Patent Application Publication

US 20150.175558A1

(19) United States

(2) Patent Application Publication (10) Pub. No.: US 2015/0175558 A1 (43) Pub. Date:

Stockwell et al.

(54) QUINAZOLINONE-BASED ONCOGENIC-RAS–SELECTIVE LETHAL COMPOUNDS AND THEIR USE

(71) Applicant: THE TRUSTEES OF COLUMBIA UNIVERSITY IN THE CITY OF

NEW YORK, New York, NY (US) (72) Inventors: Brent R Stockwell, New York, NY (US); Matthew Welsch, New York, NY (US); Wan Seok Yang, New York, NY (US) (21) Appl. No.:

14/414,669

(22) PCT Filed:

Jul. 12, 2013

(86)

PCT/US13/50244

PCT No.:

§ 371 (c)(1), (2) Date:

Jan. 13, 2015

Related U.S. Application Data (60) Provisional application No. 61/671,602, filed on Jul. 13, 2012. Publication Classification

(51) (52)

Int. Cl. C07D 239/91 U.S. CI.

(2006.01)

CPC .................................... CO7D 239/91 (2013.01)

(57)

Jun. 25, 2015

ABSTRACT

The present invention provides, interalia, compounds having the structure (1) compositions containing such compounds are also provided. Methods for using such compounds or compositions for treating or ameliorating the effects of a cancer having a cell that harbors an oncogenic RAS mutation, for modulating a lipoxygenase in a ferroptosis cell death pathway, and for depleting reduced glutathione (GSH) in a cell harboring an oncogenic RAS mutation are further pro vided. (1)

Patent Application Publication

Jun. 25, 2015 Sheet 1 of 62

Fig. 4

Metabolite Profiling

oxidized glutathione * ~ reduced glutathione

US 2015/0175558 A1


Jun. 25, 2015 Sheet 2 of 62

US 2015/0175558 A1

Fig. 4 continued

b

1001° e HT-1080

3 80

*:::,

.

§ 60

i.

•

*.

... *

£ 40

tj-2 OS

\

*

*I

•

`e

•

$º 20

`s

0–

ºx

#

*

*

*

*

S Sº Sº Sº sº sº S sº S 3 AS [Erastin], pu?/

[Erastin], HM


Jun. 25, 2015 Sheet 3 of 62

Fig. 1 continued

US 2015/0175558 A1


Jun. 25, 2015 Sheet 4 of 62

US 2015/0175558 A1

Fig. 3 continued

d

150

-*—

3.

i

*{i} 100–

§

.*

9

-

5 50 ? 0.

:

:

9 3 × 5 # ºf 3 gº

2&5# ?º

X.

*

?hq

{ }

2

gºt 26

K.

2^vite

... …

... 3.3

– 1 3. O

… tº cº, º2.

: +:

>

crºcoProM

Neº 9.

{. .} §§ 3.3%

Spearman R = 0.8638 (P = 0.0001)

1

10

100

Viability (%), 10 puM Erastin


Jun. 25, 2015 Sheet 7 of 62

US 2015/0175558 A1

Fig. 2

F------------------------------------------>

——

BJeH BJeH LI BJeLR *

— RASV12

+ RASV12


Jun. 25, 2015 Sheet 8 of 62

US 2015/0175558 A1

Hig. 2 continued

Beit *getti

§: 3:33 120

333 80&

# 60

3: 43 & 23

§{º}§ 0a &# depieters

3tºr attioxida?titäätors


Jun. 25, 2015 Sheet 9 of 62

Fig. 2 continued

1

3.

1.0 0. 0. O

US 2015/0175558 A1


Jun. 25, 2015 Sheet 10 of 62

US 2015/0175558 A1

Fig. 2 continued

d G#P-At (3×3 totation; £rastinia; 30 tº

$3%

{}


Jun. 25, 2015 Sheet 11 of 62

US 2015/0175558 A1

Fig. 2 continued

§383, 23 gift


Jun. 25, 2015 Sheet 12 of 62

Fig. 2 continued

Lipid peroxides level (fl. 1) £rastin] = 0, º, tº añº

US 2015/0175558 A1


Jun. 25, 2015 Sheet 13 of 62

Fig. 2 continued

&

tº . . | - - - - - - }~ Pi3-146476 º

|-- AA-861

|-m Zileuton T/

-

US 2015/0175558 A1


Jun. 25, 2015 Sheet 14 of 62

Fig. 3

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Jun. 25, 2015 Sheet 15 of 62

US 2015/0175558 A1

Fig. 3 continued

GFP-ALOX5 focation: RSL3] :

: {{}

-RAS”


Jun. 25, 2015 Sheet 16 of 62

US 2015/0175558 A1

Fig. 3 continued

|

º, --AA-866 / tº Zileuton ºf -

*

i_OX inh


Jun. 25, 2015 Sheet 17 of 62

Fig. 3 continued

Lipid peroxides level { F.?1) {RSL3] = 0, º, ø, 0.2,0.4 pulvi

US 2015/0175558 A1


Jun. 25, 2015 Sheet 18 of 62

US 2015/0175558 A1

Fig. 3 continued

Lipid peroxides level IsiNeg] = 6 nM [sigPX4] = 6


Jun. 25, 2015 Sheet 19 of 62

Fig. 3 continued

-Ai QX3 tºtation; isã8A3 - 6.4 n.8%

US 2015/0175558 A1


Jun. 25, 2015 Sheet 20 of 62

Fig. 3 continued

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Jun. 25, 2015 Sheet 21 of 62

US 2015/0175558 A1

Fig. 3 continued

0.4 nM

1.6 nM

[sigPX4]

6.4 nM


Jun. 25, 2015 Sheet 22 of 62

US 2015/0175558 A1

Fig. 4

Cell death rescue by ALOX inhibitors (AAUC)


Jun. 25, 2015 Sheet 23 of 62

Fig. 4 continued -A?X5 totation

US 2015/0175558 A1


Jun. 25, 2015 Sheet 24 of 62

US 2015/0175558 A1

Fig. 4 continued {{}{}

| non-RSig RSis

4.{}

2

nort-R&is RSis


Jun. 25, 2015 Sheet 25 of 62

US 2015/0175558 A1

fig. 4 continued

d

Metabolic stability ~~ ~k,’sºn 2

C. Nº ºn

Ns Solubility

N” pf (piperazine erastin)

Pºe2öe-s-s & 63 & Time imin}


Jun. 25, 2015 Sheet 26 of 62

US 2015/0175558 A1

Fig. 4 continued

ãº

ºš

w

§ég-3

#

######

§§ §§§§

£323

+ £3.4 w

~343.2


Jun. 25, 2015 Sheet 27 of 62

US 2015/0175558 A1

Fig. 4 continued

f *&

HT-1080 turnor

s 1.5 5

A

s

vehicle Vehicle

5#

A

A

{...} .*

-

3

:

.3

?º

vehicle ERA

Vehicle

PE

Vehicle

PE


Jun. 25, 2015 Sheet 28 of 62

Fig. 4 continued

14 RSLs + onc-RAS (sigPX4)

Ferroptosis

US 2015/0175558 A1


Jun. 25, 2015 Sheet 29 of 62

US 2015/0175558 A1

&

3.

|i 2

--

ºr.

