Discovery, Structure-Activity Relationship, and Biological Activity of Histone-Competitive Inhibitors of Histone Acetyltransferases P300/CBP

Fangrui Wu, Yuanda Hua, Salma Kaochar, Shenyou Nie, Yi-Lun Lin, Yuan Yao, Jingyu Wu,

Article

Discovery, Structure−Activity Relationship, and Biological Activity

of Histone-Competitive Inhibitors of Histone Acetyltransferases

P300/CBP

∇

Xiaowei Wu, Xiaoyong Fu, Rachel Schi ﬀ , Christel M. Davis, Matthew Robertson, Erik A. Ehli,

Cristian Coarfa, Nicholas Mitsiades, and Yongcheng Song *

Cite This: https://dx.doi.org/10.1021/acs.jmedchem.9b02164

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sı Supporting Information

ABSTRACT: Histone acetyltransferase (HAT) p300 and its paralog CBP acetylate histone lysine side chains and play critical roles

in regulating gene transcription. The HAT domain of p300/CBP is a potential drug target for cancer. Through compound screening

and medicinal chemistry, novel inhibitors of p300/CBP HAT with their IC₅₀values as low as 620 nM were discovered. The most

potent inhibitor is competitive against histone substrates and exhibits a high selectivity for p300/CBP. It inhibited cellular

acetylation and had strong activity with EC of 1−3 μM against proliferation of several tumor cell lines. Gene expression proﬁling in

estrogen receptor (ER)-positive breast cancer MCF-7 cells showed that inhibitor treatment recapitulated siRNA-mediated p300

knockdown, inhibited ER-mediated gene transcription, and suppressed expression of numerous cancer-related gene signatures.

These results demonstrate that the inhibitor is not only a useful probe for biological studies of p300/CBP HAT but also a

pharmacological lead for further drug development targeting cancer.

INTRODUCTION

structured domains, which share a high homology between

p300 and CBP, are connected with less conserved intrinsically

disordered regions (IDR). The CH1, KIX, CH3, and SID

domains as well as IDR of p300/CBP are transactivation

domains, which interact with a number of transcription factors

■

Histone acetylation is one of the most important post-

translational modiﬁcations. Acetylation of the side chain amino

group of a histone lysine residue neutralizes its positive charge

(

under physiological pH) and renders a more open DNA

conformation to facilitate binding of transcription factors as

well as other proteins for gene transcription, DNA replication,

(

e.g., CREB, p53, and HIF-1) and transcription coactivators

8−10

and repair. In addition, acetylated lysine can be recognized

by a number of proteins (such as bromodomain-containing

proteins) and serve as an anchor point in chromatin to form a

(e.g., steroid receptor coactivators).

Biologically, p300/

CBP not only acetylates a lysine residue of histone (e.g.,

histone H3 lysine 27, or H3K27) and certain transcription

transcription complex that regulates gene expression.

Human p300 (E1A binding protein p300) and its paralog

CBP [CREB (cAMP-response element binding protein)

binding protein] are large proteins with ∼2400 amino

Received: December 26, 2019

−7

acids,

containing multiple structured domains including

cysteine-histidine rich 1 (CH1), CREB-binding KIX, bromo-

domain, histone acetyltransferase (HAT), CH3, and steroid

receptor coactivator interaction (SID) domains. These

XXXX American Chemical Society

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

have been reported (Figure 1 ). However, except for recently

Article

,11,12

factors (e.g., p53 and c-Myc)

but also serves as a hub

RESULTS

■

protein to link and stabilize a transcription complex.

Inhibitor Discovery. A biochemical assay to determine the

activity and inhibition of recombinant HAT domain of human

p300 was developed, using the substrate histone H3 (1−21)

Previous studies have shown that HAT activity of p300/

CBP is essential for many transcription factor-mediated gene

transcription programs. For example, estrogen receptor (ER)

or androgen receptor (AR) mediated gene transcription

pathway is of importance in female or male development as

well as in breast or prostate cancer, respectively. HAT activity

of p300 has been found to be required for ER- or AR-mediated

peptide and the H-labeled cofactor Ac-CoA. We screened our

proprietary compound library containing ∼300 compounds,

which were synthesized for SAR studies of lysine speciﬁc

demethylase 1 (LSD1) whose substrate is methylated histone

H3 lysine 4 (H3K4). The rationale is that since the peptide

sequence of H3K4 (ARTKQ) is similar to those of p300

HAT’s substrate H3K27 (AARKS), there is a higher likelihood

to ﬁnd an inhibitor from the LSD1 targeting compounds. The

compound screen led to the discovery of thiophene-2-

carbamide compound 1 (Table 1) that inhibited p300 HAT

with an IC₅₀(concentration at which the enzyme activity is

inhibited by 50%) value of 8.6 μM.

Synthesis. Given its seemingly drug-like structure, more

derivatives of compound 1 were synthesized and tested for

their activities against p300 HAT. General methods for

synthesis of compounds 1−40 for SAR studies are shown in

Scheme 1.

,10,13

gene expression.

In addition, HAT of p300/CBP is a

potential drug target for cancer therapy. Although there have

been debates on whether p300/CBP alone is a tumor

suppressor or an oncogene, convincing evidence has shown

that p300/CBP HAT is essential for breast and prostate cancer

as well as other cancers with p300 overexpression or harboring

,14,15

a p300/CBP fusion oncogene.

To date, several chemotypes of p300/CBP HAT inhibitors

Synthesis of thiophene-2-carbamide compounds 1−9 was

started from a reaction between 4,5-dibromothiophene-2-

carboxylic acid (41) and 1-tert-butyloxycarbonyl (BOC)

protected 4-(aminomethyl)piperidine, together with amide

bond forming reagents 1-ethyl-3-(3-dimethylaminopropyl)-

carbodiimide (EDC) and 1-hydroxybenzotriazole. The 5- and

-Br groups of the thiophene-2-carbamide compounds (42)

can undergo two successive Suzuki coupling reactions with

diﬀerent aryl boronic acids with a high selectivity: 5-Br was

reacted ﬁrst at a lower temperature of 80 °C, followed by 4-Br

at 100 °C, to give compound 43. Removal of the BOC

protecting group using HCl produced the target compounds

−9. Synthesis of 2-aminomethylthiophene series of com-

pounds 10−12 and 14−32 was started from 4,5-dibromothio-

phene-2-carbaldehyde (44, X = S), in which the 2-CHO group

was reduced to an alcohol, followed by conversion to a -CH Cl

group by treatment with cyanuric chloride in DMF. The -Cl of

the product 45 was next substituted with 4-(aminomethyl)-1-

BOC-piperidine or other amine analogs. The resulting 2-

substituted 4,5-dibromothiophene compound 46 was sub-

jected to two selective Suzuki coupling reactions to produce,

upon deprotection of BOC, the target 2-aminomethyl-

thiophene series of compounds.

Figure 1. P300/CBP HAT catalyzed reaction and inhibitors with

^t^h^eⁱ^r^I^C⁵0 values.

Furan-containing inhibitors 34 and 35 were prepared

similarly from 4,5-dibromofuran-2-carbaldehyde (44, X = O).

All thiazole compounds were synthesized from 4-bromothia-

zole-2-carbaldehyde (48), which was treated with N-

bromosuccinimide to produce di-Br compound 49. The

aldehyde group of 48 and 49 was reduced and converted to

disclosed compound A-485, other compounds are not drug-

like probes for cellular or in vivo studies. Lys-CoA and its

7,18

analogs are not cell-permeable.

Compounds C646,

L002,

and Cpd-2c

contain a “PAINS” (pan-assay

a -CH Cl group in compound 50, which was substituted with

interfering compound)

or related structure and are

-BOC-4-(aminomethyl)piperidine followed by Suzuki cou-

pling reactions to give, after deprotection, compounds 37 and

8. Oxidation of 48 (or 49) produced thiazole-2-carboxylic

unfavorable for drug development or cell biology. A-485 is a

potent inhibitor of p300/CBP HAT, which is competitive

against the enzyme cofactor acetyl coenzyme A (Ac-CoA). It

showed strong activity against proliferation of several cancer

cells. Nevertheless, given the low success rate in drug

discovery, more chemotypes of p300 HAT inhibitors are

needed. Here, we report discovery, structure−activity relation-

ships (SAR),and biochemical and biological activities of a

novel series of inhibitors of p300/CBP HAT that are

competitive against the histone substrate.

acid 51, which was reacted with 1-BOC-4-(aminomethyl)-

piperidine followed by Suzuki coupling reactions to aﬀord

thiazole compound 39 (or 36). For the synthesis of the

pyrazine compound 40, di-4-bromobenzil (52) was reﬂuxed

with 2,3-diaminopropanoic acid in methanol to give 5,6-di(4-

bromophenyl)pyrazine-2-carboxylic acid (53), whose -CO H

group was acylated, reduced (with NaBH ) to -CH OH, and

converted to -CH Cl (54). A substitution reaction with 4-

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

inhibitory activities of compounds 1 −32 are shown in Table 1

Article

Table 1. Structure and Inhibitory Activity of Compounds 1−32

compd

R⁴

R⁵

R²

IC₅₀(μM) (% inhibition at 10 μM)

4-t-Bu-Ph

= R

-CONH-

piperidin-4-ylmethyl

same as above

piperidin-4-yl

8.6 ± 0.4

1.6 ± 0.2

2.8 ± 0.6

7.4 ± 0.8

10.0 ± 1.4

1.6 ± 0.2

>10 (12%)

>10 (36%)

>10 (31%)

7.4 ± 1.0

1.7 ± 0.2

0.62 ± 0.2

>10 (0%)

5.0 ± 1.6

2.2 ± 0.4

3.0 ± 1.0

1.4 ± 0.3

>10 (5%)

>10 (20%)

4.4 ± 1.6

7.0 ± 1.6

5.4 ± 1.4

>10 (10%)

5.8 ± 0.6

4.6 ± 1.4

2.0 ± 0.5

>10 (35%)

>10 (0%)

>10 (22%)

>10 (23%)

>10 (0%)

>10 (18%)

4-(furan-3-yl)-Ph

4-methoxybiphenyl

4-(furan-3-yl)-Ph

4-aminomethyl-Ph

4-(piperidin-1-ylmethyl)-Ph

4-(piperazin-1-ylmethyl)-Ph

furan-3-yl

4-t-Bu-Ph

4-(furan-3-yl)-Ph

4-t-Bu-Ph

pyridin-3-yl

4-t-Bu-Ph

4-methoxybiphenyl

4-(furan-3-yl)-Ph

4-t-Bu-Ph

4-i-Pr-Ph

= R

-CH NH-

-CH O-

-CH NH-

2-(piperidin-4-yl)ethyl

3-aminopropyl

6-aminohexyl

-CH NH-

-CH₂-

piperazin-1-yl

morpholin-4-yl

piperidin-4-ylmethyl

same as above

-CH N(Me)-

-CH N(CHO)-

-CH NH-

4-Bu-Ph

-CH NH-

4-(furan-3-yl)-Ph

4-t-Bu-Ph

4-aminomethyl-Ph

4-hydroxymethyl-Ph

furan-3-yl

4-t-Bu-Ph

-CH NH-

4-(piperidin-1-ylmethyl)-Ph

4-aminomethyl-Ph

4-t-Bu-Ph

-CH NH-

= R

-CH NH-

3,4-dimethoxy-Ph

3-biphenyl

-CH NH-

= R

-CH NH-

(

aminomethyl)-1-BOC-piperidine followed by Suzuki coupling

and 9 with no substituents in their 4-aromatic rings were found

to have modest activity, inhibiting p300-HAT by ∼30% at 10

μM, suggesting a substituent (e.g., furan-3-yl in 4) at this

position is favorable.

reactions produced compound 55, which was treated with HCl

to give compound 40.

Structure−Activity Relationship. The structures and

and Supporting Information Charts S1 −S3 and Figure S1.

Compound 1, having 4,5-disubstituted tert-butyl groups as well

as a piperidine-containing amide group, had a moderate IC of

Next, activity optimization of the 2-carboxamide side chain

was performed for compounds 10−21 (Table 1 and Chart S2 ).

The ﬁrst attempt was to replace the amide side chain (e.g., in 1

and 2) with a piperidin-4-yl-methylaminomethyl group in

compounds 10−12. While removal of the carbonyl group

reduces the activity of compound 10 (IC = 7.4 μM) by

.6 μM. Compound 2 with 4,5-disubstituted furan-3-yl groups

was found to have signiﬁcantly enhanced inhibitory activity

with an IC₅₀of 1.6 μM, while compound 3 with two 4-

methoxybiphenyl substituents showed slightly reduced inhib-

itory activity (IC₅₀= 2.8 μM). Compound 4 bearing a mixed

tert-butyl and furanyl group also exhibited a compromised

activity (IC₅₀= 7.4 μM). Compound 5 with a polar 4-

aminomethylphenyl substituent rendered little activity change

∼50% (as compared to 3), compound 11 with an IC of 1.7

μM exhibited a comparable activity to that of 2. Of interest is

that compound 12 showed a potent activity against p300-HAT

with an IC₅₀of 620 nM, which is ∼13× more active than its

amide analog 1. Changing the -NH- linkage (in 10−12) to an

-O- in compound 13 resulted in a complete loss of inhibitory

activity, suggesting the -NH- (which is protonated at

physiological pH) could be a key hydrogen bond donor.

Compounds 14 and 15, whose 2-substituents are one -CH₂-

shorter and longer than that of 12, respectively, were found to

possess ∼8- and 3-fold activity reduction (IC = 5.0 and 2.2

(

IC₅₀= 10 μM). However, compound 6 with a piperidine-

containing 4-substituent, which is less polar as compared to the

polar primary amine in 5, showed an improved activity (IC =

be almost inactive at 10 μM, showing an additional amino

group (as compared to 6) is likely disfavored. Compounds 8

.6 μM). Compound 7 having a piperazine group was found to

μM). Compounds 16 and 17, which carry a linear ω-

J. Med. Chem. XXXX, XXX, XXX−XXX

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Article

Scheme 1. General Synthetic Methods for Thiophene-Containing Compounds

Reagents and conditions: (i) 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, 1-hydroxybenzotriazole, 4-(aminomethyl)-1-BOC-piperidine,

CH Cl , 96% yield; (ii) Ar -B(OH) , Pd(PPh ) , Na CO , 80 °C, 80−90% yield; (iii) Ar -B(OH) , Pd(PPh ) , Na CO , 100 °C, 80−90% yield;

(

iv) HCl in 1,4-dioxane, >90% yield; (v) NaBH , MeOH; (vi) cyanuric chloride, DMF, 81% yield for the 2 steps; (vii) 4-(aminomethyl)-1-BOC-

piperidine or other amine analogs, K CO , DMF, 60−85% yield; (viii) N-bromosuccinimide, CH Cl ; (ix) NaClO , NaH PO , tert-butanol-H O,

-methyl-2-butene; (x) 2,3-diaminopropanoic acid, NaOH, MeOH, reﬂux, 55% yield; (xi) ClCO Me, (iPr) NEt, THF, 95% yield.

