Identification of small-molecule enhancers of arginine methylation catalyzed by coactivator-associated arginine methyltransferase 1

Article

pubs.acs.org/jmc

Identiﬁcation of Small-Molecule Enhancers of Arginine Methylation

Catalyzed by Coactivator-Associated Arginine Methyltransferase 1

Sabrina Castellano,^†Astrid Spannhoﬀ,^‡Ciro Milite,^†Fabrizio Dal Piaz,^†Donghang Cheng,^‡

Alessandra Tosco,^†Monica Viviano,^†Abdellah Yamani,^†,#Agostino Cianciulli,^†Marina Sala,^†

Vincent Cura,^⊥Jean Cavarelli,^⊥Ettore Novellino,^§Antonello Mai,^∥Mark T. Bedford,^‡

and Gianluca Sbardella*^,†

^†Dipartimento di Scienze Farmaceutiche e Biomediche, Epigenetic Med Chem Lab, Universita

Melillo, I-84084 Fisciano (SA), Italy

̀

degli Studi di Salerno, Via Ponte Don

^‡University of Texas M.D. Anderson Cancer Center, Science Park-Research Division, Smithville, Texas 78957, United States

^§Dipartimento di Chimica Farmaceutica e Tossicologica, Universita

Italy

̀

di Napoli “Federico II”, Via D. Montesano 49, I-80131 Napoli,

^∥Istituto PasteurFondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza Universita

̀

di Roma,

P.le A. Moro 5, I-00185 Roma, Italy

^⊥Dep

artement de Biologie Structurale Integrative, IGBMC (Institut de Genetique et Biologie Moleculaire et Cellulaire), UDS, CNRS,

́ ́ ́ ́ ́

INSERM, 67404 Illkirch Cedex, France

S

* Supporting Information

ABSTRACT: Arginine methylation is a common post-transla-

tional modiﬁcation that is crucial in modulating gene expression

at multiple critical levels. The arginine methyltransferases

(PRMTs) are envisaged as promising druggable targets, but

their role in physiological and pathological pathways is far from

being clear due to the limited number of modulators reported to

date. In this eﬀort, enzyme activators can be invaluable tools

useful as gain-of-function reagents to interrogate the biological

roles in cells and in vivo of PRMTs. Yet the identiﬁcation of

such molecules is rarely pursued. Herein we describe a series of

aryl ureido acetamido indole carboxylates (dubbed “uracando-

lates”), able to increase the methylation of histone (H3) or

nonhistone (polyadenylate-binding protein 1, PABP1) substrates induced by coactivator-associated arginine methyltransferase 1

(CARM1), both in in vitro and cellular settings. To the best of our knowledge, this is the ﬁrst report of compounds acting as

CARM1 activators.

and asymmetrically ω-N^G, N^G-dimethylated (aDMA), each of

INTRODUCTION

■

which has potentially diﬀerent functional consequences.^2,7,9To

date, nine proteins, diﬀering in sequence and in length, have

been clearly identiﬁed as PRMTs in mammals and are classiﬁed

as type I, type II, or type III enzymes.^5,7,9These enzyme types

all methylate the terminal (or ω) guanidino nitrogen atoms, but

type I PRMTs (PRMT1−4, PRMT6, and PRMT8) catalyze the

production of aDMA, whereas type II PRMTs (PRMT5/JBP1,

PRMT7) catalyze the formation of sDMA. PRMT7 has been

described as also having type III activity on certain substrates.¹⁰

In addition, a type IV enzyme that catalyzes the mono-

methylation of the internal (or δ) guanidino nitrogen atom has

been described in yeast.^7,9

The methylation of arginine residues is a prevalent post-

translational modiﬁcation found on both nuclear and

cytoplasmic proteins, which is involved in a number of diﬀerent

cellular processes, including transcriptional regulation, RNA

metabolism, and DNA damage repair.¹⁻⁴Enzymes of the

protein arginine N-methyltransferase (PRMT) family catalyze

the transfer of a methyl group from the donor S-adenosyl-^L-

methionine (SAM or AdoMet) to the guanidinium side chain

of arginine residues in the target protein, resulting in

methylated arginine and S-adenosyl-^L-homocysteine (SAH or

AdoHcy).⁵⁻⁷This post-translational modiﬁcation mainly

occurs on glycine-and arginine-rich patches (GAR motifs)

within their substrates,⁸and its complexity is enhanced by the

ability of the arginine residue to be methylated in three

diﬀerent ways on the guanidino group: ω-N^G-monomethylated

(MMA), symmetrically ω-N^G, ω-N′^G-dimethylated (sDMA),

Received: July 26, 2012

A

dx.doi.org/10.1021/jm301097p | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Chart 1. Selected Small-Molecule Inhibitors of PRMTs

A growing number of studies have recently recognized the

nearly ubiquitous expression of PRMTs (with the exception of

PRMT8, which is a neuron-speciﬁc enzyme^11,12) in most

mammalian cell types and tissues, as well as their involvement

in a wide variety of functional processes like RNA processing,

DNA damage and repair, cell signaling, and most importantly in

transcription.^9,13Moreover, arginine methylation of histones

can increase the complexity of the histone code by modulating

the interaction of nuclear factors with other nearby histone

marks.¹⁴⁻¹⁶

In mammalian cells, bulk protein arginine methylation is

likely ascribable to PRMT1, the main PRMT member. This

enzyme is involved in the dysregulation of nuclear receptor

signaling,^2,5,17,18which is a hallmark of hormone dependent

cancers, e.g., breast cancer.¹⁹⁻²²Recently, it was shown that

PRMT1 is a component of mixed lineage leukemia (MLL)

transcription complex and that its activity is required for

malignant transformation.²³Moreover, several tumors, includ-

ing breast cancer^24,25and colon cancer,^26,27are characterized by

aberrant expression of spliced forms of PRMT1, while

deregulation of the histone H4R3 methyl mark (a major

cellular substrate of PRMT1¹⁸) is a predictor of prostate

cancer.²⁸

Similarly, PRMT4 (commonly referred to as coactivator-

associated arginine methyltransferase 1 (CARM1)) plays a

crucial role in modulating gene expression at multiple critical

levels,⁵methylating many coactivators, including p300/CBP

and ampliﬁed in breast cancer-1 (AIB1),²as well as proteins

involved in splicing²⁹and RNA-binding proteins.³⁰⁻³²CARM1

has been implicated in spinal muscular atrophy pathogenesis²⁹

and has been associated with viral infections involving human T

lymphotrophic virus.³³Recently, it has been reported that

CARM1 is overexpressed in both aggressive prostate cancer

and breast tumor^34,35and that the presence of CARM1 and the

increased H3R17 methylation give an important contribution in

breast cancer (particularly estrogen receptor α dependent)

manifestation and progression.^36,37Furthermore, CARM1 and

PRMT1, together with p300/CBP and PARP1, synergistically

coactivate NF-κB-dependent gene expression³⁸and cooperate

in p53-dependent transcriptional activation.³⁹In addition to

this, PRMTs have also emerged as potential new targets for the

development of a novel therapeutic for heart disease,^{34,35,40−45}

as they are overexpressed in the hearts of patients with

coronary heart disease⁴⁶and PRMT activity appears to be

responsible for generating the majority of the aDMA, which

subsequently blocks NO production and causes many

cardiovascular conditions such as diabetes and hyperten-

sion.⁴⁷⁻⁴⁹

On the other hand, PRMT1 and CARM1, together with

PRMT5, are downregulated in senescent tissues,⁵⁰and it has

been reported that the activity of CARM1 is required for the

proper mainteinance of physiological processes like endochon-

dral ossiﬁcation and chondrocyte proliferation,⁵¹myogenesis,⁵²

glycogen metabolism,⁵³and proper control of proliferation and

diﬀerentiation of pulmonary epithelial cells.⁵⁴

Therefore, there is convincing evidence that PRMTs have a

crucial role in several serious human diseases as well as in many

important physiological processes,^55,56supporting the hypoth-

esis that targeting PRMTs would be a viable approach for drug

discovery eﬀorts.⁵⁷⁻⁶⁰

As a matter of fact, extensive research has been conducted to

better understand the role of PRMTs in physiological and

pathological pathways,^{5,17−22,34,35,40−43,45,54,61−63}to elucidate

the structure⁶⁴⁻⁶⁹of these enzymes, and to gain insights into

the mechanism of methyl transfer.^70,71Yet, the number of small

molecule modulators of the PRMTs reported to date is still

limited (Chart 1).^{13,60,67,72−99}

B

dx.doi.org/10.1021/jm301097p | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Figure 1. Indole derivatives 1a−l, 2a−h, 3a−j, and 4a−l.

a

Scheme 1. Synthesis of Derivatives 1k,l, 3a−j and 4a−l

a

Reagents and conditions: (a) (COCl)₂, Et₂O, 0 °C, overnight; (b) KOH, H₂O, reﬂux, 2 h; (c) H₂O₂(10% wt in H₂O), reﬂux, 4 h; (d) H₂SO₄,

MeOH, reﬂux, 6 h; (e) t-BuOH, DCC, TEA, DMF, 120 °C, 3h; (f) H₂(1 atm), 5% Pd/C, EtOH, 2 h; (g) Boc-Gly-OH or Fmoc-Gly-OH, DIEA,

HOBt, HBTU, THF/DMF 7:2, rt, overnight; (h) TFA/CH₂Cl₂1:3, rt, 30 min; (i) isocyanate, TEA, THF, rt, 3 h; (j) NaOH, pyridine, H₂O/THF

4:1, rt, overnight; (k) CH₂Cl₂/piperidine, rt, 30 min.

