(12) United States Patent (10) Patent No.: US 9,340,532 B2 Kirkpatrick Et Al

USOO9340532B2

(12) United States Patent (10) Patent No.: US 9,340,532 B2 Kirkpatrick et al. (45) Date of Patent: May 17, 2016

(54) METHODS AND COMPOSITIONS FOR 2007/0213378 A1 9, 2007 Thomas et al. INHIBITING CNKSR1 2007/0293516 A1 12/2007 Knight et al. 2011/0144.066 A1 6/2011 Mahadevan et al. 2012fO189670 A1 7/2012 Kirkpatricket al. (71) Applicant: PHUSIS THERAPEUTICS, INC., San 2013, O184317 A1 7/2013 Mahadevan et al. Diego, CA (US) 2015, 0126563 A1 5, 2015 Mahadevan et al.

(72) Inventors: D. Lynn Kirkpatrick, San Diego, CA FOREIGN PATENT DOCUMENTS (US); Martin Indarte, San Diego, CA (US); Nathan T. Ihle, San Diego, CA CA 77.5563 A 1, 1968 DE 2556O11 A1 6, 1977 (US) DE 3443225 A1 6, 1985 EP 2428504 A1 3, 2012 (73) Assignee: Phusis Therapeutics, Inc., San Diego, EP 2477625 T 2012 CA (US) GB 828.963. A 2, 1960 WO WO90, 15600 A2 12, 1990 (*) Notice: Subject to any disclaimer, the term of this WO WOOOf 18376 A1 4/2000 WO WOO2,083.064 A2 10, 2002 patent is extended or adjusted under 35 WO WOO3,O76436 A 9, 2003 U.S.C. 154(b) by 0 days. WO WOO3,O84473 A2 10, 2003 WO WO 2005/090461 A2 9, 2004 (21) Appl. No.: 14/649,349 WO WO 2005/OOO862. A 1, 2005 WO WO 2005.005421 A1 1, 2005 (22) PCT Filed: Dec. 16, 2013 WO WO 2005/097758 A 10/2005 WO WO 2006/046914 A1 5, 2006 WO WO 2007/039.173 A1 4/2007 (86). PCT No.: PCT/US2013/075.505 WO WO 2008/083158 A2 T 2008 WO WO 2009/129267 A2 10/2009 S371 (c)(1), WO WO 2011/032169 A2 3, 2011 (2) Date: Jun. 3, 2015 WO WO 2014/093988 A2 6, 2014

(87) PCT Pub. No.: WO2014/093988 OTHER PUBLICATIONS PCT Pub. Date: Jun. 19, 2014 PubChem Compound (http://pubchem.ncbi.nlm.nih.gov/) CID (65) Prior Publication Data 24283805 (Feb. 29, 2008). PubChem Compound (http://pubchem.ncbi.nlm.nih.gov/) CID US 2015/0307482 A1 Oct. 29, 2015 3800302 (Feb. 29, 2008). PubChem Compound (http://pubchem.ncbi.nlm.nih.gov/) CID 3268165 (Feb. 29, 2008). Related U.S. Application Data Anwar et al. “Reactions of some 5-aryl-2-thiono-1, 3, 4-thiadia (60) Provisional application No. 61/737,658, filed on Dec. Zoles' Jan. 1, 1981, Romanian Journal of Chemistry 26(8): 1127 14, 2012. 1134. Carpten et al. "A Transforming Mutation in the Pleckstrin Homology (51) Int. Cl. Domain of AKT1 in Cancer Jul 26, 2007, Nature 448(7152):439 CO7D 409/4 (2006.01) 444. Castillo et al. “Preferential Inhibition of Akt and Killing of Akt CO7D 417/4 (2006.01) dependent Cancer Cells by Rationally Designed CO7D 409/2 (2006.01) Phosphatidylinositol Ether Lipid Analogues' Apr. 15, 2004, Cancer A6 IK3I/502 (2006.01) Res. 64(8):2782-92. (52) U.S. Cl. Catley et al. “Alkyl Phospholipids Perifosine Induces Myeloid CPC ...... C07D 409/14 (2013.01); C07D409/12 Hyperplasia in a Murin Myeloma Model” Jul. 2007. Exp. Hematol. (2013.01); C07D 417/14 (2013.01) 35(7):1038-1046 (abstract). (58) Field of Classification Search Chemical Abstract Database compound (CAS RN 1022250-16-3) CPC. C07D 409/14: CO7D 417/14: CO7D 409/12: entering date May 25, 2008. A61 K31/502 Chemical Abstract Database compound (CAS RN 1022557-01-2) USPC ...... 544/327 entering date May 26, 2008. See application file for complete search history. (Continued) (56) References Cited Primary Examiner — Yevegeny Valenrod U.S. PATENT DOCUMENTS (74) Attorney, Agent, or Firm — Pepper Hamilton LLP 3,821,222 A 6, 1974 Moore 4,017,489 A 4, 1977 Lawrence (57) ABSTRACT 4,251,528 A 2f1981 Brittain et al. 4,694,015 A 9, 1987 Sebille et al. 4,939,140 A 7, 1990 Larson et al. Embodiments include compositions and methods of inhibit 6,066,311 A 5, 2000 Cheetham et al. ing CNKSR1 and methods of identifying inhibitors of 6,924,284 B2 * 8/2005 Beaton ...... A61K 41 0038 CNKSR1. 514,234.5 2004/0092524 A1 5, 2004 Perez et al. 2004.0102360 A1 5, 2004 Barnett et al. 19 Claims, 19 Drawing Sheets US 9,340,532 B2 Page 2

(56) References Cited Li"Recent Progress in the Discovery of Akt Inhibitors as Anticancer Agents' 2007, Expert Opin. Ther: Patents 17: 1077-1130. OTHER PUBLICATIONS Mahadevan et al. “Discovery of a novel class of AKT pleckstrin homology domain inhibitors' Sep. 2008, Mol. Cancer Ther: 7(9): 2621-2632. Chemical Abstract Database: compound (CAS RN 477482-99-8), Mahieu et al. “Synthesis of new thiosulfonates and disulfides from entering date Dec. 12, 2002. Sulfonyl chlorides and thiols' Jan. 1, 1986, Synthetic Communica Chemical Abstract Database: compound (CAS RN 477483-04-8), tions 16(13): 1709-1722. entering date Dec. 12, 2002. Meuillet et al. “In Vivo Molecular Pharmacology and Antitumor Chemical Abstract Database: compound (CAS RN 919458-54-1), Activity of the Targeted Akt Inhibitor PX-316' 2004. Oncol. Res. entering date Feb. 6, 2007. 14(10):513-527 (abstract). 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Du-Cuny et al. “Computational modeling of novel inhibitors targtet Office Action issued in Australian Application No. 2009236256, ing the Akt pleckstrinhomology domain Aug. 19, 2009, Bioorganic mailed May 2, 2013. & Medicinal Chemistry 17(19):6983-6992. Office Action issued in Australian Application No. 2009236256, European Search Report and Written Opinion dated Feb. 2, 2012 for mailed Jun. 11, 2014. EP 11181871. Office Action issued in U.S. Appl. No. 12/937,898, mailed Jun. 18. Feldman et al. “Novel Small Molecule Inhibitors of 2012. 3'-phosphoinositide-dependent Kinsasel (PDK-1)” Sep. 30, 2004, Office Action issued in U.S. Appl. No. 12/937,898, mailed Aug. 30. Eur: J. Cancer Supp. 2(249):77. 2012. Fengl. "Enteric Coating” Oct. 22, 2002, Enerex.ca 5 pages. Office Action issued in U.S. Appl. No. 13/789,209 dated Jun. 25. Gills et al. “Spectrum of Activity and Molecular Correlates of 2014. 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Rela 1365. tionship to nicotinamide antagonism' 1962, Cancer Res. 22:483 Powell etal. “Bile Acid Hydrophobicity is Correlated with Induction 55O. of Apoptosis and/or Growth Arrest in HCT116 Cells' 2001, Biochem International Search Report and Written Opinion dated Jun. 3, 2014 I 356:481-486. for PCT/US 2013,755.05. Rungeetal. “Ubereinige unsymmetrishce heterocyclische Disulfide, International Search Report and Written Opinion dated May 26, 2011 II' Jul. 1, 1963, J. Fuer Praktische Chemie 21(1-2):39-49 (cited in for PCT/US2O10,048813. German? no translation available). International Search Report and Written Opinion dated Oct. 19, 2009 Sassiver et al. “2-Sulfanilamido-5-methoxy-, 1.3,4-thiadiazole and for PCT/US 2009/040575. related compounds' 1966, J. Med. Chem. 9(4):541-545. International Search Report dated Dec. 15, 2009 for PCT/US2009/ Stein et al. “Discovery and structure of activity relationships of 040575. Sulfonamide ETA-selective antagonists' 1995, J. Med. Chem. Kim et al. “Targeting the Phosphatidylinositol-3 Kinase/Akt Path 38:1344-1354. way for the Treatment of Cancer” Dec. 2005, Curr: Opin. Investig. Supplementary European Search Report and Written Opinion dated Drugs 6(12): 1250-1258. Sep. 17, 2012 for EP 10816289. Komander et al. "Structural Insights into the Regulation of Pdk1 by Thomas et al. “High-resolution Structure of Pleckstrin Homology Phosphoinositides and Inositol Phosphates' 2004, EMBO J. Domain of Protein Kinase Blakt Bound to Phosphatidylinositol 23(20):3918-3928. (3,4,5)-trisphosphate” Jul. 2002, Curr. Biol. 12(14): 1256-1262. Kumar et al. “AKT Crystal Structure and AKT-specific Inhibitors' Nov. 2005. Oncogene 24(50):7493-75.01. * cited by examiner U.S. Patent May 17, 2016 Sheet 1 of 19 US 9,340,532 B2

A R C

HRAS in-KRAS48 NRAS KRAS4A

: S

fafnesy gamitate

Figtre : &tie of CN KSR is a t-KRAS sigraaiiig. RAS indergo C-terminai CAAX farthesyiation (or geranylgeranylation followed by Rice if CMi processing. A HiRAS, NRAS and KRAS4A indergo hype variabie thv} donai pain it Oyiatio and Gigi ocessing ieading to their iipid raft membrane iocaiization. B, KRAS4B does not undergo Gogi processing and its polybasic hv donnais bids to men base is aid PS in specific lipid Fafts. C. We propose that mut KRAS but bot wit-KRAS associates in a utique signaling aaoister with the Phi donai; containing protein CN KSR to bind to iii.2/3 rich membrane lipid rafis necessary for mut KRAS signating U.S. Patent May 17, 2016 Sheet 2 of 19 US 9,340,532 B2

s 2 MiaFaca-2 20 wt. KRAS rt-KrAS 4 H1373 mut-KRAS ; : 00 Weic g 8 O c st 60 s : 4 s 3. ; 2 s: CNKSR1 N O S 80 tra ag- six S e 2 w 2 if 5:my 4. in 80 g 60 s s: 4 2 20 s

is c. e ses : ; N 3 S& S. 5 3S g is2 &3 S S R5 ge. S . . . . . < ......

Figure 2 CN KSR as a target for inhibition of it-KRAS ce: growth. A., Validation ising CNKSR siRNA in Mia Patia-2 and HC - 6 isogenic wi- and aut-KRAS ceii ishes. Fified boxes are wi-KRAS and open b{xes aut-KRAS. Waities are fears of 3 tietetiinatins arid bars are S.E. * }<0.05. B.CNKSR 1 siRNA in a panel of NSCLC celi fines with fied boxes showing wi-RRAS, and opei boxes inui-KRAS ceilis. Vaities are expressed as a % relative to scrambled siRNA control. Bat's are SE. p<0.05 conpared to scrambled siRNA. C. Growth of H 373 aut-KRAS NSCLC ceil line stably transfected with closed boxes) vectorage or with (pea boxes) a CNSKR PH domai) construct that acts as a dotiinant negative inhibitor of ceili growth. Bars are S.E. ** p <},{}}. U.S. Patent May 17, 2016 Sheet 3 of 19 US 9,340,532 B2

0.7 x 0.2 - 3: 30.3 : 12 - 3.3:0. C 180

33: 1.5 s6 SS.ge 120 i-sikFASy-sicNKSR 5.2, t 108 & corpound it 60 20.2 : 0.8 2C 24, 48 72 Fire ()

83. Compound 7 (iii) C-Rai (Ser 338)

Figi'e 3 Risit is fiti i is air : CNSR iii. SR-KRAS the Wi-SRAS ce. grew it. A, inhibitors of the CN KSR PH domain predicted by the PHui ack(S. program bind to the expressert PH is main of CN KSR with Kit measured by plasmon spin fesonance binding to the expressed Pii doinain of CN KSR. Aisi shown is the calculated Gold score aid og F. B: Cei growth iahibition in wild type aid at-KRAS NSCLC celis by compounds 4 and 7 expressed as Csos; C, inhibition of A-549 NSCLC ce: growth by siRNA to KRAS, CN KSRi and 25 M compnd 7. ), Western blot showing the inhibition of dow: stream KRAS signating measured by p-C-RAF(Ser'), by compound 7. U.S. Patent May 17, 2016 Sheet 4 of 19 US 9,340,532 B2

7 g 11 13 15 17 19 21 23 25 27 2g E. days after CEE injection

Rose Campol ind 7 . 8 coinpourid 7 5. C coin pound 8 J lose COingourd 8 5 80 W compound 8 t is 60 8 ct; 4G

c. 20

O sessessessessees { 30 4. SO time (h)

Figre 4 Antii is activity at tha rackii etics {fice it is 7. A, Aitia; no activity of compound 7 administercd by gavage at 2GO agkg in {}. ini abrafii R: fabrasoi R (8:2) (Gatt.cfesse daily for 2 days show a by horizontai bar) aid criotii by gavage at {} ing/kg in G. i ni 1.2% Tween 23 daily for 20 days to female scid mice with a subcutaneous H 2:22 naut-KRAS timors. reatments were:(8) vehicle aigne: {C} c{m}{} End 7: {{a} erotinib, and {A} erotinib and compound 7. There were {} ice her group and bars are S.E. 8, Farmacokinetics of conpound 7 and compound 8 (die esterified acidic form of compound 7) in female C57B6 mice administered a single dose of conpound 7 at 25{} ag/kg 7 by gavage in {}.2 in abrafii R. ElabrasotR (8:2). (8) parent compound and (Q) acid metabolite. The acid metabolite compound S was also administered at 250 mg/kg by gavage in 0.2 mi Labrafii R: ilabrasol R (8:2) with (Y) showing its plasma leveis . There were 3 nice in each group and bai's are S.E. insert shows striciaires of compound 7 and its acii retabolite compound 8. U.S. Patent May 17, 2016 Sheet 5 Of 19 US 9,340,532 B2

Horships | CH7|H|2|'s i -

7 -s-s r- M9. >HM 0() >aM 0 gM5} :27 M FM7.

1. 52 4S 38 > 100 37 S v - " " a M M : M M FM

10 r c SV Ass, 76 HM >00 46 49 26 28 Yea l N- is -( r y an M M M M M 1 Sh; W 7 3-y i

s S6 > {}{ 3 s-s s- sy 48 - M 44 4. 56 9. 34.

> {{ i s ()() > {{ i is {}{} > (){} > (O -ss-Sr. S. c:- : M ...M i aM a M. M

Activity of cage is 7 analogs agaiast wi-KRAS as nata-RAS NSCC ceilises. Walaes are Cso for ceili growth inhibition (3 day assay), Compound 2 is a different pharmacophore U.S. Patent May 17, 2016 Sheet 6 of 19 US 9,340,532 B2

S y x & M

Figure 6. The X-Ray stricture of inositoi tetraphosphate {white sticks) overiaid with conpound 2 (green sticks). A similarity of functional groups, their orientatio; and overal shape can be seen. The moiecular surface used to retain similar computids has been aion) coiored based of ciectrostatics and hydrophobicity. U.S. Patent May 17, 2016 Sheet 7 Of 19 US 9,340,532 B2

S.M. YSS

SigE rei: Selection of the best CNKR medici to use for virtual screening. PH domain X-Ray structures with the highest siniiarity (bite ribbons) are superposed with the CNKR modici (green ribb{ns) and its tenariate X-Ray (J5F, white ribbons) with best scores for compounds 7 (white bail and sticks), 8 (green baii and sticks) and 2 (yellow bai and stickS}; co-crystai Substates {f X-ray structures are residered as white sticks and depictesi in their coTesponding binding sites.