2 2

aminopropyl and ω-aminohexyl side chain, respectively, are

also strong inhibitors with IC values of 3.0 and 1.4 μM. Loss

compound 30 suggests a smaller aromatic ring at 4- and 5-

positions is unfavored. Lack of inhibition for compounds 31

and 32 with meta-substitutions at the 4- and 5-positions

indicated that para-substitution (e.g., those in compound 10)

is more favorable for p300 HAT inhibition. Compound 33

(Chart 1) was synthesized with a -Br substituent at the 3-

position of the thiophene core. It turned out to be still a strong

inhibitor with an IC₅₀of 1.8 μM, while 3-Br reduces the

inhibitory activity by ∼3-fold (as compared to 12).

Finally, compounds 34−40 (Chart 1) were synthesized to

study SARs for the central core. Furan analogs 34 and 35 with

IC₅₀values of 4.9 and 3.0 μM, respectively, were found to be

signiﬁcantly less active than thiophene compounds 12 and 11,

suggesting a furan core is less favored. While the thiazole

of activity was found for compounds 18 and 19 bearing a 2-

substituent of piperazin-1-ylmethyl and morpholin-4-ylmethyl

group, showing shorter cyclic 2-substituents are disfavored.

Moreover, methylation and formylation of the middle -NH-

group (in compound 12) produced compounds 20 and 21,

respectively, which were found to have less inhibitory activities

with IC₅₀values of 4.4 and 7.0 μM. These results suggest an

-NH- or -CONH- linkage, which might be a hydrogen bond

donor, is of importance to the inhibition.

Compounds 22−32 (Table 1 and Chart S3) were

synthesized to evaluate eﬀects of varying 4- and/or 5-

substituents of the most potent inhibitor 12. Replacing the

tert-butyl with isopropyl groups in 22 (IC = 5.4 μM) resulted

compound 36 (IC = 2.4 μM) exhibited an improved activity

in ∼8× activity reduction, suggesting a bulkier group is

favorable for the 4- and 5-positions. Loss of activity for

compound 23 with two n-butyl groups at these positions

showed that long alkyl substituents are disfavored. Changing

the 4-substituent to a furan-3-yl group in compound 24 (IC₅₀

as compared to its thiophene analog 1, the thiazole compound

37 (IC₅₀= 8.6 μM) is more than 13× less active than its

thiophene analog 12. Loss of activity for compounds 38 and 39

shows that the 5-substituent is of importance to the inhibition

of p300 HAT. Moreover, compound 40 with a six-membered

pyrazine core was found to be signiﬁcantly less active than its

thiophene analog 11. These results suggest thiophene is the

most favorable aromatic core structure for this series of

inhibitors.

5.8 μM) as well as to a piperidin-1-ylmethyl group in 25

(

IC = 4.6 μM) resulted in reduced activities, as compared to

compounds 12 and 11. Compound 26 with a polar -CH NH

group at the 5-position (of the thiophene core) is still a strong

inhibitor with an IC of 2.0 μM, although it exhibited ∼3-fold

Compound 12 Is Competitive against Histone. We

next performed steady-state enzyme kinetics studies to

investigate the mode of inhibition for the most potent

inhibitor 12. Inhibitory activities of compound 12 were

determined with varying concentrations of the cofactor Ac-

CoA and the substrate histone. As shown in Figure 2A (left),

IC₅₀values of 12 did not change with increasing concen-

activity reduction as compared to 12. However, moving the

CH NH group to the 4-position in compound 27 suﬀered a

2 2

major activity loss. Compound 28 with the amine group at

both positions is inactive. Similarly, compound 29 with two

CH OH groups is also a weak inhibitor. Activities for

compounds 26−29 suggest polar groups are not favorable at

the 4- or 5-position. In addition, very low activity for

trations of Ac-CoA (from 0.5−8 μM, or (0.07−1.2) × K_m),

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

solution and cannot reach the free acceptor beads. The binding

Article

Chart 1. Structures and Inhibitory Activity of Compounds

between the histone peptide and p300 HAT brings the donor

and acceptor beads together, which allows the radicals to

activate the p300 HAT coated acceptor beads and produce

luminescence at 570 nm. Use of histone H4 is because the

assay with histone H3 did not produce a luminescence signal,

presumably due to a weaker binding between p300 HAT and

histone H3. As shown in Figure 2B, compound 12 can disrupt

the binding between the protein and histone H4 and reduce

the Alpha-signal in a dose-dependent manner (IC₅₀= 13.5

μM). This is in contrast to Ac-CoA-competitive inhibitor A-

3−40

485, which did not aﬀect the binding between p300 HAT

and histone H4. In addition, such binding was not interfered

by inactive compound 28 (Figure 2B). Increasing concen-

trations of compound 12 did not signiﬁcantly aﬀect the Alpha-

signals using a biotinylated His6 peptide that can directly link

the donor and acceptor beads (Figure S2), showing 12 does

not interfere with the singlet oxygen radicals or His6 tag.

Taken together, these results indicate the inhibitor 12 is

competitive against the substrate histone, which are of interest

in that due to a diﬀerent mode of inhibition, histone-

competitive inhibitor 12 might have a diﬀerent cellular activity

or selectivity, as compared to Ac-CoA-competitive inhibitors

16,20

A-485 and C646.

Docking Studies. We next performed molecular modeling

to ﬁnd possible binding structures of the most potent inhibitor

2 in p300 HAT. The program Glide in the Schrodinger small-

molecule drug discovery software suite was used for the

docking studies, with the crystal structure of human p300 HAT

in complex with Ac-CoA (PDB code 4PZS) as a template.

Since compound 12 is a competitive inhibitor against the

substrate histone, Ac-CoA was treated as an integral part of the

protein. Upon protein structure preparation as well as docking

grid generation, compound 12 was docked into the protein-Ac-

CoA complex, and the results are shown in Figure 3. The

inhibitor can be favorably docked into the protein, as the 10

distinct docking conformations of compound 12 with the

lowest energies exhibit similar binding features (Figure 3A).

One of the tert-butyl groups of these conformations is

predicted to insert into and have favorable hydrophobic

interactions with the so-called “lysine channel”, through which

the histone lysine side chain inserts into p300 HAT, attacks the

carbonyl of Ac-CoA, and gets acetylated. Residues Trp1436,

Cys1438, Tyr1446, and Ser1396 form the lysine channel of

p300 HAT (Figure 3B). As shown in Figure 3B, the tert-butyl

group is predicted to be ∼3.5 Å from these residues with

indicating compound 12 is likely a noncompetitive inhibitor

against the enzyme cofactor. Its inhibitory activities were found

to be reduced in a linear manner (Figure 2A) when increasing

concentrations of the substrate histone peptide (2−100 μM)

were used in the assay, suggesting compound 12 is a

competitive inhibitor against the substrate histone.

Next, we used a reported Alpha (ampliﬁed luminescent

proximity homogeneous assay) assay to conﬁrm this ﬁnding.

Upon excitation by a laser beam (680 nm), histone H4(1−21)

peptide coated donor beads generate singlet oxygen radicals,

which can travel for a very short distance (<200 nm) in the

Figure 2. Compound 12 is a competitive inhibitor of p300 HAT against the substrate histone. (A) Plots of IC₅₀values of 12 versus increasing

concentrations of (left) Ac-CoA (0.5−8 μM, or (0.07−1.2) × K ) and (right) histone H3 suggest the inhibitor is noncompetitive against Ac-CoA

and competitive against histone. (B) Alpha assay results show compound 12 can dose-dependently disrupt the binding between p300 HAT and

histone H4. But such binding was not aﬀected by inactive compound 28 as well as A-485 which is an inhibitor competitive against Ac-CoA.

Journal of Medicinal Chemistry

J. Med. Chem. XXXX, XXX, XXX−XXX

residues. As shown in Figure 4 , compound 12 was found to

Article

activities for compounds 30−32, which do not have a para-

substituent or have a meta-substituent which might not

favorably interact with the lysine channel residues of the

protein.

Enzyme Selectivity. Humans have three classes of

histone/protein lysine acetyltransferases with distinct con-

served motifs and structures, including p300/CBP, Gcn5-

related N-acetyltransferase (GNAT) and MYST (MOZ, Ybf2,

Sas2, and Tip60) family of HATs. Compound 12 was tested

for its activities against selected HATs from these three classes

of HATs. CBP is a homolog of p300. The HAT domains of

CBP and p300 exhibit 87% identity in sequence. PCAF (p300/

CBP associating factor) is a member of the GNAT family,

while Myst3 belongs to the MYST family of HATs. P300/CBP

HATs have a distinct sequence and three-dimensional

structure from the GNAT and MYST HAT proteins. As

shown in Table 2, compound 12 was found to be also a strong

Table 2. Inhibitory Activities of Compound 12 against

HATs

IC₅₀(μM)

P300-HAT

CBP-HAT

PCAF

0.62 ± 0.12

1.2 ± 0.13

>50

Myst3

>50

inhibitor of human CBP HAT with an IC₅₀of 1.2 μM,

comparable to its IC₅₀against p300 HAT. However,

compound 12 did not signiﬁcantly inhibit the activity of

PCAF and Myst3 even at 50 μM, showing a high selectivity for

p300/CBP HAT. In addition, since the initial hit compound 1

was for SAR studies targeting LSD1, both compounds 1 and

Figure 3. Docking results for compound 12: (A) 10 docking

conformations of 12 (with C atoms in green) with lowest energies in

the crystal structure of p300-HAT (PDB code 4PZS, shown as an

electrostatic surface) in complex with Ac-CoA (as a tube model with

C atoms in brown); (B) the lowest energy docking conformation of

2 did not signiﬁcantly inhibit activity of LSD1 at 50 μM.

Inhibition of Cellular Histone Acetylation. The ability

of compound 12 to inhibit cellular p300/CBP HAT was

evaluated. Also included in the experiment are an inactive

compound 28 and a known inhibitor A-485. Kasumi-1

leukemia cells were treated with increasing concentrations of

these compounds for 12 h. Histone was extracted and

subjected to electrophoresis separation and Western blot

staining to determine levels of acetylation at various lysine

2 (as a ball-and-stick model) with surrounding residues and Ac-CoA

in p300-HAT (PDB code 4PZS). Hydrogen bonds are shown as

yellow dotted lines.

favorable van de Waals interactions. The central thiophene

core and the two para-tert-butylphenyl groups of these

structures have extensive hydrophobic interactions with either

upper or lower surface of the protein (Figure 3A/B). In

addition, the positively charged, two amino groups of many of

these docking structures form hydrogen bonds and have

favorable electrostatic interactions with Asp1628 and Glu1505,

as exempliﬁed in Figure 3B. These favorable binding features

are consistent with the strong inhibitory activity of compound

Moreover, the docking results might be used to rationalize

some of the observed SARs. For example, H-bond/electrostatic

interactions between -NH- (positively charged at physiological

pH) and Asp1628 are predicted to contribute signiﬁcantly to

the binding of compound 12. This could explain the loss of

activity for compound 13 with an -O- linkage, which does not

favorably interact with the (negatively charged) Asp1628 side

chain. Since an amide -CONH- mimics the positively charged

Figure 4. Cellular activity of compounds 12, 28, and A-485 against

acetylation of H3K9, H3K18, and H3K27.

signiﬁcantly inhibit cellular acetylation of histone H3K9,

H3K18, and H3K27 at 5 and 10 μM in a dose dependent

manner. H3K27 seems to be the most sensitive cellular

substrate of p300 HAT. Inactive compound 28 was found to

have no signiﬁcant activity against acetylation of these histone

-NH- in 12, many of the amide compounds (e.g., compounds 2

and 6 in Table 1) are potent inhibitors. Docking results also

suggest favorable interactions of 12 with the lysine channel are

important for tight binding. This could explain weak or no

residues. As compared to A-485 (enzyme IC₅₀of 60 nM ),

compound 12 is less active against acetylation of H3K27 and

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Article

K18, which is consistent with the enzyme activities of these

compounds. However, compound 12 seems to exhibit more

potent cellular activity against acetylation of H3K9. These

diﬀerences might be due to diﬀerent mode of inhibition

Surprisingly, although A-485 exhibited more potent activity

(EC₅₀= 0.33 μM) against Kasumi-1 leukemia cells, it had no

signiﬁcant activity against proliferation of breast and pancreatic

cancer cells (Table 3). These diﬀerences might be due to

diﬀerent modes of inhibition (competitive against histone vs

Ac-CoA) or cell permeability for compound 12 and A-485.

Gene Expression Proﬁling. RNA sequencing was used to

investigate how treatment with p300 HAT inhibitor 12 aﬀects

gene expression in ER+ breast cancer MCF-7 cells. Upon

starvation (i.e., culturing in charcoal treated FBS without

hydrophobic hormones including estrogens) for 7 days, MCF-

7 cells were supplemented with estradiol (10 nM), followed by

treatment with compound 12 for 2 days. Total RNA from

three groups of MCF-7 cells, including starved, control (with

estradiol), and treated (with estradiol and compound 12)

groups, was puriﬁed, prepared, and sequenced. RNA

sequencing data were mapped onto the human genome, and

gene expression proﬁles were determined and normalized.

Genes with signiﬁcantly changed expression levels were

determined using a two-sided parametric t-test with the p

(

competitive against histone vs Ac-CoA) or cell permeability

for the two compounds.

Inhibition of Tumor Cell Proliferation. Activity of

compound 12 was tested against proliferation of several

tumor cell lines in which p300/CBP HAT is of importance.

First, ER-mediated transcription is critical to ER+ breast

cancer. It is an ordered, stepwise assembly of ER and

10,27

coactivator proteins for gene expression.

Upon binding

with an estrogen molecule in cytoplasm, ER undergoes

conformational changes, forms a homodimer, and is trans-

located into the nucleus, where ER binds to estrogen response

element, a short segment of DNA within a gene promoter.