3

#3:33.83, 3%

d :33

§

2

i: -?

º i.

3. 8: 4.

: 2.

3

§§§3%.3.8%


Jun. 25, 2015 Sheet 30 of 62

US 2015/0175558 A1

$28: -e- 3:2;

$383 & 83&

& §§33, §§

£º

3-,333333333

R3:3:33:

#######3 ºf £3:#&


Jun. 25, 2015 Sheet 31 of 62

Fig. 7

US 2015/0175558 A1


Jun. 25, 2015 Sheet 32 of 62

US 2015/0175558 A1

Fig. 8 * {3& 3.333;&

+ & 3.333;!

ox. & --

. §§3.433

£883.4%

*::$º. 8:8;&

53.3%

$33.3

* 38& : :33


Jun. 25, 2015 Sheet 33 of 62

US 2015/0175558 A1

3.2

&*:

$ 3.

...?

: :

& 3.

: 3 & 33

§

#*

§ 3:

§§§§

|| §§§33, #8%

§§§35, 38%

iš38×33, 88%


~c r ; º

*3.

Jun. 25, 2015 Sheet 34 of 62

23

Jº O

US 2015/0175558 A1


Jun. 25, 2015 Sheet 35 of 62

US 2015/0175558 A1

i 3 do?duu: ºO19 9%

3

3 $2

3

à


Jun. 25, 2015 Sheet 36 of 62

3

3

$2

s

3

3

3

US 2015/0175558 A1

3


Jun. 25, 2015 Sheet 37 of 62

15

3 3 uO!!!Quul UMO.19) 9%

3

15

3 3 uOH]]CHUul Ul/WO49 %

US 2015/0175558 A1


Jun. 25, 2015 Sheet 38 of 62

~ ?~

613 · 04

•?• •

• *?

-~ *-?~

•¿• •

US 2015/0175558 A1


Jun. 25, 2015 Sheet 39 of 62

ig. 44

US 2015/0175558 A1


Jun. 25, 2015 Sheet 40 of 62

Fig. 41 continued *.

US 2015/0175558 A1


Jun. 25, 2015 Sheet 41 of 62

##3; #38.33%

US 2015/0175558 A1


: à

*

3

§ {}

-C

Jun. 25, 2015 Sheet 42 of 62

US 2015/0175558 A1


Jun. 25, 2015 Sheet 43 of 62

Fig. 13 *

RSt. compounds

non-RSL compounds

US 2015/0175558 A1


Jun. 25, 2015 Sheet 44 of 62

Fig. 13 continued

b

US 2015/0175558 A1


Jun. 25, 2015 Sheet 45 of 62

!;:(?* &) **

US 2015/0175558 A1


Jun. 25, 2015 Sheet 46 of 62

Fig. 14 Continued

Ceil line = HT-1080 cells 15

1,5

.0

| .0

0.5

US 2015/0175558 A1


&3

33-33.3%

|

Jun. 25, 2015 Sheet 47 of 62

US 2015/0175558 A1


Jun. 25, 2015 Sheet 49 of 62

US 2015/0175558 A1

Fig. 17 Mice Bank Plasma

|+ MRM (645.3 -> 517.2) 130401-PE-Mice pla. $2 x1O 3

.

s b

:

-

: ;

4. MRM (271.1 -> 91.1) 130401-PE-Mice plas... X1 O 4

:

§

:

; :


Jun. 25, 2015 Sheet 50 of 62

US 2015/0175558 A1

Fig. 17 Continued Blank Plasma Spiked by S (Tolbutamide)

MRM (645.3 -> 517.2) 130401-PE-Mice pla. x10 3

.

:

:

* MRM (271.1 -> 91.1) 130401-PE-Mice plas.

# kio" 3

4. 3 2.

RT-2 ssa Area=167641.9046

º


Jun. 25, 2015 Sheet 51 of 62

US 2015/0175558 A1

Fig. 17 Continued Blank Sample Spiked with PE (100 ng/mL) and IS (Toibutamide)

+ MRM (645.3 -> 517.2) 130401-PE-Mice pla... ×1O 3

:

3.5 ... - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

3—

RT=2.538

Area=15646.2618

2.5 —

2 ..

MFM (271.1 -> 91.1) 130401-FE-Mice plas...


Jun. 25, 2015 Sheet 52 of 62

US 2015/0175558 A1

Fig. 17 Continued Piasma Sample from animal 108-1 Hour Following intravenous and Oral Administration

9

* MRM (645.3 -> 517.2) 130401-PE-Mice pla. x1O 3 |

RT=2.567 . .

.


Jun. 25, 2015 Sheet 53 of 62

Fig. 18

Yº?35i x&gartefatigº coefficient 38.9966%

US 2015/0175558 A1


Jun. 25, 2015 Sheet 54 of 62

Fig. 19

-e- PE 5mg/kg IV *.*.*,sº PE20mg/kg IV

PE20mg/kg PO

f Time thr)

US 2015/0175558 A1


Jun. 25, 2015 Sheet 55 of 62

US 2015/0175558 A1

Fig. 20

Mice Blank Brain

-- MRM (645.3 -> 517.2) 130403–PE-mice bra... £2 x1 O 3

s

:

.

:

;:

+ MRM (271.1 -> 91.1) 130403-PE-mice brai...

s

x10 *

º


Jun. 25, 2015 Sheet 56 of 62

US 2015/0175558 A1

Fig. 20 Continued Blank Brain Spiked by IS (Toibutamide)

c

+ MRM (645.3 -> 517.2) 130403-PE-mice bra. #2 x10 3..…

+

: 92 : C

as C

MRM (271.1 -> 91.1) 130403-PE-mice brai. x10 4

:

: . RT=2.531 6 tºArea=253480.2640

; :


Jun. 25, 2015 Sheet 57 of 62

US 2015/0175558 A1

Fig. 20 Continued Blank Sample Spiked with PE (100 ng/mL) and IS (Tolbutamide)

e

+ MRM (645.3 -> 517.2) 130403–PE-mice bra. # x10 3

& .

:C

1.1. : -

;

sº º ; :

:

RT=2.575...i.i; Area .

:

Area=264740.537

; :

6-iº,, ... ... ... ... ... ... ... ... ... ... ... ... ..., , #~~~~ sºº


Jun. 25, 2015 Sheet 58 of 62

US 2015/0175558 A1

Fig. 20 Continued Brain Sample from animal 104-0.5 Hours Following intravenous and Oral Administration

9

4. MRM (645.3 -> 517.2) 130403-PE-mice bra. x1O 3 |...........................$º RT=2697 Area=5371,6729. 1 , | --..… 1 -lºº

0.8 ..… O.7. 0.6— 0.5 ... 0.4 × O.3– 0.2. O. 1 .