E. :'''Fait S Y Exchanged fragments & S.S.

Figare 8. Schematic view of the BREED process. Active ligands are disconnected in snailief portions to be late recombined, giving birth to love igands with active fictionai g!'gaps aid scaffoids, U.S. Patent May 17, 2016 Sheet 8 of 19 US 9,340,532 B2

inhibition of 3D growth by sikkas and siCNKSR

6 na K E. 4 s es s S 2 "S

foras -Kasi wi-tas a t-Siras SSc" s: KS Figure 9 CNRSRi is secessary for Rani&AS an eitrage iniepeatient ceii growth FiCT-if colon cance ceilis (aitant-KR as i2)) (Mi-K Ras) and the same ceilis with tnutant-KRAS renovcd by honologous recombination caving at alicic of wild typc-KRas (wt-KRas were Esed for the study, siCNKSR: or siscrambied siRNA as a contri was everse transfected into the ecils 24 hr before piatig. The ce: nabe was optimized for plating for the best ceil density and found to be 2,000 ceils be ini. The id was emoved from a 96-we?t Greiter plate and turned upside down. 20 of the 20,000 ce} is per nai suspensiji was then added directly into the niddie of the circles found on the iid of the 96 wei piate forming a smaii drop. (30 of media was added into the correspoiding weiis, used is naintain the tenperatire if the drops, and the id was flipped back over carefity placing it back onto the late without disturbing the drop. The piate was the placed into the icubato for 3 tiays to a low the ceils to nigrate to the b{tion of the dip due to gravity. After 3 days, 4{{} all of media was added to the corresponding weis a SC VAX 96-wei piate. The lid from the Grcier 96-wei piate was removed and placed onto the SCEVAX 22 plate allowing the drop to coin}e in contact with the nacdia and placed back it to the incubator, After one hair, 20 i (frnetia was removed frt in the corresponding Weis carefit iiy without disturbing the spheroid aidi imaged using an N is A is S$

U.S. Patent May 17, 2016 Sheet 9 Of 19 US 9,340,532 B2

EK wt-Kas

EK fut-KaS (G13D)

Figars {} CNRSR gree caicaiacs with Ratai-K Ras resi at the pias a test rate. HEK-293 ceilis wore transfectcd with CN KSR -(GFP and nut-K Ras(G 3D} for 6 ar. two photon confocal microscopy shows that CNKSR is located at the piasia nienbraic and the cytopiasin in both wi-KRas and nut-RRas ccs. KRas tends to be fore meinbrane associated When ceii the images are merged CNKSR and wi-KRas can be seen to be colocalized (within 500 him) shown by the yelio wif orange color. Mut-K Ras cCiocalization is also seen but is more diffuse. Note the transformed phenotype of the inui-KRas ceilis. U.S. Patent May 17, 2016 Sheet 10 of 19 US 9,340,532 B2

K wi-Kras

iiietirix Six S388

EK rt-Kras

Figre Feresce ceifetire is a gig Bricrescopy {FE. Iv} showing that CN KSR hit is directiy to Rai-K&as at Bot to w-. Ras is ceilis. HER-293 ceis were transfected with CN KSR --GFP and mut-K Ras (G 3 D and 6 hr later FEM experiments were carried oit using a leica TCP SP5 inverted advanced coffocal nicroscope systein with intertai photomultiplier tube (FMT) detector for TCSPC (time-correiated single-photon counting). The samplc was cxcited with a tu nabic ficantosecond {fs) titanium-sapphire iaser with repetition rate of 8{}M is, and pulse width iess then 8{}is (Spectral Physics, Mai Tai BB). The wavelength used for two-photon excitation was 930 in and the fluorescence was detected through a 525-25 in interference filter, inhages were obtained with oil-in incision objective hutnerica; aperture NA-i.4), a iiie Scai speed of 400 Hz, with image size of 52x5: 2 pixels. For FiliM analysis the pixels were reduced to 256x256. Fi M data was collected using Becker & Hicki SPC830 data and image acquisition card for TCSPC. The fluorescence decays were fitted with a singie exposentiai decay nocici using Becker and 24 iick's SPCinage software aid the GFF fiorescence lifetimes were calculated. The ce: images in the left paiei are two typical inhages faise color for wi-KRas and hut-KRas ceils and the fuorescence ifetimes shown in the fight are for the entire ceili measured by FM. The results show a decrease in fit Forescence iifetime in the right pane caused by when it CN KSRi binds directly (i.e with a localization < 0 in) is trut-KRas bit it to wi-KRas. U.S. Patent May 17, 2016 Sheet 11 of 19 US 9,340,532 B2

(CNK KRAS GO GFP RF- merged

OWSO

Compound 7 50M

Compound 7.3 50w

Figare 2 Casa is 7 anti 9 aka 7.3 bick the Beiraise ciocaiisation if CN KSR (gree and a taat-KRas (rei) at the pias a Renahrase, and we' tetai Ratajat KR;is rteia is the cei. HEK cei is were transfected with CN KSR-GFP and nut KRas(G 3 E3) and treated for 4 hr with vehicle (EDMSO), 50 M compound 7 or 50 M compound 9. The results show colocalization (yellow/orange i.e. withi; 500 in) of CNKSR and mut-K Rasat the piasma membrane in untreated ceilis and a toss of this coica iisation in compound 7 and 9 treated ceilis. Also apparent caused by compound 7 aid 9 is a decrease in total aut-KRas protein. Figures are typical of 3 deteriniations. U.S. Patent May 17, 2016 Sheet 12 of 19 US 9,340,532 B2

DVSC

Compound 7 50M

Compound 7.3 50M

ens) 23 2: Figre 3 F.M. stadies siewing c3:43 asids 7 and 9 {sak: 7.5 inhibit the sireet is isg of {CNKSR: A Bit-RAS if seis. HEK-293 ceilis were transfected with CN KSR-GF aidi nut-K Ras (G13D) for 6 hr and then treated for 4 hr with vehicle (DMSO), 50 Mi compound 7 or 5 ( ; M coinpauni 9. The ceilinages are false coio inages with the fluorescence ifetities siwi or the right are for the entire ceili measured by F.M. The Fesuits six w that compounds 7 and 9 block the direct binding (i.e. < |{}0 nm) of CN KSR and mut-K Ras with a right was shift in the fiorescence lifetime, similar to that sect in wi-KR as cells where {CNRSR dies act bind is wi-SRas, U.S. Patent May 17, 2016 Sheet 13 Of 19 US 9,340,532 B2

2. W. KRAS oo: 8 FF is S assassi Ras S. yttant KRas

i. :

Figare 4A CNKSR siRNA is ribits growth in Arg ea cer cei is with Raiated KRas. CN KSR siRNA in a panci of NSCC cells. Ceis were treated with either on targeting control siRNA or si{CNKSR. Results are displayed as cells treated with siCNKSR divided by cci is treated with non-targeting controi. {X}{}}}

8S8.

K3S 8-8-88 S8 $38; is its 38 m ps is 38 x: Active Art 38 88 &SS

Akt Mek RaA RaiB &city: 38

Figi'e 48 CNKSR si3NA iiibits KRas signating effecters Analysis of KRas effectors after siCN KSR treatment. Raf and Akt activation were incasured asig phosphorylation specific antibodies. Rai activation was measured by pui dow; for GTF bound protein. U.S. Patent May 17, 2016 Sheet 14 of 19 US 9,340,532 B2

so if compound (20 M) : S. 3. g. :. 3. p-Akt & Active Akt

Act, if

GTP-RalA, Active Rai

Raii

------aii Ai.

Figre 5 : HT-782 Aiaiegs with increased activity. Activity of PHT-782 (Compound 7) and compouilds 8 (Analog i. 9 (Anaiag2}, {} (Analog 3}, i Ajaig 4). U.S. Patent May 17, 2016 Sheet 15 Of 19 US 9,340,532 B2

3. is 3}{ g s w

8. S 3. St. ine afte: 3 injection viay Figure : {A. initia CNK inhibitor leads demonstrate antitumor activity in A549 naut-KRAS human ton saaceti iting carcer (NSCC) Xenograft in vive antitrimoi activity of CFD 9: nice with AS49 hairman NSCLC Xenografts were dosed daily for 8 days with either vehicle, cpd 9 at 2i}{} nig/kg ip, erotini at 75 mg/kg {} or both 9 ad critinib (show; by bar), Values incan + S.E. U.S. Patent May 17, 2016 Sheet 16 of 19 US 9,340,532 B2

...is

A549 NSCC nut-KRast G12D) O mice? group

25 -

Rays after cei injection Figre 68 Modest antitanoi activity has been observed with initia inhibitors that have shown CNK binding, Compound 9 (aka 7.3) and compound 78 (aka 7.0) were admitistered at 200 ring/kg i.p. to nufu nice baring A549 non Sinai ceili iung carcer Kenografts with fast KRAS{G 21)) funnors. U.S. Patent May 17, 2016 Sheet 17 Of 19 US 9,340,532 B2

SRs 88 R 8 Sciss g 888 as:

38.

FG 7

U.S. Patent May 17, 2016 Sheet 18 of 19 US 9,340,532 B2

sai s s Sessists & S&Sigis'

ig. 8

US 9,340,532 B2 1. 2 METHODS AND COMPOSITIONS FOR Some embodiments provide a compound of formula IA: INHIBITING CNKSR1

Formula IA CLAIM OF PRIORITY R1 This application is a national phase application under 35 S U.S.C. S371 of International Application No. PCT/CN2013/ sN N R V S. 075505, filed Dec. 16, 2013 and entitled “METHODS AND 10 N N COMPOSITIONS FOR INHIBITING CNKSR1 that claims priority to U.S. Provisional Patent Application No. R2 61/737,658, filed Dec. 14, 2012 the disclosures of which are O incorporated by reference in their entirety and for all pur poses. 15 wherein R is —C-C alkyl, —NO. —NH, NHSOCH —C-Cs cylcloalkyl, or—CF; CROSS-REFERENCE R’ is Hor RandR combine as This application claims priority to U.S. Provisional Patent Application No. 61/737,658 entitled Methods and Composi tions for Inhibiting CNKSR1 filed on Dec. 14, 2013.

GOVERNMENT INTEREST 25 The United States government may have certain rights to - this invention pursuant to State of Texas; CPRIT. High Impact Grant, "KRAS the elephant in the room of cancer chemo 30 to form a bicyclic moiety with the carbons to which they are therapy: Number: RP1.00686. attached; R" is C(O), —C(O)C(C-C alkyl), —C(O)OH, - C BACKGROUND Calkyl-OH, -CH(OH)(CH), NH, -CH(OH), C(OH) 35 CNO,-(CH), NO, —CHO,-CO)NHR'. Single point mutations of at least one of the RAS genes (KRAS, HRAS, and NRAS) are found in many human can O / \ N cers, particularly in colon, lung and pancreatic cancer. RAS mutations are most commonly found in KRAS (about 85%), 40 O-\ / - -().O less commonly in NRAS (about 12%) and rarely in HRAS (about 3%). KRAS encodes two splice variants, A and B, with S O divergent C-terminal sequences due to the alternate utiliza tion of exon 4. Mutant KRAS (mut-KRAS) may be present in up to about 25% of all human tumors. Mut-KRAS may play 45 (DN --ClO O a critical role in driving tumor growth and resistance to O O therapy. An agent with even a modest effect on mut-KRAS A - 2 O, or activity, or one that exhibits selective inhibition of a subset of ly () mut-RAS could have a major impact on therapy, and decrease 50 cancer patient Suffering and morbidity. Thus, finding new agents that inhibit the growth of mut-KRAS tumors is desir n is 1, 2, 3, or 4: able. R is —C-Calkyl, -C-C alkyl-C(O)NH2, —S(O)CH: 55 provided when R is Me, R is NOT = C(O)OCHCH: SUMMARY or a pharmaceutically acceptable salt thereof. In some embodiments, R' is Embodiments are directed to small molecule drugs that may inhibit CNKSR1 through PH-domain binding and may selectively block the growth of mut-KRAS cancer cells with 60 out affecting wt-KRAS cells. In embodiments, inhibiting the CNKSR1 gene may block the growth of mut-KRAS cancer cells without affecting wt-KRAS cancer cell growth. In O ) embodiments, CNKSR1 has a PH-domain that may be critical for allowing mut-KRAS to signal tumor growth. In embodi 65 In some embodiments, R' is C(O)C(C-C alkyl). ments, iterative molecular modeling and the SPR binding In some embodiments, R is C-C alkyl-OH. approach may be used to identify PH-domain inhibitors. In some embodiments, R is C(O)NHR'. US 9,340,532 B2 3 4 In some embodiments, R1 is -continued 78 - KD

In some embodiments, R is methyl. 10 In some embodiments, R is —C1-C5 cylcloalkyl. In some embodiments, the compound is selected from: 79 CHO 15

O 25

81 35

45 82

55 11

oh 60

US 9,340,532 B2 7 8 In some embodiments, R4 is methyl. -continued 83 In some embodiments, R7 is COOH

MeO --K) V/ 10 O 1. S CrityH In some embodiments, R5 is —C2-C6alkenyl-OH, or 84 —C2-C6alkenyl-C(O)—C1-C4alkyl. COOH In some embodiments, R5 is —C1-C4alkyl-OH, or—C1 15 21

In some embodiments, R5 is MeO O O V/ O 1. S CrityH 85 COOEt 25

In some embodiments, R8 is cyclopropyl or cyclobutyl. MeO In some embodiments, R7 is H. O O In some embodiments, R5 is 30 V/ O 1. S CrityH 86 35 COOEt 2

MeO 40 V/ In some embodiments, R5 is O 1. S CrityH 45 87 COOEt

50 HO In some embodiments the compound is selected from: V/ O 1. S H 55 Crity O o1 88 COOEt 1. 21 y 21

O HO O O 's-N-Ss V/ NSr. - 65 US 9,340,532 B2 9 10 -continued -continued 89 O N COOH O 2 5 2 y

HO O O O VW O HNN /% O N1 S S 10 S H S. Crity e S S.

15 90 COOH

HO V / O N1 S S H Crity 25

91 Me 30 O-C s 21 35

HO O O V/ 40

O O 50 N

O OH 55 7N, 21 y

O 60

65 US 9,340,532 B2 11 12 -continued wherein O OMe R is —H or C-C alkyl; R' is C(O)O C-C alkyl, -C(O)OH, r-y 5 1-4 HN O O O Os N N-R / N - 10 O O O O

O O OMe N s N s 15 O O HO 21 y R12 O N HN O N N1 N C(O)NH s 2O NNN s N kn N S R11 OJ, s

25 NH, or N O OH /r O N O >> O X 30 R'' is H or C-C alkyl: O R" is C-Calkyl: N O or a pharmaceutically acceptable salt thereof. o-sa Ysaf S In some embodiments, R9 is methyl. So W \ f 35 In some embodiments, R10 is —C(O)O C1-C4alkyl. s1N O In some embodiments, the C1-C4 alkyl is ethyl. d In some embodiments, R10 is o , all

40 O O Sr., As O 21 O X N S{ X 45 O O In some embodiments, R10 is On S NH O 50 R 2 O

Some embodiments provide a compound of Formula IIIA: N 55 R11

Formula IIIA OR9 In some embodiments, R10 is

S OH 60 / O O Us N 4\o N O.

S YMe 65 O US 9,340,532 B2 13 14 In some embodiments the compound is selected from: Wherein R' is -OH or C-C alkyl: R" is H or C-C alkyl: Or a pharmaceutically acceptable salt thereof In some embodiments, R13 is methyl. No In some embodiments, R'' is methyl. In some embodiments, the compound is selected from 7 S O OH le 4. O s 107 /YN 21 N-1 SCH O H 10 SN O HN N COOEt, No S OH OH / O 15 c.OMe le 4. O 108 /YN 21 s SCH O H SN OH HN S. COOH, OH

OH 7 S O OH cOMe le 4. O s 109 /NN 21 N-1 25 SCH O H SN O HN S COOEt, and OH 30 / S O OH OH 2N M cOH 110 WN 21 OH, SCH O H SN O 35 HN N COOH. O

