Bioorganic & Medicinal Chemistry Letters 27 (2017) 217–222

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Bioorganic & Medicinal Chemistry Letters

journal homepage: www.elsevier .com/locate /bmcl

Novel 3-methylindoline inhibitors of EZH2: Design, synthesis and SAR

http://dx.doi.org/10.1016/j.bmcl.2016.11.0800960-894X/� 2016 Elsevier Ltd. All rights reserved.

⇑ Corresponding author.E-mail address: roopa.rai@hotmail.com (R. Rai).

Amantullah Ansari a, Sharad Satalkar a, Varshavekumar Patil a, Amit S. Shete a, Simranjeet Kaur a,Ashu Gupta a, Siddhartha Singh a, Mohd. Raja a, Daniel L. Severance b, Sebastián Bernales c,Sarvajit Chakravarty c, David T. Hung c, Son M. Pham c, Francisco J. Herrera c, Roopa Rai c,⇑a Integral BioSciences Pvt. Ltd., C-64, Hosiery Complex Phase II Extension, Noida, Uttar Pradesh 201306, Indiab Schrodinger, Inc., Portland, OR, USAcMedivation, 525 Market Street, 36th Floor, San Francisco, CA 94105, USA

a r t i c l e i n f o a b s t r a c t

Article history:Received 29 September 2016Revised 22 November 2016Accepted 23 November 2016Available online 25 November 2016

Keywords:EZH2IndolineAnticancerMethyltransferases

EZH2 (enhancer of zeste homologue 2) is the catalytic subunit of the polycomb repressive complex 2(PRC2) that catalyzes the methylation of lysine 27 of histone H3 (H3K27). Dysregulation of EZH2 activityis associated with several human cancers and therefore EZH2 inhibition has emerged as a promising ther-apeutic target. Several small molecule EZH2 inhibitors with different chemotypes have been reported inthe literature, many of which use a bicyclic heteroaryl core. Herein, we report the design and synthesis ofEZH2 inhibitors containing an indoline core. Partial saturation of an indole to an indoline provided leadcompounds with nanomolar activity against EZH2, while also improving solubility and oxidative meta-bolic stability.

� 2016 Elsevier Ltd. All rights reserved.

Covalent modifications of histones play pivotal roles in theorganization of the chromatin structure and the regulation of geneexpression.1,2 A large number of enzymes are known to catalyzethe addition or removal of covalent groups from specific aminoacids in histone proteins including histone methyltransferases(HMTs).3 Enhancer of Zeste Homologue 2 (EZH2) is the catalyticsubunit of Polycomb repressive complex 2 (PRC2), which catalyzesthe methylation of lysine 27 of histone 3 (H3K27).4 Tri-methyla-tion of the lysine-27 of histone 3 (H3K27) is associated with thesilencing of specific genes, including many tumor suppressorgenes.5 EZH2 has been found to be significantly overexpressed ina variety of human cancers and its expression level correlates withcancer progression and poor prognosis.6,7 In addition, point muta-tions of residues, such as Y641, A677 and A687, within the catalyticdomain of EZH2 have been found in follicular lymphomas and dif-fuse large cell B lymphomas (DLBCL).8 Several studies haverevealed that EZH2 knockdown in tumor cell lines can causedecreased cell proliferation, migration, angiogenesis and increasedapoptosis.9–11 Collectively, this evidence supports EZH2 as animportant target for cancer therapy and its inhibitors to be poten-tial anticancer agents. Therefore significant efforts have been madeon the discovery and optimization of small molecule EZH2 inhibi-tors. Through these efforts various small molecule EZH2 inhibitors

have been reported with demonstrated efficacy both in vitro andin vivo.12–14

An analysis of EZH2 inhibitors reported in the literature13 led tothe recognition that a substituted pyridone-methyl amidefragment constituted a highly optimized moiety for binding toEZH2. Examples of published molecules which have the pyridonegroup are shown in Fig. 1. Of these, EPZ643815 (7) and GSK12616

(2) are currently being evaluated in human clinical trials. We notedthat EPZ6438 and EPZ11989 are exceptions in the pyridone con-taining EZH2 inhibitors; the pyridone-methyl amide is attachedto a phenyl core in place of a bicyclic hetero-aryl core. It has beendemonstrated that increased fraction of sp3 carbons (Fsp3 = num-ber of sp3 hybridized carbons/total carbon count) correlates withimproved solubility, an experimental physical property importantto success in drug discovery.17 These observations prompted usto use a bicyclic non-planar indoline core for making novel EZH2inhibitors, where the core consists of a phenyl residue fused witha saturated 5-membered ring. Our design is drawn in Fig. 2, wherewe show that the indoline can be derived from the EPZ6438 (7)core by C7-C8 cyclization or by saturation of indole core at C-2and C-3 present in Ei1 (1) and GSK126 (2). We envisaged thatthe new molecules with a non-planar indoline core might haveimproved solubility and pharmacokinetic properties due to higherdegree of saturation and non-planarity of the core.