* MRM (271.1 -> 91.1) 130403-PE-mice brai. h

x 10


Jun. 25, 2015 Sheet 59 of 62

Fig. 21

US 2015/0175558 A1


Jun. 25, 2015 Sheet 60 of 62

US 2015/0175558 A1

Fig. 22

1 O0

; 1 i–|-----------------|-----------------|--------------------------------------------------

O

2

4

6

8

40

12

t{hr)

b

100 :

{}

2

4

6

8

{{}

12


Jun. 25, 2015 Sheet 61 of 62

Fig. 23

1000

—e– PE 5mg/kg IV

—m- PE20mg/kg IV –4– PE20mg/kg PO

Time (hr)

US 2015/0175558 A1


Jun. 25, 2015 Sheet 62 of 62

US 2015/0175558 A1

Fig. 24

10

3.

i Ö

Ž

4

§

thr)

§

30

12

US 2015/0175558 A1

QUINAZOLINONE-BASED ONCOGENIC-RAS–SELECTIVE LETHAL COMPOUNDS AND THEIR USE CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present invention claims benefit to U.S. provi sional application Ser. No. 61/671,602 filed Jul. 13, 2012, the entire contents of which are incorporated by reference. GOVERNMENT FUNDING

[0002] This invention was made with government support under grant no. R01 CA097061 awarded by the National Insti tutes of Health. The government has certain rights in the invention. FIELD OF INVENTION

[0003] The present invention provides, interalia, quinazoli none-based oncogenic-RAS-selective lethal compounds and compositions containing such compounds. Methods for using such compounds or compositions are also provided. BACKGROUND OF THE INVENTION

[0004] Synthetic lethality describes a genetic interaction in which simultaneous mutations in two genes lead to synergis tic cell death compared to individual mutations in the same genes (Kaelin, 2005; Yang et al., 2008a). The concept of synthetic lethality was originally used to study the buffering capacity of cells and organisms upon genetic variations, through which many gene-gene interactions have been dis covered in multiple organisms, including bacteria, yeasts, and nematodes (Dixon et al., 2009; Malumbres, 2003). Soon after, it was recognized that this concept can be used as a framework for discovering anti-cancer drug leads with high therapeutic indices (Kaelin, 2005; Hartwell, 1997): one can search for small molecules that are only lethal in the presence of a specific oncogenic mutation. [0005] Oncogenic RAS proteins have been targeted using this synthetic lethal screening approach, due to the wide spread importance of mutant RAS proteins in the genesis and maintenance of human cancers (Malumbres et al., 2003), as well as the challenge of targeting oncogenic RAS proteins directly (Downward, 2003). Several synthetic lethal screens using RNA-interference-based (RNAi) libraries reported genes with synthetic lethal relationships with KRAS, such as PLK1 (Luo et al., 2009), TBK1 (Barbie et al., 2009), STK33 (Scholl et al., 2009), and GATA2 (Kumar et al., 2012). Some of these results may require further verification, because some follow-up studies did not support the originally postu lated roles (Babijet al., 2011; Luo et al., 2012). The mecha nism of synthetic lethality was attributed to increased depen dence on mitotic function, NF-kB signaling, S6 kinase activity, and the GATA2 transcriptional network, respec tively. The specific death-initiating mechanisms were differ ent in these cases; however, cancer cells with oncogenic RAS mutations invariably died via apoptosis upon treatment with these RNAi reagents. [0006] A different approach to targeting oncogenic RAS uses synthetic lethal screening with small molecules. Several RAS-synthetic-lethal (RSL) compounds were identified using this strategy (Yang et al., 2009; Yagoda et al., 2007; Weiwer et al., 2012; Shaw et al., 2011; Ji et al., 2009). The lethality of these RSL compounds, such as erastin and RSL3,

Jun. 25, 2015

was significantly enhanced upon activation of RAS-RAF MEK signaling. In contrast to the results of RNAi screens, the small molecule approach yielded compounds that induced a distinct form of oxidative, non-apoptotic cell death. This mode of cell death was distinct from necrosis, and is a regu lated form of oxidative cell death termed ferroptosis due to its unique morphology, inhibitor sensitivity and strict depen dency on iron (Dixon et al., 2012). Thus, ferroptosis may be an efficient means of inducing synthetic lethality with small molecules in tumor cells harboring oncogenic RAS proteins. Defining the molecular pathways governing ferroptosis could aid in targeting RAS mutant tumors. [0007] To define the core effectors of ferroptosis, erastin and RSL3 were further investigated, because both of these RSL compounds induce ferroptotic cell death via different triggering mechanisms. Erastin binds to VDAC2/3 (Yagoda et al., 2007), and inhibits system xc- (Dixon et al., 2012) to induce ferroptotic cell death. In contrast, RSL3 is not depen dent on these proteins (Yang et al., 2008a), and its target has not been reported. Metabolomic profiling was used to evalu ate comprehensively changes in metabolism upon erastin treatment, and it was found that a common lipoxygenase mediated pathway executing ferroptotic cell death in response to RSL compounds. [0008] RAS genes are among the most commonly mutated in human cancers, but their protein products have remained intractable to therapeutic agents. Thus, there is a need for, interalia, anti-cancer drugs with high therapeutic indices that selectively target tumor cells, such as those harboring onco genic RAS mutations. The present invention is directed to meeting these and other needs. SUMMARY OF THE INVENTION

[0009] One embodiment of the present invention is a com pound that has the structure (1): (1)

ºr s^^^ R

22

R2

O º C|

wherein

[0010) R, is selected from the group consisting of H, C, a alkyl, Cia alkoxy, hydroxy, and halogen; [0011] R2 is selected from the group consisting of H, C, a alkyl, C1-4 alkoxy, Cs—s cycloalkyl, Cs—s heterocycloalkyl, aryl, heteroaryl, Cia aralkyl;

US 2015/0175558 A1

Jun. 25, 2015

[0012] Rs is selected from the group consisting of nothing, C1-4 alkyl, C1-4 alkoxy, carbonyl, Cs—s cycloalkyl, and Cs-s heterocycloalkyl; [0013] X is selected from the group consisting of C, N, and O; and

[0014] n is an integer from 0-6, [0015] with the proviso that when X is C, n=0, and Rs is nothing, RI cannot be H when R2 is CHs. or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof. [0016] Another embodiment of the present invention is a compound that has the structure (30): (30)

wherein

[0018] Ri is selected from the group consisting of H, C, a alkyl, Cia alkoxy, hydroxy, and halogen; [0019| R2 is selected from the group consisting of H, C, a alkyl, C1 a alkoxy, Csis cycloalkyl, Csis heterocycloalkyl, aryl, heteroaryl, Cia aralkyl; [0020) Rs is selected from the group consisting of nothing, C1-4 alkyl, C1-4 alkoxy, carbonyl, Cs—s cycloalkyl, and Cs-s heterocycloalkyl; [0021] X is selected from the group consisting of C, N, and O; and

[0022] n is an integer from 0-6, [0023) with the proviso that when X is C, n=0, and Rs is nothing, RI cannot be H when R2 is CHs. oran N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof. [0024] A further embodiment of the present invention is another composition. This composition comprises a pharma ceutically acceptable carrier and a compound having the structure (30): (30)

or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof. [0017] An additional embodiment of the present invention is a composition. This composition comprises a pharmaceu tically acceptable carrier and a compound having the struc ture (1): (1)

O

2. |

N S ^^ 22

R3

oran N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof. [0025] Another embodiment of the present invention is a method for treating or ameliorating the effects of a cancer comprising a cell that harbors an oncogenic RAS mutation. This method comprises administering to a subject in need thereof a therapeutically effective amount of any compound disclosed herein.