Next, a steroid receptor coactivator (SRC) protein binds to

ER, followed by recruitment of p300 through its SRC-binding

SID domain. P300 HAT activity is required for the ER-

mediated gene expression because a mutant p300 without the

enzyme function failed to activate it. Given the critical role of

p300-mediated histone acetylation in the ER signaling

pathway, compound 12 was assessed for its activity against

the ER+ breast cancer cell line MCF-7. In addition, ER

antagonist tamoxifen is widely used in the clinic to treat ER+

breast cancer by suppressing ER mediated gene transcription.

However, many patients eventually develop resistance to

value of <0.05. Gene set enrichment analysis (GSEA) was

used to analyze signiﬁcant gene expression changes between

these three groups of MCF-7 cells.

First, to determine whether the observed cellular activities of

compound 12 are due to inhibition of p300 HAT, we used

GSEA to compare the transcriptional proﬁle of the 12-treated

MCF-7 cells with a publicly available p300 transcriptional

signature (GSE31873), which was derived from siRNA-

28,29

tamoxifen and die of the cancer. In our previous work,

have developed MCF-7 derivative cells that were made

resistant to tamoxifen. Activity of compound 12 was also

tested against tamoxifen-resistant MCF-7 to ﬁnd whether

inhibition of HAT can aﬀect growth of these cells. As

summarized in Table 3, compound 12 exhibited strong

mediated p300 knockdown in C4-2B prostate cancer cells.

As shown in Figure 5A, there is a strong concordance between

the two transcriptional signatures: Genes suppressed by

compound 12 were strongly enriched among those suppressed

by p300 knockdown, with normalized enrichment score (NES)

of −3.32 and p value of <0.001. This result demonstrates that

treatment with the p300 HAT inhibitor mimics the transcrip-

tional footprint of p300 depletion and supports that p300 HAT

is the cellular target of compound 12.

Next, activity of compound 12 in the ER signaling pathway

was analyzed. As compared to starved cells, supplementation

with estradiol potently caused upregulation as well as

downregulation of several publicly available ER-related gene

signatures in the control group of MCF-7 cells (Figure S3).

Importantly, treatment with compound 12 counteracted such

estrogen-induced gene transcription: The gene set upregulated

by estradiol was strongly downregulated upon treatment with

12 (Figure 5B, NES = −9.19 and p < 0.001), and the genes

suppressed by estradiol were signiﬁcantly upregulated by

compound 12 (Figure 5C, NES = 7.41 and p < 0.001). These

results are consistent with previous studies showing p300 HAT

is essential for ER-mediated gene expression and demonstrate

that pharmacological inhibition of p300 HAT can oﬀset the

eﬀects of estrogen in gene regulation.

Moreover, we investigated gene expression changes caused

by compound 12 in the context of cancer biology and therapy.

As shown in Figure 5D, global gene expression proﬁling

analysis revealed that inhibition of p300 HAT by compound

12 counteracted estradiol and caused signiﬁcant suppression of

a number of estrogen-induced, cancer-related gene sets,

including E2F and c-Myc gene signatures as well as those

involved in cell proliferation, cell cycle, mitosis, DNA

replication, DNA repair, and self-renewal (stemness) (Figure

S4). Speciﬁcally for breast cancer, treatment with compound

12 downregulated a number of gene signatures that have

Table 3. Antiproliferative Activity EC₅₀(μM) of

Compounds 12, 28, 31, and A-485

A-485

>30

MCF-7

MCF-7 (Tam-R)

PANC-1

MDA-PANC-28

Kasumi-1

2.8 ± 0.1

3.4 ± 0.1

1.0 ± 0.2

2.8 ± 0.4

2.6 ± 0.6

>30

18.7 ± 0.1

0.33 ± 0.01

antiproliferative activity against parent MCF-7 cells (EC₅₀

.8 μM) as well as tamoxifen-resistant cells (EC₅₀= 3.4 μM).

These results show that inhibition of p300-mediated histone

acetylation can inhibit growth of MCF-7 breast cancer cells

and such activity is independent upon the status of tamoxifen

resistance, suggesting the p300 HAT inhibitor could have

potential clinical applications in breast cancer therapy.

Moreover, p300 HAT activity has been reported to play

important roles in pancreatic cancer and acute myeloid

leukemia caused by oncogene RUNX1-ETO. Activity of

compound 12 was evaluated against two pancreatic cancer cells

PANC-1 and MDA-PANC-28 and RUNX1-ETO leukemia cell

line Kasumi-1. As shown in Table 3, compound 12 showed

potent activities against proliferation of the two pancreatic

cancer cells with EC₅₀values of 1.0 and 2.8 μM. It also

inhibited growth of Kasumi-1 cells with an EC₅₀of 2.6 μM.

Inactive compounds 28 and 31 exhibited no inhibitory

activities against proliferation of these cancer cells, supporting

activity of compound 12 is related to inhibition of p300 HAT.

Journal of Medicinal Chemistry

J. Med. Chem. XXXX, XXX, XXX−XXX

Article

Figure 5. Gene set enrichment analysis (GSEA) results showing signiﬁcant gene expression changes (p < 0.001) in ER+ MCF-7 cells by treatment

with p300 HAT inhibitor 12, as compared to the starved (without estradiol) or control (with estradiol) group. (A) Inhibitor treatment caused

signiﬁcant downregulation of genes that were suppressed by siRNA-mediated p300 knockdown (GSE31873). (B−D) Results showing compound

12 counteracted estradiol in MCF-7 cells. Treatment with 12 signiﬁcantly (B) suppressed expression of genes that were upregulated by estradiol

and (C) induced expression of genes that were downregulated by estradiol. (D, E) GSEA heatmaps of normalized enrichment scores (NES) for

publicly available gene signatures (from the MSigDB database). (D) Inhibitor 12 caused signiﬁcant downregulation of cancer-related gene sets that

were induced by estradiol. (E) Inhibitor 12 suppressed expression of gene sets associated with breast cancer as well as the poor prognosis,

progression, invasion, and relapse of breast cancer.

previously been shown to be upregulated in multiple breast

cancer patient data sets (as compared to normal breast

tissues), associated with poor prognosis, cancer progression,

invasion, and relapse (Figure 5E). Collectively, these GSEA

results show the importance of p300 HAT in breast and other

types of cancer, as well as the perspective for pharmacological

inhibition of p300 HAT (by e.g., compound 12) in breast

cancer therapy.

indications. In addition, p300 and CBP are large proteins

(∼2400 amino acids) with multiple other domains playing

important physiological roles. They also share a high degree of

homology with many duplicate functions. Given these two

points, cell-permeable, small-molecule inhibitors of the HAT

domain of p300/CBP are particularly useful for studying

biological functions of p300/CBP HAT because observed

activities by genetic knockdown/knockout of p300 or CBP

might not be relevant.

CONCLUSION

Compound screening followed by medicinal chemistry

studies have led to the discovery of trisubstituted thiophene

■

Histone acetylation by the HAT domain of p300/CBP has

been found to play critical roles in many nuclear receptor-

regulated signaling pathways, such as ER, AR, and peroxisome

proliferator-activated receptors (PPARs). Dysfunction of

these gene transcription programs has been found in a number

of diseases such as cancer and obesity. Moreover, p300/CBP

has been found to directly contribute to oncogenesis (e.g., in

RUNX1-ETO leukemia) or be part of a fusion oncogene due

compound 12 that is a novel inhibitor with an IC of 620 nM

against p300 HAT and 1.2 μM against CBP HAT. It did not

signiﬁcantly inhibit two other major human HATs PCAF and

Myst3 at 50 μM, showing a high selectivity. Biochemical

studies showed that compound 12 is competitive against the

substrate histone and noncompetitive against the cofactor Ac-

CoA, showing a distinct mode of inhibition from previous

studied inhibitors C646 and A-485. Docking studies of 12 into

the histone binding pocket of the p300 HAT structure

provided possible binding models of the inhibitor in the

,14,15

to chromosome translocation.

inhibitors of p300/CBP HAT are needed, which could be

useful chemical probes and potential therapeutics for these

Therefore, drug-like

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

protein. In addition, compound 12 was found to be cell-

permeable and inhibit cellular acetylation at several histone

lysine residues, with H3K27 being the most sensitive substrate.

With EC₅₀of ∼3 μM, compound 12 strongly inhibited

proliferation of ER+ breast cancer cell line MCF-7, regardless

of its sensitivity or resistance to the commonly used estrogen

antagonist tamoxifen, suggesting the potential therapeutic

application of this compound in the context of endocrine

resistance. Compound 12 also exhibited strong activity against

growth of several pancreatic cancer cells and RUNX1-ETO

leukemia cell Kasumi-1, in which p300 HAT is known to be

5:1) was heated to 80 °C for 12 h. The reaction was then quenched

with brine (10 mL). The product was extracted with diethyl ether (3

20 mL), and the combined organic layers were washed with water

and brine, dried over Na SO . Upon removal of the solvent, the

product was puriﬁed by column chromatography (silica gel, n-

_h_e_x_a_n_e_s_/_e_t_h_y_l_a_c_e_t_a_t_e_f_r_o_m₅_:₁_t_o₁_:₂₎_t_o_g_i_v_e₅_-_A_r1 substituted

thiophene product as a white solid (60−90% yield). The product thus

obtained (0.2 mmol) was added into a solution of Ar -boronic acid or

its pinacol ester (0.21 mmol), tetrakis(triphenylphosphine)palladium

(7 mg), and sodium carbonate (42.4 mg, 0.4 mmol) in 1,4-dioxane/

H O (5/1 mL). The mixture was heated to 100 °C for 12 h. Similar

workup and puriﬁcation gave 4,5-disubstituted thiophene compound

43 as a white solid (60−90% yield).

0,31

important.

To a solution of 43 (0.1 mmol) in dichloromethane (2 mL) at 0

Global gene transcription proﬁling was performed to

investigate the cellular target of compound 12 as well as the

mechanism for its observed biological activities in MCF-7 cells.

First, the overall transcriptional changes upon treatment with

compound 12 recapitulated those caused by siRNA-mediated

p300 knockdown. This result, together with its inhibitory

activity in cellular histone lysine acetylation, support that p300

HAT is the cellular target of compound 12. Second, inhibition

of p300 HAT activity by compound 12 signiﬁcantly oﬀset

estrogen-induced gene expression, showing p300 HAT is

essential for the ER activity. Moreover, treatment with

compound 12 strongly downregulated a number of (general)

cancer-related gene signatures, as well as gene sets that have

been identiﬁed in the clinic for the progression, invasion,

relapse, and poor prognosis of breast cancer. These results,

together with the compound’s potent antiproliferative activities

against several types of cancer cells, suggest that pharmaco-

logical inhibition of p300 HAT is a useful therapeutic approach

to cancer treatment.

C, HCl (0.1−0.2 mL, 4 N in 1,4-dioxane) was added slowly and then

stirred at room temperature for 12 h. Upon removal of the solvent

carefully, the residual oil was treated with anhydrous diethyl ether and

vacuum-dried to give the target compound (white powder) as a

hydrochloric salt (90−100% yield).

General Method B (Reactions v−vii). To a solution of 4,5-

dibromothiophene-2-carboxaldehyde 44 (5.40 g, 20 mmol) in MeOH

(

40 mL), NaBH (0.79 g, 21 mmol) was added slowly 0 °C. The

reaction mixture was stirred at room temperature for 1 h and

quenched with water (50 mL). The product was extracted with

diethyl ether (3 × 100 mL), and the combined organic layers were

washed with water and brine, dried over Na SO . Upon removal of

the solvent carefully, the residual oil was dried and used in the next

step without puriﬁcation. It was dissolved in DMF (20 mL), and the

solution was cooled to 0 °C. Cyanuric chloride (3.69 g, 20 mmol) was

added slowly at 0 °C and stirred at room temperature for 10 h. The

reaction was quenched with saturated NaHCO (50 mL). The

product was extracted with diethyl ether (3 × 100 mL), and the

combined organic layers were washed with water and brine, dried over

Na SO . Removal of the solvents in vacuo aﬀorded compound 45 as a

colorless oil, which is used without puriﬁcation. To a solution of an

amine (10 mmol) and potassium carbonate (1.38 g, 10 mmol) in

DMF (10 mL), 45 (1.45 g, 5 mmol) in DMF (10 mL) was added

slowly at 0 °C, and the mixture was stirred at room temperature for 10

h. Upon quenching with saturated NaHCO (50 mL), the product

was extracted with diethyl ether (3 × 50 mL) and the combined

organic layers were washed with water and brine, dried over Na SO .

In conclusion, our results demonstrate that compound 12 is

not only a useful small-molecule probe for biological studies of

p300/CBP HAT but also a novel pharmacological lead for

further drug development targeting breast and other types of

cancer.

A column chromatography (silica gel, hexanes/ethyl acetate from 40:1

to 2:1) for the residue oil gave compound 46 as a pale-yellow oil (60−

85% yield for the three steps).

Suzuki coupling reactions of compound 46 as well as deprotection

of the BOC group were performed as described above, to produce the

target compounds.

General Method C (Reactions viii and ix). 4-Bromothiazole-2-

carbaldehyde 48 (1.92 g, 10 mmol) and N-bromosuccinimide (NBS,

1.78 g, 10 mmol) were dissolved in dichloromethane (20 mL) and

stirred at room temperature for 12 h. Upon removal of the solvent

carefully, the residue was puriﬁed by column chromatography (silica

gel, hexanes/ethyl acetate from 40:1 to 10:1) to give 4,5-

dibromothiazole-2-carbaldehyde 49 as a white solid (433 mg, 16%

yield). Conversion of aldehydes 48 and 49 to compounds 38 and 37

followed the reactions described above.

EXPERIMENTAL SECTION

■

All chemicals for synthesis were purchased from Alfa Aesar (Ward

Hill, MA) or Aldrich (Milwaukee, WI). The identity of the

synthesized compounds was characterized by H and C NMR on

a Varian (Palo Alto, CA) 400-MR spectrometer and mass

spectrometer (Shimadzu LCMS-2020). The identity of the potent

inhibitors was conﬁrmed with high resolution mass spectra (HRMS)

using an Agilent 6550 iFunnel quadrupole-time-of-ﬂight (Q-TOF)

mass spectrometer with electrospray ionization (ESI). The purities of

the ﬁnal compounds were determined to be >95% with a Shimadzu

Prominence HPLC using a Zorbax C18 (or C8) column (4.6 mm ×

50 mm) monitored by UV at 254 nm.

