociclib , abemaciclib or trilaciclib . 30 thereof three therapeutic agents selected from a compound In some embodiments , the method further comprises disclosed herein or a pharmaceutically acceptable salt administering to said patient a third therapeutic agent, such thereof, a second therapeutic agentselected from a platinum as an immune checkpoint inhibitor. In some embodiments , based therapeutic , a taxane , a nucleoside inhibitor, or a the method comprises administering to the patient in need therapeutic agent that interferes with normal DNA synthesis , thereof three therapeutic agents selected from a compound 35 protein synthesis , cell replication , or will otherwise inhibit disclosed herein or a pharmaceutically acceptable salt rapidly proliferating cells , and a third therapeutic agent thereof, a second therapeutic agent selected from an selected from an immune checkpoint inhibitor. indoleamine ( 2,3 ) -dioxygenase (IDO ) inhibitor, a Poly ADP In some embodiments , the immune checkpoint inhibitor is ribose polymerase ( PARP ) inhibitor, a histone deacetylase selected from nivolumab , pembrolizumab , ipilimumab , ave (HDAC ) inhibitor, a CDK4 /CDK6 inhibitor, or a phospha- 40 lumab , durvalumab , atezolizumab , or pidilizumab . tidylinositol 3 kinase ( PI3K ) inhibitor, and a third therapeu In some embodiments , any one of the foregoing methods tic agent selected from an immune checkpoint inhibitor . In further comprises the step of obtaining a biological sample some embodiments , the immune checkpoint inhibitor is from the patient and measuring the amount of a disease selected from nivolumab , pembrolizumab , ipilimumab , ave related biomarker . lumab , durvalumab , atezolizumab , or pidilizumab . 45 In some embodiments , the biological sample is a blood Another immunostimulatory therapeutic that may be used sample . in the present invention is recombinant human interleukin 15 In some embodiments , the disease - related biomarker is ( rhIL - 15 ) . rhIL - 15 has been tested in the clinic as a therapy selected from circulating CD8 + T cells or the ratio of CD8 + for melanoma and renal cell carcinoma (NCT01021059 and T cells : Treg cells . NCT01369888 ) and leukemias (NCT02689453 ) . Another 50 In one aspect , the present invention provides a method of immunostimulatory therapeutic that may be used in the treating an advanced cancer, comprising administering a present invention is recombinant human interleukin 12 compound disclosed herein or a pharmaceutically accept ( rhIL - 12 ) . Another suitable IL - 15 based immunotherapeutic able salt thereof or pharmaceutical composition thereof, is heterodimeric IL - 15 (hetIL - 15 , Novartis / Admune ) , a either as a single agent (monotherapy ), or in combination fusion complex composed of a synthetic form of endog- 55 with a chemotherapeutic , a targeted therapeutic , such as a enous IL - 15 complexed to the soluble IL - 15 binding protein kinase inhibitor , and /or an immunomodulatory therapy , such IL - 15 receptor alpha chain ( IL15 :SIL - 15RA ), which has as an immune checkpoint inhibitor. In some embodiments , been tested in Phase 1 clinical trials for melanoma, renal cell the immune checkpoint inhibitor is an antibody to PD - 1 . carcinoma, non - small cell lung cancer and head and neck PD - 1 binds to the programmed cell death 1 receptor (PD - 1) squamous cell carcinoma (NCT02452268 ) . Recombinant 60 to prevent the receptor from binding to the inhibitory ligand human interleukin 12 (rhlL -12 ) has been tested in the clinic PDL - 1 , thus overriding the ability of tumors to suppress the for many oncological indications , for example , as a therapy host anti -tumor immune response . for lymphoma (NM - IL - 12 , Neumedicines, Inc.) , In some embodiments , the additional therapeutic agent is (NCT02544724 and NCT02542124 ) . a kinase inhibitor or VEGF - R antagonist . Approved VEGF In some embodiments , the PI3K inhibitor is selected from 65 inhibitors and kinase inhibitors useful in the present inven idelalisib , alpelisib , taselisib , pictilisib , copanlisib , tion include: bevacizumab (Avastin® , Genentech /Roche ) an duvelisib , PQR309 , or TGR1202 . anti - VEGF monoclonal antibody ; ramucirumab US 10,624,968 B2 103 104 ( Cyramza® , Eli Lilly ) , an anti - VEGFR - 2 antibody and useful in the present invention include bortezomib ( Vel ziv -aflibercept , also known as VEGF Trap (Zaltrap® ; cade® , Takeda ); carfilzomib (Kyprolis® , Amgen ) ; and ixa Regeneron / Sanofi) . VEGFR inhibitors , such as regorafenib zomib (Ninlaro , Takeda ). ( Stivargar , Bayer ); vandetanib (Caprelsa , AstraZeneca ); In some embodiments , the additional therapeutic agent is axitinib ( Inlyta® , Pfizer) ; and lenvatinib (Lenvima® , Eisai ); 5 a histone deacetylase (HDAC ) inhibitor. Approved HDAC Raf inhibitors , such as sorafenib (Nexavar® , Bayer AG and inhibitors useful in the present invention include vorinostat Onyx ); dabrafenib ( Tafinlar® , Novartis ); and vemurafenib (Zolinza® , Merck ) ; romidepsin ( Istodax® , Celgene ); (Zelboraf® , Genentech /Roche ) ; MEK inhibitors , such as panobinostat (Farydak® , Novartis ); and belinostat (Be cobimetanib ( Cotellic® , Exelexis /Genentech /Roche ); tram leodaq® , Spectrum Pharmaceuticals ). Other HDAC inhibi etinib (Mekinist® , Novartis ) ; Ber - Abl tyrosine kinase 10 tors being studied which may be used in the present inven inhibitors , such as imatinib (Gleevec® , Novartis ); nilotinib tion include entinostat (SNDX - 275 , Syndax ( Tasignar , Novartis ) ; dasatinib (Sprycel® , BristolMyersS Pharmaceuticals ) (NCT00866333 ) ; and chidamide (Epi quibb ) ; bosutinib ( Bosulif® , Pfizer ); and ponatinib ( In daza® , HB1-8000 , Chipscreen Biosciences , China ) . clusig® , Ariad Pharmaceuticals ) ; Her2 and EGFR inhibi In some embodiments , the additional therapeutic agent is tors , such as gefitinib ( Iressa® , AstraZeneca) ; erlotinib 15 a CDK inhibitor , such as a CDK 4/6 inhibitor. Approved ( Tarceeva® , Genentech /Roche / Astellas ); lapatinib ( Tyk CDK 4/6 inhibitors useful in the present invention include erb® , Novartis ); afatinib (Gilotrif® , Boehringer Ingelheim ); palbociclib ( Ibrance® , Pfizer ); and ribociclib (Kisqali® , osimertinib (targeting activated EGFR , Tagrisso® , Astra Novartis ) . Other CDK 4/6 inhibitors being studied which Zeneca ); and brigatinib (Alunbrig® , Ariad Pharmaceuti may be used in the present invention include abemaciclib cals ) ; c -Met and VEGFR2 inhibitors , such as cabozanitib 20 (Ly2835219 , Eli Lilly ) ; and trilaciclib (G1T28 , G1 Thera (Cometriq® , Exelexis ); and multikinase inhibitors , such as peutics ). sunitinib (Sutent® , Pfizer ); pazopanib (Votrient® , Novar In some embodiments , the additional therapeutic agent is tis ) ; ALK inhibitors , such as crizotinib ( Xalkori® , Pfizer ); an indoleamine ( 2,3 ) -dioxygenase ( IDO ) inhibitor. IDO ceritinib (Zykadia® , Novartis ) ; and alectinib ( Alecenza® , inhibitors being studied which may be used in the present Genentech /Roche ) ; Bruton's tyrosine kinase inhibitors , such 25 invention include epacadostat ( INCB024360 , Incyte ) ; as ibrutinib (Imbruvica® , Pharmacyclics/ Janssen ) ; and Fit3 indoximod (NLG - 8189 , NewLink Genetics Corporation ); receptor inhibitors , such as midostaurin (Rydapt® , Novar capmanitib ( INC280 , Novartis ) ; GDC -0919 (Genentech / tis ) . Roche ); PF -06840003 (Pfizer ) ; BMS :F001287 (Bristol - My Other kinase inhibitors and VEGF - R antagonists that are ers Squibb ) ; Phy906 /KD108 (Phytoceutica ); and an enzyme in development and may be used in the present invention 30 that breaks down kynurenine (Kynase , Kyn Therapeutics) . include tivozanib (Aveo Pharmaecuticals ) ; vatalanib (Bayer / In some embodiments , the additional therapeutic agent is Novartis ) ; lucitanib (Clovis Oncology ) ; dovitinib ( TKI258 , a growth factor antagonist , such as an antagonist of platelet Novartis ); Chiauanib (Chipscreen Biosc ces ) ; CEP - 11981 derived growth factor (PDGF ), or epidermal growth factor ( Cephalon ) ; linifanib (Abbott Laboratories) ; neratinib (HKI ( EGF) or its receptor (EGFR ) . Approved PDGF antagonists 272 , Puma Biotechnology ) ; radotinib (Supect® , ?Y5511 , 35 which may be used in the present invention include olara Il - Yang Pharmaceuticals , S. Korea ) ; ruxolitinib (Jakafi® , tumab (Lartruvo? ; Eli Lilly ) . Approved EGFR antagonists Incyte Corporation ) ; PTC299 (PTC Therapeutics ); CP - 547 , which may be used in the present invention include cetux 632 (Pfizer ); foretinib (Exelexis , GlaxoSmithKline ); quizar imab (Erbitux® , Eli Lilly ); necitumumab (Portrazza® , Eli tinib (Daiichi Sankyo ) and motesanib ( Amgen / Takeda ) . Lilly ), panitumumab (Vectibix® , Amgen ); and osimertinib In some embodiments , the additional therapeutic agent is 40 (targeting activated EGFR , Tagrisso® , AstraZeneca ). an mTOR inhibitor, which inhibits cell proliferation , angio In some embodiments , the additional therapeutic agent is genesis and glucose uptake. Approved mTOR inhibitors an aromatase inhibitor. Approved aromatase inhibitors useful in the present invention include everolimus ( Afini which may be used in the present invention include exemes tor® , Novartis ); temsirolimus ( Torisel® , Pfizer ) ; and siroli tane ( Aromasin® , Pfizer) ; anastazole (Arimidex® , Astra mus (Rapamune® , Pfizer ). 45 Zeneca ) and letrozole ( Femara® , Novartis ) . In some embodiments , the additional therapeutic agent is In some embodiments , the additional therapeutic agent is a Poly ADP ribose polymerase (PARP ) inhibitor. Approved an antagonist of the hedgehog pathway . Approved hedgehog PARP inhibitors useful in the present invention include pathway inhibitors which may be used in the present inven olaparib (Lynparzar , AstraZeneca ); rucaparib (Rubracar , tion include sonidegib (Odomzor , Sun Pharmaceuticals ) ; Clovis Oncology ) ; and niraparib ( Zejula® , Tesaro ) . Other 50 and vismodegib (Erivedge® , Genentech ), both for treatment PARP inhibitors being studied which may be used in the of basal cell carcinoma . present invention include talazoparib (MDV3800 /BMN In some embodiments , the additional therapeutic agent is 673 /LT00673 , Medivation /Pfizer /Biomarin ) ; veliparib a folic acid inhibitor. Approved folic acid inhibitors useful in ( ABT- 888 , AbbVie ); and BGB - 290 (Beigene , Inc. ). the present invention include pemetrexed (Alimta® , Eli In some embodiments , the additional therapeutic agent is 55 Lilly ) . a phosphatidylinositol 3 kinase (PI3K ) inhibitor. Approved In some embodiments , the additional therapeutic agent is PI3K inhibitors useful in the present invention include a CC chemokine receptor 4 (CCR4 ) inhibitor. CCR4 inhibi idelalisib ( Zydelig® , Gilead ). Other PI3K inhibitors being tors being studied that may be useful in the present invention studied which may be used in the present invention include include mogamulizumab ( Poteligeo® , Kyowa Hakko Kirin , alpelisib (BYL719 , Novartis ); taselisib (GDC - 0032 ,Genen- 60 Japan ). tech /Roche ); pictilisib (GDC -0941 , Genentech /Roche ) ; In some embodiments , the additional therapeutic agent is copanlisib (BAY806946 , Bayer ) ; duvelisib ( formerly IPI an isocitrate dehydrogenase (IDH ) inhibitor. IDH inhibitors 145 , Infinity Pharmaceuticals ); PQR309 (Piqur Therapeu being studied which may be used in the present invention tics, Switzerland ) ; and TGR1202 ( formerly RP5230 , TG include AG120 (Celgene ; NCT02677922 ) ; AG221 ( Cel Therapeutics ) . 65 gene , NCT02677922 ; NCT02577406 ) ; BAY1436032 In some embodiments , the additional therapeutic agent is (Bayer , NCT02746081 ); IDH305 (Novartis , a proteasome inhibitor. Approved proteasome inhibitors NCT02987010 ). US 10,624,968 B2 105 106 In some embodiments , the additional therapeutic agent is Aventis ) ; and trifluridine and tipiracil ( thymidine- based an arginase inhibitor. Arginase inhibitors being studied nucleoside analog and thymidine phosphorylase inhibitor , which may be used in the present invention include Lonsurf® , Taiho Oncology ) . AEB1102 (pegylated recombinant arginase, Aeglea Bio In some embodiments , the additional therapeutic agent is therapeutics ) , which is being studied in Phase 1 clinical trials 5 a platinum -based therapeutic , also referred to as platins. for acute myeloid leukemia and myelodysplastic syndrome Platins cause cross- linking of DNA , such that they inhibit (NCT02732184 ) and solid tumors (NCT02561234 ) ; and DNA repair and /or DNA synthesis , mostly in rapidly repro CB - 1158 (Calithera Biosciences ) . ducing cells , such as cancer cells . Approved platinum -based In some embodiments , the additional therapeutic agent is therapeutics which may be used in the present invention a glutaminase inhibitor . Glutaminase inhibitors being stud- 10 include cisplatin (Platinol® , Bristol- Myers Squibb ) ; carbo ied which may be used in the present invention include platin (Paraplatin® , Bristol- Myers Squibb ; also , Teva ; CB -839 (Calithera Biosciences ). Pfizer ) ; oxaliplatin (Eloxitin® Sanofi- Aventis ) ; and neda In some embodiments , the additional therapeutic agent is platin (Aqupla® , Shionogi ). Other platinum -based therapeu an antibody that binds to tumor antigens, that is , proteins tics which have undergone clinical testing and may be used expressed on the cell surface of tumor cells . Approved 15 in the present invention include picoplatin ( Poniard Phar antibodies that bind to tumor antigens which may be used in maceuticals ) ; and satraplatin ( JM -216 , Agennix ) . the present invention include rituximab (Rituxan® , Genen In some embodiments , the additional therapeutic agent is tech /Biogen Idec ) ; ofatumumab ( anti -CD20 , Arzerra? , a taxane compound, which causes disruption of microtu GlaxoSmithKline ); obinutuzumab (anti -CD20 , Gazyva? , bules, which are essential for cell division . Approved taxane Genentech ), ibritumomab (anti - CD20 and Yttrium - 90 , 20 compounds which may be used in the present invention Zevalin® , Spectrum Pharmaceuticals ); daratumumab ( anti include paclitaxel ( Taxol® , Bristol- Myers Squibb ), doc CD38, Darzalex® , Janssen Biotech ), dinutuximab ( anti etaxel ( Taxotere® , Sanofi - Aventis ; Docefrez® , Sun Phar glycolipid GD2 , Unituxin® , United Therapeutics) ; trastu maceutical) , albumin -bound paclitaxel ( Abraxane? ; zumab ( anti- HER2 , Herceptin® , Genentech ) ; ado Abraxis / Celgene ), and cabazitaxel ( Jevtana® , Sanofi- Aven trastuzumab emtansine (anti -HER2 , fused to emtansine , 25 tis ). Other taxane compounds which have undergone clinical Kadcyla , Genentech ); and pertuzumab (anti -HER2 , Per testing and may be used in the present invention include jeta® , Genentech ); and brentuximab vedotin (anti - CD30 SID530 (SK Chemicals , Co. ) (NCT00931008 ) . drug conjugate , Adcetris® , Seattle Genetics ). In some embodiments , the additional therapeutic agent is In some embodiments , the additional therapeutic agent is an inhibitor of anti -apoptotic proteins, such as BCL - 2 . a topoisomerase inhibitor. Approved topoisomerase inhibi- 30 Approved anti- apoptotics which may be used in the present tors useful in the present invention include irinotecan (On invention include venetoclax (Venclexta® , AbbVie /Genen ivyde® , Merrimack Pharmaceuticals ) ; topotecan (Hycam tech ) ; and blinatumomab (Blincyto® , Amgen ) . Other thera tin® , GlaxoSmithKline ). Topoisomerase inhibitors being peutic agents targeting apoptotic proteins which have under studied which may be used in the present invention include gone clinical testing and may be used in the present pixantrone (Pixuvri® , CTI Biopharma) . 