R2

[0026] A further embodiment of the present invention is a method for treating or ameliorating the effects of a cancer comprising a cell that harbors an oncogenic RAS mutation. The method comprises administering to a subject in need thereofatherapeutically effective amount of any composition

N

O

disclosed herein.

Cl

[0027] Another embodiment of the present invention is a method for treating or ameliorating the effects of a cancer comprising a cell that harbors an oncogenic RAS mutation. This method comprises administering to a subject in need thereof a therapeutically effective amount of a composition comprising a pharmaceutically acceptable carrier and a com pound having the structure (30):

US 2015/0175558 A1

Jun. 25, 2015

(30)

,9

O

oran N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof. [0035] A further embodiment of the present invention is a method for depleting reduced glutathione (GSH) in a cell harboring an oncogenic RAS mutation comprising adminis tering to the cell an effective amount of a compound having the structure (1): (1)

O

~

R

N S^^ 22

or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof. [0028] An additional embodiment of the present invention is a method for modulating a lipoxygenase in a ferroptosis cell death pathway. This method comprises administering to a cell an effective amount of a compound having the structure (1): (1)

Sr.

O

2.

lº O

R2

O º C|

wherein

[0036) R, is selected from the group consisting of H, C, a alkyl, C-4 alkoxy, hydroxy, and halogen; [0037| R2 is selected from the group consisting of H, C, a alkyl, C1-4 alkoxy, Cs—s cycloalkyl, Cs—s heterocycloalkyl, aryl, heteroaryl, Cia aralkyl; [0038] Rs is selected from the group consisting of nothing, Cia alkyl, Cia alkoxy, carbonyl, Cs—s cycloalkyl, and Cs-s heterocycloalkyl; [0039] X is selected from the group consisting of C, N, and O; and

[0040] n is an integer from 0-6, [0041) with the proviso that when X is C, n=0, and Rs is nothing, RI cannot be H when R2 is CHs. [0042] or an N-oxide, crystalline form, hydrate, or pharma ceutically acceptable salt thereof. [0043] Another embodiment of the present invention is a compound having the structure (100):

N

(100)

O O

Cl

2.

|

R4 R

wherein

[0029) R, is selected from the group consisting of H, C, a alkyl, Cia alkoxy, hydroxy, and halogen; [0030) R2 is selected from the group consisting of H, C, a alkyl, C1-4 alkoxy, Cs—s cycloalkyl, Cs—s heterocycloalkyl, aryl, heteroaryl, Cia aralkyl; [0031] Rs is selected from the group consisting of nothing, C1-4 alkyl, C1-4 alkoxy, carbonyl, Cs—s cycloalkyl, and Cs-s heterocycloalkyl; [0032] X is selected from the group consisting of C, N, and O; and

[0033] n is an integer from 0-6, [0034] with the proviso that when X is C, n=0, and Rs is nothing, RI cannot be H when R2 is CHs.

R6

s^^* 3 22

R2

O º

US 2015/0175558 A1

Jun. 25, 2015

wherein

-continued (DPI4)

[0044) R, is selected from the group consisting of H, C, a alkyl, Cia alkoxy, hydroxy, and halogen; [0045] R2 is selected from the group consisting of H, C, a alkyl, C1-4 alkoxy, Cs—s cycloalkyl, Cs—s heterocycloalkyl, aryl, heteroaryl, Cia aralkyl; [0046] Rs is selected from the group consisting of nothing, H, C1-4 alkyl, C-4 alkoxy, carbonyl, Cs—s cycloalkyl, and Cs-s heterocycloalkyl; [0047] Ra and Rs are independently selected from the group consisting of H, Csis cycloalkyl, and Cais heterocycloalkyl; [0048] Re is selected from the group consisting of H, —NH2, Cia alkyl, Cia alkoxy, carbonyl, aryl, heteraryl, Cs-s cycloalkyl, and Cs—s heterocycloalkyl; [0049] X is selected from the group consisting of C, N, and

SS

S

O

º

N

C|

N

O

9s (DPI6)

N~S 2

O; and H N

[0050] n is an integer from 0-6, [0051] with the proviso that when X is C, n=0, and Rs is nothing, RI cannot be H when R2 is CHs. or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof. [0052] An additional embodiment of the present invention is a composition. This composition comprises a pharmaceu tically acceptable carrier and any compound disclosed herein. [0053] Another embodiment of the present invention is a method for treating or ameliorating the effects of a cancer comprising a cell that harbors an oncogenic RAS mutation. This method comprises administering to a subject in need thereof a therapeutically effective amount of a compound selected from the group consisting of:

N

O º O

(DPIT)

SS

H

S

O

N

N

*º

C|

O

Cl

9s

(DPI2) (DPI8)

*.

Cl l N

O

Crºr N

No

(DPI9)

o°

N Cl

N

US 2015/0175558 A1

Jun. 25, 2015

-continued

-continued (DPI10)

(51)

(DPI12)

(DPI13)

(52)

(DPI15)

(DPI17)

(40) (DPI18)

(DPI19)

º

C

US 2015/0175558 A1

Jun. 25, 2015

o

ºr

O ~

ON

N

~

O

O C|

(17)

Cl

ºr

(60)

O

O

~o

loº

O

N

C

~o ºr

O

O

!

(21)

US 2015/0175558 A1

Jun. 25, 2015

-continued

-continued (11)

O

(DPI4)

ºr

S

S

O

N

22 N

O

(DPI6)

Cl

or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof. [0054] Another embodiment of the present invention is a method for modulating a lipoxygenase in a ferroptosis cell death pathway. This method comprises administering to a cell an effective amount of any compound or composition dis

(DPIT)

closed herein.

[0055] An additional embodiment of the present invention is a method for modulating a lipoxygenase in a ferroptosis cell death pathway. This method comprises administering to a cell an effective amount of a compound selected from the group consisting of: (DPI2) Cl

Cº- º (DPI3)

S2’ o H N N

Dº CI’

o’

N

~~ O O

(DPI8)

US 2015/0175558 A1

Jun. 25, 2015 11

cºcº O ~

,9

cºoS. O

~

º

cº- sº

US 2015/0175558 A1

Jun. 25, 2015

-continued

-continued (11)

(11)

Cl

Cl

(8)

NEt3

oran N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof. [0056] A further embodiment of the present invention is a method for depleting reduced glutathione (GSH) in a cell harboring an oncogenic RAS mutation. This method com prises administering to the cell an effective amount of any compound disclosed herein. [0057] Another embodiment of the present invention is a method for depleting reduced glutathione (GSH) in a cell harboring an oncogenic RAS mutation. This method com prises administering to the cell an effective amount of a com pound selected from the group consisting of:

O

(DPI2) Cl

O

º

H

(9)

S N S

or

\

(DPI3)

SS

S O

H N N O and Cl

Asºº

ov O

US 2015/0175558 A1

Jun. 25, 2015 15

-continued

-continued (DPI4)

S

(DPI10)

S O

(DPI12)

(DPI6)

(DPI13)

(DPIT)

(DPI15)

(DPI17) (DPI8)

(DPI18)

(DPI9)

(DPI19)

US 2015/0175558 A1

Jun. 25, 2015 17

-continued

-continued (19)

(22)

) º

D

C.