General Method A (Reactions i−iv). To a mixture of 4,5-

dibromothiophene-2-carboxylic acid 41 (2.86 g, 10 mmol), 1-ethyl-3-

(

3-dimethylaminopropyl)carbodiimide (1.71 g, 11 mmol), 1-hydrox-

4,5-Dibromothiazole-2-carbaldehyde 49 (406 mg, 1.5 mmol) or 48,

ybenzotriazole (1.48 g, 11 mmol) in dichloromethane (20 mL), 1-

BOC-4-(aminomethyl)piperidine (2.57 g, 12 mmol), and triethyl-

amine (2.8 mL, 20 mmol) in CH Cl (10 mL) were added slowly at 0

sodium chlorite (190 mg, 2.1 mmol), and 2-methylbut-2-ene (1.6 mL,

15 mmol) were dissolved by BuOH/H O (5/1 mL). Sodium

dihydrogen phosphate (900 mg in 5 mL H O) was added slowly into

C. The reaction mixture was stirred at room temperature for 1 h and

the reaction mixture. The mixture was stirred at rt for 12 h. The

reaction was then quenched with brine (10 mL). The product was

extracted with diethyl ether (3 × 10 mL), and the combined organic

layers were washed with water and brine, dried over Na SO . The

quenched by adding saturated NaHCO (50 mL). The product was

extracted with dichloromethane (3 × 50 mL) and the combined

organic layers were washed with water and brine, dried over Na SO .

Upon removal of the solvent, the product was puriﬁed by column

chromatography (silica gel, n-hexanes/ethyl acetate from 10:1 to 2:1)

to give the amide 42 as a white solid (4.63 g, 96% yield).

volatiles were removed in vacuo to aﬀord acid 51 as a crude oil, which

was used directly in the next step without further puriﬁcation.

4,5-Bis(4-(tert-butyl)phenyl)-N-(piperidin-4-ylmethyl)-

thiophene-2-carboxamide Hydrochloride (1). 1 was prepared

from 4,5-dibromothiophene-2-carboxylic acid, following the general

A mixture of 42 (0.4 mmol), Ar -boronic acid or its pinacol ester

0.42 mmol), tetrakis(triphenylphosphine)palladium (13.9 mg), and

(

sodium carbonate (84.8 mg, 0.8 mmol) in 1,4-dioxane/H O (6 mL,

method A, as a hydrochloric acid salt (white powder). H NMR (400

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

MHz, DMSO-d ): δ 8.69 (br, 3H), 7.92 (s, 1H), 7.38−7.35 (m, 4H),

108.6, 58.6, 51.7, 43.9, 42.9, 33.7, 26.3, 22.2, and 21.5. MS (ESI) calcd

.26−7.21 (m, 4H), 3.26−3.23 (m, 2H), 3.17 (br, 2H), 2.85−2.79

for (C H N O S) [M + H] 540.7, found 540.6.

33 38 3 2

(

m, 2H), 1.82−1.79 (m, 3H), 1.42−1.36 (m, 2H), and 1.27 (s, 18H).

5-(4-(tert-Butyl)phenyl)-4-(4-(piperazin-1-ylmethyl)phenyl)-

N-(piperidin-4-ylmethyl)thiophene-2-carboxamide Hydro-

chloride (7). 7 was prepared from 4,5-dibromothiophene-2-

C NMR (100 MHz, DMSO-d ): 161.1, 150.8, 149.8, 141.5, 137.6

(

2), 132.7, 131.4, 130.4, 128.4, 128.2, 125.6, 125.3, 43.8, 42.8, 34.4,

carboxylic acid, following the general method A, as a hydrochloric

4.3, 33.7, 31.1, 31.0, and 26.22. MS (ESI) calcd for (C H N OS)

31 41 2

acid salt (white powder). H NMR (400 MHz, CD

OD): δ 7.83 (s,

[

M + H] 489.7, found 489.6.

H), 7.58 (d, J = 8.1 Hz, 2H), 7.42 (d, J = 8.1 Hz, 2H), 7.37 (d, J =

.4 Hz, 2H), 7.23 (d, J = 8.4 Hz, 2H), 4.44 (s, 2H), 3.61 (br, 4H),

.56 (br, 4H), 3.43 (d, J = 8.1 Hz, 2H), 3.35 (d, J = 6.0 Hz, 2H), 3.00

,5-Bis(4-(furan-3-yl)phenyl)-N-(piperidin-4-ylmethyl)-

thiophene-2-carboxamide Hydrochloride (2). 2 was prepared

from 4,5-dibromothiophene-2-carboxylic acid, following the general

(t, J = 12.2 Hz, 2H), 2.03−1.98 (m, 3H), 1.55−1.45 (m, 2H), and

method A, as a hydrochloric acid salt (white powder). H NMR (400

1.31 (s, 9H). C NMR (100 MHz, CD OD): 164.3, 153.1, 145.8,

MHz, DMSO-d ): δ 8.89 (br, 1H), 8.76 (br, 1H), 8.58 (br, 1H), 8.21

39.2, 138.5, 138.1, 134.2, 132.8, 132.3, 131.6, 130.9, 130.0, 126.9,

(

s, 2H), 8.00 (s, 1H), 7.74 (s, 2H), 7.61−7.59 (m, 4H), 7.31−7.28

1.4, 58.7, 45.4, 45.0, 42.2, 35.6, 31.6, 28.5, and 27.8. MS (ESI) calcd

for (C32H N OS) [M + H] 531.8, found 531.7.

m, 4H), 6.97 (s, 2H), 3.27−3.23 (m, 2H), 3.19 (br, 2H), 2.86−2.78

43 4

m, 2H), 1.85−1.82 (m, 3H), and 1.42−1.36 (m, 2H). C NMR

-(4-(tert-Butyl)phenyl)-4-(furan-3-yl)-N-(piperidin-4-

100 MHz, DMSO-d ): 161.0, 147.1, 144.5, 141.6, 139.9, 139.6,

ylmethyl)thiophene-2-carboxamide Hydrochloride (8). 8 was

38.6, 138.0, 137.8, 133.9, 132.08, 131.96, 131.65, 131.54, 131.0,

prepared from 4,5-dibromothiophene-2-carboxylic acid, following the

30.8, 129.2, 129.0, 125.9, 125.7, 108.6, 43.8, 42.8, 33.8, and 26.2. MS

general method A, as a hydrochloric acid salt (white powder). H

(

ESI) calcd for (C H N O S) [M + H] 509.6, found 509.5.

31 29 2 3

NMR (400 MHz, DMSO-d ): δ 8.90 (br, 1H), 8.73 (br, 1H), 8.59

,5-Bis(4′-methoxy-[1,1′-biphenyl]-4-yl)-N-(piperidin-4-

(

br, 1H), 7.97 (s, 1H), 7.65 (s, 1H), 7.44−7.34 (m, 5H), 6.29 (s,

ylmethyl)thiophene-2-carboxamide Hydrochloride (3). 3 was

H), 3.27−3.24 (m, 2H), 3.17 (br, 2H), 2.84−2.80 (m, 2H), 1.83−

prepared from 4,5-dibromothiophene-2-carboxylic acid, following the

.78 (m, 3H), 1.40−1.37 (m, 2H), and 1.29 (s, 9H). C NMR (100

general method A, as a hydrochloric acid salt (white powder). H

MHz, DMSO-d ): 161.4, 151.7, 144.1, 140.4, 138.2, 130.7, 130.5,

NMR (400 MHz, DMSO-d ): δ 8.91 (br, 1H), 8.79 (br, 1H), 8.62

29.2, 129.1, 126.3, 126.0, 120.8, 110.6, 44.2, 43.2, 34.9, 34.1, 31.4,

(

br, 1H), 8.02 (s, 1H), 7.70−7.56 (m, 8H), 7.38−7.35 (m, 4H),

and 26.6. MS (ESI) calcd for (C H N O S) [M + H] 423.6, found

.02−6.99 (m, 4H), 3.78 (s, 6H), 3.27−3.23 (m, 2H), 3.19 (br, 2H),

.87−2.79 (m, 2H), 1.85−1.82 (m, 3H), and 1.42−1.36 (m, 2H).

NMR (100 MHz, DMSO-d ): 161.1, 159.2, 159.0, 141.6, 139.6,

din-3-yl)thiophene-2-carboxamide Hydrochloride (9). 9 was

38.6, 138.1, 137.7, 133.8, 131.64, 131.57, 131.46, 131.38, 129.3,

29.2, 127.72, 127.66, 126.5, 126.2, 114.5, 114.4, 55.2, 43.8, 42.9,

prepared from 4,5-dibromothiophene-2-carboxylic acid, following the

general method A, as a hydrochloric acid salt (white powder). H

3.8, 28.1, and 26.3. MS (ESI) calcd for (C H N O S) [M + H]

NMR (400 MHz, DMSO-d ): δ 9.00 (br, 1H), 8.92 (br, 1H), 8.74

89.8, found 589.9.

-(4-(tert-Butyl)phenyl)-4-(4-(furan-3-yl)phenyl)-N-(piperi-

din-4-ylmethyl)thiophene-2-carboxamide Hydrochloride (4).

was prepared from 4,5-dibromothiophene-2-carboxylic acid,

(

br, 1H), 8.70 (br, 1H), 8.17 (br, 2H), 7.85 (s, 1H), 7.61−7.55 (m,

H), 7.40−7.26 (m, 4H), 3.27−3.18 (m, 4H), 2.83−2.80 (m, 2H),

.84−1.79 (m, 3H), 1.42−1.39 (m, 2H), and 1.26 (s, 9H). C NMR

(

100 MHz, DMSO-d ): 161.2, 152.1, 145.2, 143.8, 143.3, 142.8,

following the general method A, as a hydrochloric acid salt (white

39.2, 134.1, 132.2, 131.1, 129.5, 129.1, 126.6, 126.5, 44.3, 43.2, 34.9,

powder). H NMR (400 MHz, DMSO-d ): δ 8.90 (br, 1H), 8.76 (br,

34.1, 31.4, and 26.6. MS (ESI) calcd for (C H N OS) [M + H]

26 32 3

H), 8.61 (br, 1H), 8.21 (s, 1H), 7.97 (s, 1H), 7.92 (s, 1H), 7.74 (s,

H), 7.59 (s, 1H), 7.36−7.23 (m, 6H), 6.97 (s, 2H), 3.27−3.23 (m,

H), 3.18 (br, 2H), 2.85−2.79 (m, 2H), 1.83−1.80 (m, 3H), 1.40−

N-(piperidin-4-ylmethyl)methanamine Hydrochloride (10). 10

.36 (m, 2H), and 1.26 (s, 9H). C NMR (100 MHz, DMSO-d ):

was prepared from 4,5-dibromothiophene-2-carboxaldehyde, follow-

61.1, 151.0, 144.4, 142.0, 139.6, 137.8, 137.5, 134.1, 131.0, 129.0,

28.44, 128.40, 128.3, 125.74, 125.70, 125.4, 108.6, 43.8, 42.9, 34.4,

ing the general method B, as a hydrochloric acid salt (white powder).

H NMR (400 MHz, DMSO-d ): δ 9.46 (br, 1H), 9.39 (br, 1H), 8.90

3.8, 31.1, and 26.3. MS (ESI) calcd for (C H N O S) [M + H]

(br, 1H), 8.72 (br, 1H), 7.69 (s, 1H), 7.63−7.54 (m, 8H), 7.34 (d, J =

99.7, found 499.5.

-(4-(Aminomethyl)phenyl)-5-(4-(tert-butyl)phenyl)-N-(pi-

peridin-4-ylmethyl)thiophene-2-carboxamide Hydrochloride

5). 5 was prepared from 4,5-dibromothiophene-2-carboxylic acid,

.2 Hz, 4H), 7.01 (d, J = 8.2 Hz, 4H), 4.41 (s, 2H), 3.78 (s, 6H),

.29−3.26 (m, 2H), 2.94 (br, 2H), 2.87−2.79 (m, 2H), 2.08 (br, 1H),

.98−1.92 (m, 2H), and 1.49−1.39 (m, 2H). C NMR (100 MHz,

(

DMSO-d ): 159.2, 159.1, 139.4, 139.2, 138.6, 137.2, 134.2, 133.9,

following the general method A, as a hydrochloric acid salt (white

31.7, 131.4, 129.4, 129.2, 127.76, 127.70, 126.9, 126.6, 126.4, 126.0,

powder). H NMR (400 MHz, D O): δ 7.76 (s, 1H), 7.25 (d, J = 7.6

114.54. 114.50, 55.3, 50.7, 44.7, 42.5, 30.60, 30.55, and 26.1. MS

Hz, 2H), 7.14 (d, J = 7.6 Hz, 2H), 6.97 (d, J = 7.3 Hz, 2H), 6.90 (d, J

(ESI) calcd for (C H N O S) [M + H] 575.8, found 575.7.

37 39 2 2

7.3 Hz, 2H), 4.03 (s, 2H), 3.45 (d, J = 11.7 Hz, 2H), 3.29−3.27 (m,

-(4,5-Bis(4-(furan-3-yl)phenyl)thiophen-2-yl)-N-(piperidin-

H), 2.95 (t, J = 12.6 Hz, 2H), 1.98−1.94 (m, 3H), 1.52−1.42 (m,

4-ylmethyl)methanamine Hydrochloride (11). 11 was prepared

H), and 0.97 (s, 9H). C NMR (100 MHz, D O): 163.4, 151.3,

from 4,5-dibromothiophene-2-carboxaldehyde, following the general

43.8, 137.3, 136.1, 134.3, 131.6, 129.9, 129.4, 129.1, 128.5, 128.1,

25.4, 44.3, 43.6, 42.6, 33.9, 33.5, 30.5, and 26.1. MS (ESI) calcd for

method B, as a hydrochloric acid salt (yellow powder). H NMR (400

MHz, DMSO-d ): δ 9.54 (br, 2H), 9.03 (br, 1H), 8.88 (br, 1H), 7.46

(

C H N OS) [M + H] 462.7, found 462.6.