35 invention include navitoclax (ABT - 263 , Abbott ), a BCL - 2 In some embodiments , the additional therapeutic agent is inhibitor (NCT02079740 ) . a nucleoside inhibitor, or other therapeutic that interfere with In some embodiments , the present invention provides a normal DNA synthesis , protein synthesis , cell replication , or method of treating prostate cancer comprising administering will otherwise inhibit rapidly proliferating cells . Such to a patient in need thereof an effective amount of a nucleoside inhibitors or other therapeutics include trabect- 40 compound disclosed herein or a pharmaceutically accept edin (guanidine alkylating agent, Yondelis® , Janssen Oncol able salt thereof or pharmaceutical composition thereof in ogy ), mechlorethamine ( alkylating agent, Valchlor® , Akte combination with an additional therapeutic agent that inter lion Pharmaceuticals ) ; vincristine (Oncovin® , Eli Lilly ; feres with the synthesis or activity of androgens. Approved Vincasar® , Teva Pharmaceuticals ; Marqibo® , Talon Thera androgen receptor inhibitors useful in the present invention peutics ) ; temozolomide (prodrug to alkylating agent 5- (3- 45 include enzalutamide (Xtandi® , Astellas/ Medivation ) ; methyltriazen - 1 -yl ) -imidazole - 4 -carboxamide (MTIC ) approved inhibitors of androgen synthesis include abirater Temodar® , Merck ); cytarabine injection (ara - C , antimeta one (Zytiga® , Centocor/ Ortho ); approved antagonist of bolic cytidine analog, Pfizer ); lomustine (alkylating agent, gonadotropin - releasing hormone (GnRH ) receptor (dega CeeNU® , Bristol- Myers Squibb ; Gleostine® , NextSource ralix , Firmagon , Ferring Pharmaceuticals ) . Biotechnology ) ; azacitidine (pyrimidine nucleoside analog 50 In some embodiments , the additional therapeutic agent is of cytidine, Vidaza® , Celgene) ; omacetaxinemepesuccinate a selective estrogen receptor modulator (SERM ) , which ( cephalotaxine ester ) ( protein synthesis inhibitor, Synribo® ; interferes with the synthesis or activity of estrogens. Teva Pharmaceuticals ) ; asparaginase Erwinia chrysanthemi Approved SERMs useful in the present invention include ( enzyme for depletion of asparagine , Elspar® , Lundbeck ; raloxifene (Evista® , Eli Lilly ) . Erwinaze® , EUSA Pharma) ; eribulin mesylate (microtubule 55 In some embodiments , the additional therapeutic agent is inhibitor, tubulin -based antimitotic, Halaven® , Eisai) ; caba an inhibitor of bone resorption . An approved therapeutic zitaxel (microtubule inhibitor, tubulin -based antimitotic , which inhibits bone resorption is Denosumab (Xgeva® , Jevtana® , Sanofi- Aventis ) ; capacetrine (thymidylate syn Amgen ), an antibody that binds to RANKL , prevents bind thase inhibitor, Xeloda® , Genentech ) ; bendamustine (bi ing to its receptor RANK , found on the surface of osteo functional mechlorethamine derivative , believed to form 60 clasts , their precursors, and osteoclast- like giant cells, which interstrand DNA cross - links, Treanda® , Cephalon / Teva ) ; mediates bone pathology in solid tumors with osseous ixabepilone ( semi- synthetic analog of epothilone B , micro metastases. Other approved therapeutics that inhibit bone tubule inhibitor , tubulin -based antimitotic , Ixempra® , Bris resorption include bisphosphonates, such as zoledronic acid tol- Myers Squibb ); nelarabine ( prodrug of deoxyguanosine ( Zometa® , Novartis ) . analog , nucleoside metabolic inhibitor, Arranon® , Novar- 65 In some embodiments , the additional therapeutic agent is tis ) ; clorafabine (prodrug of ribonucleotide reductase inhibi an inhibitor of interaction between the two primary p53 tor , competitive inhibitor of deoxycytidine, Clolar® , Sanofi suppressor proteins, MDMX and MDM2. Inhibitors of p53 US 10,624,968 B2 107 108 suppression proteins being studied which may be used in the CSF, for hepatocellular carcinoma (NCT02562755 ) and present invention include ALRN -6924 ( Aileron ) , a stapled melanoma (NCT00429312 ); pelareorep (Reolysin® , Onco peptide that equipotently binds to and disrupts the interac lytics Biotech ), a variant of respiratory enteric orphan virus tion of MDMX and MDM2 with p53 . ALRN -6924 is (reovirus ) which does not replicate in cells that are not currently being evaluated in clinical trials for the treatment 5 RAS -activated , in numerous cancers, including colorectal of AML , advanced myelodysplastic syndrome (MDS ) and cancer (NCT01622543 ) ; prostate cancer (NCT01619813 ) ; peripheral T -cell lymphoma (PTCL ) (NCT02909972 ; head and neck squamous cell cancer (NCT01166542 ); pan NCT02264613 ) . creatic adenocarcinoma (NCT00998322 ) ; and non - small In some embodiments , the additional therapeutic agent is cell lung cancer (NSCLC ) (NCT 00861627 ); enadenotucirev an inhibitor of transforming growth factor- beta ( TGF -beta or 10 (NG - 348, PsiOxus, formerly known as ColoAdl) , an adeno TGFB ) . Inhibitors of TGF- beta proteins being studied which virus engineered to express a full length CD80 and an may be used in the present invention include NIS793 antibody fragment specific for the T -cell receptor CD3 (Novartis ) , an anti- TGF -beta antibody being tested in the protein , in ovarian cancer (NCT02028117 ) ; metastatic or clinic for treatment of various cancers, including breast, advanced epithelial tumors such as in colorectal cancer, lung , hepatocellular, colorectal, pancreatic , prostate and 15 bladder cancer, head and neck squamous cell carcinoma and renal cancer (NCT 02947165 ) . In some embodiments , the salivary gland cancer (NCT02636036 ) ; ONCOS -102 (Tar inhibitor of TGF -beta proteins is fresolimumab (GC1008 ; govax / formerly Oncos ), an adenovirus engineered to Sanofi -Genzyme ) , which is being studied for melanoma express GM - CSF , in melanoma (NCT03003676 ) ; and peri (NCT00923169 ) ; renal cell carcinoma (NCT00356460 ) ; and toneal disease , colorectal cancer or ovarian cancer non - small cell lung cancer (NCT02581787 ) . Additionally , in 20 (NCT02963831 ) ;GL -ONC 1 (GLV - 1h68 /GLV - 1h153, Gen some embodiments, the additional therapeutic agent is a elux GmbH ) , vaccinia viruses engineered to express beta TGF- beta trap , such as described in Connolly et al. (2012 ) galactosidase (beta -gal )/ beta -glucoronidase or beta -gal / hu Int'l J. Biological Sciences 8 : 964-978 . One therapeutic man sodium iodide symporter (HNIS ) , respectively , were compound currently in clinical trials for treatment of solid studied in peritoneal carcinomatosis (NCT01443260 ) ; fal tumors is M7824 (Merck KgaA— formerly 25 lopian tube cancer , ovarian cancer (NCT 02759588 ); or MSB0011459X ) , which is a bispecific , anti - PD - L1/ TGFB CG0070 (Cold Genesys ) , an adenovirus engineered to trap compound (NCT02699515 ) ; and (NCT02517398 ) . express GM - CSF , in bladder cancer ( NCT02365818 ). M7824 is comprised of a fully human IgG1 antibody against In some embodiments , the additional therapeutic agent is PD -L1 fused to the extracellular domain of human TGF -beta selected from JX - 929 ( SillaJen / formerly Jennerex Biothera receptor II , which functions as a TGFB “ trap .” 30 peutics ), a TK - and vaccinia growth factor- deficient vaccinia Additional Co- Administered Therapeutic Agents — Targeted virus engineered to express cytosine deaminase , which is Therapeutics and Immunomodulatory Drugs able to convert the prodrug 5 - fluorocytosine to the cytotoxic In some embodiments , the additional therapeutic agent is drug 5 - fluorouracil ; TGO1 and TGO2 ( Targovax / formerly selected from a targeted therapeutic or immunomodulatory Oncos ), peptide - based immunotherapy agents targeted for drug . Adjuvant therapies with targeted therapeutics or 35 difficult -to -treat RAS mutations; and TILT- 123 ( TILT Bio immunomodulatory drugs have shown promising effective therapeutics) , an engineered adenovirus designated : Ad5 / 3 ness when administered alone but are limited by the devel E2F -delta 24 -hTNFa - IRES -HIL20 ; and VSV -GP (ViraTh opment of tumor immunity over time or evasion of the erapeutics) a vesicular stomatitis virus (VSV ) engineered to immune response . express the glycoprotein (GP ) of lymphocytic choriomen In some embodiments , the present invention provides a 40 ingitis virus (LCMV ) , which can be further engineered to method of treating cancer , such as a cancer described herein , express antigens designed to raise an antigen - specific CD8 + comprising administering to a patient in need thereof an T cell response . effective amount of a compound disclosed herein or a In some embodiments , the present invention comprises pharmaceutically acceptable salt thereof or pharmaceutical administering to said patient a compound disclosed herein or composition thereof in combination with an additional thera- 45 a pharmaceutically acceptable salt thereof in combination peutic agent such as a targeted therapeutic or an immuno with a T - cell engineered to express a chimeric antigen modulatory drug . In some embodiments , the immunomodu receptor, or CAR . The T -cells engineered to express such latory therapeutic specifically induces apoptosis of tumor chimeric antigen receptor are referred to as a CAR - T cells . cells . Approved immunomodulatory therapeutics which may CARs have been constructed that consist of binding be used in the present invention include pomalidomide 50 domains , which may be derived from natural ligands, single ( Pomalyst® , Celgene ); lenalidomide (Revlimid® , Celgene ) ; chain variable fragments ( scFv ) derived from monoclonal ingenol mebutate (Picato® , LEO Pharma) . antibodies specific for cell -surface antigens, fused to In other embodiments , the immunomodulatory therapeu endodomains that are the functional end of the T - cell recep tic is a cancer vaccine. In some embodiments , the cancer tor ( TCR ) , such as the CD3 -zeta signaling domain from vaccine is selected from sipuleucel- T (Provenge® , Dendreo- 55 TCRs, which is capable of generating an activation signal in nNaleant Pharmaceuticals ) , which has been approved for T lymphocytes . Upon antigen binding , such CARs link to treatment of asymptomatic , or minimally symptomatic endogenous signaling pathways in the effector cell and metastatic castrate -resistant (hormone - refractory ) prostate generate activating signals similar to those initiated by the cancer; and talimogene laherparepvec ( Imlygic , Bio Vex / TCR complex . Amgen , previously known as T - VEC ) , a genetically modi- 60 For example , in some embodiments the CAR - T cell is one fied oncolytic viral therapy approved for treatment of unre of those described in U.S. Pat . No. 8,906,682 ( June ; hereby sectable cutaneous, subcutaneous and nodal lesions in incorporated by reference in its entirety ), which discloses melanoma. In some embodiments , the additional therapeutic CAR - T cells engineered to comprise an extracellular domain agent is selected from an oncolytic viral therapy such as having an antigen binding domain ( such as a domain that pexastimogene devacirepvec (PexaVec /JX -594 , Sillajen / 65 binds to CD19 ) , fused to an intracellular signaling domain of formerly Jennerex Biotherapeutics ), a thymidine kinase the T cell antigen receptor complex zeta chain ( such as CD3 ( TK-) deficient vaccinia virus engineered to express GM zeta ) . When expressed in the T cell, the CAR is able to US 10,624,968 B2 109 110 redirect antigen recognition based on the antigen binding CTLA - 4 that has been in studied in clinical trials for a specificity . In the case of CD19 , the antigen is expressed on number of indications, including : mesothelioma, colorectal malignant B cells . Over 200 clinical trials are currently in cancer, kidney cancer, breast cancer , lung cancer and non progress employing CAR - T in a wide range of indications. small cell lung cancer , pancreatic ductal adenocarcinoma, [ https://clinicaltrials.gov/ct2/results?term=chimeric+anti 5 pancreatic cancer , germ cell cancer , squamous cell cancer of gen + receptors & pg = 1 ]. the head and neck , hepatocellular carcinoma, prostate can Additional Co -Administered Therapeutic Agents — Immu cer, endometrial cancer, metastatic cancer in the liver, liver nostimulatory Drugs cancer, large B - cell lymphoma, ovarian cancer , cervical In some embodiments , the additional therapeutic agent is cancer, metastatic anaplastic thyroid cancer , urothelial can an immunostimulatory drug . For example , antibodies block- 10 cer , fallopian tube cancer, multiple myeloma, bladder can ing the PD - 1 and PD -L1 inhibitory axis can unleash acti cer, soft tissue sarcoma, and melanoma. AGEN - 1884 (Age vated tumor -reactive T cells and have been shown in clinical nus) is an anti -CTLA4 antibody that is being studied in trials to induce durable anti - tumor responses in increasing Phase 1 clinical trials for advanced solid tumors numbers of tumor histologies, including some tumor types (NCT02694822 ) . that conventionally have not been considered immuno- 15 Another paradigm for immune -stimulation is the use of therapy sensitive . See , e.g., Okazaki, T. et al . (2013 ) Nat. oncolytic viruses . In some embodiments , the present inven Immunol. 14 , 1212-1218 ; Zou et al. ( 2016 ) Sci. Transl. Med . tion provides a method for treating a patient by administer 8. The anti -PD - 1 antibody nivolumab (Opdivo® , Bristol ing a compound disclosed herein or a pharmaceutically Myers Squibb , also known as ONO -4538 , MDX1106 and acceptable salt thereof or pharmaceutical composition BMS- 936558 ), has shown potential to improve the overall 20 thereof in combination with an immunostimulatory therapy survival in patients with RCC who had experienced disease such as oncolytic viruses. Approved immunostimulatory progression during or after prior anti- angiogenic therapy. oncolytic viruses which may be used in the present invention In some embodiments , the present invention provides a include talimogene laherparepvec ( live , attenuated herpes method of treating cancer, such as a cancer described herein , simplex virus, Imlygic , Amgen ). comprising administering to a patient in need thereof an 25 In some embodiments , the additional therapeutic agent is effective amount of a compound disclosed herein or a an activator of retinoic acid receptor- related orphan receptor pharmaceutically acceptable salt thereof or pharmaceutical Y (RORyt ). RORyt is a transcription factor with key roles in composition thereof in combination with an additional thera the differentiation and maintenance of Type 17 effector peutic agent such as a immunostimulatory drug, such as an subsets of CD4 + ( Th17 ) and CD8 + ( Tc17 ) T cells , as well as immune checkpoint inhibitor. In some embodiments , the 30 the differentiation of IL - 17 expressing innate immune cell compound and the checkpoint inhibitor are administered subpopulations such as NK cells . An activator of RORyt , simultaneously or sequentially . In some embodiments , a that is being studied which may be used in the present compound disclosed herein is administered prior to the invention is LYC - 55716 (Lycera ), which is currently being initial dosing with the immune checkpoint inhibitor. In evaluated in clinical trials for the treatment of solid tumors certain embodiments , the immune checkpoint inhibitor is 35 (NCT02929862 ). administered prior to the initial dosing with the compound In some embodiments , the additional therapeutic agent is disclosed herein . an agonist or activator of a toll- like receptor ( TLR ) . Suitable In certain embodiments , the immune checkpoint inhibitor activators of TLRs include an agonist or activator of TLR9 is selected from a PD - 1 antagonist , a PD -L1 antagonist, or such as SD - 101 (Dynavax ) . SD - 101 is an immunostimula a CTLA -4 antagonist. In some embodiments , a compound 40 tory CpG which is being studied for B - cell, follicular and disclosed herein or a pharmaceutically acceptable salt other lymphomas (NCT02254772 ) . Agonists or activators of thereof administered in combination with nivolumab TLR8 which may be used in the present invention include ( anti- PD -1 antibody, Opdivo , Bristol -Myers Squibb ) ; motolimod (VTX - 2337 , VentiRx Pharmaceuticals ) which is pembrolizumab ( anti- PD - 1 antibody , Keytruda , Merck ) ; being studied for squamous cell cancer of the head and neck ipilimumab (anti -CTLA - 4 antibody , Yervoy® , Bristol- My- 45 (NCT02124850 ) and ovarian cancer (NCT02431559 ) . ers Squibb ); durvalumab (anti - PD -L1 antibody, Imfinzi® , Other checkpoint inhibitors that may be used in the AstraZeneca ) ; or atezolizumab (anti -PD -L1 antibody , present invention include inhibitors of T - cell immunoglobu Tecentriq , Genentech ). lin mucin containing protein - 3 ( TIM - 3 ). TIM -3 inhibitors Other immune checkpoint inhibitors suitable for use in the that may be used in the present invention include TSR - 022 , present invention include REGN2810 (Regeneron ), an anti- 50 LY3321367 and MBG453 . TSR -022 ( Tesaro ) is an anti PD - 1 antibody tested in patients with basal cell carcinoma TIM - 3 antibody which is being studied in solid tumors (NCT03132636 ) ; NSCLC (NCT03088540 ) ; cutaneous (NCT02817633 ) . LY3321367 (Eli Lilly ) is an anti - TIM - 3 squamous cell carcinoma (NCT02760498 ) ; lymphoma antibody which is being studied in solid tumors (NCT02651662 ) ; and melanoma (NCT03002376 ) ; pidili (NCT03099109 ) . MBG453 (Novartis ) is an anti- TIM - 3 anti zumab (CureTech ) , also known as CT- 011, an antibody that 55 body which is being studied in advanced malignancies binds to PD - 1 , in clinical trials for diffuse large B -cell (NCT02608268 ) . lymphoma and multiple myeloma; avelumab (Bavenciom , Other checkpoint inhibitors that may be used in the Pfizer/ Merck KGaA ) , also known as MSB0010718C ) , a present invention include inhibitors of T cell immunorecep fully human IgG1 anti -PD -L1 antibody , in clinical trials for tor with Ig and ITIM domains, or TIGIT , an immune non - small cell lung cancer, Merkel cell carcinoma, meso- 60 receptor on certain T cells and NK cells . TIGIT inhibitors thelioma, solid tumors , renal cancer , ovarian cancer, bladder that may be used in the present invention include BMS cancer, head and neck cancer, and gastric cancer ; and 986207 (Bristol -Myers Squibb ) , an anti - TIGIT monoclonal PDR001 (Novartis ) , an inhibitory antibody that binds to antibody (NCT02913313 ) ; OMP -313M32 (Oncomed ); and PD - 1 , in clinical trials for non - small cell lung cancer, anti - TIGIT monoclonal antibody (NCT03119428 ) . melanoma, triple negative breast cancer and advanced or 65 Checkpoint inhibitors that may be used in the present metastatic solid tumors. Tremelimumab (CP -675,206 ; Astra invention also include inhibitors of Lymphocyte Activation zeneca ) is a fully human monoclonal antibody against Gene - 3 (LAG - 3 ) . LAG - 3 inhibitors that may be used in the US 10,624,968 B2 111 112 present invention include BMS - 986016 and REGN3767 and PD -1 and co - inhibitory receptors such as cytotoxic T- lym IMP321 . BMS- 986016 (Bristol - Myers Squibb ) , an anti phocyte antigen 4 (CTLA - 4 , B and T Lymphocyte Attenu LAG -3 antibody , is being studied in glioblastoma and ator (BTLA ; CD272) , T cell Immunoglobulin and Mucin gliosarcoma (NCT02658981 ). REGN3767 (Regeneron ), is domain - 3 ( Tim - 3 ), Lymphocyte Activation Gene -3 (Lag -3 ; also an anti- LAG - 3 antibody, and is being studied in malig- 5 CD223) , and others are often referred to as a checkpoint nancies (NCT03005782 ) . IMP321 (Immutep S.A.) is an regulators . They act as molecular “ gatekeepers ” that allow LAG -3 - Ig fusion protein , being studied in melanoma extracellular information to dictate whether cell cycle pro (NCT02676869 ); adenocarcinoma (NCT02614833 ); and gression and other intracellular signalling processes should metastatic breast