º, ºr N

~

N

(60)

O

~o ~~

(23)

º

C

O

~o (24)

US 2015/0175558 A1

Jun. 25, 2015

-continued

-continued (11)

(7)

OH

O

OH

22

Cl

C|

(8)

oran N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof. NEt3

O

BRIEF DESCRIPTION OF THE DRAWINGS

Cl

(9)

[0058] FIG. 1 shows that metabolite profiling revealed cel lular glutathione (GSH) depletion as the most significant change upon erastin treatment. [0059] FIG. 1a is a graph showing the fold changes in metabolites upon erastin treatment. 264 metabolites from HT-1080 cells were analyzed. [0060] FIG. 1 b are graphs showing dose-dependent deple tion of GSH by erastin in HT-1080 cells and U-2 OS cells. Data are presented as mean:tstandard deviation (s.d.); n=3. [0061] FIG. 16 shows the structure of certain synthesized erastin analogs according to the present invention. Potency (GIso) and selectivity of each analog is shown. Selectivity is the ratio of GIso (BJeH):GIso (BJeLR). [0062] FIG. 1 dis a plot showing GSH depletion by various erastin analogs. HT-1080 cells were incubated with 10 puM of erastin analogs for 5 hours or 100 puM buthioninesulfoximine (BSO) for 12 hours before measurement of GSH concentra tion. GSH in each sample was first normalized to the DMSO sample, then box-and-whisker plots were generated (n=3-8). Mid-line, median; box, 25th to 75th percentiles; and whis kers, minimum and maximum. **, P=0.01, with respect to PYR-ERA: ***, P-0.001.

[0063] FIG. 1e shows light microscopy images (top panel) and a growth inhibition plot (bottom panel) demonstrating that BSO induces the RSL phenotype. BJeLR and DRD are

cells expressing HRAS***, whereas BJeH and BJeHLT are isogenic counterparts lacking HRAS”. Data are presented

Cl

and

as meanies.d.; n=3. Scale bars, 60 pum. [0064] FIG. 1? is a plot showing that Erastin and BSO induced the RSL phenotype through a similar mechanism. The pattern of cell death inhibition was similar between eras tin and BSO. The name of cell death inhibitors and the treat ment condition is listed in Table 1 below.

US 2015/0175558 A1

Jun. 25, 2015 20

TABLE 1

TABLE 2

Name and treatment condition of cell death inhibitors used in FIG. 1f.

List of ALOX and COX inhibitors used in this study. The indicated concentration was used in the experiment of FIG. 2g and FIG. 3c.

Concentration, Abbr.

Inhibitor

Targe

3-MA

3-methyladenine

ZVAD

z-VAD-fmk

caspases

formation of

Baf

Bafilomycin A1

autophagosome-lysosome

HM

Abbr.

Full name

1000

CDC

cinnamyl-3,4dihydroxy-a-

1

BAI

Baicalein

5

PD-

Concentration Vendor

Santa Cruz

Sc 2005.62

10 piM

Santa Cruz

Sc

PD-1461.76

5 M

Santa Cruz

Sc

AA-861

AA-861

2 plM

Santa Cruz

Sc

Zileuton

Zileuton

50 piM

Santa Cruz

Sc

Indo

Indomethacin

200 mM

Santa Cruz

preautophagosome

cyanocinnamate

50

fusion

200494

CspA

Cyclosporin A

cyclophilin D

E64D ALLN

E64D ALLN

calpains/cathepsins calpains

Nec

Necrostatin-1

RIP1 kinase

10

Chd

Chloroquine

autophagosome-lysosome

10

Wit.E

Vitamin E

lipophilic antioxidant

|UO 126

|UO126

MEK inhibitor

100 2.5

1461.76

2006.78 200570

fusion

CycH

Cycloheximide

Translation elongation

DFOM

Deferoxamine

iron chelator

Cat;

20 piM

204417

100

I-7378

10

1.5 100

[0065] FIG. 2 shows that selective activation of lipoxyge nases is responsible for the RSL phenotype of erastin. [0066] FIG.2a is a graph showing various basal ROS levels among BJ-derived cell lines, which were compared using H.DCF, a ROS detection dye, and flow cytometric analysis. The horizontal lines indicate the mean of normalized ROS

levels; n=8; *, P-0.05.

[0067] FIG.2b is a series of bar graphs showing the results of experiments in which antioxidant-targeting compounds were tested in four BJ-derived cells to determine whether they exhibited an RSL phenotype. The graphs indicate growth inhibition in the 4 BJ-derived cell lines at two different con

centrations (2x GIso and 4× G.I.so for each compound in BJeLR cells). Bargraph: meanies.d.; n=3; n.s., not significant; ***, P-0001.

[0068] FIG. 2C is a graph showing that erastin depletes cellular GSH equally in the 4 BJ-derived cell lines. Cells were treated with either DMSO or erastin for 12 hours followed by GSH quantification as described in Example 1 below. ***, P-0.001.

[0069] FIG. 2d shows a panel of microscopy images and a graph demonstrating that GFP-ALOX5 translocated to the nuclear membrane only in BJeLR cells upon 10 puM erastin treatment. Bar graph: mean+s.d.; n=3-4; ***, P-0.001. Scale bars, 60 pum. [0070] FIG. 26 shows a series of time course microscopy images of GFP-ALOX5 translocation in HT-1080 cells upon treatment with erastin or ionomycin. Scale bars, 60 pum. [0071] FIG. 2f is a series of graphs showing that erastin selectively generated lipid peroxides in BJeLR cells. The respective percentages in each graph indicate the percentage of the cell population that is BODIPY-C11 positive upon 0, 5, and 10 puM erastin treatment for 6 hours. [0072] FIG. 2g is a graph showing that ALOX inhibitors, but not a COX inhibitor, strongly suppressed erastin-induced cell death. Five different ALOX inhibitors (CDC, BAI, PD-146176, AA-861, Zileuton) and one COX inhibitor (Indo) were tested for their ability to suppress erastin lethal ity. The detailed treatment condition is listed in Table 2 below. Data are presented as meants.d.; n=3.