/ > H SCH N\ OH le S -N N sk 40 cOH /\, O , and OH Methods of treating cancer and/or inhibiting CNKSR1 OMe comprising administering an effective amount of the above O described compounds or pharmaceutical compositions con 45 taining them are also disclosed. Cl H SCH N\, BRIEF DESCRIPTION OF DRAWINGS 2n-NS N sk /\, O The file of this patent contains at least one photograph or drawing executed in color. Copies of this patent with color OH 50 drawing(s) or photograph(s) will be provided by the Patent OMe and Trademark Office upon request and payment of necessary fee. For a fuller understanding of the nature and advantages of Some embodiments provide a compound of formula IVA: the present invention, reference should be had to the follow 55 ing detailed description taken in connection with the accom panying drawings, in which: Formula IVA FIG. 1 is a scheme illustrating translational modifications OR 14 of RAS proteins, in accordance with embodiments. FIG. 2 is a collection of plots illustrating the use of OH 60 CNKSR1 as a target for inhibition of mut-KRAS growth, in accordance with embodiments. FIG. 3 contains a table of identified compounds and a HN collection of plots illustrating their use in the inhibition of mut-KRAS growth, in accordance with embodiments. 65 FIG. 4 is a collection of plots illustrating anti-tumor activ ity and pharmacokinetics of compound #7, in accordance with embodiments. US 9,340,532 B2 15 16 FIG. 5 is a table of identified compounds illustrating their ence to one or more cells and equivalents thereof known to use in the inhibition of mut-KRAS growth, in accordance those skilled in the art, and so forth. with embodiments. As used herein, the term “about” means plus or minus 10% FIG. 6 is an overlay of the X-ray structures of compound of the numerical value of the number with which it is being #12 and inositol, in accordance with embodiments. used. Therefore, about 50% means in the range of 45%-55%. FIG. 7 is an illustration of the CNKR1 model, in accor Administering when used in conjunction with a thera dance with embodiments. peutic means to administer a therapeutic directly into or onto FIG. 8 is a schematic of the BREED process, in accordance a target tissue or to administer a therapeutic to a patient with embodiments. whereby the therapeutic positively impacts the tissue to 10 which it is targeted. Thus, as used herein, the term “adminis FIG. 9 is an illustration of the inhibition of 3D growth by tering, when used in conjunction with a compound, can siKRas and siCNKSR1, in accordance with embodiments. include, but is not limited to, providing a compound into or FIG. 10 is a collection of photographs depicting CNKSR1 onto the target tissue; and/or providing a compound systemi colocalizes with mutant KRas at the plasma membranes, in cally to a patient by, e.g., intravenous injection or oral admin accordance with embodiments. 15 istration, whereby the therapeutic reaches the target tissue. FIG. 11 is a collection of photographs and lifetime histo The term “animal' as used herein includes, but is not lim grams of Fluorescence lifetime imaging microscopy (FLIM) ited to, humans and non-human vertebrates Such as wild, showing that CNKSR1 binds directly to mut-KRas but not to domestic and farm animals. wt-KRas in cells in accordance with embodiments. The term “inhibiting includes the administration of a FIG. 12 is a collection of photographs showing compounds compound of the present invention to prevent the onset of the 7 and 9 blocking the membrane colocalisation of CNKSR1 symptoms, alleviating the symptoms, or eliminating the dis and mutant-KRas at the plasma membrane, and lower total ease, condition or disorder. mutant KRas protein in the cell, in accordance with embodi By “pharmaceutically acceptable', it is meant the carrier, mentS. diluent or excipient must be compatible with the other ingre FIG. 13 is a collection of photographs and lifetime histo 25 dients of the formulation and not deleterious to the recipient grams of fluorescence lifetime imaging microscopy for com thereof. pounds 7 and 9 showing the compounds inhibit the direct As used herein, the term “therapeutic' means an agent binding of CNKSR1 to mut-KRAS in cells, in accordance utilized to treat, combat, ameliorate, prevent or improve an with embodiments. unwanted condition or disease of a patient. In part, embodi FIG. 14 is a graph and Schematic showing inhibition of 30 ments of the present invention are directed to the treatment of growth in lung cancer cells with mutated KRas and inhibition cancer or the decrease in proliferation of cells. of KRas signaling effectors. A “therapeutically effective amount” or “effective FIG. 15 shows increased activity for compounds in accor amount of a composition is a predetermined amount calcu dance with embodiments, for inhibition of KRas signaling lated to achieve the desired effect, i.e., to inhibit, block, or effectors. 35 reverse the activation, migration, or proliferation of cells. The FIG. 16 is a graph showing antitumor activity observed activity contemplated by the present methods includes both compounds in accordance with embodiments. medical therapeutic and/or prophylactic treatment, as appro FIG. 17 is a graph depicting binding of selected com priate. The specific dose of a compound administered accord pounds. ing to this invention to obtain therapeutic and/or prophylactic FIG. 18 is a sensogram comparing 3 selected compounds. 40 effects will, of course, be determined by the particular cir FIG. 19 is a sensogram comparing 3 selected compounds. cumstances Surrounding the case, including, for example, the compound administered, the route of administration, and the DETAILED DESCRIPTION condition being treated. The compounds are effective over a wide dosage range and, for example, dosages per day will Before the present compositions and methods are 45 normally fall within the range of from 0.001 to 10 mg/kg, described, it is to be understood that this invention is not more usually in the range of from 0.01 to 1 mg/kg. However, limited to the particular processes, compositions, or method it will be understood that the effective amount administered ologies described, as these may vary. It is also to be under will be determined by the physician in the light of the relevant stood that the terminology used in the description is for the circumstances including the condition to be treated, the purpose of describing the particular versions or embodiments 50 choice of compound to be administered, and the chosen route only, and is not intended to limit the scope of the present of administration, and therefore the above dosage ranges are invention which will be limited only by the appended claims. not intended to limit the scope of the invention in any way. A Unless defined otherwise, all technical and scientific terms therapeutically effective amount of compound of this inven used herein have the same meanings as commonly under tion is typically an amount Such that when it is administered stood by one of ordinary skill in the art. Although any meth 55 in a physiologically tolerable excipient composition, it is ods and materials similar or equivalent to those described Sufficient to achieve an effective systemic concentration or herein can be used in the practice or testing of embodiments local concentration in the tissue. of the present invention, the preferred methods, devices, and The terms “treat,” “treated,” or “treating as used herein materials are now described. All publications mentioned refers to both therapeutic treatment and prophylactic or pre herein are incorporated by reference in their entirety. Nothing 60 ventative measures, wherein the object is to prevent or slow herein is to be construed as an admission that the invention is down (lessen) an undesired physiological condition, disorder not entitled to antedate such disclosure by virtue of prior or disease, or to obtain beneficial or desired clinical results. invention. For the purposes of this invention, beneficial or desired clini It must also be noted that as used herein and in the cal results include, but are not limited to, alleviation of symp appended claims, the singular forms “a”, “an', and “the 65 toms; diminishment of the extent of the condition, disorder or include plural reference unless the context clearly dictates disease; stabilization (i.e., not worsening) of the state of the otherwise. Thus, for example, reference to a “cell' is a refer condition, disorder or disease; delay in onset or slowing of the US 9,340,532 B2 17 18 progression of the condition, disorder or disease; ameliora Embodiments of the invention are directed toward com tion of the condition, disorder or disease state; and remission pounds of the formula: (whether partial or total), whether detectable or undetectable, or enhancement or improvement of the condition, disorder or disease. Treatment includes eliciting a clinically significant 5 (I) response without excessive levels of side effects. Treatment also includes prolonging Survival as compared to expected Survival if not receiving treatment. Single point mutations of at least one of the RAS genes 10 (KRAS, HRAS, and NRAS) are found in many human can cers, particularly in colon, lung and pancreatic cancer. RAS mutations are most commonly found in KRAS (about 85%), less commonly in NRAS (about 12%) and rarely in HRAS (about 3%). KRAS encodes two splice variants, A and B, with 15 wherein divergent C-terminal sequences due to the alternate utiliza tion of exon 4. Mutant KRAS (mut-KRAS) may be present in W and X are each independently selected from a carbonatom up to about 25% of all human tumors. Mut-KRAS may play and a nitrogen atom; a critical role in driving tumor growth and resistance to Y is a carbon atom; therapy. An agent with even a modest effect on mut-KRAS Z is selected from methylene, an oxygen atom, and a Sulfur activity, or one that exhibits selective inhibition of a subset of atom; mut-RAS could have a major impact on therapy, and decrease cancer patient Suffering and morbidity. Thus, finding new the bond order between XandY is selected from a single bond agents that inhibit the growth of mut-KRAS tumors is argu or a double bond; ably the most important unmet need in cancer therapy today. 25 R" is selected from a hydroxyl, a thiol, optionally substituted amines, optionally Substituted ethers, optionally Substituted Early attempts to develop GTP-competitive antagonists to Sulfanes, an oxygenatom forming a ketone with Y, an option RAS protein, analogous to ATP-competitive antagonists of ally substituted nitrogenatom forming an imine with Y, and a protein-tyrosine kinases, were found to be impractical sulfur atom forming a thione with Y: because of the picomolar binding of GTP to RAS. The next 30 approach, and one that gained considerable traction, was to R’ is selected from hydroxyl, thiol, optionally substituted prevent the membrane binding of RAS by blocking RAS amines, optionally substituted ethers, optionally substituted farnesylation using cell permeable CAAX peptidomimetics sulfanes, optionally substituted alkyl chains with 1 to 3 car or small molecule farnesyl transferases (FT) inhibitors. Sev bonatoms, optionally Substituted Sulfonimidic acids, option eral potent agents were developed that showed dramatic 35 ally substituted sulfonimidates; and activity in HRAS cell lines and mouse tumor models. How R is selected from acetic acid, acetate, alkylacetate, and ever, it was found that the activity was limited to oncogenic 4-p-tolylthiazol-2-yl: HRAS which is found in only a small portion of human and wherein the compound is not: tumors, and that oncogenic NRAS and KRAS were resistant to FT inhibition because of alternative geranylgeranylation. 40 Other efforts to develop antisense or siRNA inhibitors of KRAS, or inhibitors of Rce1 and Icmt responsible for CAAX O 7 signal processing have so far not provided effective KRAS antitumor agents. The currently favored approach is to block 45 1N downstream signaling targets activated by KRAS Such as PI-3-K, RAF and mitogen activated protein kinase kinase n (MEK), and several clinical trials underway with combina S N O tions of these inhibitors. However, a limitation of the approach may be that different mut-KRAS amino acid sub 50 Nulls stitutions engage different downstream signaling effectors, and it may be necessary to have a number inhibitors available O for each of the pathways. It may be preferable to have an 12 inhibitor that works with all forms of mut-KRAS and the 7 S O approach we have adopted is to identify genes that are acti 55 le 4. vators of mut-KRAS activity to provide molecular targets for the development of selective mut-KRAS inhibitors. / YNH Following the strategy to identify genes that positively regulate mut-KRAS activity, CNKSR1 (connector enhancer 60 of kinase suppressor of RAS 1) has been identified. The OH. CNKSR1 protein is associated with KRAS in the membrane s^ signaling nanocluster, and knockdown of CNKSR1 may OH O cause inhibition of mut-KRAS tumor cell growth and signal ing without inhibition of wt-KRAS cell growth. Furthermore, 65 CNKSR1 has a potentially druggable pleckstrin homology Embodiments of the invention are directed toward com (PH) domain. pounds of the formula: US 9,340,532 B2 20 R’ is Hor R and R combine as --

10 }}- wherein to form a bicyclic moiety with the carbons to which they are W and X are each independently selected from a carbonatom attached; and a nitrogen atom; Y is a carbon atom; 15 - CH-OH, -CH(OH)(CH), NH, -CH(OH), Z is selected from methylene and a sulfur atom; —C(OH)CNO, -(CH). NO, —CHO, C(O) the bond order between X and Y is selected from a single bond NHR, or a double bond; R" is selected from a hydroxyl and an oxygenatom forming a ketone with Y: R’ is selected from an optionally substituted carbonatom and O / \ N an optionally Substituted Sulfonimidate; and R is selected from acetate and 4-p-tolylthiazol-2-yl; 2O ( ) and wherein the compound is not:

O 7 --() - Cl 1N U O n N --S O 35 n is 1, 2, 3, or 4: R is C-C alkyl, -C-C alkyl-C(O)NH2, O —S(O)CH: 12 provided when R is Me, R is NOT = C(O)CCHCH 7 S O compound 7: 40 or a pharmaceutically acceptable salt thereof. AYNH In some embodiments, R' is O

OH. - O OH O In some embodiments, R' is —C(O)C(C-C alkyl). In Some embodiments are drawn toward compounds of for 50 some embodiments, the C-C alkyl is ethyl. mula IA: In some embodiments, R' is C-C alkyl-OH. In some embodiments, R is C(O)NHR'. In some embodiments, R is Formula I RI 55 R sN S N V S. N N R2 60 --O In some embodiments, R is methyl. O In some embodiments, R is —C-C cylcloalkyl. In some embodiments C-C cycloalkyl is cyclopropyl. In some wherein 65 embodiments, C-C cycloalkyl is cyclobutyl. Ris—C-C alkyl, - NO. —NH, NHSOCH —C-Cs In some embodiments, a compound of formula IA is cylcloalkyl, or—CF; selected from: US 9,340,532 B2 21 22 -continued 8 8O N O

OH 5 O

n S N n N S N Nus N 10 Nsus N Me,

O s O 81 CONH 9 15 N1N1 2 O

OO O NN S n N S N 2O N Me, N susSN N s O O 82 y 10 25 HN1 NconH, N O

30 n N S n Nsus S N N U)S. N ()- O O 35 ON 11

O O)-O oh s/N 40 SN

78 / O NN Et N1 50 SN s/N H NN N N Nsus s Me, 55 O

79 CHO N 60 s/N NN S SN

65 US 9,340,532 B2 23 24 -continued Embodiments of the invention are directed toward com pounds of the formula:

O CF, s/N R5 sN R N y– O O 10 1n\/ O NH R6,

15 and pharmaceutically acceptable salts thereof, wherein R" is selected from a hydroxyl, a thiol, optionally substituted amines, and an optionally Substituted ether, R is selected from hydroxyl, thiol, optionally substituted amines, optionally Substituted ethers, optionally Substituted sulfanes, optionally substituted alkyl chains with 1 to 3 car 25 bonatoms, optionally Substituted Sulfonimidic acids, option NO2, ally substituted sulfonimidates; and R is selected from acetic acid, acetate, alkylacetate, and 4-p-tolylthiazol-2-yl. 30 One preferred embodiment has R selected from a hydroxyl, and an optionally substituted ether; Ras a substi tuted alkyl chains with 1 to 3 carbon atoms; and Ras 4-p- tolylthiazol-2-yl. Some embodiments are directed toward compounds of for 35 mula IIA: NH2,

40 Formula II

50 wherein R" is H. —C-Calkyl, O R is C-C alkyl-OH, -C-Calkenyl-OH, -C- 55 Calkykl-C(O)—C-C alkyl, —C-Calkenyl-C(O)—C- Calkyl, -C-C alkykl-C(O)—C-Cscycloalkyl, —C- Calkenyl-C(O)—C-C cycloalkyl,

O O O R8 N 65 N-->s --KO, or US 9,340,532 B2 25 26 -continued

R is C-C alkyl, or—C-Cs cycloalkyl; 10 R7 is Hor O O N- O N S N NNN V ---O 15 O s RI), O or a pharmaceutically acceptable salt thereof N-N Embodiments of the invention are directed toward com pounds of the formula: > SCH H 25 RS6 -N R 5 /\,MV OH, x RO; 30 R4 R'' is H or C-C alkyl: and pharmaceutically acceptable salts thereof, R' is C-C alkyl: or a pharmaceutically acceptable salt thereof wherein 35 R" is selected from a hydroxyl, a thiol, optionally substituted The compounds of some embodiments are selected from: amines, and an optionally Substituted ether, R is selected from hydroxyl, thiol, optionally substituted amines, optionally Substituted ethers, optionally Substituted COOH sulfanes, optionally substituted alkyl chains with 1 to 3 car 40 bonatoms, optionally Substituted Sulfonimidic acids, option ally substituted sulfonimidates; and R is selected from acetic acid, acetate, alkylacetate, thiazol 2-yl, and 4-p-tolylthiazol-2-yl. In one preferred embodiment, R" is selected from a hydroxyl, and an optionally substituted 45 C s1NcooH, ether; R is a substituted alkyl chains with 1 to 3 carbon OH atoms; and R is thiazol-2-yl. Some embodiments are drawn to compounds of formula IIIA: 50 COOH

Formula III OR9 55 OH

COOEt 60 wherein C \s1NcooH, 65 Ris-H or -C-C-alkyl-CH: OH R" is C(O)O C-C alkyl Et, —C(O)OH, US 9,340,532 B2 27 28 -continued -continued 4 10 COOEt 5 yN S. N

s S N 10 Nulls O , and 5 11 15 O O Xso \ 1S O N22S V O 2O NN S l N Nus N SN

O Some embodiments are directed to compounds of formula IVA: 6

30 Formula IVA OR 14

35 13 R HN 21

S N O 40 wherein R' is -OH or C-C alkyl: 8 R" is H or C-C alkyl. In some embodiment, R' is methyl. 45 OH In some embodiments, R'' is methyl. In some embodiments, a compound of formula IVA is selected from

50 107 SCH HN N-COOEt, 55 9 OH

OMe 108 60 SCH HN N-COOH,

OH 65 OMe US 9,340,532 B2 30 -continued -continued 16 109 O O SCH O O HN N-COOEt, and O N

OH 2

OH 10 110 1N, SCH HN N-COOH. 17

15 O O O OH ~s. OH

Embodiments are directed toward pharmaceutical compo sitions comprising a compound of formula I and a pharma o1 ceutically acceptable excipient. In Such embodiments, the compounds of formula I are presentinatherapeutically effec tive amount. 25 18 Embodiments are directed toward methods of treating can O O cer comprising administering an effective amount of com O O pounds of formula I. O N Embodiments are directed toward methods of inhibiting 30 CNKSR1 comprising administering an effective amount of compounds of formula I. 21 Embodiments are directed toward methods of treating can cer comprising administering an effective amount of com pounds selected from 1-12, 35

19 13 O O O O O 40 21 O O N

N N N O 45 N-s/WN-1 Co. O s O O

14 50 O O O

O ~s. 21\e 55 o1 21 O

15 O 60 s S C l,

65 US 9,340,532 B2 31 32 -continued continued 22 29

O O e b. M 10

23 30

O O)