cancer (NCT00349934 ) . Other immune - oncology agents that may be used in the 10 proceed . present invention in combination with a compound dis In one aspect, the checkpoint inhibitor is a biologic closed herein include urelumab (BMS -663513 , Bristol -My therapeutic or a small molecule . In another aspect, the ers Squibb ), an anti -CD137 monoclonal antibody; varli checkpoint inhibitor is a monoclonal antibody, a humanized lumab ( CDX - 1127 , Celldex Therapeutics) , an anti- CD27 antibody, a fully human antibody, a fusion protein or a monoclonal antibody ; BMS - 986178 (Bristol - Myers 15 combination thereof. In a further aspect , the checkpoint Squibb ) , an anti- OX40 monoclonal antibody ; lirilumab inhibitor inhibits a checkpoint protein selected from CTLA ( IPH2102/ BMS - 986015 , Innate Pharma, Bristol- Myers 4 , PDL1, PDL2 , PD1, B7 -H3 , B7 -H4 , BTLA , HVEM , Squibb ) , an anti- KIR monoclonal antibody ; monalizumab TIM3, GALI, LAG3, VISTA , KIR , 2B4 , CD160 , CGEN ( IPH2201, Innate Pharma, AstraZeneca ) an anti- NKG2A 15049 , CHK 1 , CHK2, A2aR , B - 7 family ligands or a monoclonal antibody; andecaliximab (GS -5745 , Gilead Sci- 20 combination thereof. In an additional aspect , the checkpoint ences ), an anti -MMP9 antibody ; MK -4166 (Merck & Co.) , inhibitor interacts with a ligand of a checkpoint protein an anti -GITR monoclonal antibody. selected from CTLA - 4 , PDL1 , PDL2 , PD1, B7- H3 , B7 -H4 , Other additional therapeutic agents that may be used in BTLA , HVEM , TIM3 , GAL9 , LAG3, VISTA , KIR , 2B4, the present invention include glembatumumab vedotin CD160 , CGEN - 15049 , CHK 1, CHK2 , A2R , B -7 family monomethyl auristatin E (MMAE ) ( Celldex ), an anti - gly- 25 ligands or a combination thereof. In an aspect , the check coprotein NMB ( gpNMB ) antibody (CR011 ) linked to the point inhibitor is an immunostimulatory agent, a cell cytotoxic MMAE . gpNMB is a protein overexpressed by growth factor, an interleukin , an antibody , a vaccine or a multiple tumor types associated with cancer cells ' ability to combination thereof. In a further aspect , the interleukin is metastasize . IL - 7 or IL - 15 . In a specific aspect, the interleukin is glyco A compound of the current invention may also be used to 30 sylated IL -7 . In an additional aspect , the vaccine is a advantage in combination with other antiproliferative com dendritic cell (DC ) vaccine . pounds. Such antiproliferative compounds include , but are Checkpoint inhibitors include any agent that blocks or not limited checkpoint inhibitors ; aromatase inhibitors ; inhibits in a statistically significant manner , the inhibitory antiestrogens; topoisomerase 1 inhibitors ; topoisomerase II pathways of the immune system . Such inhibitors may inhibitors ; microtubule active compounds ; alkylating com- 35 include small molecule inhibitors or may include antibodies, pounds ; histone deacetylase inhibitors ; compounds which or antigen binding fragments thereof, that bind to and block induce cell differentiation processes ; cyclooxygenase inhibi or inhibit immune checkpoint receptors or antibodies that tors; MMP inhibitors ;mTOR inhibitors; antineoplastic anti bind to and block or inhibit immune checkpoint receptor metabolites ; platin compounds; compounds targeting/ de ligands. Illustrative checkpoint molecules that may be tar creasing a protein or lipid kinase activity and further anti- 40 geted for blocking or inhibition include, but are not limited angiogenic compounds; compounds which target, decrease to , CTLA - 4 , PDL1, PDL2 , PD1 , B7 -H3 , B7 -H4 , BTLA , or inhibit the activity of a protein or lipid phosphatase ; HVEM ,GAL9 , LAG3, TIM3, VISTA , KIR , 2B4 (belongs to gonadorelin agonists ; anti -androgens ; methionine amino the CD2 family ofmolecules and is expressed on all NK , yd , peptidase inhibitors ; matrix metalloproteinase inhibitors ; and memory CD8+ (aß ) T cells ) , CD160 ( also referred to as bisphosphonates ; biological response modifiers ; antiprolif- 45 BY55) , CGEN - 15049, CHK 1 and CHK2 kinases , A2aR , erative antibodies ; heparanase inhibitors ; inhibitors of Ras and various B - 7 family ligands . B7 family ligands include , oncogenic isoforms; telomerase inhibitors ; proteasome but are not limited to , B7-1 , B7-2 , B7 -DC , B7 -H1 , B7 -H2 , inhibitors ; compounds used in the treatment ofhematologic B7 - H3, B7 -H4 , B7- H5 , B7- H6 and B7 - H7. Checkpoint malignancies ; compounds which target, decrease or inhibit inhibitors include antibodies , or antigen binding fragments the activity of Flt - 3; Hsp90 inhibitors such as 17 -AAG 50 thereof, other binding proteins, biologic therapeutics , or ( 17 -allylaminogeldanamycin , NSC330507) , 17 - DMAG ( 17 small molecules , that bind to and block or inhibit the activity dimethylaminoethylamino - 17 - demethoxy - geldanamycin , of one or more of CTLA - 4 , PDL1, PDL2 , PD1, BTLA , NSC707545 ) , IPI- 504 , CNF1010 , CNF2024 , CNF1010 HVEM , TIM3, GALI, LAG3, VISTA , KIR , 2B4 , CD 160 from Conforma Therapeutics; temozolomide ( Temodal® ) ; and CGEN - 15049 . Illustrative immune checkpoint inhibi kinesin spindle protein inhibitors, such as SB715992 or 55 tors include Tremelimumab (CTLA - 4 blocking antibody ) , SB743921 from GlaxoSmithKline, or pentamidine/ chlo anti- OX40, PD -L1 monoclonal Antibody ( Anti- B7 - H1 ; rpromazine from CombinatoRx; MEK inhibitors such as MEDI4736 ), MK - 3475 ( PD - 1 blocker) , Nivolumab ( anti ARRY142886 from Array BioPharma, Zd6244 from PD1 antibody ), CT -011 (anti -PD1 antibody ) , BY55 mono AstraZeneca , PD181461 from Pfizer and leucovorin . clonal antibody, AMP224 (anti - PDL1 antibody ) , BMS The term “ checkpoint inhibitor” as used herein relates to 60 936559 (anti - PDL1 antibody ) , MPLDL3280A (anti - PDLL agents useful in preventing cancer cells from avoiding the antibody ) , MSB0010718C (anti -PDL1 antibody ), and ipili immune system of the patient. One of the major mechanisms mumab ( anti- CTLA - 4 checkpoint inhibitor ). Checkpoint of anti -tumor immunity subversion is known as “ T -cell protein ligands include , but are not limited to PD -L1 , exhaustion ,” which results from chronic exposure to anti PD -L2 , B7 -H3 , B7 -H4 , CD28 , CD86 and TIM -3 . gens that has led to up -regulation of inhibitory receptors . 65 In certain embodiments, the immune checkpoint inhibitor These inhibitory receptors serve as immune checkpoints in is selected from a PD - 1 antagonist , a PD -L1 antagonist, and order to prevent uncontrolled immune reactions. a CTLA -4 antagonist. In some embodiments , the checkpoint US 10,624,968 B2 113 114 inhibitor is selected from the group consisting of nivolumab The term “ microtubule active agent” relates to microtu (Opdivo® ), ipilimumab ( Yervoy® ), and pembrolizumab bule stabilizing , microtubule destabilizing compounds and (Keytruda® ) . microtublin polymerization inhibitors including, but not In some embodiments , the checkpoint inhibitor is selected limited to taxanes , such as paclitaxel and docetaxel ; vinca from the group consisting of lambrolizumab (MK -3475 ), 5 alkaloids, such as vinblastine or vinblastine sulfate , vincris nivolumab (BMS - 936558 ) , pidilizumab (CT - 011 ) , AMP tine or vincristine sulfate , and vinorelbine; discodermolides ; 224 , MDX - 1105 , MEDI4736 , MPDL3280A , BMS- 936559 , cochicine and epothilones and derivatives thereof. Paclitaxel ipilimumab , lirlumab , IPH2101, pembrolizumab is marketed under the trade name TaxolTM Docetaxel is marketed under the trade name TaxotereTM Vinblastine (Keytruda® ) , and tremelimumab . 10 sulfate is marketed under the trade name Vinblastin R.PTM . The term " aromatase inhibitor” as used herein relates to a Vincristine sulfate is marketed under the trade name Farmi compound which inhibits estrogen production , for instance , stinTM the conversion of the substrates androstenedione and testos The term “ alkylating agent” as used herein includes , but terone to estrone and estradiol, respectively . The term is not limited to , cyclophosphamide , ifosfamide , melphalan includes , but is not limited to steroids, especially atames- 15 or nitrosourea (BCNU or Gliadel) . Cyclophosphamide is tane, exemestane and formestane and , in particular, non marketed under the trade name CyclostinTM . Ifosfamide is steroids, especially aminoglutethimide , roglethimide , pyri marketed under the trade name HoloxanTM . doglutethimide , trilostane, testolactone , ketokonazole , The term “ histone deacetylase inhibitors” or “ HDAC vorozole , fadrozole, anastrozole and letrozole . Exemestane inhibitors ” relates to compounds which inhibit the histone is marketed under the trade name AromasinTM . Formestane 20 deacetylase and which possess antiproliferative activity . is marketed under the trade name LentaronTM . Fadrozole is This includes , but is not limited to , suberoylanilide marketed under the trade name AfemaTM Anastrozole is hydroxamic acid (SAHA ) . marketed under the trade name ArimidexTM Letrozole is The term “ antineoplastic antimetabolite ” includes , but is marketed under the trade names FemaraTM or FemarTM . not limited to , 5 - fluorouracil or 5 - FU , capecitabine , gemcit Aminoglutethimide is marketed under the trade name Orim- 25 abine, DNA demethylating compounds, such as 5- azacyti etenTM . A combination of the invention comprising a che dine and decitabine , methotrexate and edatrexate , and folic motherapeutic agent which is an aromatase inhibitor is acid antagonists such as pemetrexed . Capecitabine is mar particularly useful for the treatment of hormone receptor keted under the trade name XelodaTM . Gemcitabine is mar positive tumors , such as breast tumors . keted under the trade name GemzarTM . The term “ antiestrogen ” as used herein relates to a com- 30 The term “ platin compound ” as used herein includes , but pound which antagonizes the effect of estrogens at the is not limited to , carboplatin , cis- platin , cisplatinum and estrogen receptor level. The term includes , but is not limited oxaliplatin . Carboplatin can be administered , e.g., in the to tamoxif fulv rant , raloxifene and raloxifene hydro form as it is mark ed , e.g. under the trademark CarboplatTM . chloride . Tamoxifen is marketed under the trade name Oxaliplatin can be administered , e.g., in the form as it is NolvadexTM . Raloxifene hydrochloride is marketed under 35 marketed , e.g. under the trademark EloxatinTM . the trade name EvistaTM . Fulvestrant can be administered The term " compounds targeting /decreasing a protein or under the trade name FaslodexTM . A combination of the lipid kinase activity; or a protein or lipid phosphatase invention comprising a chemotherapeutic agent which is an activity ; or further anti- angiogenic compounds ” as used antiestrogen is particularly useful for the treatment of estro herein includes, but is not limited to , protein tyrosine kinase gen receptor positive tumors, such as breast tumors . 40 and / or serine and /or threonine kinase inhibitors or lipid The term “ anti- androgen” as used herein relates to any kinase inhibitors , such as a ) compounds targeting , decreas substance which is capable of inhibiting the biological ing or inhibiting the activity of the platelet- derived growth effects of androgenic hormones and includes , but is not factor - receptors ( PDGFR ) , such as compounds which target , limited to, bicalutamide (CasodexTM ) . The term “ gonad decrease or inhibit the activity of PDGFR , especially com orelin agonist ” as used herein includes , but is not limited to 45 pounds which inhibit the PDGF receptor, such as an N -phe abarelix , goserelin and goserelin acetate . Goserelin can be nyl -2 -pyrimidine - amine derivative , such as imatinib , administered under the trade name ZoladexTM . SU101, SU6668 and GFB - 111; b ) compounds targeting , The term “ topoisomerase I inhibitor ” as used herein decreasing or inhibiting the activity of the fibroblast growth includes , but is not limited to topotecan , gimatecan , irino factor -receptors ( FGFR ); c ) compounds targeting , decreas tecan , camptothecian and its analogues, 9 -nitrocamptothecin 50 ing or inhibiting the activity of the insulin -like growth factor and the macromolecular camptothecin conjugate PNU receptor I ( IGF - IR ), such as compounds which target , 166148. Irinotecan can be administered , e.g. in the form as decrease or inhibit the activity of IGF - IR , especially com it is marketed , e.g. under the trademark CamptosarTM . Topo pounds which inhibit the kinase activity of IGF -I receptor , or tecan is marketed under the trade name HycamptinTM . antibodies that target the extracellular domain of IGF- I The term “ topoisomerase II inhibitor ” as used herein 55 receptor or its growth factors; d ) compounds targeting , includes , but is not limited to the anthracyclines such as decreasing or inhibiting the activity of the Trk receptor doxorubicin (including liposomal formulation , such as Cae tyrosine kinase family , or ephrin B4 inhibitors ; e) com lyxTM ), daunorubicin , epirubicin , idarubicin and nemorubi pounds targeting , decreasing or inhibiting the activity of the cin , the anthraquinones mitoxantrone and losoxantrone , and Axl receptor tyrosine kinase family ; 0 compounds targeting , the podophillotoxines etoposide and teniposide. Etoposide is 60 decreasing or inhibiting the activity of the Ret receptor marketed under the trade name EtopophosTM . Teniposide is tyrosine kinase; g ) compounds targeting, decreasing or marketed under the trade name VM 26 - Bristol Doxorubicin inhibiting the activity of the Kit/ SCFR receptor tyrosine is marketed under the trade name AcriblastinTM or Adriamy kinase , such as imatinib ; h ) compounds targeting, decreasing cinTM . Epirubicin is marketed under the trade name Farmo or inhibiting the activity of the C -kit receptor tyrosine rubicinTM Idarubicin is marketed . under the trade name 65 kinases, which are part of the PDGFR family , such as ZavedosTM . Mitoxantrone is marketed under the trade name compounds which target , decrease or inhibit the activity of Novantron . the c- Kit receptor tyrosine kinase family , especially com US 10,624,968 B2 115 116 pounds which inhibit the c -Kit receptor, such as imatinib ; i) family , including , but not limited to PI3Ka , PI3Ky, PI3Kd , compounds targeting , decreasing or inhibiting the activity of PI3KB , PI3K - C20 , PI3K - C2B , PI3K -C2y , Vps34 , p110 - a , members of the c - Abl family , their gene- fusion products p110 - B , p110 - y, p110-8, p85 - a , p85- B , p55 - y, p150, p101 , ( e.g. BCR - Abl kinase ) and mutants , such as compounds and p87 . Examples of PI3K inhibitors useful in this inven which target decrease or inhibit the activity of c - Abl family 5 tion include but are not limited to ATU -027 , SF - 1126 , members and their gene fusion products , such as an N - phe DS - 7423 , PBI- 05204 , GSK - 2126458 , ZSTK -474 , buparl nyl- 2 -pyrimidine -amine derivative, such as imatinib or nilo isib , pictrelisib , PF -4691502 , BYL - 719 , dactolisib , XL - 147 , tinib (AMN107 ) ; PD180970 ; AG957 ; NSC 680410 ; PD173955 from ParkeDavis ; or dasatinib (BMS - 354825 ) ; j ) XL -765 , and idelalisib . compounds targeting, decreasing or inhibiting the activity of 10 notThe limited term to“ Bclcompounds- 2 inhibitor having" as used inhibitory herein activityincludes against, but is members of the protein kinase C (PKC ) and Raf family of serine / threonine kinases , members of the MEK , SRC , JAK / B - cell lymphoma 2 protein (Bcl - 2 ), including but not limited pan - JAK , FAK , PDK1, PKB/ Akt, Ras/ MAPK , PI3K , SYK , to ABT- 199, ABT- 731 , ABT- 737 , apogossypol, Ascenta's TYK2, BTK and TEC family , and /or members of the cyclin pan -Bcl - 2 inhibitors , curcumin ( and analogs thereof) , dual dependent kinase family (CDK ) including staurosporine 15 Bcl- 2 /Bcl - xL inhibitors ( Infinity Pharmaceuticals /Novartis derivatives , such as midostaurin ; examples of further com Pharmaceuticals ) , Genasense (G3139 ) , HA14-1 (and ana pounds include UCN -01 , safingol, BAY 43-9006 , Bryostatin logs thereof; see WO2008118802 ), navitoclax ( and analogs 1 , Perifosine ; llmofosine; RO 318220 and RO 320432 ; GO thereof, see U.S. Pat. No. 7,390,799 ), NH — 1 (Shenayng 6976 ; Isis 3521; LY333531/ LY379196 ; isochinoline com Pharmaceutical University ), obatoclax ( and analogs thereof, pounds; FTIs ; PD184352 or QAN697 (a P13K inhibitor) or 20 see WO2004106328 ), S - 001 (Gloria Pharmaceuticals ), TW AT7519 (CDK inhibitor) ; k ) compounds targeting , decreas series compounds (Univ . of Michigan ), and venetoclax . In ing or inhibiting the activity of protein - tyrosine kinase some embodiments the Bcl - 2 inhibitor is a small molecule inhibitors , such as compounds which target , decrease or therapeutic . In some embodiments the Bcl - 2 inhibitor is inhibit the activity of protein -tyrosine kinase inhibitors peptidomimetic . include imatinib mesylate (GleevecTM ) or tyrphostin such as 25 The term “ BTK inhibitor ” as used herein includes, but is Tyrphostin A23 /RG -50810 ; AG 99 ; Tyrphostin AG 213 ; not limited to compounds having inhibitory activity against Tyrphostin AG 1748 ; Tyrphostin AG 490 ; Tyrphostin B44 ; Bruton's Tyrosine Kinase (BTK ) , including, but not limited Tyrphostin B44 ( + ) enantiomer ; Tyrphostin AG 555 ; AG 494 ; Tyrphostin AG 556 , AG957 and adaphostin ( 4 - {[ ( 2,5 to AVL -292 and ibrutinib . dihydroxyphenyl) methyl ) amino } -benzoic acid adamantyl 30 The term “ SYK inhibitor ” as used herein includes , but is ester; NSC 680410 , adaphostin ); 1 ) compounds targeting , not limited to compounds having inhibitory activity against decreasing or inhibiting the activity of the epidermal growth spleen tyrosine kinase (SYK ), including but not limited to factor family of receptor tyrosine kinases (EGFR1 ErbB2, PRT- 062070 , R - 343 , R - 333 , Excellair, PRT - 062607 , and ErbB3, ErbB4 as homo- or heterodimers ) and their mutants , fostamatinib . such as compounds which target , decrease or inhibit the 35 Further examples of BTK inhibitory compounds, and activity of the epidermal growth factor receptor family are conditions treatable by such compounds in combination especially compounds, proteins or antibodies which inhibit with compounds of this invention can be found in members of the EGF receptor tyrosine kinase family , such as WO2008039218 and WO2011090760 , the entirety of which EGF receptor, ErbB2 , ErbB3 and ErbB4 or bind to EGF or are incorporated herein by reference . EGF related ligands, CP 358774 , ZD 1839 , ZM 105180 ; 40 Further examples of SYK inhibitory compounds , and trastuzumab (HerceptinTM ), cetuximab (ErbituxTM ) , Iressa , conditions treatable by such compounds in combination Tarceva , OSI- 774 , C1-1033 , EKB - 569 ,GW - 2016 , E1.1 , E2.4 , with compounds of this invention can be found in E2.5 , E6.2 , E6.4 , E2.11, E6.3 or E7.6.3 , and 7H -pyrrolo- [ 2 , WO2003063794 , WO2005007623 , and WO2006078846 , 3 - d ]pyrimidine derivatives; m ) compounds targeting , the entirety ofwhich are incorporated herein by reference . decreasing or inhibiting the activity of the c -Met receptor, 45 Further examples of PI3K inhibitory compounds, and such as compounds which target, decrease or inhibit the conditions treatable by such compounds in combination activity of c -Met , especially compounds which inhibit the with compounds of this invention can be found in kinase activity of c -Met receptor , or antibodies that target the WO2004019973 , WO2004089925 , WO2007016176 , U.S. extracellular domain of c -Met or bind to HGF, n ) com Pat. No. 8,138,347 , WO2002088112 , WO2007084786 , pounds targeting, decreasing or inhibiting the kinase activity 50 WO2007129161, WO2006122806 , WO2005113554, and of one or more JAK family members (JAK1 / JAK2/ JAK3/ WO2007044729 the entirety of which are incorporated TYK2 and /or pan - JAK ) , including but not limited to PRT herein by reference . 