[0073] FIG. 3 shows that RSL3-induced ferroptosis acti vates an ALOX-dependent pathway. [0074] FIG. 3a is a bar graph showing that RSL3 does not deplete GSH. The level of GSH was determined in BJeLR cells after treating with 2 puM RSL3, 10 puM erastin, or 1 mM BSO. Bar graph: mean+s.d.; n=3. *, P-0.05. [0075] FIG. 3b shows microscopy images of GFP-ALOX5 and a bar graph demonstrating that GFP-ALOX5 translocated to the nuclear membrane upon RSL3 treatment (0.4 pum) in BJeLR cells, but notin BJeHLT cells. 0.4 puMRSL3 exhibited selective lethality in BJeLR cells. Bar graph: mean+s.d.; n=6 for BJeHLT, n=7 for BJeLR; ***, P-0.001. Scale bars, 60 pum [0076] FIG.3c is a graph showing that the lethality of RSL3 was suppressed by ALOXinhibitors, but not by a COXinhibi tor, in BJeLR cells. Data are presented as meants.d.; n=3. [0077|| FIG. 3d are graphs showing that RSL3 treatment generated lipid peroxides in the plasma membrane, as erastin did. The respective percentages in each graph indicate the percentage of BODIPY-C11 positive cell population upon 0, 0.1, 0.2,0.4 puM RSL3 treatment. [0078] FIG.3e is a graph showing that HT-1080 cells trans fected with a pool of siRNAs targeting GPX4 showed increased lipid peroxide level as assessed by BODIPY-C11 staining. Sineg has no homology to any known mammalian gene and was used as a negative control. [0079] FIG. 3f shows a series of microscopy images of GFP-ALOX5 and a bar graph demonstrating that GFP ALOX5 remained within the nucleus when sineg was trans fected; however, GFP-ALOX5 translocated to the nuclear

membrane upon siCPX4 transfection. Another control siRNA, called si?)eath, did not cause translocation during cell death. Bar graph: mean+s.d.; n=6, 7, 6 for sineg, si?)eath and siCPX4, respectively; n.s., not significant; ***, P-0.001. Scale bars, 60 pum [0080] FIG.3g is a bar graph showing that known inhibitors offerroptosis, 10 puMU0126, 100 pim[Vit. E, 100 puMDFOM, or 50 puM ZIL, were able to suppress siCPX4-induced cell death. Cell death induced by si?)eath could not be suppressed by any known ferroptosis inhibitor. Bar graph: meants.d.; n=3; *, P-0.05; **, P-0.01: ***, P-0.001.

[0081] FIG. 3h is a bar graph showing that knockdown of GPX4 displayed an RSL phenotype in the four BJ-derived isogenic cell lines. Bar graph: meants.d.; n=3; ***, P-0.001. [0082] FIG. 4 shows that synthetic lethality with onco genic-RAS occurs through a lipoxygenase-dependent path way.

US 2015/0175558 A1

Jun. 25, 2015 24

[0111] FIG. 20 shows chromatographic graphs for brain samples subjected to LC-MS/MS analysis. FIGS. 20a and 20b show blanksamples for testing mice brain. FIGS. 20c and 20d show blank samples before (FIG. 20c) and after the addition of an internal standard, tolbutamide (FIG.20d). FIG. 20e shows a blank sample spiked with PE (100 ng/mL), and FIG. 20fshows a blank sample spiked with the internal stan dard. FIGS. 20g and 20h show brain samples from animal 104 half an hour following intravenous and oral administration. [0112] FIG. 21 shows a calibration curve for brain samples subjected to LC-MS/MS analysis. [0113] FIG. 22 shows brain concentration-time curves of PE in male C57BL6/j mice following IV (FIG. 22a) and PO (FIG. 22b) administration of PE at 20 mg/kg (n=3 for each route of administration). Data points represent meanies.d. [0114] FIG. 23 shows, on the same axes, a set of brain concentration-time curves of PE in male C57BL6/jmice fol lowing IV and PO administration. [0115] FIG. 24 shows brain-plasma concentration ratio curves of PE in male C57BL6/j mice following IV and PO administration. Data points represent meanies.d. DETAILED DESCRIPTION OF THE INVENTION

[0116] One embodiment of the present invention is a com pound that has the structure (1): (1)

O N 22

~X

S

[0123] As used herein, the term “alkyl” refers to the radical of saturated aliphatic groups that does not have a ring struc ture, including straight-chain alkyl groups, and branched chain alkyl groups. In certain embodiments, a straight chain or branched chain alkyl has 4 or fewer carbon atoms in its backbone (e.g., C1-C4 for straight chains, Cs-Ca for branched chains). [012.4] Moreover, unless otherwise indicated, the term “alkyl” as used throughout the specification, examples, and claims is intended to include both “unsubstituted alkyls” and “substituted alkyls”, the latter of which refers to alkyl moi eties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Indeed, unless other wise indicated, all groups recited herein are intended to include both substituted and unsubstituted options. Such sub stituents can include, for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphate, a phos phonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, an aromatic, or heteroaromatic or heteroaryl moiety. It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate. For instance, the substituents of a substituted alkyl may include substituted and unsubstituted forms of amino, azido, imino, amido, phospho ryl (including phosphonate and phosphinate), sulfonyl (in cluding sulfate, sulfonamido, sulfamoyl and sulfonate), and silyl groups, as well as ethers, alkylthios, carbonyls (includ ing ketones, aldehydes, carboxylates, and esters), –CFs, —CN and the like.

R

SS-rº

[0125] The term “substituted” refers to moieties having substituents replacing a hydrogen on one or more carbons of

R2

the backbone. It will be understood that “substitution” or

“substituted with’’ includes the implicit proviso that such substitution is in accordance with the permitted valence of the

O º

substituted atom and the substituent, and that the substitution

C|

wherein

[0117 R is selected from the group consisting of H, C, a alkyl, Cia alkoxy, hydroxy, and halogen; [0118] R, is selected from the group consisting of H, C, a alkyl, C1-4 alkoxy, Cs—s cycloalkyl, Cs—s heterocycloalkyl, aryl, heteroaryl, Cia aralkyl; [0119) Rs is selected from the group consisting of nothing, C1-4 alkyl, C1-4 alkoxy, carbonyl, Cs—s cycloalkyl, and Cs-s heterocycloalkyl; [01201 X is selected from the group consisting of C, N, and O; and

[012.1] n is an integer from 0-6, [0122) with the proviso that when X is C, n=0, and Rs is nothing, RI cannot be H when R2 is CHs. or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof.

results in a stable compound, e.g., which does not spontane ously undergo transformation such as by rearrangement, cyclization, elimination, etc. As used herein, the term “sub stituted” is contemplated to include all permissible substitu ents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non aromatic substituents of organic compounds. The permissible

substituents can be one or more and the same or different for

appropriate organic compounds. For purposes of this inven tion, the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms.

[0126] The term “C.” when used in conjunction with a

chemical moiety, such as, alkyl and cycloalkyl, is meant to include groups that contain from x to y carbons in the chain.

For example, the term “C, alkyl” refers to substituted or

unsubstituted saturated hydrocarbon groups, including straight-chain alkyl and branched-chain alkyl groups that contain from x to y carbons in the chain, including haloalkyl groups such as trifluoromethyl and 2,2,2-tirfluoroethyl, etc. [0127] As used herein, “alkoxy” means an alkyl singular bonded to oxygen, or the following structure: –O-alkyl.

US 2015/0175558 A1

Jun. 25, 2015 26

wherein

-continued

[0141] Ri is selected from the group consisting of H, C, a alkyl, Cia alkoxy, hydroxy, and halogen;

(40)

[0142] Rs is selected from the group consisting of nothing,

~~

heterocycloalkyl;

O

C1-4 alkyl, C1-4 alkoxy, carbonyl, Cs—s cycloalkyl, and Cs-s

O

[0143] X is selected from the group consisting of C, N, and

O; and [0144] n is an integer from 0-6, or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof. [0145] In another aspect of this embodiment, the com-

º

'J N 22

N

N

pound has the structure (20): (20)

~~ O

N

O

2. |

N

S

X

ºO R

SS-1 3

Cl,

22

N

(50)

Sº

N

O

N

º

N

CHO

N22

O

N

C|

wherein [0146] Rs is selected from the group consisting of nothing,

C ND

heterocycloalkyl;

O

C1-4 alkyl, C1-4 alkoxy, carbonyl, Cs—s cycloalkyl, and Cs-s

O

[0147] X is selected from the group consisting of C, N, and O; and [0148] n is an integer from 0-6, or an N-oxide, crystalline form, hydrate, or pharmaceutically

Cl, and

(60)

acceptable salt thereof. [0149] In a further aspect of this embodiment, the com pound is selected from the group consisting of:

(30)

O

O

ICl O º

N

_^

N

§2.