25 31

25 30

34 US 9,340,532 B2 33 34 -continued -continued 35 40 O

10 )- ( e N

36 N D a 15 N

41 O

25 37

N N Br

a 30 N

40 38

43 45

55 39 44 O O

60 21\N-NN N S. s N

65 N N O US 9,340,532 B2 35 36 -continued -continued 45 51 O

52 46 15 N/ / O No o1 ON 47 25 O 53 O

30 S r O 35 O, O 48 O O 40 54 21 JX-N {O, N-S O 45 -N \ 2 N 49 O O 50 55 O e NH S. O 55 O O, O 50 O 56 60

65 US 9,340,532 B2 37 38 -continued -continued 63

US 9,340,532 B2 43 44 -continued -continued 89 94 COOH 21

\,A” HO O 1N 10 O

COOH

HO O O \/ 95 O 1N O l/ 91 Me M 25 -C HO

35 96

45 HO

93 97

60 HO HO

65 US 9,340,532 B2 45 46 -continued -continued 98 102 Cy>

HO 10 HO V / N 1. 15 MS 103 SCH H 99 S -N N COOEt, /M \, OH

OMe 25 104 SCH H S -N S COOH, HO M \, 30 / \, OH

OMe 105 SCH as ( N COOEt,

100 OH 40 OH 106 SCH COOH, 45 7 S S.S. OH OH 50 107 SCH HN S. COOEt, 101 55 OH N CONH OMe 21 108 60 SCH HO HN COOH, O O S. V/ OH 65 c.OMe US 9,340,532 B2 48 -continued -continued 116 109 SCH HN N-COOEt,

OH OMe 10 78 110 SCH HN N-COOH,

15 OH

OH 111 O SCH 79 CHO

25 OH

OMe

OMe 113

81 MV O O OH 45

OMe

SCH 50

S / \, 82 OH 55

OMe 115

SCH 60 ClS s1 / \, OH 65 OMe and pharmaceutically acceptable salts thereof US 9,340,532 B2 49 50 Embodiments are directed toward methods of inhibiting -continued

CNKSR1 comprising administering an effective amount of 19 compounds selected from 1-12,