062070 , SB - 1578 , baricitinib , pacritinib , momelotinib , Further examples of JAK inhibitory compounds, and VX - 509 , AZD - 1480 , TG - 101348 , tofacitinib , and ruxoli conditions treatable by such compounds in combination tinib ; o ) compounds targeting, decreasing or inhibiting the 55 with compounds of this invention can be found in kinase activity of PI3 kinase (PI3K ) including but not WO2009114512 , WO2008109943 , WO2007053452 , limited to ATU -027 , SF - 1126 , DS- 7423 , PBI- 05204 , GSK WO2000142246 , and WO2007070514 , the entirety of 2126458 , ZSTK -474 , buparlisib , pictrelisib , PF -4691502 , which are incorporated herein by reference . BYL -719 , dactolisib , XL - 147 , XL - 765, and idelalisib ; and ; Further anti- angiogenic compounds include compounds and q ) compounds targeting, decreasing or inhibiting the 60 having anothermechanism for their activity , e.g. unrelated to signaling effects of hedgehog protein (Hh ) or smoothened protein or lipid kinase inhibition e.g. thalidomide ( Thalo receptor (SMO ) pathways , including but not limited to midTM ) and TNP -470 . cyclopamine , vismodegib , itraconazole , erismodegib , and Examples of proteasome inhibitors useful for use in IPI- 926 (saridegib ) . combination with compounds of the invention include, but The term “ PI3K inhibitor” as used herein includes , but is 65 are not limited to bortezomib , disulfiram , epigallocatechin not limited to compounds having inhibitory activity against 3 -gallate ( EGCG ) , salinosporamide A , carfilzomib , ONX one or more enzymes in the phosphatidylinositol- 3 - kinase 0912 , CEP - 18770 , and MLN9708 . US 10,624,968 B2 117 118 Compounds which target , decrease or inhibit the activity compounds targeting , decreasing or inhibiting the activity of of a protein or lipid phosphatase are e.g. inhibitors of FMS- like tyrosine kinase receptors (Flt - 3R ) ; interferon , phosphatase 1 , phosphatase 2A , or CDC25 , such as okadaic 1 - B - D - arabinofuransylcytosine (ara - c ) and bisulfan ; and acid or a derivative thereof. ALK inhibitors , which are compounds which target, Compounds which induce cell differentiation processes 5 decrease or inhibit anaplastic lymphoma kinase . include, but are not limited to , retinoic acid , a- y- or Compounds which target , decrease or inhibit the activity d - tocopherol or a- y- or d - tocotrienol. of FMS- like tyrosine kinase receptors ( Flt- 3R ) are especially The term cyclooxygenase inhibitor as used herein compounds, proteins or antibodies which inhibitmembers of includes , but is not limited to , Cox -2 inhibitors , 5 -alkyl the Flt- 3R receptor kinase family , such as PKC412 , substituted 2 - arylaminophenylacetic acid and derivatives, 10 midostaurin , a staurosporine derivative , SU11248 and such as celecoxib (CelebrexTM ), rofecoxib ( VioxxTM ), etori MLN518 . coxib , valdecoxib or a 5 -alkyl - 2 -arylaminophenylacetic The term “ HSP90 inhibitors ” as used herein includes , but acid , such as 5 -methyl - 2-( 2 -chloro -6 '- fluoroanilino )phenyl is not limited to , compounds targeting , decreasing or inhib acetic acid , lumiracoxib . iting the intrinsic ATPase activity of HSP90 ; degrading , The term “ bisphosphonates ” as used herein includes , but 15 targeting , decreasing or inhibiting the HSP90 client proteins is not limited to , etridonic , clodronic , tiludronic , pamidronic , via the ubiquitin proteosome pathway. Compounds target alendronic , ibandronic , risedronic and zoledronic acid . Etri ing, decreasing or inhibiting the intrinsic ATPase activity of donic acid is marketed under the trade name DidronelTM . HSP90 are especially compounds, proteins or antibodies Clodronic acid is marketed under the trade name BonefosTM which inhibit the ATPase activity of HSP90 , such as 17 -al Tiludronic acid is marketed under the trade name SkelidTM 20 lylamino , 17 - demethoxygeldanamycin ( 17AAG ) , a geldan Pamidronic acid is marketed under the trade name ArediaTM . amycin derivative; other geldanamycin related compounds ; Alendronic acid is marketed under the trade name Fosa radicicol and HDAC inhibitors. maxTM . Ibandronic acid is marketed under the trade name The term “ antiproliferative antibodies ” as used herein BondranatTM . Risedronic acid is marketed under the trade includes , but is not limited to , trastuzumab (HerceptinTM ) , name ActonelTM Zoledronic acid is marketed under the 25 Trastuzumab -DM1 , erbitux , bevacizumab (AvastinTM ), trade name ZometaTM . The term “ mTOR inhibitors ” relates rituximab (Rituxan® ), PRO64553 (anti -CD40 ) and 2C4 to compounds which inhibit the mammalian target of Antibody . By antibodies is meant intact monoclonal anti rapamycin (mTOR ) and which possess antiproliferative bodies , polyclonal antibodies, multispecific antibodies activity such as sirolimus (Rapamune® ), everolimus (Cer formed from at least 2 intact antibodies , and antibodies ticanTM ), CCI- 779 and ABT578 . 30 fragments so long as they exhibit the desired biological The term “ heparanase inhibitor” as used herein refers to activity . compounds which target , decrease or inhibit heparin sulfate For the treatment of acute myeloid leukemia (AML ) , degradation . The term inclu but is no limited to , PI - 88 . compounds of the current invention can be used in combi The term “ biological response modifier” as used herein nation with standard leukemia therapies, especially in com refers to a lymphokine or interferons. 35 bination with therapies used for the treatment of AML . In The term “ inhibitor of Ras oncogenic isoforms” , such as particular, compounds of the current invention can be H -Ras , K -Ras , or N -Ras , as used herein refers to compounds administered in combination with , for example , farnesyl which target, decrease or inhibit the oncogenic activity of transferase inhibitors and / or other drugs useful for the Ras; for example , a “ farnesyl transferase inhibitor" such as treatment of AML , such as Daunorubicin , Adriamycin , L - 744832, DK8G557 or R115777 (ZarnestraTM ). The term 40 Ara - C , VP - 16 , Teniposide, Mitoxantrone, Idarubicin , Car “ telomerase inhibitor ” as used herein refers to compounds boplatinum and PKC412. which target , decrease or inhibit the activity of telomerase . Other anti - leukemic compounds include , for example , Compounds which target , decrease or inhibit the activity of Ara - C , a pyrimidine analog, which is the 2' - alpha -hydroxy telomerase are especially compounds which inhibit the ribose ( arabinoside ) derivative of deoxycytidine. Also telomerase receptor , such as telomestatin . 45 included is the purine analog of hypoxanthine, 6 -mercap The term “ methionine aminopeptidase inhibitor” as used topurine (6 -MP ) and fludarabine phosphate . Compounds herein refers to compounds which target, decrease or inhibit which target, decrease or inhibit activity of histone deacety the activity of methionine aminopeptidase . Compounds lase ( HDAC ) inhibitors such as sodium butyrate and sub which target, decrease or inhibit the activity of methionine eroylanilide hydroxamic acid (SAHA ) inhibit the activity of aminopeptidase include , but are not limited to , bengamide or 50 the enzymes known as histone deacetylases. Specific HDAC a derivative thereof. inhibitors include MS275 , SAHA , FK228 (formerly The term “ proteasome inhibitor ” as used herein refers to FR901228 ) , Trichostatin A and compounds disclosed in U.S. compounds which target, decrease or inhibit the activity of Pat. No. 6,552,065 including , but not limited to , N -hydroxy the proteasome . Compounds which target, decrease or 3-[ 4 -[ [ [2- ( 2 -methyl - 1H - indol- 3 -yl ) -ethyl] -amino ]methyl ] inhibit the activity of the proteasome include, but are not 55 phenyl ] -2E - 2 -propenamide , or a pharmaceutically accept limited to , Bortezomib (VelcadeTM ) and MLN 341. able salt thereof and N -hydroxy - 3- [ 4 -[ ( 2- hydroxyethyl ) { 2 The term “ matrix metalloproteinase inhibitor" or ( 1H - indol - 3 - yl ) ethyl] -amino ]methyl ]phenyl ]-2E - 2 (“ MMP ” inhibitor ) as used herein includes, but is not limited propenamide , or a pharmaceutically acceptable salt thereof, to , collagen peptidomimetic and nonpeptidomimetic inhibi especially the lactate salt . Somatostatin receptor antagonists tors , tetracycline derivatives, e.g. hydroxamate peptidomi- 60 as used herein refer to compounds which target , treat or metic inhibitor batimastat and its orally bioavailable ana inhibit the somatostatin receptor such as octreotide, and logue marimastat (BB - 2516 ) , prinomastat (AG3340 ) , SOM230 . Tumor cell damaging approaches refer to metastat (NSC 683551 ) BMS - 279251, BAY 12-9566 , approaches such as ionizing radiation . The term “ ionizing TAA211 , MMI270B or AAJ996 . radiation ” referred to above and hereinafter means ionizing The term “ compounds used in the treatment of hemato- 65 radiation that occurs as either electromagnetic rays ( such as logic malignancies ” as used herein includes, but is not X - rays and gamma rays) or particles (such as alpha and beta limited to , FMS - like tyrosine kinase inhibitors , which are particles ) . Ionizing radiation is provided in , but not limited US 10,624,968 B2 119 120 to , radiation therapy and is known in the art. See Hellman , with chemotherapy, radiotherapy , immunotherapy , photo Principles of Radiation Therapy, Cancer, in Principles and therapy, surgical intervention , or a combination of these . Practice of Oncology , Devita et al. , Eds. , 4th Edition , Vol. 1, Long -term therapy is equally possible as is adjuvant therapy pp . 248-275 ( 1993 ) . in the context of other treatment strategies , as described Also included are EDG binders and ribonucleotide reduc- 5 above . Other possible treatments are therapy to maintain the tase inhibitors . The term " EDG binders ” as used herein patient's status after tumor regression , or even chemopre refers to a class of immunosuppressants that modulates ventive therapy, for example in patients at risk . lymphocyte recirculation , such as FTY720 . The term “ ribo Those additional agents may be administered separately nucleotide reductase inhibitors” refers to pyrimidine or from an inventive compound - containing composition , as purine nucleoside analogs including , but not limited to , 10 part of a multiple dosage regimen . Alternatively , those fludarabine and /or cytosine arabinoside ( ara - C ), 6 - thiogua agents may be part of a single dosage form , mixed together nine , 5 - fluorouracil , cladribine, 6 -mercaptopurine (espe with a compound of this invention in a single composition . cially in combination with ara - C against ALL ) and/ or pen If administered as part of a multiple dosage regime, the two tostatin . Ribonucleotide reductase inhibitors are especially active agents may be submitted simultaneously , sequentially hydroxyurea or 2 - hydroxy -1H -isoindole - 1,3 -dione deriva- 15 or within a period of time from one another normally within tives . five hours from one another . Also included are in particular those compounds, proteins As used herein , the term " combination , ” “ combined , ” and or monoclonal antibodies of VEGF such as 1- ( 4 - chloroa related terms refers to the simultaneous or sequential admin nilino ) -4- (4 -pyridylmethyl ) phthalazine or a pharmaceuti istration of therapeutic agents in accordance with this inven cally acceptable salt thereof, 1- (4 -chloroanilino )-4- ( 4- 20 tion . For example , a compound of the present invention may pyridylmethyl) phthalazine succinate ; AngiostatinTM be administered with another therapeutic agent simultane EndostatinTM ; anthranilic acid amides ; ZD4190 ; Zd ,474 ; ously or sequentially in separate unit dosage forms or SU5416 ; SU6668 ; bevacizumab ; or anti - VEGF antibodies together in a single unit dosage form . Accordingly , the or anti - VEGF receptor antibodies, such as rhumAb and present invention provides a single unit dosage form com RHUFab , VEGF aptamer such as Macugon ; FLT- 4 inhibi- 25 prising a compound of the current invention , an additional tors , FLT - 3 inhibitors, VEGFR - 2 IgG1 antibody, Angiozyme therapeutic agent, and a pharmaceutically acceptable carrier , (RPI 4610 ) and Bevacizumab ( AvastinTM ) . adjuvant, or vehicle . Photodynamic therapy as used herein refers to therapy The amount of both an inventive compound and addi which uses certain chemicals known as photosensitizing tional therapeutic agent ( in those compositions which com compounds to treat or prevent cancers . Examples of photo- 30 prise an additional therapeutic agent as described above ) that dynamic therapy include treatment with compounds, such as may be combined with the carrier materials to produce a VisudyneTM and porfimer sodium . single dosage form will vary depending upon the host Angiostatic steroids as used herein refers to compounds treated and the particular mode of administration . Prefer which block or inhibit angiogenesis , such as , e.g. , anecor ably, compositions of this invention should be formulated so tave , triamcinolone, hydrocortisone, 11 - a -epihydrocotisol , 35 that a dosage of between 0.01-100 mg/ kg body weight/ day cortexolone , 17a -hydroxyprogesterone , corticosterone, des of an inventive compound can be administered . oxycorticosterone, testosterone , estrone and dexamethasone . In those compositions which comprise an additional Implants containing corticosteroids refers to compounds , therapeutic agent, that additional therapeutic agent and the such as fluocinolone and dexamethasone . compound of this invention may act synergistically . There Other chemotherapeutic compounds include, but are not 40 fore , the amount of additional therapeutic agent in such limited to , plant alkaloids , hormonal compounds and compositions will be less than that required in a mono antagonists ; biological response modifiers , preferably lym therapy utilizing only that therapeutic agent. In such com phokines or interferons; antisense oligonucleotides or oli positions a dosage of between 0.01-1,000 ug /kg body gonucleotide derivatives ; shRNA or siRNA ; or miscella weight/ day of the additional therapeutic agent can be admin neous compounds or compounds with other or unknown 45 istered . mechanism of action . The amount of additional therapeutic agent present in the The structure of the active compounds identified by code compositions of this invention will be no more than the numbers , generic or trade names may be taken from the amount that would normally be administered in a composi actual edition of the standard compendium “ The Merck tion comprising that therapeutic agent as the only active Index ” or from databases, e.g. Patents International ( e.g. 50 agent. Preferably the amount of additional therapeutic agent IMS World Publications ) . in the presently disclosed compositions will range from A compound of the current invention may also be used in about 50 % to 100 % of the amount normally present in a combination with known therapeutic processes, for composition comprising that agent as the only therapeuti example , the administration of hormones or radiation . In cally active agent. certain embodiments , a provided compound is used as a 55 The compounds of this invention , or pharmaceutical radiosensitizer, especially for the treatment of tumors which compositions thereof, may also be incorporated into com exhibit poor sensitivity to radiotherapy. positions for coating an implantable medical device, such as A compound of the current invention can be administered prostheses, artificial