O O

o” Sº

º

Cl Cl

oran N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof. Preferably, the compound has the structure (30):

US 2015/0175558 A1

Jun. 25, 2015 27

(30)

oran N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof. [0158] In one aspect of this embodiment, the compound has the structure (10): (10)

[0150] or an N-oxide, crystalline form, hydrate, or pharma ceutically acceptable salt thereof. [0151] An additional embodiment of the present invention is a composition. This composition comprises a pharmaceu tically acceptable carrier and a compound having the struc ture (1): (1)

O º C|

wherein

[0159] R is selected from the group consisting of H, C, a alkyl, Cia alkoxy, hydroxy, and halogen [0160] Rs is selected from the group consisting of nothing, C1-4 alkyl, C1-4 alkoxy, carbonyl, Cs—s cycloalkyl, and Cs-s heterocycloalkyl; [0161] X is selected from the group consisting of C, N, and O; and

[0162] n is an integer from 0-6, oran N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof. [0163] In another aspect of this embodiment, the com pound has structure (20):

O º

(20)

C|

wherein

[0152] Ri is selected from the group consisting of H, C, a alkyl, Cia alkoxy, hydroxy, and halogen; [0153| R2 is selected from the group consisting of H, C, a alkyl, C1-4 alkoxy, Cs—s cycloalkyl, Cs—s heterocycloalkyl, aryl, heteroaryl, Cia aralkyl; [0154] Rs is selected from the group consisting of nothing, C1-4 alkyl, C1-4 alkoxy, carbonyl, Cs—s cycloalkyl, and Cs-s heterocycloalkyl; [0155] X is selected from the group consisting of C, N, and O; and

[0156] n is an integer from 0-6, [0157] with the proviso that when X is C, n=0, and Rs is nothing, RI cannot be H when R2 is CHs.

US 2015/0175558 A1

Jun. 25, 2015 28

wherein

-continued

[0164] Rs is selected from the group consisting nothing, C1-4 alkyl, C1-4 alkoxy, carbonyl, Cs—s cycloalkyl, and Cs-s heterocycloalkyl; [0165] X is selected from the group consisting of C, N, and

(50) O O

O; and

N

[0166] n is an integer from 0-6, or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof. [0167] In an additional aspect of this embodiment, the com pound is selected from the group consisting of:

CHO

22

N

O N

~

O

Cl, and

~~

(30)

O

O

º

'J

N

(60)

~~ O DOJO’

22

N

O

O

N

O

º Cl

(40)

oran N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof. [0168] A further embodiment of the present invention is a composition. This composition comprises a pharmaceuti cally acceptable carrier and a compound having the structure (30): (30)

~~ O DOJO.

o O

Cl, Cl

US 2015/0175558 A1

Jun. 25, 2015 29

or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof. [0169] Another embodiment of the present invention is a method for treating or ameliorating the effects of a cancer comprising a cell that harbors an oncogenic RAS mutation. This method comprises administering to a subject in need thereof a therapeutically effective amount of any compound disclosed herein.

[0170] As used herein, the terms “treat,” “treating,” “treat ment” and grammatical variations thereof mean subjecting an individual subject to a protocol, regimen, process or remedy, in which it is desired to obtain a physiologic response or outcome in that subject, e.g., a patient. In particular, the methods and compositions of the present invention may be used to slow the development of disease symptoms or delay the onset of the disease or condition, or halt or reverse the

progression of disease development. However, because every treated subject may not respond to a particular treatment protocol, regimen, process or remedy, treating does not require that the desired physiologic response or outcome be achieved in each and every subject or subject, e.g., patient, population. Accordingly, a given subject or subject, e.g., patient, population may fail to respond or respond inad equately to treatment. [0171] As used herein, the terms “ameliorate”, “ameliorat ing” and grammatical variations thereof mean to decrease the severity of the symptoms of a disease in a subject. [0172] As used herein, “cancer” means uncontrolled growth of abnormal cells that harbor an oncogenic RAS mutation. The present invention includes those cancers selected from the following group that have one or more cells that harbor an oncogenic RAS mutation: adrenocortical car

various mutations, such as point mutations corresponding to amino acid numbers 12, 13, 59, 60 of H-RAS, may lead to impaired GTPase activity, resulting in inappropriate activa tion of RAS, such as constitutively activation of RAS. [0174] As used herein, a “subject” is a mammal, preferably, a human. In addition to humans, categories of mammals within the scope of the present invention include, for example, agricultural animals, veterinary animals, laboratory animals, etc. Some examples of agricultural animals include cows, pigs, horses, goats, etc. Some examples of veterinary animals include dogs, cats, etc. Some examples of laboratory animals include rats, mice, rabbits, guinea pigs, etc. [0175] A further embodiment of the present invention is a method for treating or ameliorating the effects of a cancer comprising a cell that harbors an oncogenic RAS mutation. This method comprises administering to a subject in need thereofatherapeutically effective amount of any composition disclosed herein.

[0176] Another embodiment of the present invention is a method for treating or ameliorating the effects of a cancer comprising a cell that harbors an oncogenic RAS mutation. The method comprises administering to a subject in need thereof a therapeutically effective amount of a composition comprising a pharmaceutically acceptable carrier and a com pound having the structure (30):

cinoma, anal cancer, bladder cancer, bone cancer, brain tumor, breast cancer, carcinoid tumor, carcinoma, cervical

cancer, colon cancer, endometrial cancer, esophageal cancer, extrahepatic bile duct cancer, Ewing family of tumors, extrac ranial germ cell tumor, eye cancer, gallbladder cancer, gastric cancer, germ cell tumor, gestational trophoblastic tumor, head and neck cancer, hypopharyngeal cancer, islet cell car cinoma, kidney cancer, laryngeal cancer, leukemia, lip and oral cavity cancer, liver cancer, lung cancer, lymphoma, malignant mesothelioma, Merkel cell carcinoma, mycosis fungoides, myelodysplastic syndrome, myeloproliferative disorders, nasopharyngeal cancer, neuroblastoma, oral can cer, oropharyngeal cancer, osteosarcoma, ovarian epithelial cancer, ovarian germ cell tumor, pancreatic cancer, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile can cer, pituitary cancer, plasma cell neoplasm, prostate cancer, rhabdomyosarcoma, rectal cancer, renal cell cancer, transi tional cell cancer of the renal pelvis and ureter, salivary gland cancer, Sezary syndrome, skin cancer (such as cutaneous t-cell lymphoma, Kaposi’s sarcoma, and melanoma), small

(30)