In 2004, a small-molecule screening study led some of us to

the identiﬁcation of the symmetric urea derivative arginine

methyltransferase inhibitor 1 (AMI-1, Chart 1) as a selective

arginine methylation speciﬁc inhibitor, noncompetitive with S-

adenosylmethionine (SAM) binding and not aﬀecting lysine

methylation.⁷⁵By molecular modeling studies, we then

proposed that AMI-1 spans both the SAM and Arg binding

sites without fully occupying them.⁷⁸This binding hypothesis,

consistent with the fact that AMI-1 does not inhibit UV cross-

linking of SAM to PRMT1 (suggesting that it does not

C

dx.doi.org/10.1021/jm301097p | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Figure 2. CARM1/PRMT4 activators 3a−j and 4a−f.

compete for the SAM binding site),⁷⁵was subsequently

supported by structure-based 3-D QSAR models¹⁰⁰and

allowed us to design and synthesize carboxy analogues of

AMI-1 that are nearly as potent as the parent compound while

retaining PRMT selectivity.⁹²

according to a slight modiﬁcation of a previously described

procedure.¹⁰⁵Curiously, any attempt to hydrolyze the

aforementioned esters failed to give the acids 4b, 4d, 4f, 4h,

4j, and 4l, which were, instead, obtained from the

corresponding tert-butyl esters 4a′, 4c′, 4e′, 4g′, 4i′, and 4h′

(prepared starting from tert-butyl 5-amino-1H-indole-3-carbox-

ylate 8′) after treatment with triﬂuoroacetic acid in dichloro-

methane (Scheme 1C).

Pursuing our eﬀorts toward the identiﬁcation of potent and

selective PRMT inhibitors, we replaced the hydroxynaphtha-

lene moiety with the bioisosteric indole framework, a

“privileged” structural motif commonly found in pharmaceut-

ical drugs and natural products.¹⁰¹Thus, we prepared 2,5-

disubstituted indole derivatives 1a−l as well as their positional

isomers 2a−h (Figure 1), and, in conformity with our previous

studies,^78,92we performed a preliminary screening of their

activities against human recombinant PRMT1 and other

methyltransferases in vitro. Surprisingly, we found that the

ureidoacetamido-indole derivative 1k, though scarcely active in

inhibiting PRMT1 (and other methyltransferases, see below),

induced a marked activation of CARM1/PRMT4. Therefore, in

this paper, we describe the synthesis of a series of

ureidoacetamido-indole derivatives 3a−j and 4a−l (Figure 1),

structurally related to 1k, and the evaluation of the eﬀects on

CARM1-induced arginine methylation.

RESULTS AND DISCUSSION

■

Identiﬁcation of CARM1 Activators. In pursuing our

campaign aimed at the identiﬁcation of potent and selective

PRMT inhibitors, we synthesized derivatives 1a−l and 2a−h

and tested them against human recombinant PRMT1

(hPRMT1) in vitro, using as a substrate the RNA-binding

nuclear shuttling protein Npl3, speciﬁcally methylated by both

yeast (HMT1)^106−108and human (PRMT1) orthologues,⁷⁵

determining the percent values of inhibition at the ﬁxed doses

of 10 and 50 μM (Tables S1 and S2, respectively, Supporting

Information). AMI-1⁷⁵and EML108⁹²were used as reference

compounds.

Even though none of the derivatives 1a−l were eﬃcient

inhibitors of PRMT1 (Supporting Information Table S1),

compounds that displayed a greater than 60% inhibition were

retested at 50 μM against recombinant PRMT1, PRMT3,

CARM1, and PRMT6, using histone H4, the glycine- and

arginine-rich (GAR) motif, and histone H3 (for both CARM1

and PRMT6) as substrates, respectively (not shown). We

noticed that, albeit scarcely active in inhibiting the other

enzymes, ureidoacetamido-indole derivative 1k induced a

marked activation of CARM1/PRMT4 (Figure 2). We repeated

the assay by testing the compound at six diﬀerent

concentrations (3, 6, 12, 25, 50, and 100 μM) against

CARM1 using as a substrate the polyadenylate-binding protein

1 (PABP1), an in vivo major substrate for CARM1.³¹Again,

compound 1k induced a marked and dose-dependent increase

in the enzyme activity, clearly noticeable even at the lowest

tested concentration (3 μM, Figure 3). A similar result was

observed when H3 was used as a substrate (Figure S1,

Supporting Information). Therefore, we prepared the analogues

3a−j and the corresponding positional isomers 4a−l (Figure 2)

and test all of them (with the exception of the acids of the

series 4)¹⁰⁹at 50 μM in an in vitro assay, to assess their potency

against CARM1, using H3 and PABP1 as substrates.

CHEMISTRY

■

The synthesis of 2,5-disubstituted indole derivatives 1a−j as

well as of 3,5-substituted analogues 2a−h is reported in

Supporting Information (Schemes S1 and S2, respectively).

Derivatives 1k,l and 3a−j were prepared (Scheme 1A)

starting from 5-amino-1H-indole-2-carboxylate, which was in

turn prepared through Fisher indole reaction and subsequent

reduction using hydrogen over palladium on activated carbon,

as previously described.^102,103

The reaction of 5-amino-1H-indole-2-carboxylate with N-

(tert-butoxycarbonyl)glycine (Boc-Gly-OH) in the presence of

the peptide coupling reagents hydroxybenzotriazole (HOBt)

and O-benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexa-

ﬂuoro-phosphate (HBTU) and Hunig’s base (N,N-diisopropy-

̈

lethylamine, DIEA) to obtain the Boc-protected glycinamide

11, which was deprotected with triﬂuoroacetic acid and then

coupled with the proper phenylisocyanate in the presence of

triethylamine to yield the esters 1k, 3a, 3c, 3e, 3g, and 3i. The

hydrolysis of the latter ﬁnally gave the corresponding acids 1l,

3b, 3d, 3f, 3h, and 3j, respectively.

A similar synthetic procedure (Scheme 1B) yielded the esters

4a, 4c, 4e, 4g, 4i, and 4h starting from 5-amino-1H-indole-3-

carboxylate 8,¹⁰⁴obtained by catalytic hydrogen reduction of

the corresponding 5-nitro derivative 7, prepared by reaction of

5-nitroindole with oxalyl chloride and subsequent alkaline

hydrolysis, oxidative decarboxylation and, ﬁnally, esteriﬁcation,

As reported in Table 1 (and also in the ﬂuorographs in

Figure S2, Supporting Information), all compounds 3 (with the

only exception of dimethylamino-substituted carboxylic acid 3j)

induced, even if with diﬀerent potency, an increase in H3

methylation catalyzed by CARM1, further substantiating the

D

dx.doi.org/10.1021/jm301097p | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Table 1. Eﬀects of Compounds 3a−j and 4a−l (50 μM) on

CARM1-Catalyzed Methylation of Histone- Or Nonhistone

a

Substrates

b

% CARM1 activity at 50 μM

c

(mean SD )

d

e

compd

3a

X

Y

H3

PABP1

OEt

OH

H

134.1 2.8

167.1 4.0

127.2 2.6

111.0 0.8

182.7 2.0

211.2 1.5

115.4 0.7

142.7 1.5

175.5 2.6

142.5 1.0

101.7 0.8

48.3 0.9

74.7 0.7

10.5 0.5

172.3 5.3

382.3 3.1

148.8 2.5

74.7 0.7

96.3 0.4

357.6 3.9

81.2 0.7

298.9 5.7

85.3 0.5

70.6 0.8

134.7 2.1

26.5 0.1

3b

3c

3d

3e

3f

OEt

OH

CH₃

OEt

OH

OCH₃

CN

3g

3h

OEt

OH

CN

Figure 3. Activation of enzymatic activity of CARM1 after treatment

with 1k (3, 6, 12, 25, 50, and 100 μM) using PABP1 as a substrate.

The bands were cut out and the eﬃciency of the enzyme was

determined by scintillation counting. DMSO was used as positive

control. N stands for negative control (no SAM added).

f

1k

OMe

OH

NO₂

f

1l

NO₂

3i

OEt

OH

N(CH₃)₂

H

3j

4a

OMe

OH

g

4b

H

ND

relationship between such eﬀect and the lead scaﬀold of this

series of molecules. The substituents decorating the benzene

moiety seem to modulate the intensity of the enzymatic

activation, the highest eﬀect being detected with the

introduction of a methoxy group (derivatives 3e and 3f),

which yielded an increase in CARM1 activity even more

signiﬁcant (183% and 211%, respectively) than what we

previously observed for nitro-substituted derivatives 1k and 1l

(Table 1).

On the other hand, the eﬀects of derivatives 3 on the

methylation of the nonhistonic substrate PABP1 were less

regular. In fact, the methyl-, the cyano-, and the nitro-

substituted esters 3c, 3g, and 1k, respectively, caused a very

strong ampliﬁcation of substrate methylation (382%, 358%, and

299%, respectively), whereas the unsubstituted acid 3b as well

as the methyl- and the dimethylamino-substituted acids 3d and

3j determined a lesser eﬀect (172%, 149%, and 135%,

respectively).

4c

OMe

OH

CH₃

39.7 1.1

4.7 0.1

g

4d

CH₃

ND

4e

OMe

OH

OCH₃

CN

62.7 0.6

28.2 0.4

g

4f

ND

4g

OMe

OH

134.9 1.5

38.8 0.2

g

4h

CN

ND

4i

OMe

OH

NO₂

134.2 2.4

114.7 2.0

g

4j

NO₂

ND

4k

OMe

OH

N(CH₃)₂

125.2 0.7

7.6 0.1

g

4l

ND

h

EML108

AMI-1

83.1 0.8

74. 1.9

NT

h

NT

a

b

c

Values are means determined for at least two separate experiments.

Enzyme activity percentage, calculated with respect to DMSO.

Standard deviation values are indicated in percentage points.

Histone H3 (1.1 μM) and SAM (0.42 μM) were used as substrates.