25 US 9,340,532 B2 51 52 -continued -continued 26 33 O

38 US 9,340,532 B2 53 54 -continued -continued 39 44 O O

21 N1 NN N S. S N 10 N N e N- O O 45 40 O O O 15 O HS)- ( e N 46 N D 25 a N 41 O

30 O

HS 47 ^)- 35 \ 2 N N Br

a 40 N 42

65 US 9,340,532 B2 55 56 -continued -continued

61 US 9,340,532 B2 57 58 -continued -continued 62 68 ity O 10

70 OH 63 15

HNN/S M O

71 25 O O 64

O, O 30 C

O 72 35 N O 65

S O 40

45 73

74 55

US 9,340,532 B2 61 62 -continued 83 89 COOH COOH 21

MeO HO \/ N H O N

84 90

MeO HO

85 91 25

MeO

86 35

92 MeO 40

87 45 HO

HO 50

55 88

60 HO HO

65 US 9,340,532 B2 63 64 -continued -continued 94 98

Et -CS,N

HO HO 10

95 99 O

N 25 O HO

30 V /

35 96

100

97 101 55

N CONH 2 60 HO HO O O V/ N1 S O H 65

US 9,340,532 B2 67 68 -continued In embodiments, the treatment of cancer or the inhibition 116 of CNKSR1 does not inhibit wild-type KRAS cancer cell growth In embodiments, the treatment of cancer or the inhibition of CNKSR1 may further comprising administering an anti cancer agent, radiation, phototherapy, or a combination thereof. Anti-cancer agents may be selected from alkylating agents, antimetabolites, anthracyclines, plant alkaloids, OMe topoisomerase inhibitors, monoclonal antibodies, tyrosine 78 10 kinase inhibitors, and hormone treatment. Embodiments are directed toward methods of identifying compounds that inhibit the activity of CNKSR1 comprising: contacting CNKSR1 with a test compound; determining the activity of CNKSR1 in the presence of the test compound; 15 and identifying the test compound as a compound that inhib its the activity of CNKSR1 if the activity of CNKSR1 is decreased in the presence of the test compound. Such embodiments may further comprise determining the activity of CNKSR1 in the absence of the test compound. Identifying 79 the test compound as a compound that inhibits the activity of CHO CNKSR1 may further comprise comparing the activity of CNKSR1 in the presence and the absence of the test com pound, wherein the compound is identified as a compound 25 that inhibits the activity of CNKSR1 if the activity of CNKSR1 is decreased in the presence of the test compound as compared to the activity of CNKSR1 in the absence of the test compound. In embodiments of the invention, cancer may include, but 80 30 is not limited to, adrenocortical carcinoma, anal cancer, blad der cancer, brain tumor, breast cancer, carcinoid tumor, gas trointestinal, carcinoma of unknown primary, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, extra hepatic bile duct cancer, Ewings family of tumors (PNET), 35 extracranial germ cell tumor, eye cancer, intraocular mela noma, gallbladder cancer, gastric cancer (stomach), germ cell tumor, extragonadal, gestational trophoblastic tumor, head and neck cancer, hypopharyngeal cancer, islet cell carcinoma, kidney cancer, laryngeal cancer, leukemia, acute lymphoblas 40 tic, adult, leukemia, acute lymphoblastic, childhood, leuke mia, lip and oral cavity cancer, liver cancer, lung cancer, 81 lymphoma, aids-related, lymphoma, central nervous system (primary), lymphoma, cutaneous T-cell, lymphoma, hodgkin’s disease, adult, lymphoma, hodgkin’s disease, 45 childhood, lymphoma, non-hodgkin’s disease, adult, lym phoma, non-hodgkin's disease, childhood, malignant mesothelioma, melanoma, merkel cell carcinoma, metasatic squamous neck cancer with occult primary, multiple myeloma and other plasma cell neoplasms, mycosis fun 50 goides, myelodysplastic syndrome, myeloproliferative disor ders, nasopharyngeal cancer, neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian epithelial can cer, ovarian germ cell tumor, pancreatic cancer, exocrine, 82 pancreatic cancer, islet cell carcinoma, paranasal sinus and 55 nasal cavity cancer, parathyroid cancer, penile cancer, pitu itary cancer, plasma cell neoplasm, prostate cancer, rhab domyosarcoma, childhood, rectal cancer, renal cell cancer, renal pelvis and ureter, transitional cell, salivary gland cancer, Sezary syndrome, skin cancer, skin cancer, cutaneous T-cell 60 lymphoma, skin cancer, kaposi's sarcoma, skin cancer, mela noma, Small intestine cancer, soft tissue sarcoma, adult, soft tissue sarcoma, child, Stomach cancer, testicular cancer, thy moma, malignant, thyroid cancer, urethral cancer, uterine cancer, sarcoma, unusual cancer of childhood, vaginal cancer, 65 vulvar cancer, Wilms Tumor and combinations thereof. In certain embodiments, the cancer is selected from colon, lung, and pharmaceutically acceptable salts thereof pancreas or combinations thereof. US 9,340,532 B2 69 70 For example, in Some aspects, the invention is directed to a cally acceptable carriers well known in the art. Such carriers pharmaceutical composition comprising a compound, as enable the compounds of the invention to be formulated as defined above, and a pharmaceutically acceptable carrier or tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, diluent, or an effective amount of a pharmaceutical compo Suspensions and the like, for oral ingestion by a patient to be sition comprising a compound as defined above. treated. Pharmaceutical preparations for oral use can be The compounds of the present invention can be adminis obtained by adding a Solid excipient, optionally grinding the tered in the conventional manner by any route where they are resulting mixture, and processing the mixture of granules, active. Administration can be systemic, topical, or oral. For after adding suitable auxiliaries, if desired, to obtaintablets or example, administration can be, but is not limited to, dragee cores. Suitable excipients include, but are not limited parenteral, Subcutaneous, intravenous, intramuscular, intrap 10 to, fillers such as Sugars, including, but not limited to, lactose, eritoneal, transdermal, oral, buccal, or ocular routes, or intra Sucrose, mannitol, and Sorbitol; cellulose preparations such vaginally, by inhalation, by depot injections, or by implants. as, but not limited to, maize starch, wheat starch, rice starch, Thus, modes of administration for the compounds of the potato starch, gelatin, gum tragacanth, methyl cellulose, present invention (either alone or in combination with other hydroxypropylmethyl-cellulose, sodium carboxymethylcel pharmaceuticals) can be, but are not limited to, Sublingual, 15 lulose, and polyvinylpyrrolidone (PVP). If desired, disinte injectable (including short-acting, depot, implant and pellet grating agents can be added, such as, but not limited to, the forms injected Subcutaneously or intramuscularly), or by use cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a of Vaginal creams, Suppositories, pessaries, Vaginal rings, salt thereof Such as sodium alginate. rectal Suppositories, intrauterine devices, and transdermal Dragee cores can be provided with Suitable coatings. For forms such as patches and creams. this purpose, concentrated Sugar Solutions can be used, which Specific modes of administration will depend on the indi can optionally contain gum arabic, talc, polyvinyl pyrroli cation. The selection of the specific route of administration done, carbopol gel, polyethylene glycol, and/or titanium and the dose regimen is to be adjusted or titrated by the dioxide, lacquer Solutions, and Suitable organic solvents or clinician according to methods known to the clinician in order solvent mixtures. Dyestuffs or pigments can be added to the to obtain the optimal clinical response. The amount of com 25 tablets or dragee coatings for identification or to characterize pound to be administered is that amount which is therapeuti different combinations of active compound doses. cally effective. The dosage to be administered will depend on Pharmaceutical preparations which can be used orally the characteristics of the Subject being treated, e.g., the par include, but are not limited to, push-fit capsules made of ticular animal treated, age, weight, health, types of concurrent gelatin, as well as Soft, sealed capsules made of gelatin and a treatment, if any, and frequency of treatments, and can be 30 plasticizer, such as glycerol or Sorbitol. The push-fit capsules easily determined by one of skill in the art (e.g., by the can contain the active ingredients in admixture with filler clinician). Such as, e.g., lactose, binders such as, e.g., starches, and/or Pharmaceutical formulations containing the compounds of lubricants such as, e.g., talc or magnesium Stearate and, the present invention and a suitable carrier can be solid dosage optionally, stabilizers. In soft capsules, the active compounds forms which include, but are not limited to, tablets, capsules, 35 can be dissolved or Suspended in Suitable liquids, such as fatty cachets, pellets, pills, powders and granules; topical dosage oils, liquid paraffin, or liquid polyethylene glycols. In addi forms which include, but are not limited to, Solutions, pow tion, stabilizers can be added. All formulations for oral ders, fluid emulsions, fluid Suspensions, semi-solids, oint administration should be in dosages suitable for Such admin ments, pastes, creams, gels and jellies, and foams; and istration. parenteral dosage forms which include, but are not limited to, 40 For buccal administration, the compositions can take the Solutions, Suspensions, emulsions, and dry powder, compris form of, e.g., tablets or lozenges formulated in a conventional ing an effective amount of a polymer or copolymer of the a. present invention. It is also known in the art that the active For administration by inhalation, the compounds for use ingredients can be contained in Such formulations with phar according to the present invention are conveniently delivered maceutically acceptable diluents, fillers, disintegrants, bind 45 in the form of an aerosol spray presentation from pressurized ers, lubricants, Surfactants, hydrophobic vehicles, water packs or a nebulizer, with the use of a Suitable propellant, e.g., soluble vehicles, emulsifiers, buffers, humectants, moisturiz dichlorodifluoromethane, trichlorofluoromethane, dichlo ers, solubilizers, preservatives and the like. The means and rotetrafluoroethane, carbon dioxide or other suitable gas. In methods for administration are known in the art and an artisan the case of a pressurized aerosol the dosage unit can be can refer to various pharmacologic references for guidance. 50 determined by providing a valve to deliver a metered amount. For example, Modern Pharmaceutics, Banker & Rhodes, Capsules and cartridges of, e.g., gelatin for use in an inhaleror Marcel Dekker, Inc. (1979); and Goodman & Gilman's The insufflator can be formulated containing a powder mix of the Pharmaceutical Basis of Therapeutics, 6th Edition, Mac compound and a suitable powder base such as lactose or Millan Publishing Co., New York (1980) can be consulted. starch. The compounds of the present invention can beformulated 55 The compounds of the present invention can also be for for parenteral administration by injection, e.g., by bolus mulated in rectal compositions such as Suppositories or reten injection or continuous infusion. The compounds can be tion enemas, e.g., containing conventional Suppository bases administered by continuous infusion Subcutaneously over a Such as cocoa butter or other glycerides. period of about 15 minutes to about 24 hours. Formulations In addition to the formulations described previously, the for injection can be presented in unit dosage form, e.g., in 60 compounds of the present invention can also be formulated as ampoules or in multi-dose containers, with an added preser a depot preparation. Such long acting formulations can be Vative. The compositions can take Such forms as Suspensions, administered by implantation (for example Subcutaneously or Solutions or emulsions in oily or aqueous vehicles, and can intramuscularly) or by intramuscular injection. contain formulatory agents such as Suspending, stabilizing Depot injections can be administered at about 1 to about 6 and/or dispersing agents. 65 months or longer intervals. Thus, for example, the com For oral administration, the compounds can be formulated pounds can be formulated with suitable polymeric or hydro readily by combining these compounds with pharmaceuti phobic materials (for example as an emulsion in an acceptable US 9,340,532 B2 71 72 oil) or ion exchange resins, or as sparingly soluble deriva (available from ISP), Manucol (available from ISP), and Pro tives, for example, as a sparingly soluble salt. tanol (available from FMC Biopolymer). In transdermal administration, the compounds of the As used herein, the term "calcium silicate” refers to a present invention, for example, can be applied to a plaster, or silicate salt of calcium. can be applied by transdermal, therapeutic systems that are As used herein, the term "calcium phosphate” refers to consequently Supplied to the organism. monobasic calcium phosophate, dibasic calcium phosphate Pharmaceutical compositions of the compounds also can or tribasic calcium phosphate. comprise Suitable solid or gel phase carriers or excipients. Cellulose, cellulose floc, powdered cellulose, microcrys Examples of Such carriers or excipients include but are not talline cellulose, silicified microcrystalline cellulose, car limited to calcium carbonate, calcium phosphate, various 10 boxyethylcellulose, carboxymethylcellulose, hydroxyethyl Sugars, starches, cellulose derivatives, gelatin, and polymers Such as, e.g., polyethylene glycols. cellulose, methylhydroxyethylcellulose, The compounds of the present invention can also be admin hydroxymethylcellulose, hydroxypropylcellulose, hydrox istered in combination with other active ingredients, such as, ypropylmethylcellulose, hydroxypropylmethylcellulose for example, adjuvants, protease inhibitors, or other compat 15 phthalate, ethylcellulose, methylcellulose, carboxymethyl ible drugs or compounds where such combination is seen to cellulose sodium, and carboxymethyl cellulose calcium be desirable or advantageous in achieving the desired effects include, but are not limited to, those described in R. C. Rowe of the methods described herein. and P. J. Shesky, Handbook of pharmaceutical excipients, In some embodiments, the disintegrant component com (2006), 5th ed., which is incorporated herein by reference in prises one or more of croscarmellose Sodium, carmellose its entirety. As used herein, cellulose refers to natural cellu calcium, crospovidone, alginic acid, Sodium alginate, potas lose. The term 'cellulose” also refers to celluloses that have sium alginate, calcium alginate, an ion exchange resin, an been modified with regard to molecular weight and/or effervescent system based on food acids and an alkaline car branching, particularly to lower molecular weight. The term bonate component, clay, talc, starch, pregelatinized starch, “cellulose” further refers to celluloses that have been chemi Sodium starch glycolate, cellulose floc, carboxymethylcellu 25 cally modified to attach chemical functionality Such as car lose, hydroxypropylcellulose, calcium silicate, a metal car boxy, hydroxyl, hydroxyalkylene, or carboxyalkylene bonate, sodium bicarbonate, calcium citrate, or calcium phos groups. As used herein, the term “carboxyalkylene' refers to phate. a group of formula -alkylene-C(O)OH, or salt thereof. As In some embodiments, the diluent component comprises used herein, the term “hydroxyalkylene' refers to a group of one or more of mannitol, lactose, Sucrose, maltodextrin, Sor 30 formula -alkylene-OH. bitol, xylitol, powdered cellulose, microcrystalline cellulose, Suitable powdered celluloses for use in the invention carboxymethylcellulose, carboxyethylcellulose, methylcel include, but are not limited to Arbocel (available from JRS lulose, ethylcellulose, hydroxyethylcellulose, methylhy Pharma), Sanacel (available from CFF GmbH), and Solka droxyethylcellulose, starch, Sodium starch glycolate, prege Floc (available from International Fiber Corp.). latinized Starch, a calcium phosphate, a metal carbonate, a 35 Suitable microcrystalline celluloses include, but are not metal oxide, or a metal aluminosilicate. limited to, the Avicel pH series (available from FMC Biopoly In some embodiments, the optional lubricant component, mer), Celex (available from ISP), Celphere (available from when present, comprises one or more of stearic acid, metallic Asahi Kasei), Ceolus KG (available from Asahi Kasei), and Stearate, sodium Stearyl fumarate, fatty acid, fatty alcohol, Vivapur (available from JRS Pharma). fatty acid ester, glyceryl behenate, mineral oil, vegetable oil, 40 As used herein, the term “silicified microcrystalline cellu paraffin, leucine, silica, silicic acid, talc, propylene glycol lose refers to a synergistic intimate physical mixture of fatty acid ester, polyethoxylated castor oil, polyethylene gly silicon dioxide and microcrystalline cellulose. Suitable silici col, polypropylene glycol, polyalkylene glycol, polyoxyeth fied microcrystalline celluloses include, but are not limited to, ylene-glycerol fatty ester, polyoxyethylene fatty alcohol ProSolv (available from JRS Pharma). ether, polyethoxylated Sterol, polyethoxylated castor oil, 45 As used herein, the term “carboxymethylcellulose polyethoxylated vegetable oil, or sodium chloride. sodium” refers to a cellulose ether with pendant groups of As used herein, the term “alginic acid refers to a naturally formula Na" ' ' , attached to the cellulose via an occurring hydrophilic colloidal polysaccharide obtained ether linkage. Suitable carboxymethylcellulose sodium poly from the various species of seaweed, or synthetically modi mers include, but are not limited to, Akucell (available from fied polysaccharides thereof. 50 Akzo Nobel), Aquasorb (available from Hercules), Blanose As used herein, the term “sodium alginate' refers to a (available from Hercules), Finnfix (available from Noviant), Sodium salt of alginic acid and can be formed by reaction of Nymel (available from Noviant), and Tylose CB (available alginic acid with a sodium containing base Such as sodium from Clariant). hydroxide or sodium carbonate. As used herein, the term As used herein, the term “carboxymethylcellulose cal "potassium alginate' refers to a potassium salt of alginic acid 55 cium” refers to a cellulose ether with a pendant groups of and can be formed by reaction of alginic acid with a potas formula —CH2—O C(O)—O/2Ca", attached to the cel sium containing base Such as potassium hydroxide or potas lulose via an ether linkage. sium carbonate. As used herein, the term "calcium alginate' As used herein, the term “carboxymethylcellulose' refers refers to a calcium salt of alginic acid and can be formed by to a cellulose ether with pendant carboxymethyl groups of reaction of alginic acid with a calcium containing base such as 60 formula HO C(O)—CH2—, attached to the cellulose via an calcium hydroxide or calcium carbonate. Suitable sodium ether linkage. Suitable carboxymethylcellulose calcium alginates, calcium alginates, and potassium alginates include, polymers include, but are not limited to, Nymel ZSC (avail but are not limited to, those described in R. C. Rowe and P. J. able from Noviant). Shesky, Handbook of pharmaceutical excipients, (2006), 5th As used herein, the term “carboxyethylcellulose' refers to ed., which is incorporated herein by reference in its entirety. 65 a cellulose ether with pendant carboxymethyl groups of for Suitable Sodium alginates, include, but are not limited to, mula HO—C(O)—CH2—CH2—, attached to the cellulose Kelcosol (available from ISP), Kelfone LVCR and HVCR via an ether linkage. US 9,340,532 B2 73 74 As used herein, the term “hydroxyethylcellulose' refers to is saturated or unsaturated. In some embodiments, the fatty a cellulose ether with pendant hydroxyethyl groups of for acid in a mixture of different fatty acids. In some embodi mula HO—CH2—CH2—, attached to the cellulose via an ments, the fatty acid has between about eight to about thirty ether linkage. Suitable hydroxyethylcelluloses include, but carbons on average. In some embodiments, the fatty acid has are not limited to, Cellosize HEC (available from DOW), about eight to about twenty-four carbons on average. In some Natrosol (available from Hercules), and Tylose PHA (avail embodiments, the fatty acid has about twelve to about eigh able from Clariant). teen carbons on average. Suitable fatty acids include, but are As used herein, the term “methylhydroxyethylcellulose' not limited to, Stearic acid, lauric acid, myristic acid, erucic refers to a cellulose ether with pendant methyloxyethyl acid, palmitic acid, palmitoleic acid, capric acid, caprylic groups of formula CH-O-CH2—CH2—, attached to the 10 acid, oleic acid, linoleic acid, linolenic acid, hydroxy Stearic cellulose via an ether linkage. Suitable methylhydroxyethyl acid, 12-hydroxyStearic acid, cetostearic acid, isostearic acid, celluloses include, but are not limited to, the Culminal MHEC sesquioleic acid, sesqui-9-octadecanoic acid, sesquiisoocta series (available from Hercules), and the Tylose series (avail decanoic acid, benhenic acid, isobehenic acid, and arachi able from Shin Etsu). donic acid, or mixtures thereof. As used herein, the term “hydroxypropylcellulose', or 15 As used herein, the term “fatty acid ester” refers to a “hypomellose”, refers a cellulose that has pendant hydrox compound formed between a fatty acid and a hydroxyl con ypropoxy groups, and includes both high- and low-substi taining compound. In some embodiments, the fatty acid ester tuted hydroxypropylcellulose. In some embodiments, the is a Sugar ester of fatty acid. In some embodiments, the fatty hydroxypropylcellulose has about 5% to about 25% hydrox acid ester is a glyceride of fatty acid. In some embodiments, ypropyl groups. Suitable hydroxypropylcelluloses include, the fatty acid ester is an ethoxylated fatty acid ester. but are not limited to, the Klucel series (available from Her As used herein, the term “fatty alcohol', employed alone or cules), the Methocel series (available from Dow), the Nisso in combination with other terms, refers to an aliphatic alcohol HPC series (available from Nisso), the Metolose series (avail that is saturated or unsaturated. In some embodiments, the able from Shin Etsu), and the LH series, including LHR-11, fatty alcohol in a mixture of different fatty alcohols. In some LH-21, LH-31, LH-20, LH-30, LH-22, and LH-32 (available 25 embodiments, the fatty alcohol has between about eight to from Shin Etsu). about thirty carbons on average. In some embodiments, the As used herein, the term “methyl cellulose' refers to a fatty alcohol has about eight to about twenty-four carbons on cellulose that has pendant methoxy groups. Suitable methyl average. In some embodiments, the fatty alcohol has about celluloses include, but are not limited to Culminal MC (avail twelve to about eighteen carbons on average. Suitable fatty able from Hercules). 30 alcohols include, but are not limited to, stearyl alcohol, lauryl As used herein, the term “ethyl cellulose' refers to a cel alcohol, palmityl alcohol, palmitolyl acid, cetyl alcohol, lulose that has pendant ethoxy groups. Suitable ethyl cellu capryl alcohol, caprylyl alcohol, oleylalcohol, linolenylalco loses include, but are not limited to Aqualon (available from hol, arachidonic alcohol, behenyl alcohol, isobelhenyl alco Hercules). hol, Selachyl alcohol, chimyl alcohol, and linoleyl alcohol, or As used herein, the term “carmellose calcium' refers to a 35 mixtures thereof. crosslinked polymer of carboxymethylcellulose calcium. As used herein, the term “ion-exchange resin' refers to an As used herein, the term “croscarmellose sodium' refers to ion-exchange resin that is pharmaceutically acceptable and a crosslinked polymer of carboxymethylcellulose Sodium. that can be weakly acidic, weakly basic, strongly acidic or As used herein, the term “crospovidone' refers to a strongly basic. Suitable ion-exchange resins include, but are crosslinked polymer of polyvinylpyrrolidone. Suitable 40 not limited to AmberliteTMIRP64, IRP88 and IRP69 (avail crospovidone polymers include, but are not limited to Poly able from Rohm and Haas) and DuoliteTM AP143 (available plasdone XL-10 (available from ISP) and Kollidon CL and from Rohm and Haas). In some embodiments, the ion-ex CL-M (available from BASF). change resin is a crosslinked polymer resin comprising As used herein, the term “crosslinked poly(acrylic acid) acrylic acid, methacrylic acid, or polystyrene Sulfonate, or refers to a polymer of acrylic acid which has been 45 salts thereof. In some embodiments, the ion-exchange resin is crosslinked. The crosslinked polymer may contain other polacrilex resin, polacrilin potassium resin, or monomers in addition to acrylic acid. Additionally, the pen cholestyramine resin. dant carboxy groups on the crosslinked polymer may be Suitable mannitols include, but are not limited to, Pharm partially or completely neutralized to form a pharmaceuti Mannidex (available from Cargill), Pearlitol (available from cally acceptable salt of the polymer. In some embodiments, 50 Roquette), and Mannogem (available from SPI Polyols). the crosslinked poly(acrylic acid) is neutralized by ammonia As used herein, the term “metal aluminosilicate” refers to or sodium hydroxide. Suitable crosslinked poly(acrylic acid) any metal salt of an aluminosilicate, including, but not limited polymers include, but are not limited to, the Carbopol series to, magnesium aluminometasilicate. Suitable magnesium (available from Noveon). aluminosilicates include, but are not limited to Neusilin As used herein, the term “an effervescent system based on 55 (available from Fuji Chemical), Pharmsorb (available from food acids and an alkaline carbonate component” refers to a Engelhard), and Veegum (available from R.T. Vanderbilt Co., excipient combination of food acids and alkaline carbonates Inc.). In some embodiments, the metal aluminosilicate is that releases carbon dioxide gas when administered. Suitable bentonite. effervescent systems are those that those utilizing food acids As used herein, the term “metal carbonate' refers to any (such as citric acid, tartaric acid, malic acid, fumaric acid, 60 metallic carbonate, including, but not limited to sodium car lactic acid, adipic acid, ascorbic acid, aspartic acid, erythor bonate, calcium carbonate, and magnesium carbonate, and bic acid, glutamic acid, and Succinic acid) and an alkaline Zinc carbonate. carbonate component (Such as sodium bicarbonate, calcium As used herein, the term “metal oxide' refers to any metal carbonate, magnesium carbonate, potassium carbonate, lic oxide, including, but not limited to, calcium oxide or ammonium carbonate, etc.). 65 magnesium oxide. As used herein, the term “fatty acid', employed alone or in As used herein, the term “metallic stearate” refers to a combination with other terms, refers to an aliphatic acid that metal salt of Stearic acid. In some embodiments, the metallic US 9,340,532 B2 75 76 Stearate is calcium Stearate, Zinc stearate, or magnesium Stear may have a free hydroxyl group at each end of the polymer ate. In some embodiments, the metallic Stearate is magnesium molecule, or may have one or more hydroxyl groups etheri Stearate. fied with a lower alkyl, e.g., a methyl group. Also suitable are As used herein, the term “mineral oil” refers to both unre derivatives of polyethylene glycols having esterifiable car fined and refined (light) mineral oil. Suitable mineral oils boxy groups. Polyethylene glycols useful in the present include, but are not limited to, the AvatechTM grades (avail invention can be polymers of any chain length or molecular able from Avatar Corp.), DrakeolTM grades (available from weight, and can include branching. In some embodiments, the Penreco), SiriusTM grades (available from Shell), and the average molecular weight of the polyethylene glycol is from CitationTM grades (available from Avater Corp.). about 200 to about 9000. In some embodiments, the average As used herein, the term “polyethoxylated castor oil', 10 molecular weight of the polyethylene glycol is from about refers to a compound formed from the ethoxylation of castor 200 to about 5000. In some embodiments, the average oil, wherein at least one chain of polyethylene glycol is molecular weight of the polyethylene glycol is from about covalently bound to the castor oil. The castor oil may be 200 to about 900. In some embodiments, the average molecu hydrogenated or unhydrogenated. Synonyms for polyethoxy lar weight of the polyethylene glycol is about 400. Suitable lated castor oil include, but are not limited to polyoxyl castor 15 polyethylene glycols include, but are not limited to polyeth oil, hydrogenated polyoxyl castor oil, mcrogolglycerolirici ylene glycol-200, polyethylene glycol-300, polyethylene noleas, macrogolglyceroli hydroxyStearas, polyoxyl 35 cas glycol-400, polyethylene glycol-600, and polyethylene gly tor oil, and polyoxyl 40 hydrogenated castor oil. Suitable col-900. The number following the dash in the name refers to polyethoxylated castor oils include, but are not limited to, the the average molecular weight of the polymer. In some NikkolTM HCO series (available from Nikko Chemicals Co. embodiments, the polyethylene glycol is polyethylene gly Ltd.), such as Nikkol HCO-30, HC-40, HC-50, and HC-60 col-400. Suitable polyethylene glycols include, but are not (polyethylene glycol-30 hydrogenated castor oil, polyethyl limited to the CarbowaxTM and CarbowaxTM Sentry series ene glycol-40 hydrogenated castor oil, polyethylene glycol (available from Dow), the LipoxolTM series (available from 50 hydrogenated castor oil, and polyethylene glycol-60 Brenntag), the LutrolTM series (available from BASF), and the hydrogenated castor oil, EmulphorTM EL-719 (castor oil 40 25 PluriolTM series (available from BASF). mole-ethoxylate, available from Stepan Products), the Cre As used herein, the term “polyoxyethylene-alkyl ether mophoreTM series (available from BASF), which includes refers to a monoalkyl or dialkylether of polyoxyethylene, or Cremophore RH40, RH60, and EL35 (polyethylene glycol mixtures thereof. In some embodiments, the polyoxyethyl 40 hydrogenated castor oil, polyethylene glycol-60 hydroge ene-alkyl ether is a polyoxyethylene fatty alcohol ether. nated castor oil, and polyethylene glycol-35 hydrogenated 30 As used herein, the term “polyoxyethylene fatty alcohol castor oil, respectively), and the Emulgin R, RO and HRE ether refers to an monoether or diether, or mixtures thereof, series (available from Cognis PharmaLine). Other suitable formed between polyethylene glycol and a fatty alcohol. polyoxyethylene castor oil derivatives include those listed in Fatty alcohols that are useful for deriving polyoxyethylene R. C. Rowe and P. J. Shesky, Handbook of pharmaceutical fatty alcohol ethers include, but are not limited to, those excipients, (2006), 5th ed., which is incorporated herein by 35 defined herein. In some embodiments, the polyoxyethylene reference in its entirety. portion of the molecule has about 2 to about 200 oxyethylene As used herein, the term “polyethoxylated sterol refers to units. In some embodiments, the polyoxyethylene portion of a compound, or mixture of compounds, derived from the the molecule has about 2 to about 100 oxyethylene units. In ethoxylation of sterol molecule. Suitable polyethoyxlated Some embodiments, the polyoxyethylene portion of the mol sterols include, but are not limited to, PEG-24 cholesterol 40 ecule has about 4 to about 50 oxyethylene units. In some ether, SolulanTM C-24 (available from Amerchol); PEG-30 embodiments, the polyoxyethylene portion of the molecule cholestanol, NikkolTM DHC (available from Nikko); Phy has about 4 to about 30 oxyethylene units. In some embodi tosterol, GENEROLTM series (available from Henkel); PEG ments, the polyoxyethylene fatty alcohol ether comprises 25 phytosterol, NikkolTM BPSH-25 (available from Nikko): ethoxylated Stearyl alcohols, cetyl alcohols, and cetylstearyl PEG-5 soya sterol, NikkolTM BPS-5 (available from Nikko): 45 alcohols (cetearyl alcohols). Suitable polyoxyethylene fatty PEG-10 soya sterol, NikkolTM BPS-10 (available from alcohol ethers include, but are not limited to, the BriTM series Nikko); PEG-20 soya sterol, NikkolTM BPS-20 (available of surfactants (available from Uniqema), which includes Brij from Nikko); and PEG-30 soya sterol, NikkolTM BPS-30 30, 35, 52, 56, 58, 72, 76, 78,93Veg, 97,98, and 721, the (available from Nikko). As used herein, the term “PEG” CremophorTM A series (available from BASF), which refers to polyethylene glycol. 50 includes Cremophor A6, A20, and A25, the EmulgenTM series As used herein, the term “polyethoxylated vegetable oil” (available from Kao Corp.), which includes Emulgen 104P. refers to a compound, or mixture of compounds, formed from 123P, 210P, 220,320P and 409P, the EthosperseTM (available ethoxylation of vegetable oil, wherein at least one chain of from Lonza), which includes Ethosperse 1A4, 1A12, TDAao, polyethylene glycol is covalently bound to the the vegetable 5120, and G26, the EthylanTM series (available from oil. In some embodiments, the fatty acids has between about 55 Brenntag), which includes Ethylan D252,253,254, 256,257, twelve carbons to about eighteen carbons. In some embodi 2512, and 2560, the PlurafacTM series (available from BASF), ments, the amount of ethoxylation can vary from about 2 to which includes Plurafac RA20, RA30, RA40, RA43, and about 200, about 5 to 100, about 10 to about 80, about 20 to RA340, the RitolethTM and RitoxTM series (available from about 60, or about 12 to about 18 of ethylene glycol repeat Rita Corp.), the VolpoTM series (available from Croda), which units. The vegetable oil may be hydrogenated or unhydroge 60 includes Volpo N 10, N 20, S2, S10, C2, C20, CS10, CS20, nated. Suitable polyethoxylated vegetable oils, include but L4, and L23, and the TexaforTM series, which includes Tex are not limited to, CremaphorTM EL or RH series (available afor A1P, AP, A6, A10, A14, A30, A45, and A60. Other from BASF), EmulphorTM EL-719 (available from Stepan suitable polyoxyethylene fatty alcohol ethers include, but are products), and EmulphorTM EL-620P (available from GAF). not limited to, polyethylene glycol (13)stearyl ether (steareth As used herein, the term “polyethylene glycol refers to a 65 13), polyethylene glycol (14)stearyl ether (steareth-14), poly polymer containing ethylene glycol monomer units of for ethylene glycol (15)stearyl ether (steareth-15), polyethylene mula —O—CH2—CH2—, Suitable polyethylene glycols glycol (16)stearyl ether (steareth-16), polyethylene glycol US 9,340,532 B2 77 78 (17)stearyl ether (steareth-17), polyethylene glycol (18) PEG-20 glyceryl oleate, TagatTM 0 (Goldschmidt); PEG-30 stearyl ether (steareth-18), polyethylene glycol (19)stearyl glyceryl oleate, Tagat TM 02 (Goldschmidt). ether (steareth-19), polyethylene glycol (20)stearyl ether As used herein, the term “propylene glycol fatty acid ester (steareth-20), polyethylene glycol (12)isostearyl ether (isos refers to an monoether or diester, or mixtures thereof, formed teareth-12), polyethylene glycol (13)isostearyl ether (isoste between propylene glycolor polypropylene glycol and a fatty areth-13), polyethylene glycol (14)isostearyl ether (isoste acid. Fatty acids that are useful for deriving propylene glycol areth-14), polyethylene glycol (15)isostearyl ether fatty alcohol ethers include, but are not limited to, those (isosteareth-15), polyethylene glycol (16)isostearyl ether defined herein. In some embodiments, the monoester or (isosteareth-16), polyethylene glycol (17)isostearyl ether diester is derived from propylene glycol. In some embodi 10 ments, the monoester or diester has about 1 to about 200 (isosteareth-17), polyethylene glycol (18)isostearyl ether oxypropylene units. In some embodiments, the polypropy (isosteareth-18), polyethylene glycol (19)isostearyl ether lene glycol portion of the molecule has about 2 to about 100 (isosteareth-19), polyethylene glycol (20)isostearyl ether oxypropylene units. In some embodiments, the monoester or (isosteareth-20), polyethylene glycol (13)cetyl ether (ceteth diester has about 4 to about 50 oxypropylene units. In some 13), polyethylene glycol (14)cetyl ether (ceteth-14), polyeth 15 embodiments, the monoester or diester has about 4 to about ylene glycol (15)cetyl ether (ceteth-15), polyethylene glycol 30 oxypropylene units. Suitable propylene glycol fatty acid (16)cetyl ether (ceteth-16), polyethylene glycol (17)cetyl esters include, but are not limited to, propylene glycol lau ether (ceteth-17), polyethylene glycol (18)cetyl ether (ceteth rates: LauroglycolTM FCC and 90 (available from Gatte 18), polyethylene glycol (19)cetyl ether (ceteth-19), polyeth fosse); propylene glycol caprylates: CapryolTM PGMC and ylene glycol (20)cetyl ether (ceteth-20), polyethylene glycol 90 (available from Gatefosse); and propylene glycol dicapry (13)isocetyl ether (isoceteth-13), polyethylene glycol (14) locaprates: LabrafacTM PG (available from Gatefosse). isocetyl ether (isoceteth-14), polyethylene glycol (15)iso Suitable sorbitols include, but are not limited to, Pharm cetyl ether (isoceteth-15), polyethylene glycol (16)isocetyl Sorbidex E420 (available from Cargill), Liponic 70-NC and ether (isoceteth-16), polyethylene glycol (17)isocetyl ether 76-NC (available from Lipo Chemical), Neosorb (available (isoceteth-17), polyethylene glycol (18)isocetyl ether (isoce 25 from Roquette), Partech SI (available from Merck), and Sor teth-18), polyethylene glycol (19)isocetyl ether (isoceteth bogem (available from SPI Polyols). 19), polyethylene glycol (20)isocetyl ether (isoceteth-20), Starch, sodium starch glycolate, and pregelatinized starch polyethylene glycol (12)oleyl ether (oleth-12), polyethylene include, but are not limited to, those described in R. C. Rowe glycol (13)oleyl ether (oleth-13), polyethylene glycol (14) and P. J. Shesky, Handbook of pharmaceutical excipients, oleyl ether (oleth-14), polyethylene glycol (15)oleyl ether 30 (2006), 5th ed., which is incorporated herein by reference in (oleth-15), polyethylene glycol (12)lauryl ether (laureth-12), its entirety. polyethylene glycol (12)isolauryl ether (isolaureth-12), poly As used herein, the term “starch” refers to any type of ethylene glycol (13)cetylstearyl ether (ceteareth-13), poly natural or modified Starch including, but not limited to, maize ethylene glycol (14)cetylstearyl ether (ceteareth-14), poly starch (also known as corn starch or maydis amylum), potato ethylene glycol (15)cetylstearyl ether (ceteareth-15). 35 starch (also known as Solani amylum), rice starch (also polyethylene glycol (16)cetylstearyl ether (ceteareth-16), known as Oryzae amylum), wheat starch (also known as tritici polyethylene glycol (17)cetylstearyl ether (ceteareth-17), amylum), and tapioca starch. The term “starch also refers to polyethylene glycol (18)cetylstearyl ether (ceteareth-18), starches that have been modified with regard to molecular polyethylene glycol (19)cetylstearyl ether (ceteareth-19), and weight and branching. The term “starch further refers to polyethylene glycol (20)cetylstearyl ether (ceteareth-20). 40 starches that have been chemically modified to attach chemi The numbers following the “polyethylene glycol term refer cal functionality Such as carboxy, hydroxyl, hydroxyalky to the number of oxyethylene repeat units in the compound. lene, or carboxyalkylene groups. As used herein, the term Blends of polyoxyethylene fatty alcohol ethers with other “carboxyalkylene' refers to a group of formula -alkylene-C materials are also useful in the invention. A non-limiting (O)OH, or salt thereof. As used herein, the term “hydroxy example of a suitable blend is ArlacelTM 165 or 165 VEG 45 alkylene' refers to a group of formula -alkylene-OH. Suitable (available from Uniqema), a blend of glycerol monostearate Sodium starch glycolates include, but are not limited to, with polyethylene glycol-100 stearate. Other suitable poly Explotab (available from JRS Pharma), Glycolys (available oxyethylene fatty alcohol ethers include those listed in R. C. from Roquette), Primojel (available from DMV Interna Rowe and P. J. Shesky, Handbook of pharmaceutical excipi tional), and Vivastar (available from JRS Pharma). ents, (2006), 5th ed., which is incorporated herein by refer 50 Suitable pregelatinized starches include, but are not limited ence in its entirety. to, Lycatab C and PGS (available from Roquette), Merigel As used herein, the term “polyoxyethylene-glycerol fatty (available from Brenntag), National 78-1551 (available from ester” refers to ethoxylated fatty acid ester of glycerine, or National Starch), Spress B820 (available from GPC), and mixture thereof. In some embodiments, the polyoxyethylene Starch 1500 (available from Colorcon). portion of the molecule has about 2 to about 200 oxyethylene 55 As used herein, the term "stearoyl macrogol glyceride' units. In some embodiments, the polyoxyethylene portion of refers to a polyglycolized glyceride synthesized predomi the molecule has about 2 to about 100 oxyethylene units. In nately from Stearic acid or from compounds derived predomi Some embodiments, the polyoxyethylene portion of the mol nately from Stearic acid, although other fatty acids or com ecule has about 4 to about 50 oxyethylene units. In some pounds derived from other fatty acids may used in the embodiments, the polyoxyethylene portion of the molecule 60 synthesis as well. Suitable Stearoyl macrogol glycerides has about 4 to about 30 oxyethylene units. Suitable polyoxy include, but are not limited to, GelucireR 50/13 (available ethylene-glycerol fatty esters include, but are not limited to, from Gattefossé). PEG-20 glyceryl laurate, TagatTML (Goldschmidt); PEG-30 As used herein, the term "vegetable oil” refers to naturally glyceryl laurate, Tagat TM L2 (Goldschmidt); PEG-15 glyc occurring or synthetic oils, which may be refined, fraction eryl laurate, GlyceroxTML series (Croda); PEG-40 glyceryl 65 ated or hydrogenated, including triglycerides. Suitable veg laurate, GlyceroxTML series (Croda); PEG-20 glyceryl Stear etable oils include, but are not limited to castor oil, hydroge ate, CapmulTM EMG (ABITEC), Aldo MS-20 KFG (Lonza): nated castor oil, sesame oil, corn oil, peanut oil, olive oil, US 9,340,532 B2 79 80 sunflower oil, safflower oil, soybean oil, benzyl benzoate, one or more halogen atoms, e.g. fluorine or chlorine or both, sesame oil, cottonseed oil, and palm oil. Other Suitable veg such as trifluoromethyl, difluoromethyl, fluorochloromethyl, etable oils include commercially available synthetic oils such and the like. The hydrocarbon chain may also be interrupted as, but not limited to, MiglyolTM 810 and 812 (available from by a heteroatom, such as N, O or S. Dynamit Nobel Chicals, Sweden) NeobeeTM M5 (available As used herein, the term “aryl' means a monovalent mono from Drew Chemical Corp.), AlofineTM (available from cyclic orbicyclic aromatic hydrocarbon radical of 6 to 10 ring Jarchem Industries), the LubritabM series (available from atoms, and optionally Substituted independently with one, JRS Pharma), the SterotexTM (available from Abitec Corp.), two or three substituents selected from alkyl, haloalkyl, SoftisanTM 154 (available from Sasol), CroduretTM (available cycloalkyl, halo, nitro, cyano, optionally Substituted phenyl, from Croda), FancolTM (available from the Fanning Corp.), 10 —OR (where R is hydrogen, alkyl, haloalkyl, cycloalkyl, Cutina TM HR (available from Cognis), SimulsoITM (available optionally substituted phenyl), acyl, -COOR (where R is from CJ Petrow), EmConTM CO (available from Amisol Co.), hydrogen or alkyl). More specifically the term aryl includes, LipvolTM CO, SES, and HS-K (available from Lipo), and but is not limited to, phenyl, 1-naphthyl 2-naphthyl, and SterotexTMHM (available from Abitec Corp.). Other suitable derivatives thereof. Vegetable oils, including sesame, castor, corn, and cottonseed 15 “Heterocycles' means a saturated, unsaturated, or aro oils, include those listed in R. C. Rowe and P. J. Shesky, matic monovalent ring of 3 to 8 ring atoms in which 1, 2, 3, or Handbook of pharmaceutical excipients, (2006), 5th ed., 4 ring atoms are heteroatoms selected from N, O, or S, the which is incorporated herein by reference in its entirety. remaining ring atoms being C. The heterocyclo ring may be Optical Isomers-Diastereomers-Geometric Isomers-Tau optionally fused to a benzene ring. The heterocyclic ring may tomers-Salts. Compounds described herein may contain an be optionally substituted independently with one or more asymmetric center and may thus exist as enantiomers. Where substituents, preferably one, two or three substituents, the compounds according to the invention possess two or selected from alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, more asymmetric centers, they may additionally exist as dias aryl, aralkyl, halo, cyano, acyl, monosubstituted amino, dis tereomers. The present invention includes all such possible ubstituted amino, carboxy, hydroxyl, oralkoxycarbonyl. The Stereoisomers as Substantially pure resolved enantiomers, 25 heterocycle ring may have 1, 2, or 3 oxo Substitution. racemic mixtures thereof, as well as mixtures of diastere Hydroxyls may exist in the keto or enol tautomer. More omers. The formulas are shown without a definitive stere specifically the term heterocyclo includes, but is not limited to ochemistry at certain positions. The present invention dioxanyl, imidazolidinyl, imidazolyl, morpholinyl, oxazo includes all stereoisomers of Such formulas and pharmaceu lidinyl, oxazinyl, oxadiazolidinyl, oxadiazolyl, piperidinyl, tically acceptable salts thereof. Diastereoisomeric pairs of 30 piperazinyl, pyrrolidinyl, pyrrolyl, dihydropyrazolyl pyra enantiomers may be separated by, for example, fractional Zolyl, tetrahydropyranyl, thiazolyl, thiomorpholinyl, triaz crystallization from a suitable solvent, and the pair of enan olyl and derivatives. tiomers thus obtained may be separated into individual stere This invention and embodiments illustrating the method oisomers by conventional means, for example by the use of an and materials used may be further understood by reference to optically active acid or base as a resolving agent or on a chiral 35 the following non-limiting examples. HPLC column. Further, any enantiomer or diastereomer of a Although the present invention has been described in con compound of the general formula may be obtained by Ste siderable detail with reference to certain preferred embodi reospecific synthesis using optically pure starting materials or ments thereof, other versions are possible. Therefore the spirit reagents of known configuration. Compounds may be neutral and scope of the appended claims should not be limited to the or pharmaceutically salts thereof. Formulas for carboxylic 40 description and the preferred versions contained within this acids and Sulfonamides may be drawn in protonated or unpro specification. tonated forms as the acid oramide, ion, or salt, and that all are RAS proteins may self assemble together with other mem encompassed by a given formula. brane-associated proteins, effectors and scaffolding proteins The term “alkyl, as used herein, unless otherwise speci into plasma membrane tethered microdomains known as fied, refers to a saturated or unsaturated Straight or branched 45 nanoclusters. The nanoclusters may be Small (about 6-20 nm hydrocarbon chain of typically C1 to C10, and specifically diameter) short lived (t1/2 less than about 0.4s) signaling includes methyl, ethyl, propyl, isopropyl, butyl, isobutyl, platforms, and may contain 6 or more proteins. Nanoclusters t-butyl, pentyl, isopentyl, neopentyl, hexyl, isohexyl, cyclo can differ depending upon the charge and covalent lipid modi hexyl, cyclohexylmethyl 3-methylpenty1, 2,2-dimethylbu fication of the C-terminal hypervariable (hv) region of the tyl, 2,3-dimethylbutyl, ethenyl, 2-propenyl, 1-butenyl, 50 individual RAS isoforms. Downstream signaling effectors 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, may be activated by the about 40% of the RAS which is 1-hexenyl, 2-hexenyl and 3-hexenyl and the like. Unsaturated associated in nanoclusters, while the remaining RAS is ran alkyls have at least one double bond, of either E or Z stere domly arrayed over the cell surface. ochemistry where applicable. The term includes both substi RAS proteins undergo several steps of translational modi tuted and unsubstituted alkyl groups. 55 fication which can determine their membrane localization The term "substituted as used herein in reference to a (FIG. 1). RAS may share a C-terminal CAAX motif that can moiety or group means that one or more hydrogen atoms in undergo cysteine residue preneylation (C15 farnesylation or the respective moiety, especially up to 5, more especially 1, 2 C20 geranylgeranylation) followed by removal of the AAX or 3 of the hydrogenatoms are replaced independently of each residues by endoplasmic reticulum (ER) Rce 1 (RAS and other by the corresponding number of the described substitu 60 a-factor converting enzyme-1) and carboxylation by Icmt ents. Alkyl groups can be optionally substituted with one or (isoprenylcysteine carboxyl methyltransferase). These more moieties selected from the group consisting of CAAX modifications by themselves may not be sufficient for hydroxyl, amino, alkylamino, arylamino, alkoxy, aryloxy, RAS plasma membrane association and a second signal may nitro, cyano, Sulfonic acid, Sulfate, phosphonic acid, phos be required. HRAS, NRAS and KRAS4A can undergo C16 phate, phosphonates, optionally Substituted heterocycles, or 65 palmitoylation on cysteine residues in their hV regions cata optionally substituted aryls. One or more of the hydrogen lyzed by ERPATs (protein acyltransferases). In KRAS4B, the atoms attached to carbon atom on alkyl may be replaces by second membrane localization signal can be provided by a US 9,340,532 B2 81 82 lysine rich polybasic amino acid sequence in its hV region that This role of CNKSR1 as a molecular target for drug devel can facilitate an interaction with the negatively charged head opment is shown in FIG. 2A where transfection with siRNA groups of and phosphatidylinositol (PI) on the inner Surface to CNKSR1 (siCNKSR1) may inhibit growth of mut-KRAS of the plasma membrane. PIP3 can be clustered in lipid raft MiaPaca-2 pancreatic cells but not the growth of MiaPaca-2 nanodomains together with high levels of PI3K protein, to give regions of high signaling activity. The binding of the cells, where an allele of mut-KRAS has been disrupted by CNKSR1 PH-domain to PIP3 could serve to position the homologous recombination. siCNKSR1 may also inhibit KRAS nanocluster in close proximity to the PI3K signaling growth of mut-KRASHCT 116 colon cancer cells but not the nanodomain leading to activation of PI3K, a downstream growth of HKE2 HCT116 cells, where mut-KRAS has been signaling effector for KRAS. Some forms of mut-KRAS can disrupted by homologous recombination. Table 1 shows that have a higher affinity for binding to PI3K than wt-KRAS, due 10 the selective inhibition of mut-KRAS cell growth can be to a mutation induced change in the structure of the KRAS validated with a second set of 4 individual siCNKSR1s from Switch 1 and 2 binding regions that form direct contact with a second manufacturer. TABLE 1. Validated hits with individual siRNAs in nut-KRAS isogenic lines MiaPaca-2 Pancreatic HCT-116 Colon % viability % viability Gene mut-RAS siRNAs mut-RAS siRNAs Symbol Name wt-KRAS positive wt-KRAS positive CNKSR1 connector 43.4 3f4 52.6 3f4 enhancer of kinase Suppressor of Ras 1