We started our lead generation effort by preparing compounds16, 17, 18 and 19 incorporating an indoline core (Scheme 1). Key

Table 1EZH2 inhibitory activity of compounds 16–19.

N

R1

O NH

HNO

R2

Compound R1 R2 EZH2 IC50a (nM)

16 C N 26,600

17 C N 571

18O

C N 15,700

19O N

O

237

a Experimental details are in the Supplemental information.

NO N

N

OHN

HNO

EPZ005687 (3)NN

N

OHN

HNO

GSK126 (2)HN

N

OHN

HNO

N

Ei1 (1)

NN

N

OHN

HNO

NN

GSK343 (4)NN

NN

OHN

HNO

N UNC1999 (5)

N

O

O HN

HN

O

O

CPI360 (6)

NO N

OHN

HNO

OEPZ6438 (7)

NO

N

OHN

HNO

NN

N

OHN

HNO

ON

O

EPZ011989 (8) NZLD1039 (9)

Fig. 1. Reported EZH2 inhibitors.13

NH

O O

Br

NH

O O

Br

N

O O

BrR1

b

N

O NH

BrR1

c

HNO

N

O NH

R2

R1

HNO

d

16:R1 = A, R2 = D17: R1 = B, R2 = D 18:R1 = B, R2 = D 19:R1 = C, R2 = E

N

O O

R2

R1N

O OH

R2

R1

a

e

f

g

10

11

12a: R1 = A12b: R1 = B12c: R1 = C

13: R1 = C, R2 = E 14: R1 = C, R2 = E

15a: R1 = A15b: R1 = B15c: R1 = C

Where, A =

B =

C =O

D = C N

E =N

O

NH2

HNO

NH2

HNO

Scheme 1. Synthesis of 16, 17, 18 and 19. Reagents and conditions: (a) TFA,triethylsilane, CH2Cl2, RT; (b) AcOH, ketone, Na(OAc)3BH, CH2Cl2, 0–RT, 16 h; (c)2 M AlMe3 in toluene, THF, 80 �C, 18 h; (d) Zn, Zn(CN)2, PdCl2(dppf)�CH2Cl2, DMA,135 �C, 18 h; (e) Boronic ester, Na2CO3, Pd(PPh3)4, dioxane-water, 90 �C, 15 h; (f) 6 NHCl, 95 �C, 16 h; (g) PyBOP, TEA, DMSO, RT, 16 h. Further details are in theSupplemental information.

N

12

345

6

7

8

9Joining C7-C8

N

Indoline corePhenyl core in 7

N 1

2

3Saturattion at

C2 and C3

Indole core in 1 and 2

Fig. 2. Design of indoline core for making EZH2 inhibitors.

218 A. Ansari et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 217–222

intermediates 12a–c were synthesized by a reductive amination ofvarious ketones with 11, which was obtained by reduction of com-mercially available indole 10. Intermediates 12a and 12b were

then converted to 15a and 15b using trimethylaluminium as thecoupling agent. Cyanation of 15a, 15b and 15c afforded 16, 17and 18 respectively. Alternatively, intermediate 12c was trans-formed to 19 via Suzuki coupling followed by hydrolysis and amidebond formation.

Compounds were tested in vitro for their ability to inhibit theenzymatic activity of EZH2, mutant EZH2 (Y641F) and the homologEZH1. The enzymatic activity was determined using the indicatedHMT complexes incubated with 3H-SAM (tritiated S-adenosenyl

L-methionine) and histone H3 and IC50 curves were determinedfor each compound (see Supplemental information). The IC50

results for inhibition of EZH2 are shown in Table 1.Based on reported compounds and published SAR,13 we main-

tained the 4,6-dimethylpyridone methyl amide as a constant. Ourfirst analogs scoped out the effect of substitution at the 1- and6-positions of the indoline. The isopropyl substituted 16 was inac-tive (EZH2 IC50 = 26.6 lM) while the cyclopentyl substituted 17had a 47-fold improvement in potency (EZH2 IC50 = 571 nM).