(s, 1H), 7.16−7.12 (m, 8H), 4.35 (s, 2H), 3.26−3.23 (m, 2H), 2.92−

-(4-(Furan-3-yl)phenyl)-4-(4-(piperidin-1-ylmethyl)-

2.88 (m, 2H), 2.84−2.79 (m, 2H), 2.54 (t, J = 7.4 Hz, 4H), 2.09 (br,

phenyl)-N-(piperidin-4-ylmethyl)thiophene-2-carboxamide

Hydrochloride (6). 6 was prepared from 4,5-dibromothiophene-2-

H), 1.97−1.93 (m, 2H), 1.52 (p, J = 7.4 Hz, 4H), 1.46−1.39 (m,

H), 1.32−1.23 (m, 4H), and 0.87 (t, J = 7.4 Hz, 6H). C NMR

carboxylic acid, following the general method A, as a hydrochloric

(100 MHz, DMSO-d ): 144.5, 144.4, 139.8, 139.6, 139.1, 137.2,

acid salt (white powder). H NMR (400 MHz, DMSO-d ): δ 10.54

134.04, 133.97, 131.79, 131.69, 131.62, 130.9, 129.2, 129.0, 126.0,

25.8, 125.3, 125.2, 108.58, 108.56, 50.6, 44.6, 42.4, 30.6, and 26.1.

(

br, 1H), 8.94 (br, 1H), 8.84 (br, 1H), 8.64 (br, 1H), 8.22 (s, 1H),

.03 (s, 1H), 7.75 (s, 1H), 7.60 (d, J = 7.8 Hz, 4H), 7.36 (d, J = 7.8

Hz, 2H), 7.28 (d, J = 7.8 Hz, 2H), 6.97 (s, 1H), 4.24 (s, 4H), 3.25−

MS (ESI) calcd for (C H N O S) [M + H] 495.7, found 495.6.

-(4,5-Bis(4-(tert-butyl)phenyl)thiophen-2-yl)-N-(piperidin-

.24 (m, 4H), 3.19−3.16 (m, 2H), 2.86−2.78 (m, 4H), 1.83−1.78

4-ylmethyl)methanamine Hydrochloride (12). 12 was prepared

(

m, 6H), 1.70−1.66 (m, 1H), and 1.48−1.34 (m, 2H). C NMR

from 4,5-dibromothiophene-2-carboxaldehyde, following the general

100 MHz, DMSO-d ): 161.0, 144.6, 142.2, 140.0, 138.2, 137.3,

method B, as a hydrochloric acid salt (white powder). H NMR (400

36.6, 132.1, 131.8, 131.6, 131.5, 129.3, 129.0, 126.0, 125.2, 115.5,

MHz, DMSO-d ): δ 9.56 (br, 2H), 9.00 (br, 1H), 8.84 (br, 1H), 7.48

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(

s, 1H), 7.36 (d, J = 7.2 Hz, 4H), 7.20 (d, J = 7.2 Hz, 4H), 4.35 (s,

1-((4,5-Bis(4-(tert-butyl)phenyl)thiophen-2-yl)methyl)-

piperazine Hydrochloride (18). 18 was prepared from 4,5-

dibromothiophene-2-carboxaldehyde, following the general method

H), 3.27−3.24 (m, 2H), 2.89 (br, 2H), 2.86−2.82 (m, 2H), 2.05 (br,

H), 1.97−1.94 (m, 2H), 1.45−1.40 (m, 2H), and 1.27 (s, 18H).

NMR (100 MHz, DMSO-d ): 150.5, 149.6, 139.0, 137.0, 134.3,

B, as a hydrochloric acid salt (white powder). H NMR (400 MHz,

32.8, 131.4, 130.6, 128.4, 128.2, 125.7, 125.4, 50.6, 44.6, 42.3, 34.42,

DMSO-d

): δ 9.61 (br, 1H), 9.50 (br, 2H), 7.44 (s, 1H), 7.38−7.34

4.34, 31.13, 31.05, 30.6, and 26.1. MS (ESI) calcd for (C H N S)

(m, 4H), 7.22−7.19 (m, 4H), 4.48 (s, 2H), 3.33 (s, 4H), 2.50 (s, 4H),

and 1.27 (s, 18H). C NMR (100 MHz, DMSO-d ): 150.6, 149.7,

[

M + H] 475.8, found 475.7; HRMS (ESI ) calcd for C H N S [M

H] 475.3147, found 475.3145.

-(((4,5-Bis(4-(tert-butyl)phenyl)thiophen-2-yl)methoxy)-

140.0, 138.6, 137.1, 132.6, 131.7, 130.4, 128.4, 128.3, 125.7, 125.4,

52.6, 47.1, 40.8, 34.4, 34.3, 31.1, and 31.0. MS (ESI) calcd for

methyl)piperidine Hydrochloride (13). Starting from 4,5-

dibromothiophene-2-carboxaldehyde, reactions v and vi gave 2-

chloromethyl-4,5-dibromothiophene, which reacted with sodium salt

of 1-BOC-4-hydroxymethylpiperidine, to give 4,5-dibromothiophen-

(C H N S) [M + H] 447.7, found 447.6.

29 39 2

4-((4,5-Bis(4-(tert-butyl)phenyl)thiophen-2-yl)methyl)-

morpholine Hydrochloride (19). 19 was prepared from 4,5-

dibromothiophene-2-carboxaldehyde, following the general method B,

-ylmethyl 1-BOC-piperidin-4-yl-methyl ether. Reactions ii and iii

as a hydrochloric acid salt (white powder). H NMR (400 MHz,

(

general method B) produced compound 13 as a hydrochloric acid

CDCl

): δ 13.34 (br, 1H), 7.45 (s, 1H), 7.30 (d, J = 7.6 Hz, 4H), 7.24

salt (white powder). H NMR (400 MHz, DMSO-d ): δ 8.72 (br,

(d, J = 7.6 Hz, 4H), 4.46 (s, 2H), 4.30 (br, 2H), 4.01 (br, 2H), 3.46

H), 8.38 (br, 1H), 7.36 (d, J = 8.1 Hz, 2H), 7.33 (d, J = 8.1 Hz, 2H),

.48 (s, 1H), 7.21 (d, J = 8.1 Hz, 2H), 7.17 (d, J = 8.1 Hz, 2H), 5.05

(br, 2H), 3.08 (br, 2H), and 1.32 (s, 18H). C NMR (100 MHz,

CDCl ): 151.3, 150.4, 142.2, 138.4, 136.6, 132.5, 130.3, 128.8, 128.6,

125.6, 125.5, 125.3, 63.8, 54.8, 50.6, 34.72, 34.64, 31.39, and 31.32.

(

s, 2H), 3.27−3.23 (m, 4H), 2.86−2.77 (m, 2H), 1.78−1.75 (m, 2H),

.62 (br, 1H), and 1.26 (s, 18H). C NMR (100 MHz, DMSO-d ):

MS (ESI) calcd for (C29H38NOS) [M + H] 448.7, found 448.6.

50.4, 149.6, 138.8, 138.6, 136.7, 132.8, 131.7, 130.7, 128.4, 128.2,

25.6, 125.3, 65.0, 42.9, 40.8, 35.9, 34.4, 34.3, 31.10, 31.02, and 25.2.

1-(4,5-Bis(4-(tert-butyl)phenyl)thiophen-2-yl)-N-methyl-N-

piperidin-4-ylmethyl)methanamine Hydrochloride (20). 20

(

was prepared from 4,5-dibromothiophene-2-carboxaldehyde, follow-

MS (ESI) calcd for (C H NOS) [M + H] 476.7, found 476.6.

ing the general method B, as a hydrochloric acid salt (white powder).

N-((4,5-Bis(4-(tert-butyl)phenyl)thiophen-2-yl)methyl)-

piperidin-4-amine Hydrochloride (14). 14 was prepared from 4,5-

dibromothiophene-2-carboxaldehyde, following the general method B,

as a hydrochloric acid salt (white powder). H NMR (400 MHz,

DMSO-d ): δ 9.85 (br, 2H), 9.24 (br, 1H), 8.99 (br, 1H), 7.50 (s,

NMR (100 MHz, DMSO-d ): 150.6, 149.7, 139.1, 137.1, 134.3,

H NMR (400 MHz, DMSO-d ): δ 9.58 (br, 1H), 9.02 (br, 1H), 8.85

(br, 1H), 7.53 (s, 1H), 7.38−7.35 (m, 4H), 7.24−7.20 (m, 4H), 4.55

(s, 2H), 3.27−3.24 (m, 2H), 3.05−2.99 (m, 2H), 2.90−2.84 (m, 2H),

2.77 (s, 3H), 2.17−2.10 (m, 2H), 1.98−1.95 (m, 1H), 1.46−1.39 (m,

H), 7.36 (d, J = 7.2 Hz, 4H), 7.20 (d, J = 7.2 Hz, 4H), 4.40 (s, 2H),

2H), and 1.27 (s, 18H). C NMR (100 MHz, DMSO-d ): 150.7,

.95 (br, 3H), 2.30 (br, 2H), 1.95 (m, 2H), and 1.27 (s, 18H).

149.7, 140.4, 137.2, 136.4, 132.6, 130.4, 128.4, 128.3, 128.2, 125.7,

125.4, 58.5, 52.6, 42.42, 42.37, 34.42, 34.33, 31.14, 31.05, 28.9, and

32.8, 131.2, 130.6, 128.4, 128.2, 125.7, 125.4, 51.4, 41.4, 34.42,

26.4. MS (ESI) calcd for (C H N S) [M + H] 489.8, found 489.6.

32 45 2

4.34, 31.14, 31.06, 28.9, and 24.9. MS (ESI) calcd for (C H N S)

N-((4,5-Bis(4-(tert-butyl)phenyl)thiophen-2-yl)methyl)-N-

(piperidin-4-ylmethyl)formamide Hydrochloride (21). 21 was

prepared from 4,5-dibromothiophene-2-carboxaldehyde, following the

[

M + H] 461.7, found 461.6.

N-((4,5-Bis(4-(tert-butyl)phenyl)thiophen-2-yl)methyl)-2-

piperidin-4-yl)ethanamine Hydrochloride (15). 15 was pre-

(

general method B, as a hydrochloric acid salt (white powder). H

pared from 4,5-dibromothiophene-2-carboxaldehyde, following the

general method B, as a hydrochloric acid salt (white powder). H

NMR (400 MHz, CDCl ): δ 9.34 (br, 1H), 9.01 (br, 1H), 7.29−7.25

(m, 4H), 7.20−7.17 (m, 4H), 6.99 (s, 1H), 4.65 (s, 2H), 3.31−3.27

(m, 2H), 3.14−3.11 (m, 2H), 2.93−2.89 (m, 2H), 1.83−1.76 (m,

NMR (400 MHz, DMSO-d ): δ 9.55 (br, 2H), 8.99 (br, 1H), 8.81

(

br, 1H), 7.47 (s, 1H), 7.35 (br, 4H), 7.20 (br, 4H), 4.35 (s, 2H),

3H), 1.62−1.59 (m, 2H), and 1.29 (s, 18H). C NMR (100 MHz,

.23 (br, 2H), 2.98 (br, 2H), 2.80 (br, 2H), 1.78 (br, 1H), 1.65 (br,

H), 1.35 (br, 2H), and 1.26 (s, 18H). C NMR (100 MHz, DMSO-

CDCl ): 162.9, 154.8, 150.1, 138.7, 137.0, 136.5, 131.2, 130.8, 130.3,

128.7, 128.6, 125.5, 125.4, 52.7, 47.6, 47.5, 41.1, 34.6, 31.44, 31.36,

d₆): 150.5, 149.6, 138.9, 137.0, 134.1, 132.7, 131.4, 130.5, 128.4,

29.8, and 28.5. MS (ESI) calcd for (C H N OS) [M + H] 503.8,

32 43 2

28.2, 125.7, 125.4, 44.0, 43.8, 43.0, 34.39, 34.31, 31.3, 31.1, 31.0,

found 503.6.

0.6, and 28.0. MS (ESI) calcd for (C H N S) [M + H] 489.8,

1-(4,5-Bis(4-isopropylphenyl)thiophen-2-yl)-N-(piperidin-4-

ylmethyl)methanamine Hydrochloride (22). 22 was prepared

from 4,5-dibromothiophene-2-carboxaldehyde, following the general

found 489.7.

N -((4,5-Bis(4-(tert-butyl)phenyl)thiophen-2-yl)methyl)-

propane-1,3-diamine Hydrochloride (16). 16 was prepared from

method B, as a hydrochloric acid salt (white powder). H NMR (400

,5-dibromothiophene-2-carboxaldehyde, following the general meth-

MHz, DMSO-d ): δ 9.67 (br, 2H), 9.13 (br, 1H), 9.03 (br, 1H), 7.50

od B, as a hydrochloric acid salt (white powder). H NMR (400 MHz,

CDCl ): δ 9.70 (br, 2H), 8.24 (br, 3H), 7.34 (s, 1H), 7.17−7.07 (m,

(s, 1H), 7.20−7.15 (m, 8H), 4.34 (s, 2H), 3.26−3.23 (m, 2H), 2.90−

2.83 (m, 4H), 2.75 (sep, J = 7.0 Hz, 2H), 2.10 (br, 1H), 1.99−1.94

H), 7.24 (d, J = 7.6 Hz, 4H), 4.22 (s, 2H), 3.29−3.20 (m, 2H), 2.47

(m, 2H), 1.47−1.40 (m, 2H), and 1.18 (d, J = 7.0 Hz, 12H).

(

br, 2H), 1.34−1.32 (m, 2H), and 1.19 (s, 18H). C NMR (100

NMR (100 MHz, DMSO-d ): 148.2, 147.3, 139.0, 137.1, 134.2,

MHz, CDCl ): 150.6, 149.8, 140.8, 137.7, 135.0, 132.9, 130.8, 129.0,

133.1, 131.3, 130.9, 128.6, 128.5, 126.8, 126.5, 50.5, 44.5, 42.3, 33.1,

28.8, 128.7, 125.5, 125.4, 45.5, 44.0, 37.8, 34.62, 34.57, 31.44, 31.36,

30.6, 28.1, 26.1, 23.8, and 23.7. MS (ESI) calcd for (C H N S) [M

+ H] 447.7, found 447.6.

and 29.8. MS (ESI) calcd for (C H N S) [M + H] 435.7, found

35.6.