PABP1 was used as a nonhistone substrate, SAM as cofactor (see

d

e

f

Experimental Section). Compounds 1k and 1l were inserted for

Regarding compounds 4, positional isomers of 3, in general

they produced a reduction of methylation of both substrates,

more evident in the case of PABP1 (Table 1). The only

exceptions are the cyano- and the dimethylamino-substituted

esters 4g and 4k, which yielded a moderate gain in H3

methylation (135% and 125%, respectively) while inhibiting

PABP1 methylation, and the nitro-substituted ester 4i, which

determined a slight augmentation of the methylation on both

substrates (134% for H3 and 115% for PABP1). It is

noteworthy that derivatives 3a, 3j, and 4k seem to be endowed

with a unique activity proﬁle, apparently being capable of

amplifying the methylation levels of one substrate while

inhibiting the CARM1 induced methylation of the other

(Table 1). All tested derivatives were basically inactive against

PRMT1/Npl3p even at 50 μM concentration (Table S4,

Supporting Information).

comparison and their activities were obtained from previous

experiments. Not detectable (see ref 111.). Not tested.

g

h

directions for the two substrates, (b) binding of the small

molecule to an allosteric site resulting in a conformational

change of the enzyme structure, leading to a better aﬃnity for

one substrate and a worse aﬃnity for the other, (c) interaction

of the small molecule with the substrates themselves, thus

improving or worsening their molecular recognition by the

enzyme catalytic site.

As a matter of fact, Zheng and co-workers recently proposed

that AMI-1 and structurally related compounds preferentially

interact with the histone peptide rather than the enzyme.⁸⁷

Similarly, Kundu and co-workers found that ellagic acid (Chart

1), one component of pomegranate extract, speciﬁcally inhibits

CARM1-induced methylation at arginine 17 of histone H3

(H3R17) by binding to a “KAPRK” motif of the substrate.⁹⁰

Therefore, we resolved to ascertain if the observed activity of

the compounds on protein methylation could be due (at least

SPR-Based Binding Assay. The diﬀerent eﬀects of

ureidoacetamido indole derivatives 3 and 4 on the methylation

of histone or nonhistone substrates could be due to various

reasons, among which are (a) diﬀerent enzyme approaching

E

dx.doi.org/10.1021/jm301097p | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

partially) to a direct interaction with the substrate. To this aim,

we took advantage of a surface plasmon resonance-based (SPR-

based) binding assay as implemented with Biacore technology,

recently set up and successfully employed by us to study in real-

time kinetic and thermodynamic parameters of ligand−protein

interactions between KAT3B and small-molecule deriva-

tives.^110,111

In a ﬁrst set of experiments, calf thymus histone H3 or a

peptide from PABP1 comprising residues 437−488 (thus

containing the two arginine residues, R455 and R460, that have

been reported to be methylated by CARM1)³¹were

immobilized (17000 RU, response units)¹¹²on diﬀerent ﬂow

cells of the biosensor chip and diﬀerent concentrations (50−

10000 nM) of derivatives 1k, 3c, 3g, and 4e were injected over

the peptide surface. Both AMI-1⁷⁵and ellagic acid⁹⁰were used

as reference compounds.

absence (not shown) of SAH. Conversely, the presence of 4

μM 1k or 3g aﬀects the aﬃnity of the substrate for the complex

enzyme/SAH (panels H,K and I,L). Similar results were

obtained with compounds 3c and 4e (Figure S5, Supporting

Information). It is noteworthy that, under the same conditions

AMI-1 binds to the protein with a K_Dvalue of 810 nM (Figure

S4, Supporting Information). Together with the aforemen-

tioned results from the analysis of the interaction with the

substrates, this outcome suggests that the inhibition of CARM1

activity showed by AMI-1⁷⁵may be not completely due to an

interaction with the substrate as recently proposed,⁸⁷at least

when PABP1 is used as a substrate. To evaluate possible

unspeciﬁc bindings, all compounds were also injected on an

immobilized bovine serum albumin (BSA), and no interaction

was observed (data not shown).

Cellular Activity of CARM1 Activators. Then, we

resolved to determine whether the increase of CARM1 induced

arginine methylation after treatment with compounds 3 was

maintained within a cellular context. The cell-based reporter

system we previously developed for PRMT1 activity^75,92was

inadequate to this purpose, as CARM1 does not methylate

Npl3p. On the contrary, PABP1 is a major CARM1 substrate³¹

and in CARM1 knockout cells it is hypomethylated.¹¹⁷

Moreover, CARM1 is the only enzyme that methylates

PABP1. An accurate gauge of CARM1 activity should be

performed starting from unmethylated PABP1, yet the methyl

mark is extremely stable, as no arginine demethylases have been

hitherto discovered,⁹thus a cell-based assay could require days

of treatment to allow for the methylated substrates to turnover.

Recently, we circumvented this problem by using human

embryonic kidney 293 cells (HEK293) to develop a PABP1

inducible cell line, that, upon treatment with tetracycline (Tet),

express a FLAG-tagged form of full-length PABP1 (fPABP1,

Figure 4A).⁹⁸This tag allows the induced form of PABP1 to

migrate more slowly by SDS-PAGE, thus making easy to

distinguish between the endogenous PABP1 and the newly

synthesized fPABP1. When fPABP1 expression is induced with

Tet in the presence of a potential CARM1 modulator, the

degree of fPABP1 methylation can be measured using a methyl-

speciﬁc antibody against the PABP1 sequence CGAIR*-

PAAPR*PPFS (where R* represents an asymmetrically

dimethylated arginine residue).⁹⁸

Therefore, a Tet-inducible 3XFLAG-tagged PABP1 (fPABP)

was stably transfected into HEK293 cells and post-transfection

the cells were treated for 24 h with 50 μM concentrations of

selected compounds 3c and 3g, which had triggered the highest

increase of PABP1 methylation (in vitro, Table 1, 382% and

358% activation, respectively). The antimethyl-PABP1 antibody

(α-Me-PABP1) was used as methylation sensor and the anti-

FLAG antibody (α-FLAG, M2) to detect the induction of

fPABP1 and to show protein levels. As shown in Figure 4C,

when fPABP signal intensities of lane 1 (control) are compared

with those of lane 3 (treatment with 3c) or of lane 4 (treatment

with 3g), both derivatives strongly increase the methylation of

fPABP1 in cells, thus conﬁrming the in vitro activity of the

compounds as well as their cell permeability.

To reduce false positives from detergent-sensitive nonspeciﬁc

aggregation-based binding,¹¹³0.005% NP40 was added to the

running buﬀer in all the experiments.¹¹⁴To evaluate possible

unspeciﬁc bindings, all compounds were also injected on an

immobilized bovine serum albumin (BSA). The binding of each

compound was read out in real time as the change in mass at

the sensor surface. After injection, running buﬀer was ﬂowed

over the surface and dissociation of the compounds from the

surface was observed (Figure S3, for derivatives 1k and 3g, and

Figure S5, for derivatives 3c and 4e, Supporting Information).

Consistently with previous reports,^87,90both reference

compounds, AMI-1 and ellagic acid, interacted eﬃciently

(aﬃnity constant K_D= 671 nM and 172 nM, respectively)

with the immobilized histone H3 substrate, as shown by the

concentration-dependent responses and the clearly evident

exponential curves during both the association and dissociation

phases (Figure S4, Supporting Information). Yet, the

interaction with PABP1(437−488) was barely perceptible (if

any; not shown). SPR experiments carried out on tested

compounds also produced good sensorgrams but showed very

low and/or concentration-independent responses, thus suggest-

ing negligible interactions of such molecules with either histone

(Figure S3, panels A,D, Supporting Information) or nonhistone

substrates (Figure S3, panels B,E, Supporting Information).

Next, we turned our attention to the investigation of the

binding to CARM1. It has been reported that SAM binding to

CARM1 induces the formation of a helix that serves as a

binding ridge for the protein substrate.^64,67Therefore, the

presence of the cofactor is crucial for the formation of the

enzyme active complex. Yet, commercially available SAM is

contaminated by various amounts of S-adenosylhomocysteine

(SAH) and, moreover, a nonenzymatic pseudo-ﬁrst-order

formation of SAH spontaneously occurs in aqueous medium

by the transfer of methyl groups from SAM to water forming

methanol.^115,116On the other hand, the commercially available

and inexpensive SAH is quite stable and binds eﬃciently to the

enzyme, thus being an excellent replacement for the cofactor in

biophysical studies.⁶⁷Therefore, we immobilized full length

GST-tagged CARM1 on the biosensor chip (12000 RU) and

validated the immobilization by injecting either histone H3 or

PABP1(437−488) at diﬀerent concentrations (50−4000 nM),

in the presence of 200 μM SAH, obtaining K_Dvalues of 6.5 and

158 nM, respectively (Figure S3, panels G and J, Supporting

Information). As shown in Figure S3C,F, Supporting

Information, no interaction was observed after injection of

compounds 1k and 3g (in the range 50−10000 nM) on the

surface of the immobilized enzyme, both in the presence and in

CONCLUSION

■

PRMT-mediated arginine methylation regulates nucleosomal

remodeling, gene expression, DNA repair, RNA processing and

shuttling, and other cellular processes, thus the development of

small molecule modulators of PRMT activity is a lively research

endeavor in the ﬁeld of epigenetics. Such molecules will be

F

dx.doi.org/10.1021/jm301097p | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

approaches the enzyme catalytic site from a diﬀerent direction,

the binding of the tested compounds could diﬀerently aﬀect

substrate recognition. Otherwise, derivatives could bind to an

allosteric site, thus promoting a conformational change of the

enzyme structure resulting in the stabilization of a more active

state but with diﬀerent aﬃnity for the two substrates, in

accordance with the Monod−Wyman−Changeux (MWC)

model.^118,119Moreover, Zheng and co-workers recently

published a very insightful work in which they report that a

series of naphthalene-sulfo derivatives (NS), bearing structural

similarities with AMI-1, directly target H4(1−20) and GAR

peptide substrates instead of PRMT1, and the binding

subsequently blocks the recognition of the substrates by the

enzyme, which is largely responsible for the observed PRMT1

inhibition eﬀect.⁸⁷With the aim to investigate if tested

compounds could (at least partially) recognize and directly

bind speciﬁc sequences on the substrates themselves, thus

enhancing or impairing their molecular recognition by the

enzyme catalytic site, we performed a surface plasmon

resonance-based (SPR-based) analysis of the interactions of

derivatives 1k, 3c, 3g, and 4e with the enzyme, the substrate

(both histone and nonhistone) peptide, and the ternary

complex CARM1/substrate/SAH. We found that no inter-

action occur when the compounds are injected on the surface

of the substrates or of the enzyme, even in the presence of

SAH. Nonetheless, all the molecules signiﬁcantly aﬀect the

aﬃnity of H3 or PABP1 to CARM1/SAH complex. Moreover,

we found that AMI-1 binds to CARM1 as well as to H3,

whereas no binding interaction with PABP1 is detectable.