A. Ansari et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 217–222 219

However, a tetrahydropyran (THP) substitution led to a drastic lossof potency (18 EZH2 IC50 = 15.7 lM). We also made compound 19with a large phenyl-methyl morpholine at the 6-position, and sur-prisingly, while this compound has a THP group at the 1-position,its activity improved to EZH2 IC50 = 237 nM. With some uncer-tainty about our SAR conclusions at this point, we turned ourattention to substitution at the 3-position.

For compounds containing a 3-position methyl substituent, wesynthesized several analogs using different R1 and R2 groups,including those reported in the literature13 for molecules havingan indole core. R2 groups included nitrile, phenyl-methyl morpho-line, piperazino-pyridine, piperazino-pyrimidine and piperazino-isoquinoline. R1 groups included isopropyl, cyclopentyl, cyclo-propylmethyl and THP. The 3-position methyl group resulted inenantiomeric pairs of compounds after separation by chiralchromatography.

Scheme2describes the synthesis of compounds 27, 28, 30, 31, 33and 34. Briefly, intermediate 24a and 24bwere synthesized from 10by alkylation followed by formylation and reduction while 24c and24d were synthesized by reductive amination of 23 which wasobtained from 10 via 22. Cyanation of 24a and 24b resulted in 25aand 25b which after hydrolysis followed by amide bond formationwith 3-(aminomethyl)-4,6-dimethylpyridin-2(1H)-one gave 27

NH

O O

Br

N

O O

BrR1

N

O O

BrR1

O

b

b c

O

BrN

O O

R2

R1N

O

R2

R1

NH

HNO

NH

O O

Br

O

10 22

20a: R1 = A20b: R1 = B

Where, A =

B =

C =O

D = C N

E =N

21a: R1 = A21b: R1 = B

29a29b29c

F =

33: R1 = B, R2 = E34: R1 = C, R2 = E

32a: R1 = B, R2 = E32b: R1 = C, R2 = E

c

a

i

h

Scheme 2. Synthesis of 27, 28, 30, 31, 33 and 34. Reagents and conditions: (a) NaH, aNaCNBH3, DCE, RT, 16 h (ii) TFA, triethylsilane, DCE, RT, 16 h; (d) AcOH, aldehyde/ketonLiOH, THF, MeOH, Water, RT, 6 h; (g) 3-(Aminomethyl)-4,6-dimethylpyridin-2(1H)-one,2 M AlMe3 in toluene, THF, 80 �C, 18 h; (i) Boronic ester, Na2CO3, Pd(PPh3)4, dioxane, wa

and 28, respectively. Compound 29a, 29b and 29c were obtainedby amide bond formation of 3-(aminomethyl)-4,6-dimethylpyri-din-2(1H)-one with 24a, 24d and 24c respectively, using trimethy-laluminium as coupling agent. Suzuki coupling of 29a and 29bwith arylboronic ester yielded 30 and 31 respectively. Suzuki cou-pling of 24b and 24c respectively gave 32a and 32bwhich after ami-dation afforded 33 and 34 respectively.

Scheme 3 describes the synthesis of compounds 36, 39, 40, 41and 45. Boc-piperazine-isoquinoline boronic ester (synthesizedby a method described in Supplemental information) was coupledwith 29c to give 35 which after Boc-deprotection afforded 36.Compound 24a, 24b and 24c were coupled with commerciallyavailable Boc-piperazine-pyridine boronic ester to afford 37a,37b and 37c which after amide bond formation followed by Boc-deprotection afforded 39, 40 and 41 respectively. Suzuki couplingof Boc-piperazine-pyrimidine boronic ester with 24c resulted 42which was converted to 45 via 43 and 44.

With this set of methyl-substituted compounds in hand, wetested for activity against EZH2; the active analogs were addition-ally tested for activity against EZH1 and the Y641F EZH2 mutant.The Y641 residue is frequently mutated in certain lymphomas.Moreover, lymphomas with this mutation have increased H3K27methylation.16 The SAR is illustrated in Table 2.