second manufacturers individual siRNAs the PI-3-K catalytic domain causing allosteric activation. The effect of siCNKSR1 is further shown in FIG. 2B where This could explain the greater sensitivity of mut-KRAS to transfection with siCNKSR1 can inhibit the growth of a panel inhibition by siRNA knockdown of CNKSR1 or PH-domain 30 of 10 mut-KRAS non small cell lung cancer (NSCLC) cell inhibition, than wt-KRAS. lines but not of 4 NSCLC cell lines with wt-KRAS. The PH-domain is an about 100 to about 120 amino acid In order to demonstrate whether the pleckstrin homology three dimensional superfold found in over 500 human pro (PH) domain of CNKSR1 plays a role in facilitating the effect teins. The core of each PH-domain consists of seven B-strands of CNKSR1 on mut-KRAS activity we over expressed the and a C-terminal a-helix. While PH-domains may show a 35 PH-domain in H1373 mut-KRAS NSCLC cells and found highly conserved 3 dimensional organization, the sequence that it acted as a dominant negative and inhibited cell growth. identities among different proteins are only about 7% to about We suggest that the PH-domain fragment competes with the 23%. This is important because with this sequence diversity, full length CNKSR1 in the cell (FIG. 2C). selective agents can be identified that will be specific for each In embodiments, a homology model for the PH-domain of 40 CNSKR1 based on known PH-domain crystal structures can protein. PH-domains can bind to phosphotyrosine and be developed. The docking program PHuDockR) can be used polyproline sequences, GBY Subunits of heterotrimeric G pro to identify potential inhibitors of CNKSR1. Using an in silico teins and phosphoinositides (PIs). While for the majority of library of over 3 million compounds, seven compounds have PH-domain proteins PI binding is weak and non-specific, the been identified as potential inhibitors of CNKSR1 and, thus, PH-domains of many proteins that are components of signal 45 of mut-KRAS cell lines (FIG. 3A). The binding of the com transduction pathways regulating cancer cell growth and Sur pounds to the expressed PH-domain of CNKSR1 (KDobs) vival show high affinity for PIP3 and sometimes PIP2. can be measured by Surface plasmon resonance (SPR) spec CNKSR is one such protein that has high affinity binding for troscopy. Two of the seven identified compounds (com PIP3. In embodiments, the binding of a small molecule to a pounds #4 and #7) exhibit low micromolar inhibition of mut PH-domain may inhibit protein function. 50 KRAS cell growth (FIG.3B). The most active compound was In other embodiments, identifying small molecule PH #7, which inhibited mut-KRAS cell growth as effectively as domain inhibitors using a computational platform may speed siRNA to KRAS or CNKSR1 (FIG. 3C). identification of potential inhibitors and the decrease the costs In embodiments, the binding of identified compounds to of optimizing a drug lead. In such embodiments, the in silico the crystal structures of other PH-domain signaling proteins, molecular docking of libraries of several million chemical 55 AKT, PDPK1, Btk, and Tiam1 can be predicted. In such structures using the known crystal or homology model struc embodiments, the Kds exceed about 100 uM. In other tures of the PH-domain of the protein of interest may be used embodiments, SPR can measure the binding of identified to identify inhibitors of CNKSR1. Surface plasmon reso compounds to the expressed PH-domains of AKT, PDPK1 nance (SPR) spectroscopy can measure the extent of binding and Tiam1. No measurable binding was found for #4 and #7. of the compounds to the PH-domain of the protein, and in 60 Thus, the identified compounds appear have, at least, about vitro cellular assays can determine biological efficacy. Once 50 to about 100 fold selectivity for CNKSR1 compared to the active moieties are identified there may be recursive refine other PH-domains studied. ment of the model through repeated in silico docking and SPR In embodiments, a homology model can predict Small mol spectroscopic measurements of binding until lead com ecules that bind to the PH-domain of CNKSR1, and identify pounds are obtained. Such embodiments may be used to 65 compounds that exhibit selective inhibition of mut-KRAS discover highly specific and potent PH-domain inhibitors of cell proliferation. CNKSR1 inhabitation of K-RAS signaling CNKSR1. can be measured by Western blotting of the down stream US 9,340,532 B2 83 84 target phospho-c-RAF(Ser338) which is specifically phos the pocket of the PH-domain. Thus, providing a competing phorylated by KRAS (FIG. 3D). molecule for that site could diminish its activity in a notice In embodiments, identified compounds may be nontoxic at able manner. about 200 mg/day for about 20 days with no weight loss and The X-Ray structure of a PH-domain target bound to inosi no observable toxic effects for the animal, and may have tol tetraphosphate (IP4) was retrieved from the RCSB (code: antitumor activity (FIG. 4 A). Compound #7 may have anti 1UNQ) and prepared within MAESTRO utilizing the Protein tumor activity against a mut-KRAS H2122 NSCLC tumor Preparation Wizard module. The ligand was extracted and Xenograft in scid mice, where the growth rate of vehicle used as a query for virtual screening using the shape Screening treated tumors (n=10 mice per group) may be about 55 mm/ module of the aforementioned software. Databases of com day and that of compound 7 treated tumors may be about 30 10 mercial vendors were downloaded as SDF files and converted mm/day giving a tumor growth rate inhibition of about 45%. into 3D structures with Ligprep at pH 7 and calculating pro In order to understand better the reasons for the antitumor tomers and tautomers using EPIK. Basic Lipinski’s rules of activity of compound #7 pharmacokinetic studies were con drug-likeness were used to filter out offending compounds. ducted. It was found that compound #7, which is an ethyl The phase shape program within MAESTRO was employed 15 to screen the aforementioned databases. Briefly, conformers ester, administered orally at a dose of 200 mg/kg was rapidly were generated “on the fly' and up to 1000 low energy con de-esterified to the acid metabolite in vivo (FIG.4B), and also formations per each entry in the commercial databases were by mouse plasma (Table 1). Following oral administration in retained and screened. The atom type used was Phase QSAR Vivo plasma concentrations of the parent compound were low, Model due to its remarkable good shape Screening capabili about 3 g/ml (7 uM) whereas the de-esterified acid form ties seen in previous cases. Conformers with similarity below (compound 8) was present at high peak concentrations of 0.7 were discarded and the results ranked based on shape around 50 g/ml (128 uM). When compound 8 was adminis similarity. A large database of approximately 3000 com tered orally to mice at the same dose even higher peak con pounds was retrieved from the method and every single com centrations of 90 ug/ml (230 uM) were achieved. Compound pound was visually inspected to maximize hydrogen bond #7 was eliminated with a halflife of 6 hr and compound 7 with 25 pattern, maximum overlap of functional groups and overall a half life of 13 hr. Because compound #8 is inactive in cells shape and geometry of the molecules. A Subset of these com in culture (see FIG. 5) it is likely that the rapid conversion of pounds were tested; the compounds are as follows: compound #7 to its inactive metabolite compound 8 limits its in vivo activity. It is noteworthy that compound #7 was more stable in dog 30 13 and bovine serum and completely stable in human plasma O which might lead to less metabolism in human, although it was broken down by human carboxylesterases 1 and 2 (Table 21 O 2) which are found in human intestine, liver and tumor. 35 N In order to develop more stable analogs of compound 7 we N N further modeled and Nus/ TABLE 2 40 Stability of compound 7 in biological media 14 Halflife (min) Mouse plasma 6.3 Dog plasma (beagle) 558 Human plasma stable 45 10% fetal bovine serum 790 rHuman carboxyesterase 1 24 Uml# 19 rHuman carboxyesterase 2 24 Uml# 99 *compound 7 concentration 50 ugml, temperature 37°C., it pH 7.4, 1 U = 1 nmol/min 15 synthesized a group of compounds with a rigid non-hydrolys 50 able group in place of the ester functionality (FIG. 5, com pounds 9, 10, 11). All three compounds inhibited the growth of mut-KRAS NSCLC cell lines but also inhibited wt-KRAS cell growth. Compound #10 was the most potent and showed 55 approximately two fold selectivity for mut-KRAS cells com pared to wt-KRAS cells. 16 Through further modeling and screening using an optimal CNKR1 model (FIG. 7) a new pharmacophore was identified (Table 3 compound 35) identified as #12 in FIG. 5, that 60 inhibits the growth of wt-KRAS and mut-KRAS cells more potently than compound 7 or its analogs. In this study a ligand-based method that takes into account molecular shape of a query molecule and the pharmacophoric features (accep tor, donor, hydrophobic, aromatic, etc.) of its functional 65 groups was launched. The underlying hypothesis is that molecular entities such as inositol X-phosphate may bind in US 9,340,532 B2 85 86 -continued -continued