Cl

intestine cancer, soft tissue sarcoma, stomach cancer, testicu

lar cancer, thymoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, Wilms’ tumor. Prefer ably, the cancer is a sarcoma. [0173] As used herein, an “oncogenic RAS mutation” means a cellular change that results in the abnormal activation of any of the RAS family of genes (such as, e.g., H-RAS, K-RAS 4A, K-RAS 4B, M-RAS, N-RAS and R-RAS). RAS serves as a molecular switch in a large network of signaling pathways in cells. It cycles between the GDP-bound inactive form and the GTP-bound active form. Mutations in RAS have

been found in about 30% of all human cancers. For example,

oran N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof. [0177] An additional embodiment of the present invention is a method for modulating a lipoxygenasein a ferroptosis cell death pathway. This method comprises administering to a cell an effective amount of a compound having the structure (1):

US 2015/0175558 A1

Jun. 25, 2015 30

(1)

O

[0186] In one aspect of this embodiment, the compound has the structure (10): (10)

2. |

s’s”Suz" 22

R2

N

GD

O º C|

wherein wherein

[0178] Ri is selected from the group consisting of H, C, a

alkyl, Cia alkoxy, hydroxy, and halogen;

[0179| R2 is selected from the group consisting of H, C, a alkyl, C1 a alkoxy, Csis cycloalkyl, Cs—s heterocycloalkyl, aryl, heteroaryl, Cia aralkyl; [0180] Rs is selected from the group consisting of nothing, C1 a alkyl, Cia alkoxy, carbonyl, Cs—s cycloalkyl, and Cs-s heterocycloalkyl; [0181] X is selected from the group consisting of C, N, and

[0187] R1 is selected from the group consisting of H, C, a alkyl, Cia alkoxy, hydroxy, and halogen [0188] Rs is selected from the group consisting of nothing, Cia alkyl, Cia alkoxy, carbonyl, Cs—s cycloalkyl, and Cs-s heterocycloalkyl; [0189] X is selected from the group consisting of C, N, and O; and

[0190] n is an integer from 0-6, oran N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof. [0191] In another aspect of this embodiment, the com pound has the structure (20): (20)

O; and

[0182] n is an integer from 0-6, [0183) with the proviso that when X is C, n=0, and Rs is nothing, RI cannot be H when R2 is CHs. or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof. [0184] As used herein, the terms “modulate”, “modulat ing” and grammatical variations thereof mean to change, such as increasing the activity or expression of lipoxygenase. A “lipoxygenase” means an enzyme that catalyzes the oxidation of unsaturated fatty acids with oxygen to form peroxides of the fatty acids. Lipoxygenases according to the present inven tion include those polypeptides encoded by the ALOX genes, including ALOX5, ALOX12, ALOX12B, ALOX15, ALOX15B, and ALOXE3. Preferably, the ALOX gene is the ALOXE3 gene as set forth in more detail below. [0185] As used herein, “ferroptosis” means regulated cell death that is iron-dependent. Ferroptosis is characterized by the overwhelming, iron-dependent accumulation of lethal lipid reactive oxygen species. Ferroptosis is distinct from apoptosis, necrosis, and autophagy. Assays for ferroptosis are as disclosed, for instance, in Dixon et al., 2012.

O º C|

wherein

[0192] Rs is selected from the group consisting nothing, Cia alkyl, Cia alkoxy, carbonyl, Cs—s cycloalkyl, and Cs-s heterocycloalkyl; [0193] X is selected from the group consisting of C, N, and O; and

[0194] n is an integer from 0-6,

US 2015/0175558 A1

Jun. 25, 2015 31

[0195] or an N-oxide, crystalline form, hydrate, or pharma ceutically acceptable salt thereof. Preferably, the compound is selected from the group consisting of:

-continued (60)

(30)

,9

O.

N

Cl

(40)

oran N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof. More preferably, the compound has the structure (30): (30)

O Cl,

(50)

oran N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof. [0196) In another aspect of this embodiment, the modula tion comprises activation of one or more polypeptides encoded by ALOX genes. As used herein, “ALOX” refers to arachidonate lipoxygenase such as, e.g., those identified above.

Cl, and

[0197] A further embodiment of the present invention is a method for depleting reduced glutathione (GSH) in a cell harboring an oncogenic RAS mutation comprising adminis tering to the cell an effective amount of a compound having the structure (1):

US 2015/0175558 A1

Jun. 25, 2015

-continued (30)

(40)

C|

(50)

oran N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof. [0215] Another embodiment of the present invention is a compound having the structure (100): (100) O

O R6

N 22

2. |

R4

S_2^ Sir R3 R2

O º

(60)

wherein

Cl

or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof. More preferably, the compound has the structure (30):

[0216] R1 is selected from the group consisting of H, C, a alkyl, Cia alkoxy, hydroxy, and halogen; [0217| R2 is selected from the group consisting of H, C, a alkyl, C1 a alkoxy, Csis cycloalkyl, Csis heterocycloalkyl, aryl, heteroaryl, Cia aralkyl; [0218] Rs is selected from the group consisting of nothing, H, Cia alkyl, Cia alkoxy, carbonyl, Cs—s cycloalkyl, and Cs-s heterocycloalkyl; [0219) Ra and Rs are independently selected from the group consisting of H, Csis cycloalkyl, and Cs—s heterocycloalkyl; [0220) Re is selected from the group consisting of H, —NH2, C1-4 alkyl, Cia alkoxy, carbonyl, aryl, heteraryl, Cs-s cycloalkyl, and Csis heterocycloalkyl;

US 2015/0175558 A1

Jun. 25, 2015 34

[0221] X is selected from the group consisting of C, N, and

wherein

O; and

[0230] Rs is selected from the group consisting of nothing, C1-4 alkyl, C1-4 alkoxy, carbonyl, Cs—s cycloalkyl, and Cs-s heterocycloalkyl; [0231] n is an integer from 0-6, oran N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof. [0232] In a further aspect of this embodiment, the com pound is selected from the group consisting of:

[0222] n is an integer from 0-6, [0223) with the proviso that when X is C, n=0, and Rs is nothing, RI cannot be H when R2 is CHs. or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof. [0224] In one aspect of this embodiment, the compound has the structure (200). (200)

O N

O

º

Cl

C|

wherein

[0225] Rs is selected from the group consisting of nothing, C1-4 alkyl, C1-4 alkoxy, carbonyl, Cs—s cycloalkyl, and Cs-s heterocycloalkyl; [0226] Re is selected from the group consisting of H, —NH2, Cia alkyl, Cia alkoxy, carbonyl, aryl, heteraryl, Cs-s cycloalkyl, and Cs—s heterocycloalkyl; [0227] X is selected from the group consisting of C, N, and

cºO

~

O; and

[0228] n is an integer from 0-6, or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof. [0229] In another aspect of this embodiment, the com pound has the structure (300): (300)

Y.

O O

cºO

R3 N

(52)

#

22

O º

N

Cl

O

US 2015/0175558 A1

Jun. 25, 2015 36

-continued

-continued (21)

ºr

O

(24)

O

Cº.JJ*>~on

ON

º

*s-s-s-OH

*

O

C

~o

sº

~~

O

|C

(23)

~

cº-0 Ocº

(1a)