N

O O

BrR1

e

N

O O

R2

R1

N

O OH

R2

R1

N

O

R2

R1

f

g

NH

HNO

NR1

NH

HNO

N

O

R2

R1

NH

HNO

NH

O O

Br23

O

24a: R1 = A24b: R1 = B24c: R1 = C24d: R1 = F

25a: R1 = A, R2 = D25b: R1 = B, R2 = D

26a: R1 = A, R2 = D26b: R1 = B, R2 = D

27: R1 = A, R2 = D28: R1 = B, R2 = D

: R1 = A: R1 = F: R1 = C

30: R1 = A, R2 = E31: R1 = F, R2 = E

d

i

h

lkyl bromide, DMF, 0-RT, 16 h; (b) (i) POCl3,DMF, 2.5 h (ii) 2 N NaOH; (c) (i) ZnI2,e, Na(OAc)3BH, 0–RT, 16 h (e) Zn, Zn(CN)2, PdCl2(dppf)�DCM, DMA, 135 �C, 18 h; (f)PyBOP, TEA, DMSO, RT, 16 h; (h) 3-(Aminomethyl)-4,6-dimethylpyridin-2(1H)-one,ter, 90 �C, 15 h. Further details are in the Supplemental information.

N

O O

BrR1

N

O O

R1

NNN

O

O N

O NH

R1

NNN

O

O

HNO

N

O NH

R1

NNHN

HNO

N

O O

N

NNN

O

O

N

O OH

N

NNN

O

O

N

O NH

N

NNHN

HNO

N

O

Br

NH

HNO

N

O NH

HNO

N

NN

O

O

N

O NH

HNO

N

NHN

36OO O29c

a b

a c b

a

de

24a: R1 = A24b: R1 = B24c: R1 = C

O

O

O

35

37a: R1 = A37b: R1 = B37c: R1 = C 38a: R1 = A

38b: R1 = B38c: R1 = C

45

42

43

39: R1 = A40: R1 = B41: R1 = C

N

O NH

N

NNN

HNO

OO

O

b

44

Scheme 3. Synthesis of 36, 39, 40, 41 and 45. Reagents and conditions: (a) PdCl2(dppf)�dcm, K2CO3, boronic ester, dioxane, water, 90 �C, 16 h; (b) TFA, DCM, RT, 16 h; (c) 3-(Aminomethyl)-4,6-dimethylpyridin-2(1H)-one, 2 M AlMe3 in toluene, THF, 80 �C, 18 h; (d) LiOH, THF, MeOH, water, RT, 6 h; (e) 3-(Aminomethyl)-4,6-dimethylpyridin-2(1H)-one, PyBOP, TEA, DMSO, RT, 16 h. Further details are in the Supplemental information.

220 A. Ansari et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 217–222

The methyl substituent had a dramatic effect on potency in theseries. As an illustrative example, 16 has EZH2 IC50 of 26.6 lM,while the methyl substituted active enantiomer 27e1 has EZH2IC50 of 88 nM - a 302 fold improvement. The inactive enantiomer27e2 has no measurable activity against EZH2. This interesting phe-nomenon is an example of the ‘‘magic methyl” effect that has beendescribed in the literature.18 During the prosecution of this pro-gram, a patent application describing indoline core containingEZH2 inhibitors was published.19 While the broad claims includedmultiple 3- substituted indolines and some compounds containing3,3-dimethyl substituted indolines were described, no compoundscontaining 3-monomethyl substituted indolines were exemplified.

We found that for all R1 and R2 groups, the 2 enantiomers of apair had very disparate activity. This difference in activity in anenantiomeric pair was more pronounced in the case of a small R2

substituent (-CN, in 27e1, 27e2, 28e1 and 28e2). In these examples,the difference in activity between enantiomers is >100 fold, withthe less potent enantiomer (27e2 and 28e2) having no measurableIC50 against EZH2 up to 100 lM. With the larger R2 substituentsshown in Table 2, the less active enantiomer had measurableactivity, albeit in the micromolar range. This indicates that in the3-methyl substituted indoline series, there appears to be somecross talk between the 3- and 6-position substituents. In an effortto understand the ‘‘magic methyl” effect, we used the publishedcrystal structure of EZH220 to dock both enantiomers of compound

34 (see Supplemental information). This pair of enantiomers pre-sented the dimethyl pyridone moiety to the protein in a manneridentical to the published bound inhibitor, forming 2 H-bonds withthe backbone of W624. The active enantiomer (34e1, predicted tobe the R enantiomer based on docking) indeed has the methylgroup sitting in a groove lined by T678, F665, and the face ofR685, possibly displacing an isolated water molecule. The inactiveenantiomer (34e2) is seen to rotate the indoline group by 180�,while still anchored to the protein through the pyridone. This likelyhappens in an attempt to avoid a steric clash with the protein. Inthis state, R685 and F665 interactions with the ligand are lost,and a high entropy stranded water is likely interacting withR685, thus explaining the potency loss.