Therefore, the inhibition of CARM1 activity showed by this

molecule⁷⁵may be not completely due to an interaction with

the substrate as proposed,⁸⁷at least when PABP1 is used as a

substrate.

On the basis of the outcomes of the SPR analysis, it is

possible to rule out the direct interaction of uracandolates with

the substrates. Similarly, their negligible binding to the full-

length enzyme suggests an allosteric interaction as unlikely. As a

matter of fact, the mechanism underlying their biological eﬀects

remains unclear, in particular for the compounds exhibiting

remarkable diﬀerences in potency or in the activity proﬁle when

histone or nonhistone substrates were used. A possible

hypothesis could be that the binding of the substrate (and

maybe also of the cofactor) induce structural changes allowing

the binding of uracandolates, which aﬀect methylation.

Ongoing additional experiments will shed more light on this

issue.

In conclusion, in this study we found that the methylation

activity of CARM1 is enhanced by a series of aryl ureido

acetamido indole carboxylates, both in in vitro and cellular

settings. To the best of our knowledge, this is the ﬁrst report of

compounds acting as activators of CARM1-induced methyl-

ation.¹²⁰While most chemical biology and drug discovery

eﬀorts are focused on identifying small molecules that inhibit

enzyme function, it is quite infrequent to come across enzyme

activators, yet there are signiﬁcant advantages to ﬁnding them.

Enzyme activators can serve as gain-of-function reagents that

can be used to interrogate the biological roles of PRMTs in

cells and in vivo. These activators could be used in combination

with inhibitors to identify new substrates. Also, CARM1

activators may simulate the eﬀects of CARM1 overexpression

that is seen in cancer cells, and these compounds could possibly

promote cellular transformation. Furthermore, it has recently

been reported that the enzymatic activity of CARM1 is crucial

Figure 4. Eﬀects of compounds 3c and 3g on arginine methylation of

induced fPABP1. (A,B) The depiction of a cell-based reporter system

for CARM1 activity. HEK293 cells were stably transfected with a Tet-

inducible 3XFLAG-tagged PABP1 (fPABP). Orange dots represent

arginine methylation on both endogenous PABP1 (sky blue) and

induced fPABP1 (cyan). (A) Low levels of methylation are observed

when induction takes place in the absence of activators. (B) Higher

levels of methylation are observed with fPABP1 when induction takes

place in the presence of an activator. (C) The fPABP1 was induced

with tetracycline (1 μg/mL) in the presence of compounds 3c or 3g at

50 μM. The degree of methylated PABP1 was gauged by using a

methyl-speciﬁc anti-PABP1 antibody (top panel). The anti-FLAG

antibody (α-FLAG, M2) was used to detect the induction of fPABP1

as well as protein levels (bottom panel).

invaluable tools to study the function of protein arginine

methylation and the pathways in which PRMTs are involved.

After replacing the hydroxynaphthalene moiety contained in

selective PRMT inhibitors previously identiﬁed by us^75,78,92

with the bioisosteric indole framework, with the aim to obtain a

more versatile and synthetically handy scaﬀold for the future

development of more potent inhibitors, we prepared 2-carboxy-

and 3-carboxy-substituted indole derivatives 1a−l and 2a−h,

respectively. We noticed that ethyl 5-(2-(3-(4-nitrophenyl)-

ureido)acetamido)-1H-indole-2-carboxylate 1k was endowed

with an hitherto unobserved marked and dose−dependent

activating eﬀect against CARM1. Therefore, we prepared a

series of ureidoacetamido indole derivatives 3a−j, structurally

related to 1k, and found that nearly all of them increased

CARM1 induced arginine methylation in vitro. We dubbed

these compounds “uracandolates” (ureidoacetamido indole-2-

carboxylates). The activity of the two most potent derivatives,

namely 3c and 3g, was also conﬁrmed in a cellular assay. Again,

the simple formal shift of the carboxy function from the

position 2 to position 3 of the indole ring signiﬁcantly aﬀected

the activity, as the resulting derivatives 4a−l were consistently

less potent as activators or even displayed an inhibitory eﬀect.

Interestingly, a few derivatives exhibited signiﬁcant diﬀerences

in potency or in the activity proﬁle when histone or nonhistone

substrates were used. Such diﬀerences could be attributed to a

number of possible reasons. For example, if each substrate

G

dx.doi.org/10.1021/jm301097p | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

for endochondral ossiﬁcation and chondrocyte proliferation,⁵¹

for the control of myogenesis,⁵²for glycogen metabolism,⁵³for

proper control of proliferation and diﬀerentiation of pulmonary

epithelial cells,⁵⁴and that CARM1 as well as other PRMTs

(namely PRMT1 and PRMT5) are downregulated in senescent

tissues.⁵⁰Therefore, small molecules able to enhance the

catalytic activity of CARM1 could be useful chemical tools for

the mechanistic study of arginine methylation and its

implications in physiological and/or pathological processes.

in the next step. A solution of 4-nitrophenyl isocyanate (163 mg, 0.99

mmol) in dry THF (8 mL) was added dropwise to a solution of

deprotected compound 11 (308 mg, 0.82 mmol) and triethylamine

(0.34 mL, 2.46 mmol) in dry THF (20 mL) under N₂atmosphere.

The reaction was stirred at room temperature for 3 h. The precipitate

was collected by ﬁltration and rinsed with water and diethyl ether

successively. Puriﬁcation by crystallization (DMF/H₂O) gave the title

compound as a white solid (244 mg, 70%); mp 290−291 °C. IR

(KBr): ν = 3375, 3329, 2978, 1695, 1659, 1597, 1551, 1533 cm⁻¹. ¹H

NMR (300 MHz, DMSO-d₆): δ 11.84 (s, 1H, exchangeable with

deuterium oxide), 9.99 (s, 1H, exchangeable with deuterium oxide),

9.74 (s, 1H, exchangeable with deuterium oxide), 8.18 (d, 2H, J = 9.1

Hz), 8.02 (s, 1H), 7.67 (d, 2H, J = 9.1 Hz), 7.43−7.36 (m, 2H), 7.14−

7.11 (m, 1H), 6.84 (br t, 1H, J = 5.0 Hz, exchangeable with deuterium

oxide), 4.35 (q, 2H, J = 7.0 Hz), 3.99 (d, 2H, J = 5.0 Hz), 1.35 (t, 3H, J

= 7.0 Hz). ESI-MS m/z: 426 (M + H)⁺. Anal. (C₂₀H₁₉N₅O₆) C, H, N.

Synthesis of 5-(2-(3-(4-Nitrophenyl)ureido)acetamido)-1H-in-

dole-2-carboxylic Acid (1l). NaOH (376 mg, 9.40 mmol) and

pyridine (0.15 mL, 1.88 mmol) were added to a solution of ester 1k

(200 mg, 0.47 mmol) in THF−H₂O (4:1, 40 mL). The reaction was

stirred at room temperature overnight and then concentrated in vacuo.

The residue was dissolved in water (20 mL), washed with CH₂Cl₂(3

× 10 mL), and acidiﬁed to pH 2 with 12N HCl. The precipitate was

collected by ﬁltration and washed with water. Puriﬁcation by

crystallization (DMF/H₂O) gave the title compound as a white solid

(168 mg, 90%); mp 283−284 °C (decomp). IR (KBr): ν = 3404,

3348, 3283, 3080, 1684, 1645, 1610, 1544, 1521 cm⁻¹. ¹H NMR (300

MHz, DMSO-d₆): δ 11.71 (s, 1H, exchangeable with deuterium

oxide), 9.98 (s, 1H, exchangeable with deuterium oxide), 9.67 (s, 1H,

exchangeable with deuterium oxide), 8.18 (d, 2H, J = 9.0 Hz), 8.00 (s,

1H), 7.67 (d, 2H, J = 9.0 Hz), 7.40−7.33 (m, 2H), 7.06 (s, 1H), 6.74−

6.73 (m, 1H, exchangeable with deuterium oxide), 4.00 (d, 2H, J = 4.9

Hz), the carboxylic acid proton could not be detected. ESI-MS m/z:

396 (M − H)⁻. Anal. (C₁₈H₁₅N₅O₆) C, H, N.

Synthesis of Methyl 5-Nitro-1H-indole-3-carboxylate (7). Oxalyl

chloride (8.52 mL, 98.7 mmol) was added dropwise to a solution of 5-

nitroindole (2.00 g, 12.3 mmol) in dry EtOEt (150 mL) at 0 °C under

N₂atmosphere, and the resulting mixture was stirred at room

temperature overnight. The yellow precipitate was collected by

ﬁltration, washed with cold diethyl ether, and dissolved in water (80

mL). KOH (3.25 g, 58.0 mmol) was added portionwise, and the

reaction was heated to reﬂux for 2 h. The reaction solution was ice-

cooled and acidiﬁed to pH 1 with HCl 12N. The precipitate was

collected by ﬁltration, washed with HCl 1N, and dried under vacuum.