valves, vascular grafts , stents and cath alone or in combination with one or more other therapeutic eters . Vascular stents , for example , have been used to compounds , possible combination therapy taking the form 60 overcome restenosis ( re -narrowing of the vessel wall after of fixed combinations or the administration of a compound injury ). However, patients using stents or other implantable of the invention and one or more other therapeutic com devices risk clot formation or platelet activation . These pounds being staggered or given independently of one unwanted effects may be prevented or mitigated by pre another, or the combined administration of fixed combina coating the device with a pharmaceutically acceptable com tions and one or more other therapeutic compounds. A 65 position comprising a kinase inhibitor. Implantable devices compound of the current invention can besides or in addition coated with a compound of this invention are another be administered especially for tumor therapy in combination embodiment of the present invention . US 10,624,968 B2 121 122 EXEMPLIFICATION -continued The following Examples illustrate the invention described Day Protocol Description above ; they are not, however, intended to limit the scope of concentration starts at 1 uM , 3 - fold dilution and 10 doses . the invention in any way. The beneficial effects of the 5 The final I - 1a concentration starts at 5 uM , 5 - fold dilution pharmaceutical compounds, combinations, and composi and 10 doses. The final DM1- Sme, MMAE and SN - 38 concentration starts at 0.5 uM , 5 - fold dilution and 10 doses. tions of the present invention can also be determined by Toxins treat cells for 72 hours . other test models known as such to the person skilled in the Day3 Equilibrate the assay plate to room temperature for nearly 30 pertinent art. minutes . Add 50 UL CellTiter Glo reagent to each well. List of common abbreviations used in the experimental 10 Detect the plate after 10 mins by EnVision (luminescence ). section . DMA : N , N - dimethylacetamide Details of the initial cell plating procedure are described DMSO : dimethyl sulfoxide in Table 3. All cell lines met the criteria that the viability of EDCI: 1-( 3 -dimethylaminopropyl ) -3 -ethylcarbodiimide adhere /mixed cells > 90 % and the viability of suspension hydrochloride 15 cells > 85 % during the initial cell plating. All cell plates in eqv : equivalents this assay met the criteria thatMax CV < 10 % . The reference HPLC : high performance liquid chromatography compound paclitaxelworked well on all the tested cell lines LCMS: liquid chromatography -mass spectrometry M : molar and the data of paclitaxel were consistent with the historical mg: milligrams 20 data as shown in Table 5 , below . min : minutes TABLE 3 mL: milliliters mM : millimolar Cell Plating Details mmol : millimoles 25 Initial cell NHS: N -hydroxysuccinimide Cell plating 0 C .: degrees Celsius PBS : phosphate buffered saline Line Tumor Cell Seed Pas Via RP- HPLC : reverse phase high performance liquid chro Name Types Type Media Density sage bility matography 30 HCT15 Colorectal adherent RPMI 1640 + 1500 P8 89.9 % RP- HPLC -MS : reverse phase high performance liquid 10 % FBS chromatography mass spectrometry HT29 Colorectal adherent RPMI 1640 + 4000 P19 96.7 % 10 % FBS rt: room temperature NCI Lung (Non adherent RPMI 1640 + 2000 P10 95.6 % TFA : trifluoracetic acid H292 small cell) 10 % FBS 35 MDA Breast adherent RPMI 1640 + 3000 P15 98.4 % Example 1 MB - 231 10 % FBS HCC1806 Breast adherent RPMI 1640 + 3000 P10 97.7 % 10 % FBS Evaluation of 1-1a , DM1- Sme, MMAE and SN38 EBC - 1 Lung (Non adherent RPMI 1640 + 3000 P22 96.5 % Against a Panel of 11 Cancer Cell Lines small cell ) 10 % FBS SNU - 16 Gastric suspen RPM1640 + 5000 P15 89.0 % 40 sion 10 % FBS The procedure for the cancer cell line panel is provided in HT1080 Sarcoma adherent RPMI 1640 + 2000 P17 97.2 % Table 2 . 10 % FBS DM1- Sme is DM1 wherein the free thiol is covalently MOLP - 8 Myeloma suspen RPMI 1640 + 10000 P13 86.5 % attached to methanethiol via a disulfide bond . sion 20 % FBS PC - 3 Prostate adherent RPMI 1640 + 2000 P21 98.0 % 45 10 % FBS Table 2. Procedure NCI Lung (Non adherent RPMI 1640 + 4000 P25 98.6 % H1975 small cell ) 10 % FBS
Day Protocol Description Tested cell lines were evaluated for expression of MT1 Day - 1 Adjust the cell density to the recommend information . Detailed 50 MMP. Results are shown in Table 4. Although the relation information for each seeding cell line can be found in Table 3 . ship between expression and efficacy in the cytotoxicity Add 90 uL cells to two plates as the cells seeding plate -map . assay is not linear, there is the potential of a threshold level Add 100 uL media and 100 uL PBS to the plates as the cells of MT1 expression for efficacy despite the range of tumor seeding plate- map . Dayo Toxins preparation : types, growth rates and model formats present in the 11 cell Diluted I - la with DMSO and adjusted the stock concentration 55 line panel. to 10 mM . DM1- Sme & SN - 38 stock concentration is 10.6 mM and 10 mM , respectively . MMAE stock concentration is 10 mM . TABLE 4 The stock concentration of the reference compound Paclitaxel is 10 mM . Prepare the stock plate of test compounds: For Expression of MT1 -MMP in Tested Cell Lines Paclitaxel , the starting concentration is 1 mM , 3 - fold dilution in DMSO , 10 doses . For I- 1a , the starting concentration is 60 Expression of MT1 -MMP 5 mM , 5 - fold dilution in DMSO , 10 doses . For DM1- Sme, Cell Line Name ( > 5 implies expression ) MMAE and SN - 38 , the starting concentration is 500 M , 5 - fold dilution in DMSO , 10 doses. For all compounds , HT1080 8.7 100 - fold dilution of the dose compounds from the compound PC - 3 7.6 stock plate by the complete medium in the mid plate ( 2 ul , MDA -MB - 231 7.1 compound + 198 uL medium ) . Transfer 10 uL diluted 65 NCI- H292 7.0 compounds to the assay plate . The final paclitaxel NCI- H1975 6.7 US 10,624,968 B2 123 124 TABLE 4 - continued The IC50 curves were generated by 4 Parameter Logistic Model — Sigmoidal Dose- Response Model as follows: Expression of MT1 -MMP in Tested Cell Lines Y = Bottom + ( Top - Bottom )/ ( 1 + 10 ^ ( ( log EC50 - x ) * Hill Slope ) ) Expression of MT1 -MMP 5 Cell Line Name ( > 5 implies expression ) Example 2 HCC1806 6.6 EBC - 1 6.5 Evaluation of the Efficacy of I - 1A in EBC - 1 SNU - 16 6.2 10 Xenograft Model in Female Balb / C Nude Mice HCT15 5.8 HT29 5.7 Study Objective MOLP - 8 5.6 The objective of this study was to evaluate the anti- tumor efficacy of I- la , in EBC - 1 xenograft model in female The IC50 results and top inhibition percentage for I - la and 15 BALB /c nude mice . The experimental design is shown in the reference compound paclitaxel is shown in Table 5 . Table 7 . TABLE 5 TABLE 7 Experimental Design 11 Cell Line Panel Cytotoxicity Testing Results for I- la 20 and Paclitaxel (Reference Compound ) Dose Dose volume Conc . Dosing I - 1a Paclitaxel Gra nb Treatment (mg / kg ) (ml /kg ) (mg /ml ) Route Schedule Re Top IC50 IC50 * 1 3 Vehicle 10 IV 3x weekly for Cell Line Name IC50 (nM ) inhibitions ( NM ) (NM ) 25 2 weeks HT1080 21.4 82 6.6 ( D 0 , 2 , 4 , 7 , PC - 3 102 59 9.2 12 9 , 11 , 14 ) MDA -MB - 231 211 71 4.8 9 2 3 I - 1a 10 0.1 IV 3x weekly for NCI- H292 56.2 82 2.9 2 2 weeks NCI- H1975 24.2 60 7.2 5 ( D 0 , 2 , 4 , 7 , HCC1806 15.2 88 2.7 2 30 9 , 11 , 14 ) EBC - 1 35.7 70 4.1 4 3 3 I - 1a 3 10 0.3 IV 3x weekly for SNU - 16 63.3 96 2.5 2 2 weeks HCT15 > 5000 < 50 112 128 ( D 0 , 2 , 4 , 7 , HT29 219 81 4.1 2 9 , 11 , 14 ) MOLP - 8 197 89 6.9 4 4 3 I- 1a 10 10 IV 3x weekly for 35 2 weeks * Historical IC50 observed for paclitaxel ( D 0 , 2 , 4 , 7 , 9 , 11 , 14 ) The IC50 results and top inhibition percentage for DM1 agroup , bnumber Sme, MMAE and SN38 are shown in Table 6 . 40 Materials TABLE 6 Animals and Housing Conditions 11 Cell Line Panel Cytotoxicity Testing Results Animals for DM1- Sme, MMAE and SN38 Species : Mus Musculus. DM1- Sme MMAE SN38 45 Strain : BALB / c nude . Age : 6-8 weeks. Re Top Re Top Re Top Cell Line IC50 inhibi IC50 inhibi IC50 inhibi Sex : Female . Name (nM ) tion % (nM ) tion % (nM ) tion % Body weight: 18-22 g . Number of animals : 42 mice . HT1080 0.22 99 0.68 97 1.35 96 50 PC - 3 0.18 54 0.85 58 48.7 78 Housing Conditions MDA -MB - 231 0.79 49 0.62 61 25.1 78 NCI- H292 0.27 77 0.34 86 3.48 92 The mice were kept in individual ventilation cages at NCI- H1975 1.52 64 0.38 65 272 116 constant temperature and humidity with 3 animals in each HCC1806 0.62 88 0.26 91 4.69 96 cage . EBC - 1 0.32 65 0.35 67 2.34 87 SNU - 16 0.77 98 0.18 99 2.14 76 55 Temperature : 20-26 ° C. HCT15 0.94 94 21.4 100 11.5 80 Humidity 40-70 % . HT29 0.68 82 0.42 81 23.7 98 MOLP - 8 0.21 92 0.65 93 3.59 88 Cages : Made of polycarbonate . The size is 300 mmx180 mmx150 mm . The bedding material is corn cob , which is changed twice per week . Three toxins (DM1 - Sme , MMAE and SN38 ) work well 60 Diet : Animals had free access to irradiation sterilized dry on all 11 tumor cell lines ( IC50 < 300 nM ) . I - la works well on granule food during the entire study period . all cell lines except HCT- 15 . I - la has elevated HCT- 15 Water: Animals had free access to sterile drinking water . IC50's ( IC50 > 5000 nM ) . Cage identification : The identification labels for each cage Data Details for Tables 5 and 6 . 65 contained the following information : number of animals , sex , strain , date received , treatment, study number, group Inhibition % = (Max - Sample value) / ( Max -Min )* 100 . number and the starting date of the treatment. US 10,624,968 B2 125 126 Animal identification : Animals were marked by ear cod Tumor Measurements and the Endpoints ing . The major endpoint was to see if the tumor growth could Test and Positive Control Articles be delayed or mice could be cured . Tumor sizes were measured three times per week in two dimensions using a Testing article 5 caliper , and the volume was expressed in mm using the Product identification : 1-1a formula : V = 0.5 axb2 where a and b were the long and short Physical description : Clear solution ( in DMSO ) diameters of the tumor, respectively . Purity : > 95 % The tumor size was used for calculations of T /C values . Package and storage condition : stored at -80 ° C. The T / C value in percent) is an indication of antitumor Experimental Methods and Procedures effectiveness ; T and C are the mean volumes of the treated Cell Culture 10 and control groups. The EBC - 1 tumor cells were maintained as a monolayer TGI was calculated for each group using the formula : TGI culture in RPMI1640 medium supplemented with 10 % fetal ( % )= [1- ( Ti- T0 )/ ( Vi - VO ) ]x100 ; Ti is the average tumor calf serum at 37 ° C. in an atmosphere of 5 % CO , in air. The volume of a treatment group on a given day, TO is the tumor cells were routinely subcultured twice weekly by 15 averagetreatment tumor start , volume Vi is the of averagethe treatment tumor group volume on ofthe vehicleday of trypsin - EDTA treatment. The cells growing in an exponen control group on the same day with Ti, and VO is the average tial growth phase were harvested and counted for tumor tumor volume of vehicle group on the day of treatment start . inoculation . Sample Collection Tumor Inoculation Plasma was collected from all the mice 5 min , 15 min , 30 Each mouse was inoculated subcutaneously at the right 20 min , 1 h and 2 h after dosing on day 14. Study end blood flank with the EBC - 1 tumor cells ( 5x10 ° ) in 0.2 ml of PBS sample ( 30-50 ul) was taken via orbital sinus vein puncture for tumor development. The treatments were started on Day with EDTA - 2K as anticoagulant ( 1.5 ul 0.5 M EDTA - 2K ) . 10 ul plasma was pipetted to a new tube for PK analysis . 7 after the tumor inoculation when the average tumor size Statistical Analysis reached approximately 152mm ». Each group consisted of 3 Summary statistics , including mean and the standard error tumor- bearing mice. The testing articles were administrated 25 of the mean (SEM ), were provided for the tumor volume of to the mice according to the predetermined regimen as each group at each time point. shown in Table 7 . Statistical analysis of difference in tumor volume among the groups was conducted on the data obtained at the best TABLE 8 therapeutic time point after the final dose . 30 A one- way ANOVA was performed to compare tumor Testing Article Formulation Preparation volume among groups , and when a significant F -statistics ( a Concentration ratio of treatment variance to the error variance ) was Compound Preparation (mg /ml ) Storage obtained , comparisons between groups were carried out with Vehicle 50 mM Hepes pH 7.0 , 20 % PEG 0.1 -80 ° C. Games -Howell test. All data were analyzed using Prism . 400 35 P < 0.05 was considered to be statistically significant. I- la , Dilute 14 uL 100 mg/ml I -la stock Do not Results 1 mg/ kg into 1386 ul formulation buffer to store Mortality , Morbidity , and Body Weight Gain or Loss make the 1 mg /ml mother solution . Animal body weight was monitored regularly as an indirect Dilute 100 ul mother solution into 900 ul formulation buffer , mix measure of toxicity . Body weight change in female BALB /c immediately with the pipette / gentle 40 nude mice bearing EBC - 1 dosed with I- la is shown in Table vortexing. The solution should be 9 . completely clear to the eye and free of particulates . I- la , Dilute 300 ul mother liquid into 0.3 Do not TABLE 9 3 mg/kg 700 ul formulation buffer, mix store immediately with the pipette / gentle 45 Body Weight ( g ) vortexing. The solution should be completely clear to the eye and free Days after the start of treatment of particulates. I- la , The remaining mother liquid 1 Do not Group Treatment 0 2 4 7 9 11 14 10 mg/kg store 50 Vehicle 22.0 + 22.0 + 22.7 + 22.9 + 23.9 = 23.7 + 23.9 + i.v. tiw * 0.2 0.3 0.1 0.2 0.1 0.1 0.0 x2 w Observations 2 I - la 23.4 + 23.3 + 23.6 + 23.8 + 24.0 + 24.0 + 23.8 + All the procedures related to animalhandling , care and the 1 mg/kg 1.4 1.3 1.6 1.8 1.9 1.7 1.8 treatment in the study were performed according to the i.v. tiw x2 w guidelines of the Association for Assessment and Accredi 55 3 I - la 21.9 21.4 = 21.7 + 22.3 22.4 + 22.0 + 22.0 + tation of Laboratory Animal Care ( AAALAC ) . At the time 3 mg/ kg 1.1 1.0 1.1 1.0 1.1 1.1 1.3 i.v. tiw of routine monitoring , the animals were daily checked for x2 w any effects of tumor growth and treatments on normal 4 I - la 23.1 + 21.8 + 20.8 + 19.3 + 22.0 + 20.6 = 22.4 + behavior such as mobility , food and water consumption (by 10 mg/ kg 0.8 0.7 0.7 1.0 0.9 1.2 1,0 looking only ) , body weight gain / loss (body weights were 60 i.v. tiw measured every day ), eye /hair matting and any other abnor x2 w mal effect as stated in the protocol. Death and observed clinical signs were recorded on the basis of the numbers of * tiw = 3 times per week . animals within each subset. Animals that were observed to Tumor Volume Trace be in a continuing deteriorating condition or their tumor size 65 Mean tumor volumes over time in female BALB / c nude exceeding 2000 mm were euthanized prior to death or mice bearing EBC - 1 xenografts dosed with 1-1a are shown before reaching a comatose state . in Table 10 . US 10,624,968 B2 127 128 TABLE 10 TABLE 12 Tumor Volume Trace over Time Cytotoxicity in HT1080 cells in the presence and absence of 1 mM bacitracin Tumor volume Days 5 No block + 1 mM bacitracin ( PDI inhibitor ) ( mm3ja 0 2 4 7 9 11 14 I - 1a IC50 = 5.3 nM IC50 = 7.3 nM ( 1.4 x shift ) Vehicle 146 + 309 + 443 + 730 + 925 1061 + 1195 + tiwo 24 3 18 56 35 118 91 I - la 162 + 239 + 308 + 427 + 395 + 380 + 271 + 10 1 mg/ kg 41 92 78 188 174 171 99 Example 4 tiw I - la 144 + 126 + 105 + 63 + 22 + 0 + 0 + 3 mg/ kg 15 22 8 3 6 0 0 Plasma Stability of 1-1A (LC -MS / MS Assay ) tiw I -1a 165 + 110 + 74 + 28 + 5+ 0 + 0 + 15 10 mg/ kg 33 8 17 6 3 0 0 l- la plasma stability studies were conducted in human tiw and mouse plasma. Note : BACKGROUND Mean + SEM , btiW = 3 times per week Tumor Growth Curve 20 In vitro plasma stability assay measures the extent of Tumor growth curves are shown in Table 11 . degradation of compounds in plasma . Determination of the stability of new chemical entities in plasma is important in drug discovery , as compounds which TABLE 11 rapidly degrade in plasma generally show poor in vivo Tumor Growth Inhibition 25 efficacy. Tumor Size (mm3 ) TGI TIC Peptides or BDCs are incubated at 37 ° C. in plasma Treatment at Day 14 ( % ) ( % ) p value (mouse or human ) at low micromolar concentrations for Vehicle 1195 + 91 several hours . The stability is quantitatively measured by I - la , 1 mg/kg TIW 271 + 99 90 23 30 LC -MS / MS . I - 1a , 3 mg/ kg TIW 0 + 0 114 0 *** Preparation of Dmso Stock Solutions I - 1a , 10 mg/ kg TIW 0 0 116 0 The preparation of stock solutions for the plasma stability Mean + SEM . assay is as follows. Prepare a 160 uM DMSO stock of a standard peptide Tumor Growth Inhibition Analysis 35 (containing all D - AAs) to use as an internal standard . Tumor Growth Inhibition is calculated by dividing the Prepare DMSO (CC grade, hybrimax ) stock for each group average tumor volume for the treated group by the peptide or BDC at 1 mM . group average tumor volume for the control group ( T / C ) . Dilute the 1 mM DMSO stock for each peptide at 160 uM For a test article to be considered to have anti -tumor activity, (64 ul of 1 mM stock + 168 * 2 uL of DMSO ( CC grade, T / C must be 50 % or less . Dunnett's One -way ANOVA was 40 hybrimax ) ) . conducted to analysis the statistical differences of com Prepare scalar dilutions from 160 uM to 5 uM ( 160 UM , pound -treatment groups vs Vehicle group on day 14 , *** 80 uM , 40 uM , 20 uM , 10 UM , 5 uM ). indicates p < 0.001. 