We probed the role of substituent at the N1 position of indoline(R1) and found that compounds with cyclopentyl (28e1, 33e1) andTHP group (34e1) had better potency compared to their corre-sponding isopropyl analogs 27e1 and 30e1 (Table 2). These resultsindicated that bulkier groups are needed at this position for betterpotency. The compound with cyclopropylmethyl group at N1 (31e1)was found to be 8-fold less potent compared to its isopropyl analog(30e1) (Table 2). This result highlights the role of a-branching atthis position on potency.

Next, we decided to study effect of substituent at C-6 position ofindoline on activity (Table 2). We used different substituents atthis position and pyridine-2-yl-piperizine residue was found to

Table 3In vitro ADME dataa for 7, 15e1 and 21e1.

Compound Sol (lM)b hLM (% R)c mLM (% R)c

7 28 25 3934e1 75 62 5841e1 75 83 95

a Experimental details are in the Supplemental information.b Kinetic solubility.c Percent parent remaining after 30 min.

Table 2SAR of 3-methlindoline based compounds.

N

R1

O NH

HNO

R2

Me

Compound R1 R2 EZH1 IC50a (nM) EZH2 IC50

a (nM) EZH2 (Y641F) IC50a (nM)

27e1 C N 7750 88 47827e2 ND >100,000 ND

28e1 C N 4440 27 18528e1 ND >100,000 ND

30e1

N

O

2710 47 51830e2 ND 4140 ND

31e1

N

O

ND 375 ND31e2 ND 15,200 ND

33e1

N

O

2650 24 90233e2 ND 448 ND

34e1

ON

O

3340 29 97534e2 ND 6980 ND

39e1

2

NN NH

1870 17 62239e2 ND 1082 ND

40e1

NN NH

1350 21 50540e2 ND 378 ND

41e1

ON

N NH1980 11 578

41e2 ND 2150 ND

45e1

O

N

NN NH

ND 98 ND45e2 ND 2160 ND

36e1

O

NN NH

1560 17 30436e2 ND 540 ND

e1/e2Active/inactive enantiomer.ND: not determined.

a Experimental details are in the Supplemental information.

A. Ansari et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 217–222 221

be the best substituent at this position as far as the biologicalactivity against WT EZH2 inhibition is concerned (Compounds39–41, Table 2).

The pyrimidine analog of 41e1�(45e1) was found to be 9-fold lesspotent than 41e1. We also explored a 3-ethyl substituted analog of41e1 and found it to be 13-fold less potent (EZH2 IC50 = 145 nM,data not shown).

While these compounds maintained relatively good potency forEZH2, they did not display appreciable inhibitory activity againstEZH1 (IC50 > 1.35 lM), indicating their relative selectivity forEZH2 versus the homolog EZH1. Regarding Y641F EZH2 activity,overall, this series suffered a 7–40-fold loss in potency relative toWT EZH2. Compound 28e1 with a cyano group at the R2 positionhad the best potency against Y641F EZH2.

We tested EPZ6438 (7, EZH2 IC50 = 2 nM in our assay) and twoof our lead compounds with the indoline core (34e1 and 41e1) insolubility and microsomal stability assays; the data are shown inTable 3. These two compounds demonstrated superior kinetic sol-ubility in PBS (pH 7.5) and improved oxidative stability in human

and mouse liver microsomes (HLM and MLM) compared toEPZ6438 (7).

In conclusion, we describe the indoline as a novel core for mak-ing EZH2 inhibitors. These findings provide a foundation for futureanaloging and SAR; lead compounds will be tested in cell assays,in vivo for optimization of pharmacokinetic properties and evalua-tion in pharmacology models.

222 A. Ansari et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 217–222

Funding: This research did not receive any specific grant fromfunding agencies in the public, commercial or not-for-profit sec-tors. It was funded solely by Medivation.

Acknowledgements

The authors thank Kevin Quinn for his advice and expertise onthe in vitro metabolism experiments and the Analytical and Purifi-cation group at Integral Bioscience for purification of compoundsand for acquiring NMR data.

A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.bmcl.2016.11.080.

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