The solid was then resuspended in 10% hydrogen peroxide aqueous

solution (150 mL) and heated to reﬂux for 4h. Resulting white

precipitate was collected by ﬁltration, washed with cold water, and

dried under vacuum to give 5-nitro-1H-indole-3-carboxylic acid (2.27

g, 95% over two steps), which was used in the esteriﬁcation step

without further puriﬁcation. Spectroscopic data of the title compound

are consistent with those reported in the literature.¹⁰⁵Sulphuric acid

(1 mL) was added to a solution of 5-nitro-1H-indole-3-carboxylic acid

(2.27 g, 11.0 mmol) in methanol (150 mL). The reaction was heated

at reﬂux for 6 h and then concentrated in vacuo. The residue was

dissolved in water, basiﬁed to pH 8 with NaHCO₃saturated solution,

and extracted with EtOAc (3 × 80 mL). The combined organic layers

were washed with brine, dried (Na₂SO₄), ﬁltered, and concentrated in

vacuo. The crude compound was recrystallized from methanol yielding

a light-yellow solid (2.18 mg, 90%); mp 288−289 °C (lit. 282−284

°C).¹⁰⁵¹H NMR (300 MHz, DMSO-d₆): δ 12.59 (br s, 1H,

exchangeable with deuterium oxide), 8.88 (d, 1H, J = 2.2 Hz), 8.39 (s,

1H), 8.13 (dd, 1H, J = 9.0, 2.2 Hz), 7.70 (d, 1H, J = 9.0 Hz), 3.88 (s,

3H). ESI-MS m/z: 221 (M + H)⁺.

EXPERIMENTAL SECTION

■

Chemistry. All chemicals were purchased from Aldrich Chimica

(Milan, Italy) or from Alfa Aesar GmbH (Karlsruhe, Germany) and

were of the highest purity. All solvents were reagent grade and, when

necessary, were puriﬁed and dried by standard methods. All reactions

requiring anhydrous conditions were conducted under a positive

atmosphere of nitrogen in oven-dried glassware. Standard syringe

techniques were used for anhydrous addition of liquids. Reactions

were routinely monitored by TLC performed on aluminum-backed

silica gel plates (Merck DC, Alufolien Kieselgel 60 F254) with spots

visualized by UV light (λ = 254, 365 nm) or using a KMnO₄alkaline

solution. Solvents were removed using a rotary evaporator operating at

a reduced pressure of ∼10 Torr. Organic solutions were dried over

anhydrous Na₂SO₄. Chromatographic separations were performed on

silica gel (silica gel 60, 0.015−0.040 mm; Merck DC) columns.

Melting points were determined on a Stuart SMP30 melting point

apparatus in open capillary tubes and are uncorrected. Infrared (IR)

spectra (KBr) were recorded on a Shimadzu IR Aﬃnity-1 FTIR

spectrophotometer at room temperature. ¹H NMR spectra were

recorded at 300 MHz on a Bruker Avance 300 spectrometer. Chemical

shifts are reported in δ (ppm) relative to the internal reference

tetramethylsilane (TMS). Mass spectra were recorded on a Finnigan

LCQ DECA TermoQuest (San Jose, USA) mass spectrometer in

electrospray positive and negative ionization modes (ESI-MS). Purity

of tested compounds was established by combustion analysis,

conﬁrming a purity ≥95%. Elemental analyses (C, H, N) were

performed on a Perkin-Elmer 2400 CHN elemental analyzer at the

laboratory of microanalysis of the Department of Chemistry and

Biology, University of Salerno (Italy); the analytical results were within

0.4% of the theoretical values. When the elemental analysis is not

included, compounds were used in the next step without further

puriﬁcation.

The synthesis of 2,5-disubstituted indole derivatives 1a−j as well as

of 3,5-substituted analogues 2a−h is reported in Supporting

Information.

Synthesis of Ethyl 5-(2-((tert-Butoxycarbonyl)amino)acetamido)-

1H-indole-2-carboxylate (11). A solution of HOBt (347 mg; 2.57

mmol), HBTU (974 mg, 2.57 mmol), Boc-Gly-OH (375 mg, 2.14

mmol), and DIEA (0,90 mL; 5.14 mmol) in dry THF−DMF (7:2, 9

mL) was added to a solution of ethyl 5-amino-1H-indole-2-carboxylate

(436 mg, 2.14 mmol) in dry THF (3 mL) under N₂atmosphere. The

reaction was stirred at room temperature overnight and then

concentrated in vacuo. The residue was dissolved in EtOAc (100

mL) and washed with NaHCO₃saturated aqueous solution (3 × 30

mL), citric acid 10% aqueous solution (3 × 30 mL), and brine. The

organic solution was dried (Na₂SO₄), ﬁltered, and concentrated in

vacuo. The residue was puriﬁed by silica gel chromatography (EtOAc/

hexanes, 7:3) to give the title compound as a white solid (626 mg,

81%); mp 193−194 °C. ¹H NMR (300 MHz, DMSO-d₆): δ 11.78 (s,

1H exchangeable with deuterium oxide), 9.78 (s, 1H exchangeable

with deuterium oxide), 7.98 (s, 1H), 7.38−7.33 (m, 2H), 7.08 (d, 1H,

J = 2.1 Hz), 6.99 (br t, 1H, J = 6.0 Hz, exchangeable with deuterium

oxide), 4.31 (q, 2H, J = 7.1 Hz), 3.70 (d, 2H, J = 6.0 Hz), 1.38 (s, 9H),

1.32 (t, 3H, J = 7.1 Hz). ESI-MS m/z: 362 (M + H)⁺.

Synthesis of tert-Butyl 5-Nitro-1H-indole-3-carboxylate (7′). 5-

Nitro-1H-indole-3-carboxylic acid (0.500 g, 2.42 mmol), TEA (1.01

mL, 7.26 mmol), tert-butanol (3.40 mL, 36.3 mmol), and DCC (1.50

g, 7.26 mmol) were dissolved in dry DMF (5 mL) under N₂

atmosphere. The vessel was sealed, and the reaction was stirred at

120 °C for 3 h and then concentrated in vacuo. The residue was

dissolved in EtOAc (100 mL) and washed with NaHCO₃saturated

Synthesis of Ethyl 5-(2-(3-(4-Nitrophenyl)ureido)acetamido)-1H-

indole-2-carboxylate (1k). A mixture of TFA and CH₂Cl₂(1:3, 12

mL) was added to compound 11 (300 mg, 0.83 mmol), and the

reaction was stirred at room temperature for 30 min. Vacuum

evaporation of the solvents gave a white solid, which was directly used

H

dx.doi.org/10.1021/jm301097p | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

aqueous solution (3 × 60 mL) and brine. The organic solution was

dried (Na₂SO₄), ﬁltered, and concentrated in vacuo. The residue was

puriﬁed by silica gel chromatography (CH₂Cl₂/EtOAc, 9:1) to give the

title compound as a light-brown solid (585 mg, 92%); mp 212−213

°C. ¹H NMR (300 MHz, DMSO-d₆): δ 9.11 (s, 1H, exchangeable with

deuterium oxide), 8.86 (s, 1H), 8.18 (dd, 1H, J = 8.9, 2.1 Hz), 8.01 (d,

1H, J = 2.1 Hz), 7.46 (d, 1H, J = 8.9 Hz), 1.67 (s, 9H). ESI-MS m/z:

261 (M + H)⁺.

Synthesis of Methyl 5-Amino-1H-indole-3-carboxylate (8). Pd/C

(5 wt % on activated carbon, 0.1 equiv) was added to a solution of 7

(500 mg, 2.27 mmol) in EtOH (100 mL), and the reaction was stirred

under H₂(1 atm, balloon) for 2 h. The reaction mixture was ﬁltered

and concentrated to give the title compound as a light-yellow solid

(427 mg, 99%); mp 144−146 °C. ¹H NMR (300 MHz, DMSO-d₆): δ

11.49 (s, 1H, exchangeable with deuterium oxide), 7.82 (s, 1H), 7.17

(d, 1H, J = 2.2 Hz), 7.13 (d, 1H, J = 9.0 Hz), 6.55 (dd, 1H, J = 9.0, 2.2

Hz), 4.74 (br s, 2H, exchangeable with deuterium oxide), 3.75 (s, 3H).

ESI-MS m/z: 191 (M + H)⁺.

s, 2H), the carboxylic acid proton could not be detected. ESI-MS m/z:

353 (M + H)⁺. Anal. (C₁₈H₁₆N₄O₄) C, H, N.

Synthesis of Ethyl 5-(2-(3-(p-Tolyl)ureido)acetamido)-1H-indole-

2-carboxylate (3c). Compound 3c was obtained as a white solid (274

mg, 84%) from compound 11 (300 mg, 0.83 mmol) and the proper

phenyl isocyanate, according to the procedure used for 1k (vide

supra); mp 267−268 °C. IR (KBr): ν = 3387, 3327, 2980, 1699, 1670,

1

1639, 1597, 1544, 1533 cm⁻¹. H NMR (300 MHz, DMSO-d₆): δ

11.80 (s, 1H, exchangeable with deuterium oxide), 9.92 (s, 1H,

exchangeable with deuterium oxide), 8.70 (s, 1H, exchangeable with

deuterium oxide), 7.99 (s, 1H), 7.40−7.33 (m, 2H), 7.28 (d, 2H, J =

8.1 Hz), 7.11−7.08 (m, 1H),7.02 (d, 2H, J = 8.1 Hz), 6.37 (br t, 1H, J

= 5.1 Hz, exchangeable with deuterium oxide), 4.36 (q, 2H, J = 7.0

Hz), 3.93 (d, 2H, J = 5.1 Hz), 2.21 (s, 3H), 1.35 (t, 3H, J = 7.0 Hz).