1-(4,5-Bis(4-butylphenyl)thiophen-2-yl)-N-(piperidin-4-

ylmethyl)methanamine Hydrochloride (23). 23 was prepared

from 4,5-dibromothiophene-2-carboxaldehyde, following the general

N -((4,5-Bis(4-(tert-butyl)phenyl)thiophen-2-yl)methyl)-

hexane-1,6-diamine Hydrochloride (17). 17 was prepared from

,5-dibromothiophene-2-carboxaldehyde, following the general meth-

method B, as a hydrochloric acid salt (white powder). H NMR (400

od B, as a hydrochloric acid salt (white powder). H NMR (400 MHz,

MHz, DMSO-d ): δ 9.54 (br, 2H), 9.03 (br, 1H), 8.88 (br, 1H), 7.46

DMSO-d ): δ 9.34 (br, 2H), 7.87 (br, 3H), 7.44 (s, 1H), 7.38 (d, J =

(s, 1H), 7.16−7.12 (m, 8H), 4.35 (s, 2H), 3.26−3.23 (m, 2H), 2.90

(br, 2H), 2.84−2.79 (m, 2H), 2.54 (t, J = 7.4 Hz, 4H), 2.09 (br, 1H),

1.97−1.93 (m, 2H), 1.52 (p, J = 7.4 Hz, 4H), 1.46−1.39 (m, 2H),

(

.2 Hz, 2H), 7.36 (d, J = 8.2 Hz, 2H), 7.21 (d, J = 8.2 Hz, 2H), 7.19

d, J = 8.2 Hz, 2H), 4.35 (s, 2H), 2.94 (br, 2H), 2.76 (p, J = 7.4 Hz,

H), 1.66 (p, J = 7.4 Hz, 2H), 1.54 (p, J = 7.4 Hz, 2H), 1.35−1.33

1.32−1.23 (m, 4H), and 0.87 (t, J = 7.4 Hz, 6H). C NMR (100

(

m, 4H), and 1.27 (s, 18H). C NMR (100 MHz, DMSO-d ): 150.6,

MHz, DMSO-d ): 142.5, 141.5, 139.3, 137.3, 134.2, 133.0, 131.2,

49.7, 139.0, 137.0, 134.0, 132.7, 131.4, 130.5, 128.3, 128.2, 125.6,

25.4, 46.7, 46.1, 44.1, 34.4, 34.3, 31.1, 31.0, 26.7, 25.5, 25.3, and

130.8, 128.86, 128.77, 128.62, 128.56, 50.7, 44.7, 42.5, 34.6, 33.09,

33.00, 30.6, 26.1, 24.8, 21.9 (2), and 13.9 (2). MS (ESI) calcd for

(C H N S) [M + H] 475.8, found 475.7.

31 43 2

5.2. MS (ESI) calcd for (C H N S) [M + H] 477.8, found 477.6.

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

-(5-(4-(tert-Butyl)phenyl)-4-(4-(furan-3-yl)phenyl)-

(br, 1H), 7.47 (s, 1H), 7.28 (d, J = 7.1 Hz, 2H), 7.26 (d, J = 7.1 Hz,

2H), 7.19 (d, J = 7.1 Hz, 2H), 7.17 (d, J = 7.1 Hz, 2H), 4.47 (s, 4H),

thiophen-2-yl)-N-(piperidin-4-ylmethyl)methanamine Hydro-

chloride (24). 24 was prepared from 4,5-dibromothiophene-2-

.37 (s, 2H), 3.27−3.24 (m, 2H), 2.92 (br, 2H), 2.89−2.82 (m, 2H),

carboxaldehyde, following the general method B, as a hydrochloric

acid salt (white powder). H NMR (400 MHz, DMSO-d ): δ 9.51

.07 (br, 1H), 1.97−1.93 (m, 2H), and 1.48−1.39 (m, 2H).

NMR (100 MHz, DMSO-d ): 142.6, 141.7, 139.5, 137.4, 134.2,

(

br, 2H), 8.95 (br, 1H), 8.79 (br, 1H), 8.08 (s, 1H), 7.89 (d, J = 8.1

Hz, 1H), 7.61−7.58 (m, 1H), 7.52 (s, 1H), 7.39−7.36 (m, 2H), 7.32

d, J = 8.1 Hz, 1H), 7.24−7.19 (m, 4H), 6.97 (s, 1H), 4.38 (s, 2H),

34.0, 131.8, 131.4, 128.7, 128.4, 127.0, 126.8, 62.7, 62.6, 50.8, 44.8,

2.5, 30.6, and 26.1. MS (ESI) calcd for (C H N O S) [M + H]

(

23.6, found 423.5.

.28−3.25 (m, 2H), 2.93−2.88 (m, 2H), 2.86−2.82 (m, 2H), 2.08

-(4,5-Di(furan-3-yl)thiophen-2-yl)-N-(piperidin-4-

(

br, 1H), 1.98−1.95 (m, 2H), 1.47−1.42 (m, 2H), and 1.26 (s, 9H).

ylmethyl)methanamine Hydrochloride (30). 30 was prepared

C NMR (100 MHz, DMSO-d ): 146.5, 144.5, 139.6, 136.7, 134.1,

from 4,5-dibromothiophene-2-carboxaldehyde, following the general

(

33.9, 131.6, 131.4, 129.0, 128.8, 128.4, 128.3, 127.4, 125.8, 125.7,

08.5, 50.6, 44.6, 42.4, 34.4, 31.0, 30.5, and 26.1. MS (ESI) calcd for

method B, as a hydrochloric acid salt (white powder). H NMR (400

MHz, DMSO-d ): δ 9.61 (br, 2H), 9.09 (br, 1H), 8.96 (br, 1H), 7.88

C H N OS) [M + H] 485.7, found 485.6.

(s, 1H), 7.78 (s, 2H), 7.71 (s, 1H), 7.48 (s, 1H), 6.52 (s, 1H), 6.48 (s,

-(4-(4-(Furan-3-yl)phenyl)-5-(4-(piperidin-1-ylmethyl)-

1H), 4.32 (s, 2H), 3.27−3.24 (m, 2H), 2.87−2.82 (m, 4H), 2.08 (br,

phenyl)thiophen-2-yl)-N-(piperidin-4-ylmethyl)methanamine

Hydrochloride (25). 25 was prepared from 4,5-dibromothiophene-

H), 1.97−1.94 (m, 2H), and 1.45−1.40 (m, 2H). C NMR (100

MHz, DMSO-d ): 144.2, 143.7, 140.9, 140.2, 133.1, 131.1, 129.8,

-carboxaldehyde, following the general method B, as a hydrochloric

acid salt (white powder). H NMR (400 MHz, DMSO-d ): δ 10.50

29.2, 120.1, 118.1, 111.1, 110.3, 50.4, 44.4, 42.3, 30.5, and 26.0. MS

(ESI) calcd for (C H N O S) [M + H] 343.5, found 343.4.

19 23 2 2

(

br, 1H), 9.60 (br, 2H), 8.91 (br, 1H), 8.78 (br, 1H), 8.21 (s, 1H),

.75 (s, 1H), 7.64−7.54 (m, 5H), 7.35 (d, J = 8.1 Hz, 2H), 7.24 (d, J

8.1 Hz, 2H), 6.97 (s, 1H), 4.40 (s, 2H), 4.24 (s, 2H), 3.28−3.24

-(4,5-Bis(3,4-dimethoxyphenyl)thiophen-2-yl)-N-(piperi-

din-4-ylmethyl)methanamine Hydrochloride (31). 31 was

prepared from 4,5-dibromothiophene-2-carboxaldehyde, following

m, 4H), 2.94−2.90 (m, 2H), 2.86−2.81 (m, 4H), 2.09 (br, 1H),

the general method B, as a hydrochloric acid salt (white powder).

.98−1.95 (m, 2H), 1.77 (br, 4H), 1.71−1.67 (m, 2H), and 1.48−

H NMR (400 MHz, DMSO-d ): δ 9.40 (br, 2H), 8.94 (br, 1H), 8.77

.39 (m, 2H). ¹³C NMR (100 MHz, DMSO-d ): 144.5, 139.6, 138.4,

(

br, 1H), 7.47 (s, 1H), 7.17−7.14 (m, 1H), 7.01−6.91 (m, 2H), 6.84

37.6, 134.4, 134.1, 133.7, 132.1, 132.0, 131.5, 131.4, 129.0, 128.8,

28.7, 125.8, 108.6, 51.6, 50.6, 44.5, 42.3, 30.7, 30.5, 26.1, 22.1, and

1.4. MS (ESI) calcd for (C H N OS) [M + H] 526.8, found

d, J = 8.4 Hz, 1H), 6.78 (s, 1H), 6.74 (s, 1H), 4.34 (s, 2H), 3.75 (s,

H), 3.73 (s, 3H), 3.56 (s, 3H), 3.54 (s, 3H), 3.28−3.25 (m, 2H),

.92 (br, 2H), 2.88−2.82 (m, 2H), 2.07 (br, 1H), 1.98−1.91 (m, 2H),

26.6.

and 1.48−1.38 (m, 2H). C NMR (100 MHz, DMSO-d ): 148.8,

-(5-(4-(Aminomethyl)phenyl)-4-(4-(tert-butyl)phenyl)-

48.51, 148.48, 148.1, 136.9, 131.6, 131.5, 128.9, 128.8, 125.9, 121.4,

21.0, 112.54, 112.52, 112.0, 111.9, 55.69, 55.62, 55.59, 55.36, 50.7,

thiophen-2-yl)-N-(piperidin-4-ylmethyl)methanamine Hydro-

chloride (26). 26 was prepared from 4,5-dibromothiophene-2-

4.8, 42.4, 30.6, and 26.1. MS (ESI) calcd for (C H N O S) [M +

carboxaldehyde, following the general method B, as a hydrochloric

acid salt (white powder). H NMR (400 MHz, D O): δ 7.63 (s, 1H),

H] 483.6, found 483.5.

-(4,5-Di([1,1′-biphenyl]-3-yl)thiophen-2-yl)-N-(piperidin-4-

.45 (d, J = 9.2 Hz, 2H), 7.42−7.39 (m, 4H), 7.28 (d, J = 9.2 Hz,

H), 4.54 (s, 2H), 4.18 (s, 2H), 3.50−3.46 (m, 2H), 3.14 (br, 2H),

.03 (t, J = 14.4 Hz, 2H), 2.15 (br, 1H), 2.07−2.04 (m, 2H), 1.57−

ylmethyl)methanamine Hydrochloride (32). 32 was prepared

from 4,5-dibromothiophene-2-carboxaldehyde, following the general

method B, as a hydrochloric acid salt (white powder). H NMR (400

.48 (m, 2H), and 1.29 (s, 9H). C NMR (100 MHz, D O): 144.2,

MHz, DMSO-d ): δ 9.50 (br, 2H), 8.88 (br, 1H), 8.68 (br, 1H), 7.65

39.8, 138.3, 137.4, 134.0, 132.4, 130.2, 129.8, 129.1, 128.7, 125.6,

08.8, 50.7, 44.4, 43.1, 42.6, 30.7, 30.3, 25.8, and 22.1. MS (ESI) calcd

for (C H N S) [M + H] 448.7, found 448.8.

(

s, 2H), 7.52−7.31 (m, 17H), 4.42 (s, 2H), 3.29−3.26 (m, 2H), 2.95

(br, 2H), 2.90−2.82 (m, 2H), 2.08 (br, 1H), 1.98−1.95 (m, 2H), and

.48−1.39 (m, 2H). C NMR (100 MHz, DMSO-d ): 140.7, 140.5,

-(4-(4-(Aminomethyl)phenyl)-5-(4-(tert-butyl)phenyl)-

39.8, 139.5, 139.4, 137.6, 136.0, 133.9, 129.7, 129.4, 128.95 (2),

28.90 (2), 127.9, 127.7, 127.6 (2), 127.24, 127.17, 126.6 (2), 126.5,

25.8, 50.5, 44.5, 42.3, 30.5, and 26.1. MS (ESI) calcd for

thiophen-2-yl)-N-(piperidin-4-ylmethyl)methanamine Hydro-

chloride (27). 27 was prepared from 4,5-dibromothiophene-2-

carboxaldehyde, following the general method B, as a hydrochloric

acid salt (white powder). H NMR (400 MHz, DMSO-d ): δ 9.73

br, 1H), 9.12 (br, 1H), 9.00 (br, 1H), 8.50 (br, 2H), 8.28 (br, 2H),

.55 (s, 1H), 7.47 (d, J = 7.9 Hz, 2H), 7.37 (d, J = 7.9 Hz, 2H), 7.28

d, J = 7.9 Hz, 2H), 7.19 (d, J = 7.9 Hz, 2H), 4.35 (s, 2H), 4.00 (s,

(C H N S) [M + H] 515.7, found 515.5.

35 35 2

-(3-Bromo-4,5-bis(4-(tert-butyl)phenyl)thiophen-2-yl)-N-

(

piperidin-4-ylmethyl)methanamine Hydrochloride (33). 33

was prepared from 4,5-dibromothiophene-2-carboxaldehyde, follow-

ing the general method B, as a hydrochloric acid salt (white powder).

H), 3.24 (d, J = 12.7 Hz, 2H), 2.89−2.83 (m, 4H), 2.10 (br, 1H),

H NMR (400 MHz, DMSO-d ): δ 9.65 (br, 1H), 9.00 (br, 1H), 8.84

.97 (d, J = 12.3 Hz, 2H), 1.50−1.42 (m, 2H), and 1.27 (s, 9H).

(

br, 1H), 8.10 (br, 1H), 7.44 (d, J = 8.2 Hz, 2H), 7.31 (d, J = 8.2 Hz,

NMR (100 MHz, DMSO-d ): 150.7, 139.6, 136.6, 135.7, 134.2,

H), 7.14 (d, J = 8.2 Hz, 2H), 7.11 (d, J = 8.2 Hz, 2H), 4.41 (s, 2H),

.28−3.25 (m, 2H), 2.97 (br, 2H), 2.89−2.82 (m, 2H), 2.09 (br, 1H),

.99−1.96 (m, 2H), 1.49−1.43 (m, 2H), 1.30 (s, 9H), and 1.22 (s,

33.0, 130.5, 129.2, 128.7, 128.4, 125.7, 115.3, 50.5, 44.4, 42.3, 41.8,

4.4, 31.0, 30.5, and 26.1. MS (ESI) calcd for (C H N S) [M +

H] 448.7, found 448.8.

(5-(((Piperidin-4-ylmethyl)amino)methyl)thiophene-2,3-

diyl)bis(4,1-phenylene))dimethanamine Hydrochloride (28).