29 US 9,340,532 B2 87 88 -continued -continued 36

40 US 9,340,532 B2 89 90 -continued -continued 41 46

O O ON/ / No O 10 OS 47 Br O

15 O

51 45

65 US 9,340,532 B2 91 92 -continued -continued

63 US 9,340,532 B2 93 94 -continued -continued 64 N 72 O O

O S O 10 65

15 73 O O

S 66 O X-OH N

O 25 74

67 30 ON YS Br 35 r 21 O | 68 N

40 75

CN 45

70 N21 O

50 “rs N O | N

71 60

65 and

US 9,340,532 B2 97 98 TABLE 4 flasks via trypsinization and plated into 96-well plates at an initial density range of 2000 cells per well. Cells were allowed Compounds modeled and identified based on hybridization 24 hours to attach, and then the agents were added to the of Compound 7 and 12 culture media at a range of concentrations from 0-100 uM. COOH 1 Cells were incubated for 72 hours with the drugs, and then viability was assessed using an MTS viability assay. Cells were exposed to MTS reagent (Promega) dissolved in PBS COOH (Hyclone) at a concentration of 200 uL reagent/mL media for 2 hours. Absorbance was then read at 490 nm, and viability 10 was expressed as a percentage normalized between the nega - tive control (no cells plated) and the condition of cells with no drug added (100% viability) normalized as the upper limit of OH viability. COOH 2 Screening of Compounds Against NSCLC Cell Line Panel 15 (FIGS. 2 and 3B). Our panel of 30 cell lines and an extensive characterization COOH were obtained from Dr. John Minna (UTSW). All cell lines were cultured in RPMI 1640 with 10% FBS. Cells were O treated with concentrations of agents at concentrations 0.01 to - 50 uM and evaluated as described above. ICsos were deter OH mined using Excelfit. siRNA Screening COOEt 3 MiaPaca-2 and M27 were confirmed mycoplasma and maintained in DMEM with 10% FBS. Optimization was car 25 ried out using in house optimization methods in house. A parallel screen was then carried out with a genome wide O COOH siRNA library (Dharmacon). Individual siRNA and Plasmid Transfection (FIG.3C). - For transfection in a six well plate, cells were plated at OH 30 100,000 cells per well in 2 mls media and allowed to attach overnight. Per well 5 ul of Dharmafect 2 (Dharmacon) was COOEt 4 added to 200 ul OptiMEM (Gibco) and 4 ul of the siCNKSR1 smartpool Dharmacon (M-0122 17-01-0020) or individual NN siCNKSR1 siRNAs (Qiagen SIO2665411) was added to 200 35 uL to OptiMEM in parallel and allowed to sit for 5 minutes. These tubes were mixed and incubated at room temperature Ns1Ncoon for 20 minutes. 1.6 of the appropriate media was then added to this mixture. and then media in the wells removed. This O mixture was then added to the cells in a dropwise fashion and O. O. S 5 40 the cells were incubated for 48-72 hours. For the GFP control and CNK1 PH-domain plasmids 175,000 cellsper well plated in a 6 well plate. Perwell 2.5ul of lipofectamine 2000 (Gibco) and 125 ul of OptiMEM were combined and 2.5 ug of the appropriate plasmid and 125ul of OptiMEM were combined 45 in separate tubes and allowed to incubate at room temperature for 5 minutes. These two tubes were then combined and allowed to incubate for 20 minutes. 200 ul of this mixture was then added to 1 ml of fresh media already in the appropriate well and allowed to incubate for 5 hours. The transfection 50 efficiency was determined through the expression of GFP after 24 hours and the cells were counted with a hemocytom eter after 72 hours to determine viability. Spheroid Formation (FIG.9) The plates were optimized for the best cell density and 55 found to be 20,000 cells per mL. The lid was removed from a 96-well Greiner plate and turned upside down. 20 uL of the 20,000 cells per mL suspension was then added directly into the middle of the circles found on the lid of the 96-well plate forming a small drop. 100 uL of media was added into the 60 corresponding wells, used to maintain the temperature of the Experimental Description drops, and the lid was flipped back over carefully placing it Screening of Compounds Against Isogenic Mutant KRAS back onto the plate without disturbing the drop. The plate was Lines (FIG. 2). then placed into the incubator for 3 days to allow the cells to The isogenic KRAS lines harboring G12D, G12C, and migrate to the bottom of the drop due to gravity. After 3 days, G12V were obtained from Horizon Discovery labs on a one 65 400 uL of media was added to the corresponding wells a year lease. These cells were cultured in McCoys media with SCIVAX96-well plate. The lid from the Greiner 96-well plate 10% FBS to 80% confluency. Cells were then released from was removed and placed onto the SCIVAX plate allowing the US 9,340,532 B2 99 100 drop to come in contact with the media and placed back into for 40 min. Proteins were electrophoretically transferred to a the incubator. After one hour, 200 uL of media was removed nitrocellulose membrane; preincubated with a blocking from the corresponding wells carefully without disturbing the buffer of 137 mmol/L NaCl, 2.7 mmol/L KC1, 897 mmol/L spheroid and imaged. CaCl2, 491 mmol/L MgCl2, 3.4 mmol/L Na2HPO4, 593 Confocal Imaging (FIGS. 10 and 12) 5 mmol/L KH2PO4, and 5% bovine serum albumin; and incu HEK293T cells were co-transfected with CNK and either bated overnight with anti-phosphorylated Thr308-Akt, wild type or G12D mutant KRAS. Twenty-four hours post Ser473-Akt, anti-CRaf Ser 338 Mapk Thr202/Tyr204, p70 transfection, cells were seeded on glass coverslips and S6K Thr389 or anti-Akt. (Cell Signaling 1:1000), anti allowed to grow a further 24 h and then serum deprived CNKSR1 (Signal Transduction labs) anti-lamin A/C and anti overnight. Cells were fixed with 4% (w/v) paraformaldehyde 10 B-actin (Santa Cruz, Biotechnology 1:2000Donkey anti-rab pH 8.0 for 20 minat room temperature. Following 6-7 washes bit IgG peroxidase-coupled secondary antibody (GE with PBS (pH 8.0) the coverslip was mounted onto a slide with mounting medium (0.1% p-phenylenediamine/75% Healthcare) was used for detection). For measurement of glycerol in PBS at pH 7.5-8.0). Confocal laser scanning active RalA and RalB, Ral and RalB activation kits were used microscopy was performed with a Leica SP5 confocal micro 15 (Biorad). Band density was measured using the Renaissance Scope system with 63x oil-immersion objective (numerical chemiluminescence system on Kodak X-Omat Blue ML aperture NA=1.4), a line scan speed of 600 Hz, with image films (Eastman Kodak). size of 1024x1024 pixels. GFP was excited with an argon A commercially available docking package, GOLD visible light laser tuned to 488 nm, mRFP were excited with (GOLD 3.2), CCDC: Cambridge, UK, 2007) was used to a krypton laser tuned to 543 nm GFP and RFP fluorescence evaluate the docking of compounds 1-7 into the binding emissions were collected using a photomultiplier tube via pocket, see e.g. Table 5. Other docking was performed using 514/10 nm and 595/10 nm band selections respectively. modeling algorithms with state-of-the-art commercial drug Fluorescence Lifetime Imaging Microscopy (FLIM) (FIGS. discovery software (Schrodinger suite). GLIDE was chosen 11 and 13) as the docking algorithm used to select and optimize com FLIM experiments were carried out using a Leica TCPSP5 25 inverted advanced confocal microscope system with internal pounds, providing a GlideScore as a rough estimate of bind photomultiplier tube (PMT) detector for TCSPC (time-cor ing affinity that was used to rank and select the best com related single-photon counting). The sample was excited with pounds. Additionally, ligand-based approaches provided an a tunable femtosecond (fs) titanium-sapphire laser with rep alternative to structure based drug discovery. Ligand-based etition rate of 80 MHz and pulse width less then 80 fs (Spec 30 virtual screening methodologies can take into account shape tral Physics, Mai Tai BB). The wavelength used for two and electrostatics (like ROCS) and the pharmacophoric fea photon excitation was 930 nm and the fluorescence was tures (acceptor, donor, hydrophobic, aromatic, etc.) of its detected through a 525+25 nm interference filter. Images functional groups. Inositol tetraphosphate (IP4) binding to were obtained with oil-immersion objective (numerical aper the PH-domain of CNKSR1 provided a good starting point ture NA=1.4), a line scan speed of 400 Hz, with image size of 35 for shape screening. Both structure-based and ligand-based 512x512 pixels. For FLIM analysis the pixels were reduced to approaches were used to find novel compounds (Table 7) and 256x256. FLIM data was collected using Becker & Hickl to refine and improve lead compounds (Tables 8, and 9). SPR SPC830 data and image acquisition card for TCSPC. The interaction analyses for Compounds 1 through 7 were per fluorescence decays were fitted with a single exponential formed with a Biacore 2000, using Biacore2000 Control Soft decay model using Becker and Hickl’s SPCImage software 40 ware v3.2 and BIAevaluation v4.1 analysis software (Bia and the GFP fluorescence lifetimes were displayed in a false core) as described in Mol Cancer Ther 7:2621 (2008). SPR colour map. interaction analyses of all other compounds was undertaken Surface Plasmon Resonance Spectroscopy Binding Assays using a Biacore T100 with Control and Evaluation software (Binding Scores for all Agents) kit. All interaction analyses were done with a Biacore T100 45 Control Software v3.2, and BIAevaluation v4.1 analysis soft ware (Biacore). The PH-domain His-fusion proteins (CNK1 and AKT1) were expressed and immobilized on a NTA chip Synthetic Scheme I to a level of 10,000 response units or less. Small molecule CHO CHO analytes at concentrations ranging from 50 uM to 0.010 LM 50 were injected at a high flow rate (30 uL/min) DMSO concen He 1. He trations in all samples and running buffer were 1-5% (v/v) (30 OH O O LL/min) DMSO concentrations in all samples and running buffer were 1-5% (v/v). OMe OMe Immunoblots and Immunoprecipitations (FIGS. 3D, 14 and 55 I II 15) Cells were washed twice with ice-cold PBS and lysis buffer containing 50 mmol/L HEPES (pH 7.5), 50 mmol/L NaCl, 0.2 mmol/L NaF, 0.2 mmol/L sodium orthovanadate, 1 Br Br mmol/L phenylmethylsulfonyl fluoride, 201g/mL aprotinin, 60 CHO CHO 20 g/mL leupeptin, 1% NP40, and 0.25% sodium deoxycho late. Protein concentration was determined by bicinchoninic --- acid assay (Pierce Biotechnology) and 50 lug of cell lysate O lsO OH protein were boiled for 5 min with denaturing buffer contain ing 0.25 mol/L Tris (pH 6.8), 35% glycerol, 8% SDS, and 65 OMe OMe 10% 2-mercaptoethanol, loaded on a 10% acrylamide/ III IV bisacrylamide gel, and separated by electrophoresis at 150 V US 9,340,532 B2 101 102 -continued Br SCH Synthetic Scheme II NO CHO NO CHO CN