80 uM stock : 100 uL of 160 uM + 100 uL of DMSO (CC Results Summary and Discussion grade, hybrimax ) , In this study, the therapeutic efficacy of I -la in the EBC - 1 45 40 uM stock : 100 ul of 80 uM + 100 uL of DMSO (CC xenograft model was evaluated . The measured body weight grade , hybrimax ), is shown in Table 9. Tumor sizes of all treatment groups at 20 uM stock : 100 uL of 40 uM + 100 uL of DMSO (CC various time points are shown in Table 10 . grade , hybrimax ) As shown in Table 10 , the mean tumor size of vehicle 10 uM stock : 100 uL of 20 uM + 100 uL of DMSO (CC treated mice reached 1195 mm on Day 14 ,mice treated I -la 50 grade, hybrimax ) at 1 mg/ kg ( TGI= 90 % ), 3 mg/ kg ( TGI= 114 % ) , 10 mg/ kg 5 um stock : 100 ul of 10 uM + 100 uL of DMSO (CC ( TGI= 116 % ) showed significant tumor growth inhibition grade, hybrimax ) effect . For each stock solution , mix thoroughly with pipette and gently vortex . Example 3 55 Plasma Stability Experiment Time Course Add 8 uL of the 160 UM DMSO stock of peptide or BDC Specificity of Toxin Release and add 8 uL of 160 UM DMSO stock of 06-34-18 -N182 as internal standard to 304 uL of plasma ( 152 uL * 2 ) for a final I - 1a, which possesses a AA -AA ? linker, is more resistant concentration of 4 uM ( 5 % DMSO ). to non - specific release of toxin versus BDCs with disulfide 60 Incubate at 37 ° C. in 100 % humidity . linkers , as demonstrated by a reduced susceptibility to Pipette out 40 uL for 7 timepoints : time 0 , 1 h , 2 h , 4 h , blocking with bacitracin , a protein disulfide isomerase 6 h , 8 h , 24 h into a 1.5 mL Eppendorf. Store at -80 ° C. (PD1 ) inhibitor. Measurement of cytotoxicity in HT 1080 immediately . (total volume of samples ? x40 = 280 ML ) . cells after 24 hours incubation with 1-1a showed a 1.4 - fold Plasma Stability Experiment Standard Curve shift in IC50 when comparing no bacitracin versus 1 mM 65 Add 5 uL of each DMSO stock to 95 uL of plasma. For bacitracin as shown in Table 12. The AA - AAP linker each sample, mix thoroughly with pipette and gently vortex . chemistry results in less non - specific toxin release . Store at -80 ° C. US 10,624,968 B2 129 130 The concentrations of the standard curve samples are TABLE 16 shown in Table 13 , below . Recommended Gradient 2 TABLE 13 Time Flow Rate Concentrations of the Standard Curve Samples (min ) (mL / mi n ) C % D % Initial 95 5 Extract 1.50 0.6 95 5 DMSO Stock Concentration 5.50 0.6 40 60 Dilution (UM ) Prespike (UM ) ( 1 : 4 dilution ) UM 6.00 0.6 5 95 10 6.50 0.6 5 95 80 80 4 1 6.70 0.6 95 5 40 40 2 0.5 8.00 0.6 95 5 20 20 1 0.25 10 10 0.5 0.125 5 5 0.25 0.0625 Where the mobile phase A through D is as follows : 15 A = 0.1 % HCOOH in water; B = 0.1 % HCOOH in MeCN ; Preparation for LCMS Experiments C = 5 mM ammonium bicarbonate in water ; D = 5 mM ammo nium bicarbonate 10 % water in acetonitrile Thaw the plasma samples in a water bath at room tem The recommended injection volume is 5 uL perature . Sample temperature should be set up at 21 ° C. Add to the sample the extraction solvent. The extraction 20 The column temperature is 40 ° C. solvent contains 1 part of water , 3 parts of methanol, and 3 The LC -MS method is called “ Default ” and has the parts of acetonitrile . following settings: To the time course samples add 120 uL of extraction Polarity : ES + solvent, mix thoroughly with pipette and gently vortex . CalibrationDynamic 1 To the standard curve samples add 2 * 150 ul , mix thor- 25 Capillary (kV ) : 2.51 oughly with pipette and gently vortex . Cone ( V ) : 20.00 Extractor ( V ) : 3.00 Centrifuge for 40 minutes at 14000 rpm at 4 ° C. RF (V ) : 0.10 Transfer the supernatant to the LC -MS vials and run the Source Temperature ( ° C.) : 150 experiment immediately afterwards. 30 Desolvation Temperature (° C.) : 400 LC -MS / MS Experiments Cone Gas Flow ( L / Hr ): 2 It is recommend to follow one transition for the peptide of Desolvation Gas Flow ( L /Hr ) : 800 BDCs to study, and one transition for the internal standard Collision Gas Flow (mL /Min ) : 0.20 06-34-18 -N182 at the same time. The most relevant Selected LM 1 Resolution : 12.00 Ion Reaction (SIR ) and Multiple Reaction Monitoring 35 HM 1 Resolution : 12.00 (MRM ) transitions were determined for each peptide or Ion Energy 1: 1.00 BDC . The recommended transitions for the study is shown MS Mode Entrance : 50.00 MS Mode Collision Energy : 3.00 in Table 14 , below . MS Mode Exit: 50.00 40 MSMS Mode Entrance : 1.00 TABLE 14 MSMS Mode Collision Energy : 20.00 Recommended Transitions for the Standard , I - 1a and MMAE MSMS Mode Exit : 0.50 LM 2 Resolution : 14.00 Compound Parent Cone Collision Name (m /z ) Daughter ( m / z ) Dwell ( s ) ( V ) ( V ) HM 2 Resolution : 14.00 45 Ion Energy 2: 1.00 Standard 871 871.1 0.160 35 10 Gain : 1.00 I - 1a 1293.1 213 0.160 30 60 I- 1 a 1293.1 1293.7 0.160 30 10 The suggested order of the samples is : from time course MMAE 718 506.5 0.160 10 20 7 ( 24 h ) to time course 0 to reduce carrying over of the MMAE 718 718 0.160 10 10 samples ; and from dilution 5 uM to 80 uM for the standard 50 curve samples for the same reason . The analysis of the data has been performed using “ Quan The recommended gradients as shown in Tables 15 and Lynks ” and “ Sigmaplot” software . 16 , below . Results for the plasma stability testing in mouse and human plasma are shown in FIGS. 1-3. TABLE 15 55 For the studies depicted in FIGS. 1-3 , the following conditions were used . Recommended Gradient 1 Column : Waters ACQUITY CSH Phenyl- Hexyl 1.7 um Time Flow Rate 2.1x50 mm (min ) (mL / min ) A % B % Mobile Phase : C = 5 mM ammonium bicarbonate in water; Initial 95 5 60 B = 5 mM ammonium bicarbonate 10 % water in acetonitrile . 1.50 0.6 95 5 Gradient: 5 to 60 % B in 4 minutes at 0.6 mL /min flow . 5.50 0.6 40 60 FIG . 1 shows plasma stability curves for I- la in mouse 6.00 0.6 5 95 6.50 0.6 5 95 and human plasma ( 1 uM in plasma, pH 7.4 at 37 ° C.) . After 6.70 0.6 95 5 24 hours incubation in human plasma, 96 % I - la remained 8.00 0.6 95 5 65 which corresponds to a t1/ 2 = >> 24 h . After 24 hours incu bation in mouse plasma, 57 % I - la remained which corre sponds to a t1 /2 = 6.4 h . US 10,624,968 B2 131 132 Without being bound to any particular theory , it is incubation in mouse plasma + AEBSF, 100 % I - la remained believed that the lower stability observed in mouse is likely which corresponds to a t12 >> 24 h . due to mouse carboxyesterase 1c ( Ces1c ) and the generally Additionally , formation of the MMAE metabolite in more aggressive proteolytic environment found in mouse . mouse plasma ( release of the toxin ) was monitored both in To test the hypothesis that plasma instability in mouse was 5 the presence and absence of AEBSF . As shown in FIG . 3 , in due to Cesic, I- la was incubated in mouse plasma both in the presence of AEBSF , degradation of I - la is inhibited as the presence and absence of AEBSF, shown by the lack of formation of free MMAE . In the absence of AEBSF, I- la degradation tracks with the appear ance of free MMAE . 10 Example 5
H?N Target Specificity of In Vitro Cytotoxicity of I- la 15 The target dependence of cytotoxicity of I - 1a in cell lines a serine protease inhibitor that is cross- reactive with expressing or lacking MT1- MMP was assessed by measur Ceslc . ing ATP levels in an endpoint assay after 24 hours exposure FIG . 2 shows plasma stability curves for l - la in mouse to toxin , in the presence or absence of an excess of binding plasma ( 4 uM in plasma, pH 7.4 at 37 ° C.) . After 24 hours peptide or non - binding control peptide. incubation in mouse plasma without AEBSF , 50 % I- 1a 20 The structure of the binding peptide , which is the remained which corresponds to a t1 /2 = 24 h . After 24 hours Bicycle + Spacer of l - la , is US 10,624,968 B2 133 134
HO' HO
NH HO HN to-CH3 NH HO NH NH H2 -?? NH -S— H2 HN H2N NH .HN ??. H3C H2C H2C NH H2C OH NH HN
N
N
o
H2N Toattachto AA2AA1- moiety US 10,624,968 B2 135 136 where all amino acids are in the natural L - form except the TABLE 17 two alanines which are in the D - form . The non -binding peptide is ( B - Ala )-Sar10 - ACVPCADF HT1080 Cytotoxicity IC50 shift in the presence PIWYC (SEQ ID NO : 17 ). and absence of binding peptide The non - binding peptide showed no inhibition in MT1 5 MMP fluorescent polarization competition experiments up I - 1a MMAE to 20 UM . IC50 Maximum Shift in IC50 Maximum Shift in Methods ( NM ) kill ( % ) IC 50 (nM ) kill ( % ) IC50 HT1080 cells were seeded in 96 well plates ( 75 uL / well) No block 13.7 + 95 % 0.06 + 95 % and incubated overnight at 37 ° C.+ 5 % CO2. The next day, 10 5.5 0.07 peptides were prepared in buffer from a DMSO stock . 10 uL +250 UM 160.7 + 95 % x12.5 + 0.05 + 95 % x1.2 + 0.5 of buffer or peptide was added to the cells and incubated for binding 83.4 7.6 0.06 1 hour. Toxins were diluted in cell culture media and 15 peptide +250 UM 18.7 + 95 % x1.7 + 0.05 + 95 % x1.0 + 0.3 UL / well was added to the cells (< 0.5 % final DMSO ). Cells non -binding 9.9 1.2 0.07 were approximately 30-50 % confluent upon dosing . Plates peptide were sealed with gas permeable seals and incubated for 24 15 hours at 37 ° C.+ 5 % CO2. After 24 hours, media was Data( % ) comparedis represented to a lysisas mean control IC50 and ( NM average) standard shift deviation in IC50 of , averageI- la in maximumpresence ofcell peptide killing : removed , cells were washed with PBS and 100 ?L fresh standard deviation compared to no block . Number of experimental repeats = 3 media was added per well. Cells were incubated for a further 48 hours at 37 ° C. + 5 % CO2. ATPLite reagent (Perkin Elmer ) was equilibrated to room 20 Example 6 temperate and reconstituted in buffer as per the manufac turer's instructions . 100 uL reagent was added per well. Plates were sealed and shaken gently for 20 minutes to Cell Toxicity of l - la and Toxins in HT1080 and ensure complete cell lysis before luminescence was read L540 Cells in the Presence / Absence of Protein using a BMG Pherastar. Luminescence counts correlate with 25 Disulphide Isomerase Inhibitors Bacitracin at 1 Mm ATP levels and hence cell viability . Data was analyzed using One advantage of the AA - AA ? linker is its resistance to GraphPad Prism . Non -linear regression fit was used to cleavage by protein disulphide isomerase which can readily calculate an IC50 for each toxic agent: Y = Bottom + ( Top cleave disulfide bonds . This can result in the non -selective BottomResults )/ ( 1 +are 10 °shown (( Log IC50in -Table X )* HillSlope 17, below )) . . A shift in the 30 release of toxin for BDCs that utilize a disulfide linkage . cytotoxicity IC50 of 13 - fold was observed for 1-1a in the As mentioned previously , non -selective delivery of toxin presence of 250 uM binding peptide , whereas the presence is termed bystander activity . One contributing factor in some of 250 uM non -binding peptide resulted in only a 2- fold BDCs may be the enzyme protein disulphide isomerase shift, indicating that I- la can be competed with the binding (PD1 ) which can cleave the SS bond to release toxin . stratepeptide the but specificity not an unrelated of l- la peptidebinding. Thesein targeting results HT1080demon- 35 In order to determine the effect of PD1 in the delivery of cells . The toxin MMAE shows a 1.2 - fold shift in the toxin , PD1 is inhibited with bacitracin , a known inhibitors of cytotoxicity IC5, in the presence of 250 um binding peptide PD1. and no shift in IC50 in the presence of 250 uM non -binding Bacitracin is a cyclic peptide that interferes with cell peptide consistent with its lack of a MT1- MMP binding wall/ peptidoglycan synthesis . It has been used as a PD1 moiety . inhibitor and has the following structure:
OH
NH
H2N NH HN
HN Ph
HO . HN .
HN"
H2N N
NH2 US 10,624,968 B2 137 138 Materials This gives a 5 uM top concentration for BDCs in assay DM1- SMe, 10.58 mM stock in DMSO . This gives a 2 uM top concentration for DM1- SMe & I - 1a , 100 mg/ ml , 25.8 mM . MMAE in assay MMAE , 10 mm 5 Add 104 ofmedia + bacitracin ( 2 plates of each ) -leave for ATPlite 1 - step kit (Perkin Elmer 6016731) 1 hr HT1080 cells ( ATCC , via LGC , BIC918 ) Maintained in EMEM media (Sigma M2279 ) + 10 % FBS (Sigma Code Bacitracin = 1 mM final concentration . This requires a 10 F7524 ) +4 mM L -Glu ( Invitrogen Cat 25030-024 ) mM concentration with no DMSO . L540 cells (DSMZ # ACC72 , BIC5121) , maintained in 10 For HT1080 & L540 media this requires 3 mlmedia + 1 ml RPMI- 1640 medium (Sigma # R8758 ) + 20 % heat - inacti 40 mM bacitracin to prepare the required concentration . vated serum The solution of 40 mM bacitracin was prepared by Clear tissue -culture treated 96 well plates dissolving 0.2 g bacitracin in 3514 ul water . Gas permeable adhesive seal ( VWR 732-0077 ) Add 15 uL treatment per well as in Table 19 , below . TABLE 19 Plate Map of Concentrations Tested (nM unless indicated otherwise )
1 2 3 4 5 6 7 8 9 10 11 12 I - 1a A 5000 1000 200 40 8 1.6 0.32 0.064 0.0128 0 5 Lysis B 5000 1000 200 40 8 1.6 0.32 0.064 0.0128 0 UM SS * DM1 ? 500 100 20 4 0.8 0.16 0.032 0.0064 0.00128 0 SMe D 500 100 20 4 0.8 0.16 0.032 0.0064 0.00128 0 MMAE E 500 100 20 4 0.8 0.16 0.032 0.0064 0.00128 0 F 500 100 20 4 0.8 0.16 0.032 0.0064 0.00128 0
* SS = staurosporin
X -clear advanced polyolefin starseal (Starlabs E2796 30 Seal plates with gas permeable membrane and incubate at 9795 ) 37 ° C. Staurosporine 1 mM ( Sigma 56942-200UL ) Washout HT1080 plates with 2x150 ?L PBS at 24 h and Method add 100 uL fresh media I - 1a and Toxins with Bacitracin — 24 or 72 Hour Exposure in At t = 72 h , equilibrate ATPlite reagent to room tempera 96 Well Plates 35 ture . Add 10 ml buffer to each vial of lyophilized substrate . DB seeded cells in normal media , 75 uL in 96 well clear Add 100 uL reagent per well. Seal with PCR seal and cover plates, ~ 24 hours previous. to protect from light. Shake for 20 mins before reading HT1080—7.5 k cells /well : 50 % confluent upon dosing . luminescence L54020 k cells /well : 40 % confluent upon dosing . Luminescence Settings Preparation of staurosporine : 1 mM stock in DMSO : 40 Endpoint Settings Dilute 1:50 (5 uL + 245 uL ) to 20 uM in media or imaging Measurement interval time [ s ] : 1.00 buffer (4x final conc 5 uM . Final DMSO conc = 0.5 % ). Optic Settings Lyis reagent made from R & D DYC002 : (Final 1 % NP40 , Optic module : LUM plus 20 mM Tris pH8, 137 mM NaCl, 10 % glycerol , 2 mM Gain : 3000 EDTA , 1 mM NaOrthovanadate , 10 ug /ml leupeptin , 10 45 Focal height [mm ] : 10.0 ug/ ml aprotinin ). Combine 750 uL water and 7504 DYC002 . General settings Stock solutions were prepared as shown in Table 18 . Setting time [ s ] : 0.1 Target temperature [ ° C. ]: 25 TABLE 18 To allow comparison of different kinetic windows all Stock Solution Preparation 50 measurement values are normalized to 1 second . Results stock Dilution The results are summarized in Table 20 , below . mM factor mM mg/ mL mg/mL TABLE 20 Amount required for 1428 55 UM in DMSO Summary of I - 1a and Toxin ICsos with Bacitracin I - 1a 25.8 100 18.07 1.11 18.89 HT1080 cells , L540 cells , Amount required for 571 24 hour exposure 72 hour exposure uM in DMSO (high MT1 expression ) ( low /no MT1 expression ) 60 DM1- SMe 10.58 18.53 1.08 18.92 IC50 Fold IC50 Fold MMAE 10 17.51 1.14 18.86 with shift with shift Standard 1 mM with Standard 1 mM with Dilute above 1 in 5 by adding 4 mL above to 16 mL DMSO (columns 1-9 , DMSO in 10 ) IC50 bacitracin baci IC50 bacitracin baci (nM ) (nM ) tracin (nM ) ( nM ) tracin HT1080 and L540 Cells 65 1-1a 5.3 7.3 x1.4 1088 2551 x2.3 Dilute in media 1 :42.85 = 5 uL DMSO dilute + 209 uL DM1 0.07 0.41 X5.9 9.6 12 x1.2 media = > use 15 uL per well US 10,624,968 B2 139 140 TABLE 20 - continued Example 7 Summary of I- la and Toxin ICsos with Bacitracin Evaluation of the Efficacy of I- 1A in Ht1080 HT1080 cells , L540 cells , Xenograft Model 24 hour exposure 72 hour exposure 5 ( high MT1 expression ) (low /no MT1 expression ) I -la was evaluated in a HT1080 xenograft model at 1 , 3 , IC 50 Fold IC50 Fold and 10 mg/ kg . Additionally the ability of the binding peptide with shift with shift ( the Bicycle + Spacer of I - la , structure shown in Example 6 ) Standard 1 mM with Standard 1 mM with 10 IC50 bacitracin baci IC50 bacitracin baci to abrogate activity when dosed at a 100 - fold excess imme (NM ) (nM ) tracin ( NM ) ( NM ) tracin diately before treatment with I - la in the HT1080 xenograft SMe model was assessed . MMAE 0.083 0.15 x1.8 0.49 0.28 x0.6 The results of the xenograft studies are shown in FIGS. 15 4-6 . I - 1a IC50s were shifted 1.4 - fold with bacitracin treatment FIG . 4 shows the efficacy with I - la in the HT1080 in HT1080 cells . l - 1a ICsOs were shifted 2.3 - fold with xenograft model. Complete clearance of tumor was bacitracin treatment in L540 cells . These data indicate that observed at 1 mg/ kg . No dose- related effects on body weight 20 were observed at any of the doses tested . cleavage of the AA - AA linker is unaffected by the pres FIG . 5 shows the displacement of I - la activity with ence of absence of PD1 activity . Similarly MMAE which 100 - fold excess of the binding peptide at 1 mg/ kg 1-1a . At has no linker shows negligible shifts in the presence of 1 mg/ kg , significant displacement of efficacy is seen with bacitracin in both HT1080 and L540 cells . DM1- SMe which 25 co -dosing of the binding peptide . possesses a disulfide linkage to methanethiol shows a small shift of 5.9 - fold in HT1080 cells and no shift in L540 cells . Example 8
SYNTHESIS OF I- la SUMMARY
HO H2N H3Ci i NH -NH NH NH
NH H?N 0= H2C NH S CH3 H2C H2 H2 NH 0
OH H2C
HN HC NH OH HN to NH HN . -??