8 was prepared from 4,5-dibromothiophene-2-carboxaldehyde,

H). C NMR (100 MHz, DMSO-d ): 151.1, 150.4, 141.2, 137.0,

(

132.0, 129.95, 129.84, 128.0, 126.6, 125.6, 125.3, 118.0, 50.9, 44.7,

43.0, 42.3, 34.44, 34.40, 31.1, 30.9, and 26.0. MS (ESI) calcd for

following the general method B, as a hydrochloric acid salt (white

powder). H NMR (400 MHz, D O) δ 7.43 (s, 1H), 7.40 (br, 8H),

.57 (s, 2H), 4.19 (s, 4H), 3.50 (d, J = 12.8 Hz, 2H), 3.15 (d, J = 6.8

Hz, 2H), 3.05 (t, J = 12.0 Hz, 2H), 2.18 (s, 1H), 2.08 (d, J = 14.0 Hz,

H), and 1.59−1.50 (m, 2H). C NMR (100 MHz, D O) 140.6,

37.9, 136.1, 133.9, 133.8, 132.6, 131.8, 130.5, 129.9, 129.6, 129.1,

29.0, 50.8, 45.4, 43.1, 42.64, 42.57, 30.7, and 25.8. MS (ESI) calcd

(C31H42BrN S) [M + H] 554.7, found 554.6.

1-(4,5-Bis(4-(tert-butyl)phenyl)furan-2-yl)-N-(piperidin-4-

ylmethyl)methanamine Hydrochloride (34). 34 was prepared

from 4,5-dibromofuran-2-carboxaldehyde, following the general

method B, as a hydrochloric acid salt (white powder). H NMR

(400 MHz, DMSO-d ): δ 9.54 (br, 2H), 8.97 (br, 1H), 8.78 (br, 1H),

7.46−7.33 (m, 8H), 6.88 (s, 1H), 4.28 (s, 2H), 3.28−3.25 (m, 2H),

2.94 (br, 2H), 2.85−2.83 (m, 2H), 2.06 (br, 1H), 1.97−1.94 (br,

for (C H N S) [M + H] 421.6, found 421.5.

(

(5-(((Piperidin-4-ylmethyl)amino)methyl)thiophene-2,3-

2H), 1.48−1.42 (m, 2H), 1.30 (s, 9H), and 1.27 (s, 9H). C NMR

diyl)bis(4,1-phenylene))dimethanol Hydrochloride (29). 29

(100 MHz, DMSO-d ): 150.9, 150.0, 148.4, 144.8, 130.2, 127.8,

was prepared from 4,5-dibromothiophene-2-carboxaldehyde, follow-

127.5, 125.8, 125.7, 125.5, 122.2, 116.2, 50.7, 42.8, 42.4, 34.49, 34.39,

ing the general method B, as a hydrochloric acid salt (white powder).

31.15, 31.02, 30.5 and 26.0. MS (ESI) calcd for (C H N O) [M +

H NMR (400 MHz, DMSO-d ): δ 9.42 (br, 2H), 8.91 (br, 1H), 8.74

H] 459.7, found 459.6.

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

-(4,5-Bis(4-(furan-3-yl)phenyl)furan-2-yl)-N-(piperidin-4-

mL, 3.5 mmol). The reaction mixture was stirred at room temperature

for 5 h. Methanol (1.8 mL) was then added followed by NaBH₄

(537.2 mg, 14.2 mmol) in small portions at 0 °C. Stirring was

continued at 0 °C for 1 h. The product was extracted with ether and

puriﬁed with ﬂash chromatography (silica gel, hexane/ethyl acetate =

5/1) to give the corresponding alcohol (509 mg, 52% for two steps),

which was converted to compound 40 as a pale yellow powder using

ylmethyl)methanamine Hydrochloride (35). 35 was prepared

from 4,5-dibromofuran-2-carboxaldehyde, following the general

method B, as a hydrochloric acid salt (yellow powder). H NMR

400 MHz, DMSO-d ): δ 9.57 (br, 2H), 8.96 (br, 1H), 8.78 (br, 1H),

.24−8.21 (m, 1H), 7.78−7.74 (m, 2H), 7.70−7.58 (m, 5H), 7.55 (d,

J = 8.0 Hz, 2H), 7.41 (d, J = 8.0 Hz, 2H), 6.99 (s, 1H), 6.96 (d, J =

(

.0 Hz, 2H), 4.31 (s, 2H), 3.27−3.23 (m, 2H), 2.95 (br, 2H), 2.89−

the reactions vi, vii, ii, and iv described in general methods A and B.

.83 (m, 2H), 2.08 (br, 1H), 1.98−1.92 (m, 2H), and 1.49−1.38 (m,

H NMR (400 MHz, DMSO-d ): δ 8.94 (br, 2H), 8.72 (br, 1H), 8.69

H). ¹³C NMR (100 MHz, DMSO-d ): 148.5, 145.2, 144.6, 139.9,

(s, 1H), 8.24 (br, 2H), 7.92 (d, J = 7.1 Hz, 2H), 7.75 (s, 2H), 7.62 (d,

J = 7.7 Hz, 4H), 7.45 (d, J = 4.5 Hz, 2H), 7.43 (d, J = 4.5 Hz, 2H),

6.99 (s, 2H), 5.26 (s, 2H), 3.21 (d, J = 10.2 Hz, 2H), 2.94 (br, 2H),

2.79−2.75 (m, 2H), 1.76 (d, J = 14.2 Hz, 2H), 1.69 (br, 1H), and

39.7, 131.6, 131.5, 128.9, 128.8, 128.7, 126.6, 126.1, 125.8, 125.4,

24.3, 122.6, 116.1, 114.7, 108.67, 108.61, 50.7, 42.9, 42.4, 30.6, and

6.1. MS (ESI) calcd for (C H N O ) [M + H] 479.6, found

79.5.

1.33−1.26 (m, 2H). C NMR (100 MHz, DMSO-d ): 156.0, 150.48,

,5-Bis(4-(tert-butyl)phenyl)-N-(piperidin-4-ylmethyl)-

150.44, 149.5, 144.5, 140.4, 140.02, 139.99, 136.63, 136.57, 132.3,

130.1, 130.0, 129.7, 128.0, 126.4, 125.3, 125.2, 108.58, 108.56, 53.4,

45.3, 42.7, 33.9, and 26.0. MS (ESI) calcd for (C H N O ) [M +

thiazole-2-carboxamide Hydrochloride (36). 36 was prepared

from 4-bromothiozole-2-carboxaldehyde, following the general

method A, as a hydrochloric acid salt (white powder). H NMR

H] 491.6, found 491.5.

(

400 MHz, DMSO-d ): δ 8.96 (br, 1H), 8.85 (br, 1H), 8.53 (br, 1H),

Inhibition of p300-HAT and Other HATs. The HAT domain

(1195−1673) of human p300 was cloned, inserted into pGEX-KG

vector, and the DNA sequence was veriﬁed by sequencing. The p300-

HAT expression plasmid was transformed into E. coli BL21-

CodonPlus strain (Agilent) and cultured at 37 °C in LB medium

containing ampicillin (50 μg/mL) and chloramphenicol (34 μg/mL).

After the optical density of the bacterial culture reached ∼0.9 at 600

nm, p300 HAT expression was induced by adding 300 μM

isopropylthiogalactoside (IPTG) at 18 °C for 48 h. Cells were

collected, lysed, and centrifuged for 20 min at 20 000 rpm. The

supernatant was subjected to column chromatography with

glutathione sepharose resins. The recombinant GST-p300-HAT

fusion protein was obtained in ∼90% purity (SDS−PAGE) by

elution with 10 mM glutathione solution, which was further puriﬁed

using a Superdex 200 gel ﬁltration column chromatography. CBP,

PCAF, and Myst3 HATs were obtained using similar methods.

To determine inhibitory activity, a compound with concentrations

ranging from 100 nM to 10 μM was incubated with p300-HAT (10

nM) in 20 μL of 50 mM phosphate buﬀer (pH = 7.0) containing

0.01% Brij-35 for 10 min at 25 °C. Histone H3 peptide

(ARTKQTARKSTGGKAPRKQLA) (20 μM) and acetyl-CoA (1

.48−7.42 (m, 4H), 7.38−7.33 (m, 4H), 3.26−3.20 (m, 4H), 2.82

(

br, 2H), 1.87 (br, 1H), 1.82−1.76 (m, 2H), 1.42−1.36 (m, 2H), and

.28 (s, 18H). ¹³C NMR (100 MHz, DMSO-d ): 161.0, 152.1, 151.3,

50.4, 137.8, 137.5, 131.7, 129.4, 128.9, 128.3, 126.4, 125.6, 44.2,

3.2, 34.9, 34.8, 34.1, 31.5, 31.4, and 26.6. MS (ESI) calcd for

(

C H N OS) [M + H] 490.7, found 490.6.

30 40 3

-(4,5-Bis(4-(tert-butyl)phenyl)thiazol-2-yl)-N-(piperidin-4-

ylmethyl)methanamine Hydrochloride (37). 37 was prepared

from 4-bromothiozole-2-carboxaldehyde, following the general

method B, as a hydrochloric acid salt (white powder). H NMR

(

400 MHz, DMSO-d ): δ 9.83 (br, 2H), 9.13 (br, 1H), 8.98 (br, 1H),

.44−7.31 (m, 8H), 4.57 (s, 2H), 3.27−3.24 (m, 2H), 3.03 (br, 2H),

.85 (br, 2H), 2.10 (br, 1H), 1.98−1.95 (m, 2H), 1.55−1.50 (m, 2H),

and 1.27 (s, 18H). C NMR (100 MHz, DMSO-d ): 151.4, 150.6,

48.5, 134.2, 131.7, 131.3, 129.0, 128.2, 128.0, 126.0, 125.2, 51.3,

6.8, 42.3, 34.52, 34.42, 31.07, 31.02, 30.6, and 28.1. MS (ESI) calcd

for (C H N S) [M + H] 476.7, found 476.5.

-(4-(4-(tert-Butyl)phenyl)thiazol-2-yl)-N-(piperidin-4-

ylmethyl)methanamine Hydrochloride (38). 38 was prepared

from 4-bromothiozole-2-carboxaldehyde, following the general

method B, as a hydrochloric acid salt (white powder). H NMR

μM H-Ac-CoA and 19 μM Ac-CoA) were added to initiate the

(

400 MHz, DMSO-d ): δ 9.77 (br, 2H), 9.01 (br, 2H), 8.01 (s, 1H),

.86 (d, J = 8.4 Hz, 2H), 7.42 (d, J = 8.4 Hz, 2H), 4.55 (s, 2H), 3.27−

.24 (m, 2H), 3.03 (br, 2H), 2.85 (br, 2H), 2.10 (br, 1H), 1.98−1.95

reaction. After 30 min at 25 °C, the reaction was stopped by adding 6

N formic acid (5 μL). 20 μL of reaction mixture was then transferred

to a small piece of P81 ﬁlter paper (Whatman) that binds histone H3

(

m, 2H), 1.47−1.39 (m, 2H), and 1.25 (s, 9H). C NMR (100 MHz,

peptide. The ﬁlter paper was washed 3× with 50 mM NaHCO , dried,

DMSO-d ): 160.0, 154.3, 150.9, 131.0, 125.9, 125.6, 116.0, 51.2, 46.8,

and transferred into a scintillation vial containing 2 mL of scintillation

cocktail. Radioactivity on the ﬁlter paper was measured with a

Beckman LS-6500 scintillation counter. IC₅₀values were obtained by

using a standard sigmoidal dose−response curve ﬁtting in Prism

(version 5.0, GraphPad Software, Inc., La Jolla, CA). IC values were

2.3, 34.4, 31.1, 30.6, and 26.0. MS (ESI) calcd for (C H N S) [M

20 30 3

H] 344.5, found 344.6.

-(4-(tert-Butyl)phenyl)-N-(piperidin-4-ylmethyl)thiazole-2-

carboxamide Hydrochloride (39). 39 was prepared from 4-

bromothiozole-2-carboxaldehyde, following the general method A, as

a hydrochloric acid salt (white powder). H NMR (400 MHz,

DMSO-d ): δ 8.99 (br, 1H), 8.90 (br, 1H), 8.68 (br, 1H), 8.34 (s,

the mean values from at least three experiments.

Alpha Assay. We followed a published method to investigate

whether compound 2 can disrupt the binding of p300 HAT and

histone H4 peptide. In brief, the assay was performed in a 384-well

plate using His6-tagged P300 HAT (125 nM) coated nickel chelate

acceptor beads (5 μL, PerkinElmer), biotinylated H4 peptide

H), 7.98 (d, J = 8.2 Hz, 2H), 7.49 (d, J = 8.2 Hz, 2H), 3.27−3.19

(

m, 4H), 2.88−2.79 (m, 2H), 1.91 (br, 1H), 1.83−1.79 (m, 2H),

.67−1.63 (m, 2H), and 1.31 (s, 9H). C NMR (100 MHz, DMSO-

d ): 163.5, 159.4, 153.9, 151.2, 130.8, 126.1, 125.5, 118.8, 43.8, 42.9,

[SGRGKGGKGLGKGGAKRHRKVLRGG-K(biotin)-NH ] (30

4.4, 31.1, 28.1, and 26.3. MS (ESI) calcd for (C H N OS) [M +

nM) coated streptavidin donor beads (5 μL, PerkinElmer), and

varying concentrations of 12 in a PBS buﬀer with 0.5% BSA (ﬁnal

volume of 25 μL). Upon incubation for 1 h, the Alpha signal was

determined (laser excitations at 680 nm and reading at 570 nm) using

a Tecan Spark microplate reader. The IC₅₀values were determined

using the sigmoidal dose−response ﬁtting in the program of Prism 5.0

(GraphPad). The biotinylated His6 peptide (PerkinElmer no.

6760302) was used as a reference to eliminate the possible

interference of compounds on the signal generated by singlet oxygen

transfer.

H] 358.5, found 358.4.

N-((5,6-Bis(4-(furan-3-yl)phenyl)pyrazin-2-yl)methyl)-

piperidin-4-amine Hydrochloride (40). A mixture of 2,3-

diaminopropanoic acid hydrochloride (600.4 mg, 4.26 mmol), 1,2-

bis(4-bromophenyl)ethane-1,2-dione 52 (1.745 g, 4.26 mmol), and

NaOH (677.2 mg, 16.93 mmol) in methanol (25 mL) was reﬂuxed

for 2 h. The mixture was cooled to 25 °C and air was bubbled through

the solution for 2 days, after which the pH was adjusted to 2 by HCl.