ESI-MS m/z: 395 (M + H) ⁺, 417 (M + Na)⁺. Anal. (C₂₁H₂₂N₄O₄) C,

H, N.

Synthesis of 5-(2-(3-(p-Tolyl)ureido)acetamido)-1H-indole-2-car-

boxylic Acid (3d). Compound 3d was obtained as a white solid (187

mg, 96%) from compound 3c (210 mg, 0.53 mmol), according to the

procedure used for 1l (vide supra); mp 242−244 °C (decomp). IR

(KBr): ν = 3453, 3319, 3265, 3072, 1707, 1670, 1654, 1599, 1560,

1541 cm⁻¹. ¹H NMR (300 MHz, DMSO-d₆): δ 11.80 (s, 1H

exchangeable with deuterium oxide), 9.92 (s, 1H exchangeable with

deuterium oxide), 8.70 (s, 1H exchangeable with deuterium oxide),

7.99 (s, 1H), 7.40−7.33 (m, 2H), 7.28 (d, 2H, J = 8.1 Hz), 7.04−7.01

(m, 3H), 6.38−6.36 (m, 1H, exchangeable with deuterium oxide), 3.93

(d, 2H, J = 4.8 Hz), 2.21 (s, 3H), the carboxylic acid proton could not

be detected. ESI-MS m/z: 367 (M + H) ⁺, 389 (M + Na)⁺. Anal.

(C₁₉H₁₈N₄O₄) C, H, N.

Synthesis of tert-Butyl 5-Amino-1H-indole-3-carboxylate (8′).

Compound 8′ was obtained as a light-yellow solid (420 mg, 95%)

from compound 7′ (500 mg; 1.90 mmol) according to the procedure

1

used for 8; mp 61−63 °C. H NMR (300 MHz, DMSO-d₆): δ 11.62

(s, 1H, exchangeable with deuterium oxide), 8.32 (s, 1H), 7.90 (d, 1H,

J = 2.1 Hz), 7.62 (br s, 2H, exchangeable with deuterium oxide), 7.36

(d, 1H, J = 9.0 Hz), 6.93−6.87 (m, 1H), 1.56 (s, 9H). ESI-MS m/z:

233 (M + H)⁺.

Synthesis of Ethyl 5-(2-((tert-Butoxycarbonyl)amino)acetamido)-

1H-indole-3-carboxylate (12). Compound 12 was obtained as a white

solid (462 mg, 70%) from compound 8 (631 mg, 1.90 mmol),

according to the procedure used for 11 (vide supra); mp 218−220 °C.

¹H NMR (300 MHz, DMSO-d₆): δ 11.85 (s, 1H, exchangeable with

deuterium oxide), 9.85 (s, 1H, exchangeable with deuterium oxide),

8.22 (s, 1H), 8.03 (d, 1H, J = 2.3 Hz), 7.46 (dd, 1H, J = 8.8, 2.3 Hz),

7.39 (d, 1H, J = 8.8 Hz), 7.01 (br t, 1H, J = 6.0 Hz, exchangeable with

deuterium oxide), 3.79 (s, 3H), 3.73 (d, 2H, J = 6.0 Hz), 1.41 (s, 9H).

ESI-MS m/z: 348 (M + H)⁺.

Synthesis of Ethyl 5-(2-(3-(4-Methoxyphenyl)ureido)acetamido)-

1H-indole-2-carboxylate (3e). Compound 3e was obtained as a white

solid (262 mg, 77%) from compound 11 (300 mg, 0.83 mmol) and

the proper phenyl isocyanate, according to the procedure used for 1k

(vide supra); mp 253−254 °C. IR (KBr): ν = 3377, 3325, 2995, 1692,

1665, 1638, 1597, 1545, 1533 cm⁻¹. ¹H NMR (300 MHz, DMSO-d₆):

δ 11.80 (s, 1H, exchangeable with deuterium oxide), 9.91(s, 1H,

exchangeable with deuterium oxide), 8.61 (s, 1H, exchangeable with

deuterium oxide), 7.99 (s, 1H), 7.40−7.37 (m, 2H), 7.30 (d, 2H, J =

8.8 Hz), 7.12−7.09 (m, 1H), 6.81 (d, 2H, J = 8.8 Hz), 6.31 (br t, 1H, J

= 5.0 Hz, exchangeable with deuterium oxide), 4.36 (q, 2H, J = 7.0

Hz), 3.93 (d, 2H, J = 5.0 Hz), 3.69 (s, 3H), 1.35 (t, 3H, J = 7.0 Hz).

Synthesis of tert-Butyl 5-(2-((((9H-Fluoren-9-yl)methoxy)-

carbonyl)amino)acetamido)-1H-indole-3-carboxylate (13). Com-

pound 13 was obtained as a white solid (680 mg, 70%) from

compound 8′ (442 mg, 1.90 mmol), according to the procedure used

for 11 (vide supra) except that Fmoc-Gly-OH was used in place of

Boc-Gly-OH; mp 95−96 °C. ¹H NMR (300 MHz, DMSO-d₆): δ

11.75 (s, 1H, exchangeable with deuterium oxide), 9.85 (s, 1H,

exchangeable with deuterium oxide), 8.32 (s, 1H), 7.91−7.88 (m, 3H),

7.77−7.73 (m, 2H), 7.66−7.62 (m, 1H), 7.45−7.31 (m, 6H, one

exchangeable with deuterium oxide), 4.32−4.26 (m, 3H), 3.81 (d, 2H,

J = 6.0 Hz), 1.59 (s, 9H). ESI-MS m/z: 534 (M + Na)⁺, 550 (M + K)⁺.

Synthesis of Ethyl 5-(2-(3-Phenylureido)acetamido)-1H-indole-2-

carboxylate (3a). Compound 3a was obtained as a white solid (236

mg, 75%) from compound 11 (300 mg, 0.83 mmol) and the proper

phenyl isocyanate, according to the procedure used for 1k (vide

supra); mp 263−264 °C (decomp). IR (KBr): ν = 3380, 3318, 2981,

+

ESI-MS m/z: 411(M + H) . Anal. (C₂₁H₂₂N₄O₅) C, H, N.

Synthesis of 5-(2-(3-(4-Methoxyphenyl)ureido)acetamido)-1H-

indole-2-carboxylic Acid (3f). Compound 3f was obtained as a

white solid (223 mg, 96%) from compound 3e (250 mg, 0.61 mmol),

according to the procedure used for 1l (vide supra); mp 260−262 °C

(decomp). IR (KBr): ν = 3399, 3321, 3267, 3096, 1670, 1654, 1603,

1

1541, 1508 cm⁻¹. H NMR (300 MHz, DMSO-d₆): δ 11.67 (s, 1H,

exchangeable with deuterium oxide), 9.89 (s, 1H, exchangeable with

deuterium oxide), 8.61 (s, 1H, exchangeable with deuterium oxide),

7.98 (s, 1H), 7.38−7.35 (m, 2H), 7.30 (d, 2H, J = 8.8 Hz), 7.03 (s,

1H), 6.81 (d, 2H, J = 8.8 Hz), 6.34−6.31 (m, 1H exchangeable with

deuterium oxide), 3.93 (d, 2H, J = 4.7 Hz), 3.69 (s, 3H), the carboxylic

acid proton could not be detected. ESI-MS m/z: 383 (M + H)⁺. Anal.

(C₁₉H₁₈N₄O₅) C, H, N.

1

1694, 1663, 16356, 1598, 1556, 1532 cm⁻¹. H NMR (300 MHz,

DMSO-d₆): δ 11.81 (s, 1H, exchangeable with deuterium oxide), 9.94

(s, 1H, exchangeable with deuterium oxide), 8.82 (s, 1H, exchangeable

with deuterium oxide), 7.99 (s, 1H), 7.41−7.38 (m, 4H), 7.25−

7.20(m, 2H), 7.10 (s, 1H), 6.92−6.87 (m, 1H), 6.43 (br t, 1H, J = 5.1

Hz, exchangeable with deuterium oxide), 4.36 (q, 2H, J = 7.0 Hz), 3.94

(d, 2H, J = 5.1 Hz), 1.35 (t, 3H, J = 7.0 Hz). ESI-MS m/z: 381 (M +

Synthesis of Ethyl 5-(2-(3-(4-Cyanophenyl)ureido)acetamido)-

1H-indole-2-carboxylate (3g). Compound 3g was obtained as a

white solid (242 mg, 72%) from compound 11 (300 mg, 0.83 mmol)

and the proper phenyl isocyanate, according to the procedure used for

1k (vide supra); mp 279−280 °C (decomp). IR (KBr): ν = 3375,

3280, 2985, 2222, 1700, 1669, 1595, 1551, 1533 cm⁻¹. ¹H NMR (300

MHz, DMSO-d₆): δ 11.80 (s, 1H, exchangeable with deuterium

oxide), 9.95 (s, 1H, exchangeable with deuterium oxide), 9.38 (s, 1H,

exchangeable with deuterium oxide), 7.99 (s, 1H), 7.67 (d, 2H, J = 8.6

Hz), 6.58 (d, 2H, J = 8.6 Hz), 7.38−7.37 (m, 2H), 7.11−7.08 (m, 1H),

6.64 (br t, 1H, J = 5.0 Hz, exchangeable with deuterium oxide), 4.36

(q, 2H, J = 7.0 Hz), 3.97 (d, 2H, J = 5.0 Hz), 1.35 (t, 3H, J = 7.0 Hz).

+

H) , 403 (M + Na) . Anal. (C₂₀H₂₀N₄O₄) C, H, N.