Hos 5 -e- OH OH ON OMe V o: O XI s1 O 10

NO N OEt --

OH 15 C XIII OMe VII s1 O

NH2 N OEt XII --

OH 25 OMe VIII

S 2 O O 30 se s1 O HN N OCH5 --- 35 OH XIV OMe IX O

40 S 2 HN1 \ S O O se s1 O NN N HN S. OH 45 O Pl OH S N N

OMe 50 X

Compounds in accordance with embodiments may be pro duced as shown in Synthetic Scheme I. The 2-hydroxy-3- 55 methoxybenzaldehyde I was protected by acylation to give XVI compound II, then brominated to give compound III, and deprotected to give compound IV. Nitration of compound IV Compounds in accordance with embodiments may be pro gave the nitrobenzene V, which reacted with an alkylsulfide to duced as shown in Synthetic Scheme II. The methyl 2-cy give the thiol ether VI. Wittig reaction of the aldehyde to the 60 anobenzoate XI was reacted with a hydrazine equivalent to unsaturated ester VII followed by a reduction gave the aniline give the azaisoquinolone XII. The azaisoquinoline was alky ester VIII (compound 107). The aniline was sulfonylated to lated with the chloride XIII to give the coupled compound give the thioamide IX (compound 103), and the ester hydro XIV. The free amine of the coupled compound XIV was lyzed to give acid X (compound 104). Synthesis of analogs 65 sulfonylated with the acid chloride XV to give the thioamide 103-110 may be readily prepared by a person of skill in the art XVI (compound 5). Synthesis of analogs may be readily of organic synthesis. prepared by a person of skill in the art of organic synthesis. US 9,340,532 B2

Synthetic Scheme III ->O OoN C. O NH2 OEt EtO O O S N e XVII LiAlH4, THF -e- Br EtOH S / 0° C. 1 hr reflux, 2 hr 78% 70% XVIII XIX OH C

N N e SOCl e / HerTHF / r. t. 2 hr 82% XX XXI EtOC H O 2 O

O (Ph)P=CHCOEt O NH-NH2 t reflux, 18 hr, CHCl3 EtOH, N 54% reflux, 3 hr 2 NaH 89% DMF O O Or.t. 1 hr 73% r XXII XXIII O O O 10%KOHA N1Ne.N. 1-N EtOH ^-N - N S N/ - N S-/ dioxane - N S-/ r.t. 2 hr. O O O () OH r N

XXVI XXV XXIV

Compounds in accordance with embodiments may be pro out in 73% yield by the action of sodium hydride in dimeth duced as shown in Synthetic Scheme III. The bromoketone ylformamide at room temperature for one hour to give the XVIII was reacted with the thioamide XVII in ethanol at N-alkylated isoquinazaline XXIV. Sapolification with 10% reflux for two hours to give the thiazole ester XIX in 70% 55 potassium hydroxide in ethanol and dioxane at room tem yield. The thiazole ester was reduced with lithium aluminum perature for two hours gave the carboxylic acid XXV (com hydride in tetrahydrofuran at 0°C. for one hour to give the pound 8). Amide formation with N-methylpiperazine gave benzylic alcohol XX in 78% yield. The benzylic alcohyl was the amide XXVI (compound 123). Compound 123: Molecu displaced to give benzylic chloride XXI in 82% yield by 60 reaction with thionyl chloride in tetrahydrofuran at room lar Formula C.H.N.O.S: Melting Point: 135.8° C.; NMR temperature for two hours. Wittig reaction of phthalic anhy Analysis: "H NMR (600 MHz, CDC1) & 8.54 (d. J=7.8, 1H), dride in chloroform at reflux for 18 hours provided the unsat 8.07 (d. J=7.9, 1H), 7.91 (t, J=7.6, 1H), 7.86 (t, J=7.5, 1H), urated lactone XXII in 54% yield. Reaction of the unsaturated 7.70 (d. J=7.7, 2H), 7.46 (s, 1H), 7.22 (d. J–7.7, 2H), 5.81 (s, lactone with hydrazine in ethanol at reflux for three hours 65 1H), 5.68 (s, 1H), 4.65 (d. J=12.1, 1H), 4.32 (d. J=12.0, 1H), provided the oXoisoquinazaline XXIII. Coupling the oXoiso 4.08 (s. 2H), 3.63 (s, 1H), 3.40 (d. J=10.7, 1H), 3.20 (s, 1H), quinazaline XXIII with the benzyl chloride XXI was carried 3.12 (s, 1H), 2.50 (s, 1H), 2.38 (d. J=14.8, 4H), 2.30 (s.3H). US 9,340,532 B2 105 106 -continued Synthetic Scheme IV CHO CHO O OH O 5 O OMe

OH O O XXV XXVI 3. OH OH 10 O O CHO O NH

O O 15 XXVII XXVIII a Ns OP OP

CHO O CHO O XXXIII OH He- 2O

O O O OMe

NO O XXIX XXX 25 u O 's -NH OMe 30 so O a S

X XXXIV O p = Bn 35 NO XXXI Compounds in accordance with embodiments may be pro duced as shown in Synthetic Scheme IV. 2,3-Dihydroxyben Zaldehyde XXV was ketalized with formaldehyde to give the O 40 aryl dioxole XXVI, and the aldehyde oxidized to give the phenol XXVII. Acylation of the benzyl protected phenol with O OMe a formate equivalent gave the benzaldehyde XXIX, which was nitrated to give the nitrobenzaldehyde XXX. The alde O hyde was conjugated to give the unsaturated ester XXXI, and 45 reduced to the anilino ester XXII. Sulfonylation gave the thioamide XXXIII (compound 85), which was saponified to O the carboxylic acid XXXIV (compound 83). Similarly, ana logs 80-90 may be prepared by a person of skill in the art of NH2 organic synthesis. A person of skill in the art of organic XXXII 50 synthesis can readily prepare other claimed compounds by processes similar to those in Schemes I-IV. TABLE 5

Initial Screening Hits

Mean ICso Biacore ICso mut- nut Gold Log KD KRAS KRAS Cpd Fitness P Structure (IM) (IM) witKRAS

117 51.97 3.89 O.7 O.2 21.95.7 O.9

C US 9,340,532 B2 107 108 TABLE 5-continued

Initial Screening Hits

Mean ICso Biacore ICso mut nut Gold Log KD KRAS KRAS Cpd Fitness Structure (IM) (IM) witKRAS

118 56.06 4.28 3O3 + 1.2 49.3 O.6 1.O

119 52.68 S.21 1.O

C 120 52.33 2.26 in O 3.31.2 47.O-52 O Me

S NN-O NS/N

121 51.98 3.43 5.2 2.6 46.73.3 N1 NSN \ N/ N / O O O

122 SO.16 3.92 F 20.2 - 0.8 >50 1.O CN O

51.72 4.57 COOEt 18 0.6 23.92.5

US 9,340,532 B2 115 116 TABLE 6-continued Analogs of Compound 7

NSCL cell line cytotoxicity* Mouse CNKS AKT ICso (LIM Pharmkinetics (Iv KD KD wt- mut- to C Cpd Structure (IM) (M) KRAS KRAS min ml/min Kg

123 O ND ND l8 l 327 31

Me - N rty-OnS/

O C Me 4-(2-(4-methylpiperazin-1-yl)-2- oxoethyl)-2-((4-p-tolylthiazol-2- yl)methyl)phthalazin-1 (2H)-one

ND no binding determined; iv intravenous; UN unstable is plasma; * mean of 3 wt-KRAS and 3 mut-KRAS; no not analyzed

TABLE 7 TABLE 7-continued 35 Curated Diversity Set Curated Diversity Set ICso ICso ICso ICso Com- CNKKD U- AKT KID U- nut- WT- Com- CNKKD U- AKT KID U- nut- WT pound M Value M Value KRAS KRAS pound M Value M Value KRAS KRAS 61 101* 106 12 128 * 106 43 >100 >100 40 76 113 * 10 20 Bound Bound >100 >100 34 ND 95 Bound Bound 12 3.64 * 106 4 Bound Bound 17 12 67 1.96 * 10 43 Bound Bound 56 5.34 * 10-6 2 4.74 * 104 2 >1OO >100 64 ND 95 Bound Bound 66 ND 73 Bound Bound ND = no binding determined

TABLE 8

Analogs modeled from Second Series Hits

CNK PLE AKT CV Mol KD KD KD Structure No WT (IM) (M) (M) IUPAC Name

COOH 83 385 l8 l8 l 3-(4-methoxy-7- (thiophene-2- Sulfonamido)benzod1, 3dioxol-5-yl)propanoic acid MeO O\/ O N 1 S H

US 9,340,532 B2 123 124 TABLE 8-continued

Analogs modeled from Second Series Hits

CNK PLE AKT CV Mol KD KD KD Structure No WT (IM) (M) (IM) IUPAC Name

ON 12S 436

HN 126 406

127 469 US 9,340,532 B2 125 126 TABLE 8-continued

Analogs modeled from Second Series Hits

CNK PLE AKT CV Mol KD KD KD Structure No WT (IM) (IM) (IM) IUPAC Name

91 437 OH y O

OES NH So S N

O 128 568 OH -N y 21 O

1 N-s OS No S N

O OMe 129 411 21 y O

OES NH so S N

O OMe 130 411 21 y

HN O s s so US 9,340,532 B2 127 128 TABLE 8-continued

Analogs modeled from Second Series Hits

CNK PLE AKT CV Mol KD KD KD Structure No WT (IM) (IM) (IM) IUPAC Name

OMe 131 369 HO 21 y

HN O s NS No

470 SCH

OMe

457 SCH

OMe

100 424 OH

US 9,340,532 B2 141 142 -continued

Summary Table 10 for CNKSR1 inhibitors Proliferation Assay (IC50) IM Surface Plasmon Resonance

KRAS RU * 100, Da

KRAS mutant wildtype CNKSR1 CNKSR1 cell lines cell lines 11 26 13 12 09 13 compound A549 H1373 H2122 H1975 H226 binding stability binding stability

28 - OO OO OO OO 1.409 O456 -0.541 -0.304 29 & OO OO OO OO -0.133 -0.041 30 3 OO OO OO -O.251 -018O 31 2 OO OO OO -0.789 -0.044 32 72 60 OO 60 O.457 O.906 33 > OO OO OO 95 -1537 -0.123 34 OO OO OO OO O.383 O.152 35 OO OO OO OO -OOOS O.046 36 45 OO OO OO -2.252 -O.O79 O.O72 0.06S 37 OO OO OO OO -0.712 O.O76 38 OO OO OO OO 1874 -O.O85 39 OO OO OO OO 3.026 O.151 40 OO OO OO OO 4.572 O.06S 41 OO OO OO OO 4.246 O.239 42 OO OO 23 OO 2.690 O.124 43 OO OO OO OO 2.62O O.O13 44 OO OO 75 OO O.2OS O.122 45 OO OO OO OO 2.973 O.108 46 OO OO OO OO 2.842 O.O17 47 75 OO OO OO 3.085 -O.O87 48 47 77 OO 75 2.304 O.167 49 8O 40 OO OO 4.228 1.S2O 50 > OO OO OO OO 3.695 O.236 51 OO OO OO OO 0.585 O485 52 > OO OO 19 OO 4.8O8 O.458 53 2O 45 11 72 11.460 O.245

Proliferation: N = 2 experiments per cell line (except H226 and H1975 are n = 1 currently) binding =RU max, stability = RU max post-injection SPRRU data obtained under different conditions on each date. “blank cell means not evaluated that day,

What is claimed is: 45 1. A compound of Formula IIA: O O O

Formula IIA 50 x -sk O N\,

55 sk s R is C-C-alkyl, or—Cs-Cs cycloalkyl; R7 is Hor wherein 60 R" is -H, -C-C alkyl, S R5 is ---O 65 Cscycloalkyl, Cscycloalkyl, or a pharmaceutically acceptable salt thereof. US 9,340,532 B2 143 144 2. The compound of claim 1, wherein R is methyl. -continued 84 3. The compound of claim 1, wherein R is COOH 21

MeO & Q J O O \/ 10 N1 S2 4. The compound of claim 1, wherein R is C 85 Calkenyl-OH, or—C Calkenyl-C(O)—C-C alkyl. COOEt

5. The compound of claim 1, wherein R is 15

MeO O O

N \/1 S

86 COOEt 6. The compound of claim 5, wherein R is cyclopropyl or 25 cyclobutyl. 21

7. The compound of claim 1, wherein R is H. MeO 8. The compound of claim 1, wherein R is O O 30 \/ N1 S

87 x-R 35 COOEt

HO 40 9. The compound of claim 1, wherein R is O\/ O N H O N\, 45 88 x -sk 50 HO

10. The compound of claim 1, selected from:

55 83 89 COOH

60 MeO HO

65 US 9,340,532 B2 145 146 -continued -continued 90 O OMe COOH Ds, 21 y

HO O

O=S( NH 10

91 15 -->

Crity5. \,/ S 25

30 ^ O

35 OES NH prico So Y SO S S N 40

O OMe

45 1No 21 y

OS -NH 50 So S N

O OMe

rHN O , s s so 65 US 9,340,532 B2 147 148 -continued -continued OMe HO 21 y

HN O s s so 10 -N

O OH -N 15 JC- xts/- O

x RO;

25 R'' is H or C-C alkyl: R'' is C-Calkyl: or a pharmaceutically acceptable salt thereof. O 12. The compound of claim 11, wherein R is methyl. A, 21 O 30 13. The compound of claim 11, wherein R' is C(O)C) C-C alkyl. 14. The compound of claim 13, wherein the C-C alkyl is ethyl.

s S NH 35 15. The compound of claim 11, wherein R' is

O O

40 NY. 11. A compound of Formula IIIA: >>{O Formula IIIA 45 OR9 16. The compound of claim 11, wherein R' is / S OH O R12 le canS N 4\o 50 H S n Me

55 wherein Ris-H or C-C alkyl: R' is –C(O)O C-C alkyl, -C(O)OH, 60 O NY. 65 US 9,340,532 B2 149 150 18. The compound of claim 11 selected from: -continued OH

S OH No 5 Clle /O S / > OH / nSN 21 OH, le / s S O S O N /NN 21 N-1 10 O O H / is SCH SN O H NN N le S -N S. isk d O / \, O , all / > OH 15 OH O le 4. 21 O OMe n s O

n OH 2O le S -N N SK OH / \, O / S OH OH O le 4. O s 25 OMe WYN 21 N-1 O H 19. The compound of claim 1, wherein Ris—C-Calkyl SN O OH, or—C-Calkyl-C(O)—C-C alkyl. k k k k k