Upon removal of the solvents, the product was extracted with diethyl

ether (3 × 50 mL) and the combined organic phases were dried,

ﬁltered, and evaporated under reduced pressure. The crude product

Molecular Modeling. Docking studies were performed with

Schro

dinger, LLC, New York, NY, 2017), using our previous published

dinger small-molecule drug discovery software suite (Schro-

̈ ̈

3 was dissolved in THF (22 mL) followed by addition of methyl

23,35

chloroformate (0.22 mL, 2.8 mmol) and diisopropylethylamine (0.6

methods.

The crystal structure of p300-HAT in complex with Ac-

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

The Supporting Information is available free of charge at

Article

CoA (PDB code 4PZS) was prepared using the module “Protein

Preparation Wizard” in Maestro with the default protein parameters.

Hydrogen atoms were added, and water molecules were extracted. Ac-

CoA was included in the protein structure for docking. Hydrogen

bonds were optimized, the partial charges were assigned, and the

protein structure was energy-minimized using OPLS-2005 force ﬁeld.

A receptor grid was generated using the program Glide without any

constraints. Compound 12 was constructed, energy-minimized using

OPLS-2005 force ﬁeld in Maestro, and then docked into the prepared

protein structure using Glide (docking parameters: standard-precision

and dock ﬂexibly).

chemistry with paired-end 101 bp reads and a 6 bp index read

culminating in an average output of 20 million paired-end reads (or

40 million total reads) per sample. Sequence read data were processed

and converted to FASTQ format by Illumina BaseSpace analysis

software (v2.0.13).

Bioinformatics Analysis. RNAseq data ﬁles were trimmed using

39,40

Trim Galore!

Then the data were mapped using Hisat2 onto the

human reference genome build UCSC hg38/GRCh38 and sorted

41,42

using samtools.

Gene expression was assessed, and fragments per

kilobase of transcript per million (FPKM) values were determined

using Stringtie. Gene expression proﬁles were normalized using the

quantile normalization method. Signiﬁcantly changed genes were

determined using a two-sided parametric t test, with signiﬁcance

assessed at p < 0.05; only genes for which at least one sample

exceeded 1 FPKM in expression were considered. Enriched pathways

were inferred using the gene set enrichment (GSEA) method and the

molecular signature database (MSigDB) pathway collection.

Western Blot. 10 Kasumi-1 cells/well were incubated with

compound 12 at 0, 5, and 10 μM for 12 h. Histone proteins were

extracted using EpiQuik total histone extraction kit (Epigentek),

according to the manufacturer’s protocol. Equal amounts of histones

(

2 μg) were separated on SDS−PAGE and transferred to PVDF

membranes. The blots were probed with primary antibodies against

H3K9Ac, H3K18Ac, H3K27Ac, and H3 (Cell Signaling), followed by

anti-rabbit IgG (Thermo Scientiﬁc) secondary antibodies.

ASSOCIATED CONTENT

sı Supporting Information

https://pubs.acs.org/doi/10.1021/acs.jmedchem.9b02164.

■

Cell Growth Inhibition. The antiproliferation assays for non-

6−38

breast cancer cells were performed using our previous method.

In brief, for Kasumi-1 cells, 10 cells per well were added into 96-well

plates and cultured with increasing concentrations of a compound in

RPMI-1640 medium supplemented with 20% fetal bovine serum and

Charts S1−S3, Figures S1−S4, representative HPLC

penicillin (100 U/mL) and streptomycin (100 μg/mL) at 37 °C in a

traces, and H and C NMR spectra for compound 12

% CO atmosphere with 100% humidity. For solid tumor cells, 10

(PDF )

cells per well were added into 96-well plates and cultured in

Dulbecco’s modiﬁed Eagle’s medium (DMEM) supplemented with

Molecular formula strings and some data (CSV )

PDB ﬁles for docking models (ZIP)

0% fetal bovine serum and penicillin (100 U/mL) and streptomycin

(

100 μg/mL) overnight. Upon addition of increasing concentrations

of a compound, cells were incubated for 5 days. Cell viability was

assessed by using an XTT assay kit (Roche) for leukemia cells or an

MTT assay (Sigma) for attachment cells. For the breast cancer MCF-

AUTHOR INFORMATION

Corresponding Author

■

Yongcheng Song − Department of Pharmacology & Chemical

Biology and Dan L. Duncan Cancer Center, Baylor College of

Medicine, Houston, Texas 77030, United States; orcid.org/

0000-0003-2611-2476 ; Phone: 713-798-7415;

Email: ysong@bcm.edu

cells, culture and inhibition assays were performed according to our

28,29

previous methods.

Compound EC₅₀values were calculated from

dose−response curves using Prism 5.0.

RNA Extraction. Upon starvation (culturing in phenol red-free

DMEM medium with 10% charcoal treated FBS to remove

hydrophobic hormones such as estrogens) for 7 days, MCF-7 cells

were treated with the p300 inhibitor (6 μM) or DMSO control, in the

presence or absence of (10 nM) estradiol (E2) for 48 h. Total RNA

was extracted using RNeasy Plus Mini Kit (Qiagen), according to the

manufacturer’s instructions.

Authors

Fangrui Wu − Department of Pharmacology & Chemical

Biology, Baylor College of Medicine, Houston, Texas 77030,

United States

Yuanda Hua − Department of Pharmacology & Chemical

Biology, Baylor College of Medicine, Houston, Texas 77030,

United States

Salma Kaochar − Department of Medicine and Dan L. Duncan

Cancer Center, Baylor College of Medicine, Houston, Texas

77030, United States

Shenyou Nie − Department of Pharmacology & Chemical

Biology, Baylor College of Medicine, Houston, Texas 77030,

United States

Yi-Lun Lin − Department of Pharmacology & Chemical Biology,

Baylor College of Medicine, Houston, Texas 77030, United

States

Yuan Yao − Department of Pharmacology & Chemical Biology,

Baylor College of Medicine, Houston, Texas 77030, United

States

Jingyu Wu − Department of Pharmacology & Chemical Biology,

Baylor College of Medicine, Houston, Texas 77030, United

States

Xiaowei Wu − Department of Pharmacology & Chemical

Biology, Baylor College of Medicine, Houston, Texas 77030,

United States; orcid.org/0000-0001-7248-9149

Xiaoyong Fu − Department of Molecular & Cellular Biology

and Lester and Sue Smith Breast Center, Baylor College of

Medicine, Houston, Texas 77030, United States

Library Preparation. The RNA integrity for each sample was

assessed with a RNA 6000 Nano chip on a 2100 BioAnalyzer

(

Agilent; Santa Clara, CA). The average RNA integrity score for the

sample set was 6.48. Sequencing libraries were prepared using the

TruSeq Stranded Total RNA Library Prep Kit (Illumina, Inc.; San

Diego, CA). Concisely, rRNA (rRNA) was depleted from total RNA

where the remaining (ribo-depleted RNA) was puriﬁed, fragmented

for 7.5 min, and primed for cDNA synthesis. Blunt-ended cDNA was

generated after ﬁrst and second strand synthesis. Adenylation of the 3′

blunt-ends was followed by adapter ligation prior to the enrichment of

the cDNA fragments. Final library quality control was carried out by

measuring the fragment size on a DNA1000 chip on a 2100

BioAnalyzer (Agilent; Santa Clara, CA). The average library yielded

an insert bp size of 258. The concentration of each library was

determined by quantitative PCR (qPCR) by the KAPA Library

Quantiﬁcation Kit for Next Generation Sequencing (KAPA

Biosystems; Woburn, MA).

Sequencing. Libraries were normalized to 50 nmol/L in 10 mM

Tris-Cl, pH 8.5, with 0.1% Tween 20 and then pooled evenly. The 50

nmol/L library pool was diluted to 2 nmol/L with 10 mM Tris-Cl, pH

.5, with 0.1% Tween 20. The pooled libraries were denatured with

.05 N NaOH and diluted to 20 pmol/L. Cluster generation of the

denatured libraries was performed according to the manufacturer’s

speciﬁcations (Illumina, Inc.; San Diego, CA) utilizing the HiSeq

Rapid PE Cluster Kit v2 chemistry and ﬂow cell. Libraries were

clustered appropriately with a 1% PhiX spike-in. Sequencing-by-

synthesis (SBS) was performed on a HiSeq 2500 utilizing v2

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

(4) Yang, Z.; Yik, J. H.; Chen, R.; He, N.; Jang, M. K.; Ozato, K.;

Article

Rachel Schiﬀ − Department of Medicine, Department of

Molecular & Cellular Biology, and Lester and Sue Smith Breast

Center, Baylor College of Medicine, Houston, Texas 77030,

United States

Christel M. Davis − Avera Institute for Human Genetics, Sioux

Falls, South Dakota 57108, United States

Matthew Robertson − Dan L. Duncan Cancer Center, Baylor

College of Medicine, Houston, Texas 77030, United States

Erik A. Ehli − Avera Institute for Human Genetics, Sioux Falls,

South Dakota 57108, United States

Cristian Coarfa − Dan L. Duncan Cancer Center, Baylor

College of Medicine, Houston, Texas 77030, United States

Nicholas Mitsiades − Department of Medicine and Dan L.

Duncan Cancer Center, Baylor College of Medicine, Houston,

Texas 77030, United States

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Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.9b02164

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Author Contributions

F.W. and Y.H. contributed equally.

∇

(10) Kraus, W. L.; Manning, E. T.; Kadonaga, J. T. Biochemical

analysis of distinct activation functions in p300 that enhance

transcription initiation with chromatin templates. Mol. Cell. Biol.

Notes

The authors declare no competing ﬁnancial interest.

11) Gu, W.; Shi, X. L.; Roeder, R. G. Synergistic activation of

999, 19, 8123−8135.

transcription by CBP and p53. Nature 1997, 387, 819−823.

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ACKNOWLEDGMENTS

■

(

This work was supported by the Cancer Prevention and

Research Institute of Texas (CPRIT) Grants RP150129 and

RP180177 to Y.S. It was also partly supported by the Breast

Cancer Research Foundation Grants 16-142, 17-143, and 18-

R.; Walsemann, G.; Dohmann, K.; Austen, M.; Luscher, B.

Stimulation of c-MYC transcriptional activity and acetylation by

recruitment of the cofactor CBP. EMBO Rep. 2003, 4, 484−490.

(13) Ma, H.; Hong, H.; Huang, S. M.; Irvine, R. A.; Webb, P.;

45 (to R.S.), Susan G. Komen for the Cure Foundation

Promise Grant PG12221410 (to R.S.), Department of Defense

Grant W81XWH-14-1-0326 to X.F.), the NCI SPORE Grants

P50CA58183 and P50CA186784 (to R.S. and N.M.), the NCI

grant U54CA233223 (to S.K. and N.M.), and the NIEHS

grants 1P30ES030285 and 1P42ES0327725 (C.C.). The gene

expression proﬁling was supported by the CPRIT Grant

RP170005, the Prostate Cancer Foundation Grant 18YOUN09

Kushner, P. J.; Coetzee, G. A.; Stallcup, M. R. Multiple signal input

and output domains of the 160-kilodalton nuclear receptor

coactivator proteins. Mol. Cell. Biol. 1999, 19, 6164−6173.

(

(14) Lavau, C.; Du, C.; Thirman, M.; Zeleznik-Le, N. Chromatin-

like syndrome that evolves into myeloid leukemia. EMBO J. 2000, 19,

(

655−4664.

15) Gervais, C.; Murati, A.; Helias, C.; Struski, S.; Eischen, A.;

Lippert, E.; Tigaud, I.; Penther, D.; Bastard, C.; Mugneret, F.; Poppe,

B.; Speleman, F.; Talmant, P.; VanDen Akker, J.; Baranger, L.; Barin,

C.; Luquet, I.; Nadal, N.; Nguyen-Khac, F.; Maarek, O.; Herens, C.;

Sainty, D.; Flandrin, G.; Birnbaum, D.; Mozziconacci, M. J.; Lessard,

M. Acute myeloid leukaemia with 8p11 (MYST3) rearrangement: an

integrated cytologic, cytogenetic and molecular study by the groupe

francophone de cytogenetique hematologique. Leukemia 2008, 22,

(

to S.K.), and NCI SPORE Grant P50 CA186784 (to N.M.).

The authors also acknowledge the assistance of the Dan L

Duncan Cancer Center Core Resources (supported by the

NCI Cancer Center Support Grant P30CA125123).

ABBREVIATIONS USED

■

(

567−1575.

Ac-CoA, acetyl coenzyme A; AR, androgen receptor; CBP,

CREB (cAMP-response element binding protein) binding

protein; CH1, cysteine-histidine rich 1; CoA, coenzyme A; ER,

estrogen receptor; GNAT, Gcn5-related N-acetyltransferase;

GSEA, gene set enrichment analysis; H3K4, histone H3 lysine

16) Lasko, L. M.; Jakob, C. G.; Edalji, R. P.; Qiu, W.; Montgomery,

D.; Digiammarino, E. L.; Hansen, T. M.; Risi, R. M.; Frey, R.;

Manaves, V.; Shaw, B.; Algire, M.; Hessler, P.; Lam, L. T.; Uziel, T.;

Faivre, E.; Ferguson, D.; Buchanan, F. G.; Martin, R. L.; Torrent, M.;

Chiang, G. G.; Karukurichi, K.; Langston, J. W.; Weinert, B. T.;

Choudhary, C.; de Vries, P.; Kluge, A. F.; Patane, M. A.; Van Drie, J.

H.; Wang, C.; McElligott, D.; Kesicki, E.; Marmorstein, R.; Sun, C.;

Cole, P. A.; Rosenberg, S. H.; Michaelides, M. R.; Lai, A.; Bromberg,

K. D. Discovery of a selective catalytic p300/CBP inhibitor that

targets lineage-specific tumours. Nature 2017, 550, 128−132.

; H3K9, histone H3 lysine 9; H3K27, histone H3 lysine 27;

LSD1, lysine speciﬁc demethylase 1; MYST, MOZ, Ybf2, Sas2,

and Tip60p300; NES, normalized enrichment score; E1A,

binding protein p300; PCAF, p300/CBP associating factor;

SAR, structure−activity relationship; SID, steroid receptor

coactivator interaction; SRC, steroid receptor coactivator

(17) Lau, O. D.; Kundu, T. K.; Soccio, R. E.; Ait-Si-Ali, S.; Khalil, E.

M.; Vassilev, A.; Wolffe, A. P.; Nakatani, Y.; Roeder, R. G.; Cole, P. A.

HATs off: selective synthetic inhibitors of the histone acetyltrans-

ferases p300 and PCAF. Mol. Cell 2000, 5, 589−595.

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