HO . OH US 10,624,968 B2 141 142 -continued OH
HN H2N
NHS activated VcMMAE glutarate
OH
N
HN H?N HO H2N H3C ?NH NH NH NH
NH H HC NH S HC CH3 H2 H2 NH C - S OH H2C
HN H?C NH OH HN
NH HN NH
HO OH 1-1
Bicyclic peptide 1, MW( . 2658.95 Da) , was coupled to an 60 had been selected and to develop an HPLC gradient capable auristatin cytotoxic drug linker, monomethyl auristatin E of resolving starting materials /products /by -products . ( VCMMAE ) via a glutarate linker to give VcMMAE - Bicy clic peptide conjugate (I - 1a, MW.3877.94 ) after appropriate Two RP- HPLC methodswere developed the first using 10 purification . mM ammonium acetate using a Hichrom ACE 3 column 65 ( 2.1x50 mm , 3 um , 100 Å , C18) and the second using 10 An Initial small scale reaction ( 10 mg of bicyclic peptide ) mM ammonium acetate with a waters xselect column ( 2.1x was carried out to confirm suitable conjugation conditions 50 mm , 3.5 um , peptide CSHTM , C18 , 130 Å ) . US 10,624,968 B2 143 144 Final analysis was carried out on the ACE 3 column using The buffer was prepared as follows: HEPES (5.96 g ) , pH the TFA method . adjustment with 0.5M NaOH to pH 7.0 and made up in A single 114 mg preparative conjugation was performed ELGA water (500 mL ). and purified using the Gilson 20:20 preparative HPLC in Key Methods multiple 20 mg passes after translation from the analytical 5 Reverse Phase HPLC with UV and /or MSD method using a waters xselect prep column (10x100 mm , 10 Initial quality control data for the peptide was obtained on um , peptide CSHTM , C18 , 130 Å ) to yield pure product . a Hichrom ACE 3 C18 column with the mass spectrometer Fractions containing pure product were pooled , on a negative polarity setting . A summary of the HPLC and lyophilized / reconstituted and quantified gravimetrically LCMS methods are described in Table 23 , below . before being separated into 5 mg aliquot vials . 10 A single nominal 5 mg vial ( 3.4 mg actual) was recon TABLE 23 stituted in DMSO for QC analysis The results of the QC tests and product vials supplied are HPLC and LCMS Methods summarized in Table 21 , below . Method 2 Method 3 The process from starting materials to final product vials 15 Method 1 ( TFA ) ( NHOAC ) (NH OAC) gave 72 % yield of l -la . Column Ace 3 C18 , 50 mm x Ace 3 C18 , 50 mm x 2.1 mm 2.1 mm Buffer A 0.05 % v / v TFA in 10 mM NH4OAc in H20 at pH 5.7 TABLE 21 H2O 20 Buffer B 0.05 % v / v TFA in Acetonitrile QC Test Data Summary MeCN QC TEST DATA Gradient : Time Time Time TEST SPECIFICATION RESULT (min ) % B (min ) % B (min ) % B Visual White lyophilized solid White lyophilized 25 0 1 0 1 0 1 solid 15 99 15 80 5 30 > 95 % 99.5 % 17 17 1 25 50 Headline Purity (HPLC ) 19 19 1 26 80 < 0.5 % Not detected 80 Salt state from Based on 10 mM NH4OÁc 28 30 1 purification NH40Ac buffer system 35 1 30 Flow Rate : 0.4 mL /min Wavelength : 214 nm , 220 nm , 254 nm & 280 nm PRODUCT SUPPLIED Injection 10 uL (unless otherwise stated ) No. Vials and Volume: Nominal Vial Size HPLC and Agilent 1100 and Agilent 1100 Agilent 1100 QC Value Total Mass (mgs ) LCMS Agilent G1946D 35 5.00 + 0.5 mgs 24 x 5.00 system 5 mgs Column 25 ° C. TOTAL MASS OF PRODUCT SUPPLIED 118.7 mgs Temperature : MSD ES Positive unless N / A N / A Materials and Methods stated otherwise Key Materials 40 Bicyclic Peptide 1 Once qualified the same sample was run on a separate Bicyclic Peptide 1, ( 196.74 mg, 73.9 umol) quantified by HPLC system with the same mobile phase but using a UV A280 was provided as a lyophilized powder as follows Waters xselect CSH , C18 column . Analytical monitoring of in Table 22 , below . the reaction progress was again carried out on both columns 45 with the xselect column giving the best peak shape and separation . Final product analysis was carried out using TABLE 22 water acetonitrile (0.05 % TFA ) as this gave the best quality Bicyclic Peptide 1 chromatography. The final HPLC conditions developed are described in Table 23 . Quantity Quantity MW by A280 by weight (Average Extinction 50 Biotage ISOLERATM Prime Peptide Tubes (mg ) (mg ) mass ) coefficient The Biotage Purification RP -HPLC method involved 1/1 Falcon 196.74 244.47 2658.95 7290 increasing the method duration for the developed methods to TOTAL 196.74 244.47 3 or 4 times as described in Table 24 . QUANTITY 55 TABLE 24 VcMMAE RP -HPLC Method on Biotage VcMMAE was purchased from Levena Biopharma. Lot no . PI5707040144 Method 1 ( TFA ) NHS Activated VCMMAE Glutarate 60 Time (min ) % B VcMMAE glutarate was prepared and activated in house Gradient 0 5 with a purity of 80 % mid scale and 60 % large scale ( 40 % 40 99 non activated glutarate present) . Column SNAP Ultra C18 12 g / 30 g and 60 UV Analysis Buffers Buffer A Water ( 0.05 % TFA additive ) 50 mM HEPES , PH7 ( for UV measurement, for starting 65 Buffer B Acetonitrile ( 0.05 % TFA additive ) peptide concentration by UV ) was used for UV measure Flow Rate 12/18 mL /min ments . US 10,624,968 B2 145 146 Freeze Drying (Lyophilization ) TABLE 24 - continued Lyophilization used after purification used the conditions RP -HPLC Method on Biotage described below . Wavelength 214 nm & 254 nm System : Thermo PL3000 Heto Powerdry Loading onto column Direct loading via luer lock syringe 5 Vacuum : 0.80 hPa System Biotage Isolera TM Prime Condenser temp: –63 ° C. Length of time: 2-4 days Preparative HPLC Uv Measurements at A280 and A320 Preparative HPLC was conducted on a Gilson 20:20 The absorbance at both 280 nm and 320 nm was deter HPLC system with a Waters xselect, CSH , C18, prep column 10 withmined 50 for mM each HEPES sample , pH after 7.0 the . The instrument average hadA280 been nm zeroedvalue ( 10 um , 130 Å ) with 10 mM ammonium acetate (pH 5.7 ) as was then converted into a concentration and the total product this system was capable of resolving the peptide conjugate per vial was based on the equation below . from other crude reaction components as summarized in Concentration (mg / mL ) = ( (( A280 nm average ) x Table 25 . 15 D - F ** )/ ? * )xmolecular weight [where * extinc tion coefficient and ** is the dilution factor] . TABLE 25 Bicyclic peptide 1 has an estimated extinction coefficient of 7290 M - cm- in aqueous neutral buffered solution . Preparative HPLC Method Reagent and Reaction Characterization Method 1 10 mM NH40AC (pH 5.7 ) 20 Using both optimized HPLC methods the reagents and conjugation reaction were characterized as listed below . Time (min ) % B Bicyclic peptide 1 stock solution . Gradient 0 10 NHS activated VcMMAE glutarate stock solution . 12 10 A trial conjugation reaction between bicyclic peptide 1 18 20 50 40 25 and NHS activated VcMMAE glutarate . 60 80 Full scale conjugation of bicyclic peptide 1 and NHS 70 80 activated VcMMAE glutarate . The preparation of the reagents and conjugation reaction Column Waters Xselect CSH TM peptide column conditions are described below . Buffer A 10 mM NH4Ac in H2O Buffer B MeCN 30 Stock solution of bicyclic peptide 1 . Flow Rate 4.7 mL /min Bicyclic peptide 1 (244.47 mg) was provided in a 50 mL Wavelength 214 nm & 254 nm falcon tube. The tube was centrifuged at 3900 rpm for 2 System Gilson 20:20 minutes . DMA (2.29 mL) was added dissolve the peptide giving a 40 mM stock solution which was stored at -80 ° C. until required .
SYNTHESIS OF VCMMAE GLUTARATE
OH
N
NH2 Dimethylacetamide , HN ??? H2N 0 ° C. to room VcMMAE temperature
OH
oferter VcMMAE glutaratetreyler US 10,624,968 B2 147 148 A 7 mL screw top vial containing VcMMAE (250 mg) frozen at -80 ° C. overnight before being lyophilized to was purged using a nitrogen balloon . 1.72 mL of anhydrous dryness . Meanwhile pure product containing fractions were dimethylacetamide was added with stirring and the solution identified using LCMS and were combined in a pre -weighed 100 mL round - bottom flask by passing 2x5 mL methanol was cooled to 0 ° C. in an ice water bath . DIPEA (52.24 mg, 5 through pure product containing vials . The methanol was 0.40 mmol, 2.0 eqv. ) was then added as a stock solution in then removed by rotary evaporation with a maximum water anhydrous dimethyl acetamide ( 1.05 mL at 50 mg/ mL ) and bath temperature of 20 ° C. to give the product as an off white the reaction was stirred at 0 ° C. for 10 minutes . solid ( 203 mg, 0.18 mmol, 91 % yield ) .
SYNTHESIS OF NHS ACTIVATED VcMMAE GLUTARATE (MID SCALE )
OH
OH 1 ) Dimethylacetamide , CH2Cl2, HN N -hydroxysuccinimde 2 ) EDCI H2N 3 ) 0 ° C. to room VcMMAE glutarate temperature
HN ornOn. N sher HN H2N NHS activated VcMMAE glutarate
Glutaric anhydride ( 46.12 mg, 0.40 mmol, 2.0 eqv. ) was VcMMAE glutarate ( 50 mg, 0.040 mmol) of the material then added as a solution in anhydrous dimethyl acetamide described above was weighed into a 7 mL screw top vial (922.4 uL at 50 mg/ mL ) . 40 which was then purged using a nitrogen balloon . Anhydrous The ice bath was then removed and the reaction was dimethylacetamide ( 1.55 mL ) and anhydrous dichlorometh stirred to r.t. over the course of 1 h . The progress of the ane (550 uL ) were then added with stirring before adding reaction was monitored by LCMS (method 1 ) and on N -hydroxysuccinimide (5.58 mg, 0.049 mmol, 1.2 eqv .) was completion the reaction was quenched with saturated aque then added as a solid and the solution was cooled to 0 ° C. ous ammonium sulphate ( 4 mL ) ensuring that the mixture 45 in an ice water bath . was at a neutral pH before continuing . EDCI ( 9.30 mg, 0.049 mmol, 1.2 eqv. ) was then added as Pure water ( 20 mL ) was added causing a precipitate a solid before removing the ice water bath and allowing the which formed on neutralization to re -dissolve . The aqueous reaction to warm to r.t. overnight. Monitoring of the reaction mixture was then transferred into a 100 mL separating using LCMS (method 1 ) showed that the reaction had funnel rinsing the reaction vessel into the funnel with 50 proceeded to approximately 33 % . The reaction mixture was dichloromethane ( 25 mL ) . The layers were separated and the cooled to 0 ° C. and a further 3.0 eqv. of each N -hydroxy aqueous phase was extracted twice more with fresh dichlo succinimide and EDCIwas then added before removing the romethane (2x25 mL ) before drying the combined organic ice bath and allowing the reaction to warm to r.t. over a phases by passing through a Biotage® phase separation further 20 h giving 98 % conversion to the product . cartridge into a clean 250 mL round- bottom flask . 55 Dichloromethane was removed by gentle rotary evapora The solvent was removed by rotary evaporation with a tion before injecting the resulting dimethyl acetamide solu maximum water bath temperature of 30 ° C. to give the crude tion onto a 12 g Biotage® cartridge which had been equili material as a sticky yellow oil which was taken up into 1 : 5 brated into 5 % acetonitrile in water (0.05 % TFA buffer ) dimethylacetamide : acetonitrile ( 4 mL ) and loaded as liquid before the cartridge was eluted with 5-99 % acetonitrile in onto a 60 g C18 Biotage cartridge which had been equili- 60 water ( 0.05 % TFA ) over 40 minutes . brated into 1 % acetonitrile in water with 0.05 % TFA buffer. Pure product containing fractions were identified using The cartridge was eluted with 1-99 % acetonitrile in water LCMS and transferred into individual 50 mL freeze dry vials with 0.05 % TFA over 40 minutes . Individual fractions were rinsing the collection tubes with 2 mL 50:50 acetonitrile : transferred into 50 mL freeze dry vials rinsing the collection water (0.05 % TFA ) before filling to 25 mL with pure water tubes with 2 mL 50:50 acetonitrile in water with 0.05 TFA . 65 to ensure an organic content less than 30 % . The vials were then filled to 25 mL using pure water to Vials were then frozen in the -80 ° C. freezer overnight ensure a solvent content less than 30 % . The vials were then before being dried by lyophilization . Pure product contain US 10,624,968 B2 149 150 ing vials were then combined by passing 2x5 mL of 50:50 a HPLC sample for method development. The reaction acetonitrile : water (0.05 % TFA ) through all vials into a mixture was stored at -80 ° C. single pre weighed vial. The vial was then topped up to 25 HPLC analysis using method 3 gave 1.3 minutes of mL with pure water to give an organic content less than 30 % separation between product and the closest migrating impu before freezing at -80 ° C. overnight and drying by 5 rity . The closest impurity to the product at 12.92 mins in 10 lyophilization to give 80 % pure material as a free flowing off mM ammonium acetate (pH 5.7 ) was identified as the NHS white solid (33 mg, 0.025 mmol, 61 % yield ). ester . Manufacture of Nhs Activated Vcmmae Glutarate (Large The analytical method was translated onto the Gilson Scale) 20:20 preparative HPLC system to give the preparative A 50 mL round bottom flask which contained VcMMAE 10 HPLC method shown in Table 25 . glutarate (136.6 mg, 0.11 mmol) (material described above ) The material was purified in two 8.77 mg passes by was purged using a nitrogen balloon . Anhydrous dimethyl adding 123 uL of the crude reaction mixture carefully to 123 acetamide (4.20 mL ) and anhydrous dichloromethane ( 1.5 uL of 20 % acetonitrile in 10 mM ammonium acetate (pH mL ) were then added with stirring before adding N -hy 5.7 ) followed by vortex mixing. The full aqueous mixture droxysuccinimide ( 38.4 mg, 0.033 mmol, 3.0 eqv. ) was then 15 was then loaded in one portion onto the prep HPLC system added as a solid and the solution was cooled to 0 ° C. in an running preparative gradient . ice water bath . Product containing fractions were identified using HPLC EDCI ( 63.51 mg, 0.33 mmol, 3.0 eqv. ) was then added as method 2 before being transferred to 20 mL lyophilization a solid before removing the ice water bath and allowing the vials , washing collection tubes with a further 2 mL of 50:50 reaction to warm to r.t. overnight. Monitoring of the reaction 20 acetonitrile : 10 mM ammonium acetate . Vials were then using LCMS (method 1 ) showed that the reaction had topped up to 10 mL using pure water, then frozen in the -80 ° proceeded to completion . C. freezer overnight. Vials were then lyophilized to dryness Dichloromethane was removed by gentle rotary evapora against a control vial which contained ammonium acetate tion before injecting the resulting dimethyl acetamide solu only . tion onto a 30 g Biotage® cartridge which had been equili- 25 5 mL of 50:50 acetonitrile : 10 mM ammonium acetate brated into 5 % acetonitrile in water (0.05 % TFA buffer ) was then passed through product containing vials in two 2.5 before the cartridge was eluted with 5-99 % acetonitrile in mL portions into a single pre weighed vial . 5 mL of pure water (0.1 % TFA ) over 40 minutes. water was then added and the mixture was frozen overnight Pure product containing fractions were identified using in the —80 ° C. freezer and lyophilized to dryness to give the LCMS and transferred into individual 50 mL freeze dry vials 30 pure product as a free flowing white powder (8.7 mg, rinsing the collection tubes with 2 mL 50:50 acetonitrile : 2.24x10-3 mmol, 60 % yield ) . water (0.1 % TFA ) before filling to 25 mL with pure water to Synthesis ensure an organic content less than 30 % . 166 mg Scale Up Batch Vials were then frozen in the -80 ° C. freezer overnight A 7 mL screw top vial which contained stock peptide before being dried by lyophilization . Pure product contain- 35 solution 1 (32 mm , 1.34 mL in DMA , 42.9 umol, 114 mg) ing vials were then combined by passing 2x5 mL of 50:50 was purged using a nitrogen balloon . DIPEA ( 37.4 uL , 27.1 acetonitrile : water (0.1 % TFA ) through all vials into a single mg, 0.22 mmol , 5 eqv. ) was then added and the reaction was pre weighed vial . The vial was then topped up to 25 mL with stirred at r.t. for 10 minutes. NHS activated VcMMAE pure water to give an organic content less than 30 % before glutarate ( 85.9 mg, 64.4 umol, 1.5 eqv. ) in DMA ( 1.85 mL , freezing at -80 ° C. overnight and drying by lyophilization to 40 34.8 mM solution , 60 % pure ) was then added and the give 60 % pure material as a free flowing off white solid ( 118 reaction was stirred under a positive nitrogen atmosphere mg, 0.089 mmol, 80 % yield ). overnight at r.t. Analysis of the reaction showed a similar On freeze drying the ice cake melted on a number of profile to the 10 mg trial reaction with a trace of peptide occasions a trace of the pool sample is shown below and remaining . The crude reaction mixture was purified by indicates that degradation to the acid has taken place during 45 reverse phase preparative chromatography (8x20 mg passes the problematic lyophilization . on the crude material plus 3 re -passed impure material ) Bicyclic Peptide 1 using the preparative method shown in section 3.2.2 . Prod 5 uL of the peptide stock solution was added to 195 uL of uct containing collection tubes were transferred to individual DMA . The solution was then vortexed and split into two 100 50 mL lyophilization vials washing collection tubes with a ?L HPLC samples. The samples were analyzed with a 5 uL 50 further 4 mL of 50:50 acetonitrile : 10 mM ammonium injection on separate machines fitted with ACE 3 and xselect acetate . Vials were then topped up to a volume of 25 mL columns with 10 mM NH_Ac as mobile phase in each case . using pure water, then frozen in the -80 ° C. freezer over Analysis of the peptide by LCMS confirmed its purity and night. Vials were then lyophilized to dryness against a identity . control vial which contained ammonium acetate only . Trial Reaction Analysis 55 10 mL of 50:50 acetonitrile : 10 mM ammonium acetate A 10 mg scale trial reaction between the peptide and NHS was then passed through product containing vials in two 5 activated VcMMAE glutarate was carried out to allow for mL portions into a single pre weighed 50 mL lyophilization analytical method development. vial . 15 mL of pure water was then added and the mixture Stock peptide 1 (32 mm , 116.8 ul in DMA, 3.76 umol) was frozen overnight in the -80 ° C. freezer and lyophilized was transferred to a 1.5 mL vial ( septum cap ) which had 60 to dryness to give the pure product as a free flowing white been purged using a nitrogen balloon . DIPEA ( 3.33 uL , 2.43 powder ( 122.1 mg, by weight ) . The material was taken back mg, 18.8 umol , 5 eqv. ) was then added and the solution was up into 50:50 acetonitrile : 10 mM ammonium acetate (24.42 stirred at r.t. for 10 minutes before adding NHS activated mL , 5 mg per mL ) . 