Synthesis of 5-(2-(3-Phenylureido)acetamido)-1H-indole-2-car-

boxylic Acid (3b). Compound 3b was obtained as a white solid

(189 mg, 93%) from compound 3a (220 mg, 0.58 mmol), according to

the procedure used for 1l (vide supra); mp 252−254 °C (decomp). IR

(KBr): ν = 3379, 3298, 3057, 1694, 1670, 1627, 1595, 1541 cm⁻¹. ¹H

NMR (300 MHz, DMSO-d₆): δ 11.68 (s, 1H, exchangeable with

deuterium oxide), 9.93(s, 1H, exchangeable with deuterium oxide),

8.84 (s, 1H, exchangeable with deuterium oxide), 7.98 (s, 1H), 7.41−

7.35 (m, 4H), 7.25−7.20 (m, 2H), 7.05−7.03 (m, 1H), 6.92−6.87 (m,

1H), 6.48−6.45 (m, 1H, exchangeable with deuterium oxide), 3.94 (br

+

ESI-MS m/z: 406 (M + H) . Anal. (C₂₁H₁₉N₅O₄) C, H, N.

Synthesis of 5-(2-(3-(4-Cyanophenyl)ureido)acetamido)-1H-in-

dole-2-carboxylic Acid (3h). Compound 3h was obtained as a white

I

dx.doi.org/10.1021/jm301097p | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

solid (173 mg, 93%) from compound 3g (200 mg, 0.49 mmol),

according to the procedure used for 1l (vide supra); mp 269−270 °C

(decomp). IR (KBr): ν = 3352, 3291, 3107, 2960, 2216, 1690, 1643,

deuterium oxide), 3.95 (d, 2H, J = 4.9 Hz), 1.57 (s, 9H). ESI-MS m/z:

409 (M + H)⁺.

Synthesis of 5-(2-(3-Phenylureido)acetamido)-1H-indole-3-car-

boxylic Acid (4b). Compound 4b was obtained as a white solid

(112 mg, 99%) from compound 4a′ (130 mg, 0.32 mmol), according

to the procedure used for 2f (vide supra); mp 249−250 °C (decomp).

IR (KBr): ν = 3387, 3311, 3213, 2934, 1676, 1662, 1595, 1551 cm⁻¹.

¹H NMR (300 MHz, DMSO-d₆): δ 11.74 (s, 1H, exchangeable with

deuterium oxide), 9.96 (s, 1H, exchangeable with deuterium oxide),

8.81 (s, 1H, exchangeable with deuterium oxide), 8.24 (s, 1H), 7.95 (d,

1H, J = 2.4 Hz), 7.48−7.37 (m, 4H), 7.25−7.20 (m, 2H), 6.92−6.97

(m, 1H), 6.42 (br t, 1H, J = 5.1 Hz, exchangeable with deuterium

oxide), 3.94 (d, 2H, J = 5.1 Hz), the carboxylic acid proton could not

be detected. ESI-MS m/z: 353 (M + H)⁺, 357 (M + Na)⁺. Anal.

(C₁₈H₁₆N₄O₄) C, H, N.

Synthesis of Methyl 5-(2-(3-(p-Tolyl)ureido)acetamido)-1H-in-

dole-3-carboxylate (4c). Compound 4c was obtained as a white

solid (271 mg, 83%) from compound 12 (300 mg, 0.86 mmol) and

the proper phenyl isocyanate, according to the procedure used for 1k

(vide supra); mp 271−273 °C (decomp). (KBr): ν = 3395, 3250,

3092, 1672, 1647, 1603, 1541, 1526 cm⁻¹. ¹H NMR (300 MHz,

DMSO-d₆): δ 11.86 (s, 1H, exchangeable with deuterium oxide), 9.98

(s, 1H, exchangeable with deuterium oxide), 8.70 (s, 1H, exchangeable

with deuterium oxide), 8.25 (s, 1H), 8.03 (d, 1H, J = 1.9 Hz), 7.45−

7.38 (m, 2H), 7.29 (d, 2H, J = 8.1 Hz), 7.03 (d, 2H, J = 8.1 Hz), 6.38

(br t, 1H, J = 5.1 Hz, exchangeable with deuterium oxide), 3.94 (d, 2H,

J = 5.1 Hz), 3.79 (s, 3H), 2.21 (s, 3H). ESI-MS m/z: 381 (M + H)⁺,

403 (M + Na)⁺. Anal. (C₂₀H₂₀N₄O₄) C, H, N.

1

1591, 1533 cm⁻¹. H NMR (300 MHz, DMSO-d₆): δ 11.68 (s, 1H,

exchangeable with deuterium oxide), 9.94(s, 1H, exchangeable with

deuterium oxide), 9.39 (s, 1H, exchangeable with deuterium oxide),

7.97 (s, 1H), 7.67 (d, 2H, J = 8.6 Hz), 6.58 (d, 2H, J = 8.6 Hz), 7.38−

7.31 (m, 2H), 7.04 (s, 1H), 6.64 (br t, 1H, J = 4.9 Hz, exchangeable

with deuterium oxide), 3.97 (d, 2H, J = 4.9 Hz), the carboxylic acid

proton could not be detected. ESI-MS m/z: 378 (M + H)⁺. Anal.

(C₁₉H₁₅N₅O₄) C, H, N.

Synthesis of Ethyl 5-(2-(3-(4-(Dimethylamino)phenyl)ureido)-

acetamido)-1H-indole-2-carboxylate (3i). Compound 3g was

obtained as a white solid (250 mg, 71%) from compound 11 (300

mg, 0.83 mmol) and the proper phenyl isocyanate, according to the

procedure used for 1k (vide supra); mp 252−253 °C (decomp). IR

(KBr): ν = 3323, 3264, 2984, 1709, 1659, 1597, 1566, 1535 cm⁻¹. ¹H

NMR (300 MHz, DMSO-d₆): δ 11.70 (s, 1H, exchangeable with

deuterium oxide), 9.90 (s, 1H, exchangeable with deuterium oxide),

8.45 (s, 1H, exchangeable with deuterium oxide), 7.99 (s, 1H), 7.43−

7.33 (m, 2H), 7.22 (d, 2H, J = 9.0 Hz), 7.09 (s, 1H), 6.66 (d, 2H, J =

9.0 Hz), 6.29 (br t, 1H, J = 5.4 Hz, exchangeable with deuterium

oxide), 4.32 (q, 2H, J = 7.0 Hz), 3.90 (d, 2H, J = 5.4 Hz), 2.80 (s, 6H),

1.33 (t, 3H, J = 7.0 Hz). ESI-MS m/z: 424 (M + H) ⁺. Anal.

(C₂₂H₂₅N₅O₄) C, H, N.

Synthesis of 5-(2-(3-(4-(Dimethylamino)phenyl)ureido)-

acetamido)-1H-indole-2-carboxylic Acid (3j). Compound 3j was

obtained as a white solid (181 mg, 81%) from compound 3i (240 mg,

0.57 mmol), according to the procedure used for 1l (vide supra); mp

241−243 °C (decomp). IR (KBr): ν = 3505, 3370, 3294, 2932, 1701,

Synthesis of tert-Butyl 5-(2-(3-(p-Tolyl)ureido)acetamido)-1H-

indole-3-carboxylate (4c′). Compound 4c′ was obtained as a white

solid (392 mg, 80%) from compound 13 (vide supra, 600 mg, 1.17

mmol) and the proper phenyl isocyanate, according to the procedure

used for 4a′; mp 243−245 °C. (KBr): ν = 3366, 3323, 3267, 2978,

1

1670, 1654, 1611, 1544 cm⁻¹. H NMR (300 MHz, DMSO-d₆): δ

11.69 (s, 1H, exchangeable with deuterium oxide), 9.95 (s, 1H,

exchangeable with deuterium oxide), 9.15 (s, 1H, exchangeable with

deuterium oxide), 7.99 (s, 1H), 7.55−7.45 (m, 4H), 7.39−7.32 (m,

2H), 7.04−7.02 (m, 1H), 6.54 (br t, 1H, J = 4.8 Hz, exchangeable with

deuterium oxide), 3.94 (d, 2H, J = 4.8 Hz), 3.07 (s, 6H), the carboxylic

acid proton could not be detected. ESI-MS m/z: 396 (M + H)⁺. Anal.

(C₂₀H₂₁N₅O₄) C, H, N.

Synthesis of Methyl 5-(2-(3-Phenylureido)acetamido)-1H-indole-

3-carboxylate (4a). Compound 4a was obtained as a white solid (198

mg, 63%) from compound 12 (300 mg, 0.86 mmol) and the proper

phenyl isocyanate, according to the procedure used for 1k (vide

supra); mp 260−262 °C (decomp). (KBr): ν = 3258, 3086, 2945,

1681, 1641, 1595, 1547, 1525 cm⁻¹. ¹H NMR (300 MHz, DMSO-d₆):

δ 11.87 (s, 1H, exchangeable with deuterium oxide), 9.99 (s, 1H,

exchangeable with deuterium oxide), 8.82 (s, 1H, exchangeable with

deuterium oxide), 8.25 (s, 1H), 8.06−8.03 (m, 1H), 7.47−7.39 (m,

4H), 7.25−7.20 (m, 2H), 6.90−6.85 (m, 1H), 6.42 (br t, 1H, J = 4.9

Hz, exchangeable with deuterium oxide), 3.95 (d, 2H, J = 4.9 Hz), 3.79

(s, 3H). ESI-MS m/z: 367 (M + H)⁺, 389 (M + Na)⁺. Anal.

(C₁₉H₁₈N₄O₄) C, H, N.

1

1670, 1643, 1597, 1545 cm⁻¹. H NMR (300 MHz, DMSO-d₆): δ

11.75 (s, 1H, exchangeable with deuterium oxide), 9.93 (s, 1H,

exchangeable with deuterium oxide), 8.72 (s, 1H, exchangeable with

deuterium oxide), 8.32 (s, 1H), 7.92−7.90 (m, 1H), 7.38−7.35 (m,

2H), 7.29 (d, 2H, J = 8.0 Hz), 7.03 (d, 2H, J = 8.0 Hz), 6.38 (br t, 1H,

J = 4.9 Hz, exchangeable with deuterium oxide), 3.94 (d, 2H, J = 4.9

Hz), 2.21 (s, 3H), 1.57 (s, 9H). ESI-MS m/z: 423 (M + H)⁺, 445 (M

+ Na)⁺.