1 mL portions of the stock solution were VcMMAE glutarate (7.53 mg, 5.64 uL , 1.5 eqv. ) in DMA divided between 24 clean , dry 10 mL lyophilization vials ( 129.7 uL , 58.02 mM solution ). The reaction was stirred at 65 (pre weighed ) before adding further 50:50 acetonitrile : 10 r.t. under a positive nitrogen atmosphere for 18 hours . A mM ammonium acetate ( 24.42 mL ) to the parent vial and 1 : 100 dilution in 1 : 1 water: acetonitrile was used to produce again portioning between the 10 mL vials in 1 mL portions. US 10,624,968 B2 151 152 3 mL of water was then added to each vial to give a final of peptide . Translation of this method to the Gilson 20:20 5 mL per vial (a single vial was not filled to 5 mL ; this was preparative HPLC system gave reproducible resolution and used as the analysis vial ) . separation All vials were frozen in the —80 ° C. freezer overnight then Yield lyophilized to dryness against a control vial which contained 5 A total of 1x3.4 mg, 3x4.5 mg, 2x4.6 mg, 4x4.8 mg, ammonium acetate only . After the ice cake has disappeared 3x4.9 mg, 5x5.0 mg, 1x5.1 mg, 2x5.2 mg, 2x5.3 mg, 1x5.4 the vials were lyophilized for a further 48 h to give 23 vials mg and 1x5.6 mg product vials were filled . This is a total of containing 5 mg of product (+0.5 mg ) and a single vial 122.1 mgs from 166 mgs achievable from the two starting which contained 3.4 mg. The total yield was 122.1 mg ( 32 conjugations. The overall yield from starting materials to umol, 73 % yield ) of pure product see Table 26 . 10 vialled product is 73 % . 3x4.5 mg, 2x4.6 mg, 4x4.8 mg, 3x4.9 mg, 5x5.0 mg, TABLE 26 1x5.1 mg, 2x5.2 mg, 2x5.3 mg, 1x5.4 mg and 1x5.6 mg Nominal Vials with Fill Volumes and Amounts were isolated . A vial containing 3.4 mg in DMSO ( 5 mm ) 15 was retained for future QC analysis along with a vial Nominal Vial Size Quantity of Vials Fill Volume (mL ) containing 8.7 mg of product from the trial scale . 5 mgs 24 5.00 Part filled 1 4.5 SUMMARY The target purity and yield of I- la was achieved without Product QC Analysis 20 the presence of MMAE derivatives . Preparative HPLC in 10 RP -HPLC and RP -HPLC -MS for I - 1a Purity and Identity mM ammonium acetate (pH 5.7 ) gave the best separation of A single representative nominal 5 mg vial (actual 3.4 mg) compounds, but gave poor product peak shape. HPLC was reconstituted in DMSO ( 175.5 uL ) such that the con analysis in TFA gave superior peak shape and chromatog centration was at 5 mM , 2 uL of this stock solution was 25 raphy quality . added to 198 uL of DMSO and the sample analyzed by RP -HPLC and RP- HPLC -MS with a 5 uL to confirm purity Example 9 and identity respectively . RP - HPLC using method 2 (NH4OAc , method 2) (see Table 23 ) was used initially as Efficacy of 1-1A in MT1 -MMP Expressing NSCLC this method gave clear resolution of the product from 30 PDX Model VcMMAE glutaryl and the VcMMAE glutaryl NHS ester and confirmed that VcMMAE related impurities were not Study Objective present. A TFA method (method 1 , see Table 23 ) was then The objective of this study was evaluate the in vivo used as this method gave the best peak shape compared to anti- tumor efficacy of l - la in the treatment of MT1 -MMP NHOÀc. 35 high expressing LU - 01-0046 PDX model in female Balb / C MS analysis using Method 1 ( TFA ) ( see Table 23 ) showed nude mice . the presence of four major m / z ion series . The [ M ] 2+ series Materials : Animals and Housing Conditions has the expected mass species of 1939.4 ( (2x1939.4 ) Animals 2 = 3876.8 ) . The [ M ] 3+ series also has the expected mass Species : Mus Musculus species of 1293.4 (( 3x1293.4 )–3 = 3877.2 ). Finally , the 40 Strain : Balb / C nude [M ]++ has the mass of 970.2 (( 970.2x4 ) -4- = 3976.8 . The Age : 6-8 weeks 718 Da adduct which is consistent with MMAE is an artefact Sex : female of the MS analysis where its intensity is dependent on Body weight: 18-22 g collision energy . Number of animals : 18 mice . Discussion and Conclusion 45 Housing Conditions Product Quality The mice were kept in individual ventilation cages at The final product is 99.5 % target peptide - VcMMAE constant temperature and humidity with 3 animals in each conjugate with minimal quantities of unknown impurities as cage . shown in Table 27 , below . Temperature : 20-26 ° C. 50 Humidity 40-70 % . TABLE 27 Cages : Made of polycarbonate . The size is 300 mmx180 mmx150 mm . The bedding material is corn cob , which is Final QC analysis using Method 1 ( TFA ) with Hichrom ACE 3 changed twice per week . Diet: Animals had free access to irradiation sterilized dry Ret. Time Width Area Height 55 granule food during the entire study period . Peak No. (min ) (min ) (MAU ) (mAU ) Area % Water : Animals had free access to sterile drinking water. 80749 0.0835 26.30051 5.24981 0.4949 Cage identification : The identification labels for each cage 2 10.273 0.1184 5288.19580 744.12122 99.5051 contained the following information : number of animals , sex , strain , the date received , treatment, study number, group Synthesis 60 number and the starting date of the treatment. The synthesis of I -la required little optimization such that Animal identification : Animals were marked by ear cod the conversion of starting peptide to product was high ing . without the need to add a large excess of VcMMAE glutaryl Tumor Inoculation NHS ester . Each mouse was inoculated subcutaneously at the right Purification 65 flank with LU -01-0046 of tumor fragment ( ~ 30 mm ) for Analytical RP -HPLC resulted in a good separation where tumor development. The treatment was started when the all unwanted impurities were resolved from the product average tumor volume reaches 163 mm3. The test article US 10,624,968 B2 153 154 administration and the animal numbers in each group were Example 10 shown in the experimental design table . Efficacy & Tolerability of l -la in MT1- MMP Observations Low -Expressing NSCLC PDX Model All the procedures related to animal handling, care and the 5 treatment in the study were performed following the guid Study Objective ance of the Association for Assessment and Accreditation of The objective of this study was to evaluate the in vivo Laboratory Animal Care (AAALAC ) . At the time of routine anti - tumor efficacy of l - la in the treatment of MT1- MMP any effects low - expressing LU -01-0486 PDX model in female Balb / C monitoring, the animals were daily checked for nude mice of tumor growth and treatments on normal behavior such as 10 Materials : Animals and Housing Conditions mobility , food and water consumption (by looking only ) , Animals body weight gain / loss (body weights were measured twice Species: Mus Musculus. weekly ) , eye /hair ting and any other abnormal effect as Strain : Balb / C nude . stated in the protocol. Death and observed clinical signs Age : 6-8 weeks. were recorded on the basis of the numbers of animals within 15 Sex : female . each subset. Body weight: 18-22 g . Number of animals : 18 mice . Tumor Measurements and the Endpoints Housing Conditions The major endpoint was to see if the tumor growth could The mice were kept in individual ventilation cages at be delayed or mice could be cured . Tumor size was mea- 20 constant temperature and humidity with 3 animals in each sured two times weekly in two dimensions using a caliper , cage . and the volume was expressed in mm using the formula : Temperature: 20-26 ° C. V = 0.5 axb ? where a and b are the long and short diameters Humidity 40-70 % . of the tumor, respectively . The tumor size was then used for Cages : Made of polycarbonate . The size is 300 mmx180 calculations of T/ C value . The T / C value in percent ) is an 25 mmx150changed twicemm . perThe week bedding . material is corn cob, which is indication of antitumor effectiveness ; T and C are the mean Diet : Animals had free access to irradiation sterilized dry volumes of the treated and control groups , respectively , on granule food during the entire study period . a given day . Water: Animals had free access to sterile drinking water . TGIwas calculated for each group using the formula : TGI 30 Cage identification : The identification labels for each cage ( % ) = [ 1- ( Ti - T0 )/ (Vi - VO ) ] x100 ; Ti is the average tumor contained the following information : number of animals , volume of a treatment group on a given day, TO is the sex , strain , the date received , treatment, study number, group average tumor volume of the treatment group on the day of number and the starting date of the treatment. treatment start, Vi is the average tumor volume of the vehicle ingAnimal. identification : Animals were marked by ear cod control group on the same day with Ti, and VO is the average 35 Experimental Methods and Procedures tumor volume of the vehicle group on the day of treatment Tumor Inoculation start . Each mouse was inoculated subcutaneously at the right flank with LU -01-0486 of tumor fragment (30 mm ) for Sample Collection tumor development. The treatments was started when the Mice in group 2 was re -dosed and plasma was collected 40 average tumor volume reaches 164 mm ”. at 5 min , 15 min , 30 min , 60 min and 120 min on day 56 . Observations All the procedures related to animal handling , care and the The tumor samples were collected from group 2 and fixed treatment in the study were performed following the guid in 10 % formalin , then embedded in paraffin and stored at ance of the Association for Assessment and Accreditation of ambient temperature . Laboratory Animal Care (AAALAC ) . At the time of routine 45 monitoring , the animals were daily checked for any effects Statistical Analysis of tumor growth and treatments on normal behavior such as Summary statistics , including mean and the standard error mobility , food and water consumption ( by looking only ) , of the mean (SEM ) , are provided for the tumor volume of body weight gain / loss (body weights were measured twice each group at each time point . weekly ), eye / hair matting and any other abnormal effect as 50 stated in the protocol. Death and observed clinical signs Statistical analysis of difference in tumor volume among were recorded on the basis of the numbers of animals within the groups was conducted on the data obtained at the best each subset. therapeutic time point after the final dose . Tumor Measurements and the Endpoints A one - way ANOVA was performed to compare tumor The major endpoint was to see if the tumor growth could volume among groups, and when a significant F - statistics ( a 55 besured delayed two times or mice weekly could in betwo cured dimensions . Tumor usingsize wasa caliper mea, ratio of treatment variance to the error variance ) was and the volume was expressed in mm using the formula : obtained , comparisons between groups were carried out with V = 0.5 axb2 where a and b are the long and short diameters Games -Howell test . All data were analyzed using Prism . of the tumor, respectively . The tumor size was then used for P < 0.05 was considered to be statistically significant. calculations of T / C value . The T / C value ( in percent) is an As shown in FIG . 6 , the mean tumor size of vehicle- 60 indication of antitumor effectiveness ; T and C are the mean volumes of the treated and control groups, respectively , on treated animals reached 2304 mm on day 28, mice treated a given day. with l- la at 1 mg/ kg showed significant growth inhibition , TGI was calculated for each group using the formula : TGI comparable to that seen with the clinically used agent ( % ) = [ 1-( Ti - T0 ) / (Vi - VO ) ]x100 ; Ti is the average tumor Docetaxel. Mice treated with 1-1a at 3 or 10 mg/ kg showed 65 volume of a treatment group on a given day, TO is the significant growth inhibition , greater than that seen with average tumor volume of the treatment group on the day of docetaxel. treatmentstart , Vi is the average tumor volumeof the vehicle US 10,624,968 B2 155 156 control group on the same day with Ti, and VO is the average Separation column: Luna 200 * 25 mm 10 um , C18 , 110 A tumor volume of the vehicle group on the day of treatment and Gemin150 * 30 mm , C18 , 5 um , start . 110 A , connection , 50 ° C. Dissolve method : DMF Sample Collection 5 Separation purity : 95 % Mice in group 2 , 3 were re -dosed and plasma was ( 2 ) The Reaction Scheme of 1-12 , 1-15 , 1-16 and 1-17 is collected at 5 min , 15 min , 30 min , 60 min and 120 min on Shown in FIG . 13 . day 24 . The tumor samples were collected and fixed in 10 % formalin , then embedded in paraffin and stored at ambient 10 General procedure for preparation of compound 3 temperature before sending to client. Statistical Analysis ???. H2N . Summary statistics , including mean and the standard error OH of the mean (SEM ), are provided for the tumor volume of 15 OH each group at each time point. Statistical analysis of difference in tumor volume among NH the groups was conducted on the data obtained at the best 20 NH2 therapeutic time point after the final dose . 2 A one -way ANOVA was performed to compare tumor OH volume among groups , and when a significant F - statistics ( a ratio of treatment variance to the error variance ) was Boc obtained , comparisons between groups were carried out with 25 Games -Howell test. All data were analyzed using Prism . P < 0.05 was considered to be statistically significant. As shown in FIGS . 7 and 8 , the mean tumor size of NH vehicle - treated animals reached 1201 mm on day 24 , mice 30 treated with the clinically used agent Docetaxel showed significant inhibition of tumor growth , but with severe NH2 weight loss, leading to the humane sacrifice of animals at day 24. Mice dosed with I - 1a at 3 mg/ kg showed greater inhibition of tumor growth without significant effect on body 35 To a solution of Compound 2 ( 7.00 g, 18.70 mmol, 1.00 weight. Mice dosed with 1-1a at 1 mg/ kg showed limited eq ) in DCM (80.00 mL ) and MeOH (40.00 mL ) was added inhibition of tumor growth and no significant effect on body ( 4 - aminophenyl) methanol (2.53 g , 20.56 mmol, 1.10 eq ) and weight. EEDQ ( 9.25 g , 37.39 mmol, 2.00 eq) in the dark . And the mixture was stirred at 25 ° C. for 8 hr. LC -MS showed Example 11 40 Compound 2 was consumed completely and one main peak with desired MS was detected . The resulting reaction mix Synthesis of I - 10 to 1-17 ture was concentrated under reduced pressure to remove the Exemplary synthesis methods are described below . solvent to give a residue . The residue was purified by flash ( 1 ) Separation : silica gel chromatography (ISCO® ; 120 g SepaFlash® Separation Condition : A phase : 0.075 % TFA in H2O , B 45 Silica Flash Column, Eluent of 0-10 % MeOH / DCM @ 85 phase : MeCN mL /min ). Compound 3 ( 7.00 g , 14.60 mmol, 78.06 % yield ) Separation method : 18-48-55 min , RT = 53.5 min was obtained as a white solid .
General procedure for preparation of Compound 4
OH NO2 Boc syid
NH
NH2 US 10.624,968 B2 157 158 -continued NO2
Boc IZ
NH
NH2 4
To a solution of Compound 3 (4.00 g , 8.34 mmol, 1.00 eq ) A mixture of Compound 4 ( 1.10 g, 1.71 mmol, 1.00 eq ) and 4 -nitrophenyl carbonochloridate (6.72 g , 33.36 mmol, 20 and DIEA (2.21 g , 17.06 mmol, 2.98 mL , 10.00 eq ) in DMF 4.00 eq ) in THF ( 20.00 mL ) and DCM (10.00 mL ) was ( 10.00 mL ) was stirred under nitrogen at 0 ° C. for 30 mins . added PYRIDINE (2.64 g , 33.36 mmol, 2.69 mL , 4.00 eq ) . MMAE (900.00 mg, 1.25 mmol, 0.73 eq ) and HOBt (230.56 And the reaction mixture was stirred at 25 ° C. for 5 hr. mg, 1.71 mmol, 1.00 eq ) was added to the mixture and the LC -MS showed Compound 3 was consumed completely and 25 resulting reaction mixture was stirred under nitrogen at 0 ° C. one main peak with desired MS was detected . The reaction for 10 min and at 30 ° C. for additional 18 hr. LC -MS showed mixture was concentrated under reduced pressure to give a Compound 4 was consumed completely and one main peak residue, which was purified by flash silica gel chromatog with desired MS was detected . The reaction mixture was raphy (ISCO® ; 120 g SepaFlash® Silica Flash Column, purified by flash C18 gel chromatography ( ISCO® ; 120 g Eluent of 0-20 % DM /MeOH @ 85 mL/ min ) . Compound 4 30 SepaFlash® C18 Flash Column, Eluent of 0-50 % MeCN / ( 2.20 g , 3.41 mmol, 40.92 % yield ) was obtained as a white H20 @ 85 mL /min ). Compound 5 (850.00 mg ,694.71 umol , solid . 40.63 % yield ) was obtained as a white solid .
General procedure for preparation of Compound 5 NO2
Boc MMAE
que?? NH2
Boc
HN
H2N ortable 5 US 10,624,968 B2 159 160 General procedure for preparation of Compound 6 OH
Boc TFA aly K2CO3 HN H2N 5
OH N
NH2
HN
H2N 6 ???? 40 To a solution of Compound 5 (850.00 mg, 694.71 umol, stirred at 25 ° C. for 12 hr. LC -MS showed Compound 5 was 1.00 eq ) in DCM (36.00 mL ) was added TFA ( 12.18 g , consumed completely and one main peak with desired MS 106.80 mmol, 7.91 mL , 153.73 eq ) and the mixture was was detected . The resulting reaction mixture was purified by stirred at room temperature for 2 hr. The mixture was flash C18 gel chromatography (ISCO® ; 120 g SepaFlash® concentrated under reduced pressure to give a residue, which 45 C18 Flash Column, Eluent of 0-50 % MeCN / H , 0 @ 85 was dissolved in THF ( 10.00 mL ), and K2CO3 ( 2.40 g , 17.37 mL/ min ). Compound 6 ( 560.00 mg, 498.48 umol, 71.75 % mmol, 25.00 eq) was added to the mixture. The reaction was yield ) was obtained as a white solid .
General procedure for preparation of Compound 7
osssee mahaHN .H2N US 10,624,968 B2 161 162 oinson -continuedhaythe HN H2N 7
A 50 mL round flask containing Compound 7 (400.00 mg, 356.06 umol, 1.00 eq ) was purged using a nitrogen balloon . 10 mL of anhydrous DMA was added with stirring and the solution was cooled to 0 ° C. in an ice water bath . DIEA 20 ( 92.03 mg, 712.11 umol, 124.37 uL , 2.00 eq ) was then added as a stock solution in anhydrous DMA and the reaction was stirred at 0 ° C. for 10 min . Tetrahydropyran - 2,6 -dione (81.25 mg, 712.11 umol, 2.00 eq ) was added as a solution in DMA . The ice bath was then removed and the reaction was 25 stirred at room temperature over the course of 1 hr. LC -MS showed Compound 6 was consumed completely and one main peak with desired MS was detected . The reaction mixture was purified by flash C18 gel chromatography ( ISCO® ; 120 g SepaFlash® C18 Flash Column , Eluent of 30 0-50 % MeCN /H20 @ 85 mL/ min ). Compound 7 (300.00 mg, 242.42 umol, 68.08 % yield ) was obtainde as a white solid .
General procedure for preparation of Compound 8 OH OH ????1
HN H2N the 7
H2N otros material8 thang mer