Synthesis of 5-(2-(3-(p-Tolyl)ureido)acetamido)-1H-indole-3-car-

boxylic Acid (4d). Compound 4d was obtained as a white solid (120

mg, 99%) from compound 4c′ (140 mg, 0.33 mmol), according to the

procedure used for 2f (vide supra); mp 219−221 °C (decomp). IR

(KBr): ν = 3372, 3282, 3117, 2920, 1662, 1647, 1599, 1551 cm⁻¹. ¹H

NMR (300 MHz, DMSO-d₆): 11.86 (s, 1H, exchangeable with

deuterium oxide), 9.95 (s, 1H, exchangeable with deuterium oxide),

8.70 (s, 1H, exchangeable with deuterium oxide), 8.24 (s, 1H), 7.96 (d,

1H, J = 2.6 Hz), 7.46 (d, 1H, J = 9.6 Hz), 7.38−7.35 (m, 1H), 7.28 (d,

2H, J = 8.2 Hz), 7.03 (d, 2H, J = 8.2 Hz), 6.38 (br t, 1H, J = 5.2 Hz,

exchangeable with deuterium oxide), 3.94 (d, 2H, J = 5.2 Hz), 2.21 (s,

3H), the carboxylic acid proton could not be detected. ESI-MS m/z:

367 (M + H)⁺, 389 (M + Na)⁺. Anal. (C₁₉H₁₈N₄O₄) C, H, N.

Synthesis of Methyl 5-(2-(3-(4-Methoxyphenyl)ureido)-

acetamido)-1H-indole-3-carboxylate (4e). Compound 4e was

obtained as a white solid (276 mg, 81%) from compound 12 (300

mg, 0.86 mmol) and the proper phenyl isocyanate, according to the

procedure used for 1k (vide supra); mp 250−252 °C (decomp).

(KBr): ν = 3389, 3285, 3080, 1680, 1639, 1599, 1554 cm⁻¹. ¹H NMR

(300 MHz, DMSO-d₆): δ 11.85 (s, 1H, exchangeable with deuterium

oxide), 9.96 (s, 1H, exchangeable with deuterium oxide), 8.61 (s, 1H,

exchangeable with deuterium oxide), 8.25 (s, 1H), 8.03 (d, 1H, J = 2.6

Hz), 7.45−7.40 (m, 2H), 7.30 (d, 2H, J = 8.6 Hz), 6.82 (d, 2H, J = 8.6

Hz), 6.32 (br t, 1H, J = 5.0 Hz, exchangeable with deuterium oxide),

3.93 (d, 2H, J = 5.0 Hz), 3.70 (s, 3H), 3.70 (s, 3H). ESI-MS m/z: 397

(M + H)⁺, 419 (M + Na)⁺. Anal. (C₂₀H₂₀N₄O₅) C, H, N.

Synthesis of tert-Butyl 5-(2-(3-Phenylureido)acetamido)-1H-in-

dole-3-carboxylate (4a′). Piperidine (2 mL) was added to a

suspension of 13 (vide supra, 600 mg, 1.17 mmol) in CH₂Cl₂(8

mL), and the resulting mixture was stirred at room temperature for 30

min. Vacuum evaporation of the solvent gave a solid which was washed

with hexane. The white solid obtained was directly used in the next

step. A solution of phenyl isocyanate (166 mg, 1.40 mmol) in dry THF

(8 mL) was added dropwise to a solution of deprotected compound

13 (336 mg, 1.16 mmol) and triethylamine (0.48 mL, 3.48 mmol) in

dry THF (20 mL) under N₂atmosphere. The reaction was stirred at

room temperature for 3 h. The precipitate was collected by ﬁltration

and rinsed with water and diethyl ether, successively. Puriﬁcation by

crystallization (DMF/H₂O) gave the title compound as a white solid

(307 mg, 65%); mp 238−239 °C. (KBr): ν = 3375, 3287, 2976, 1665,

1

1641, 1597, 1545 cm⁻¹. H NMR (300 MHz, DMSO-d₆): δ 11.76 (s,

1H, exchangeable with deuterium oxide), 9.94 (s, 1H, exchangeable

with deuterium oxide), 8.82 (s, 1H, exchangeable with deuterium

oxide), 8.32 (s, 1H), 7.91 (d, 1H, J = 2.5 Hz), 7.42−7.37 (m, 5H),

7.25−7.20 (m, 2H), 6.89 (br t, 1H, J = 4.9 Hz, exchangeable with

Synthesis of tert-Butyl 5-(2-(3-(4-Methoxyphenyl)ureido)-

acetamido)-1H-indole-3-carboxylate (4e′). Compound 4e′ was

obtained as a white solid (343 mg, 67%) from compound 13 (vide

J

dx.doi.org/10.1021/jm301097p | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

supra, 600 mg, 1.17 mmol) and the proper phenyl isocyanate,

according to the procedure used for 4a′; mp 238−240 °C. (KBr): ν =

3373, 3325, 3273, 2976, 1670, 1641, 1600, 1551 cm⁻¹. ¹H NMR (300

MHz, DMSO-d₆): δ 11.75 (s, 1H, exchangeable with deuterium

oxide), 9.92 (s, 1H, exchangeable with deuterium oxide), 8.62 (s, 1H,

exchangeable with deuterium oxide), 8.32 (s, 1H), 7.92−7.90 (m, 1H),

7.37−7.34 (m, 2H), 7.29 (d, 2H, J = 8.9 Hz), 6.81 (d, 2H, J = 8.9 Hz),

6.33 (br t, 1H, J = 5.4 Hz, exchangeable with deuterium oxide), 3.94

(d, 2H, J = 5.4 Hz), 3.69 (s, 3H), 1.57 (s, 9H). ESI-MS m/z: 439 (M +

H)⁺, 461 (M + Na)⁺.

Synthesis of 5-(2-(3-(4-Methoxyphenyl)ureido)acetamido)-1H-

indole-3-carboxylic Acid (4f). Compound 4f was obtained as a

white solid (112 mg, 99%) from compound 4e′ (130 mg, 0.30 mmol),

according to the procedure used for 2f (vide supra); mp 271−273 °C

(decomp). IR (KBr): ν = 3366, 3282, 3012, 2933, 1670, 1654, 1609,

1541 cm⁻¹. ¹H NMR (300 MHz, DMSO-d₆): δ 11.74 (s, 1H,

exchangeable with deuterium oxide), 9.95 (s, 1H, exchangeable with

deuterium oxide), 8.61 (s, 1H, exchangeable with deuterium oxide),

8.22 (s, 1H), 7.94 (d, 1H, J = 2.6 Hz), 7.45−7.34 (m, 2H), 7.28 (d,

2H, J = 8.9 Hz), 6.80 (d, 2H, J = 8.9 Hz), 6.33 (br t, 1H, J = 5.2 Hz,

exchangeable with deuterium oxide), 3.93 (d, 2H, J = 5.2 Hz), 3.69 (s,

3H), the carboxylic acid proton could not be detected. ESI-MS m/z:

381 (M − H)⁻. Anal. (C₁₉H₁₈N₄O₅) C, H, N.

= 2.7 Hz), 7.67 (d, 2H, J = 9.1 Hz), 7.60−7.38 (m, 2H), 7.01 (br t, 1H,

J = 4.8 Hz, exchangeable with deuterium oxide), 3.73 (d, 2H, J = 4.8

Hz), 3.81 (s, 3H). ESI-MS m/z: 412 (M + H)⁺. Anal. (C₁₉H₁₇N₅O₆)

C, H, N.

Synthesis of tert-Butyl 5-(2-(3-(4-Nitrophenyl)ureido)acetamido)-

1H-indole-3-carboxylate (4i′). Compound 4i′ was obtained as a

yellow solid (377 mg, 71%) from compound 13 (vide supra, 600 mg,

1.17 mmol) and the proper phenyl isocyanate, according to the

procedure used for 4a′; mp 237−239 °C. (KBr): ν = 3368, 3271,

2980, 1676, 1647, 1597, 1551 cm⁻¹. ¹H NMR (300 MHz, DMSO-d₆):

δ 11.76 (s, 1H, exchangeable with deuterium oxide), 9.98 (s, 1H,

exchangeable with deuterium oxide), 9.65 (s, 1H, exchangeable with

deuterium oxide), 8.32 (s, 1H), 8.15 (d, 2H, J = 9.0 Hz), 7.91 (d, 1H, J

= 2.5 Hz), 7.65 (d, 2H, J = 9.0 Hz), 7.40−7.37 (m, 2H), 6.72 (br t, 1H,

J = 5.0 Hz, exchangeable with deuterium oxide), 4.00 (d, 2H, J = 5.0

Hz), 1.57 (s, 9H). ESI-MS m/z: 476 (M + Na)⁺.

Synthesis of 5-(2-(3-(4-Nitrophenyl)ureido)acetamido)-1H-in-

dole-3-carboxylic Acid (4j). Compound 4j was obtained as a yellow

solid (104 mg, 99%) from compound 4i′ (120 mg, 0.26 mmol),

according to the procedure used for 2f (vide supra); mp 283−285 °C

(decomp). IR (KBr): ν = 3345, 3309, 3138, 2922, 1662, 1599, 1541

1

cm⁻¹. H NMR (300 MHz, DMSO-d₆): δ 11.89 (s, 1H, exchangeable

with deuterium oxide), 11.75 (s, 1H, exchangeable with deuterium

oxide), 10.00 (s, 1H, exchangeable with deuterium oxide), 9.63 (s,

1H), 8.16 (d, 2H, J = 9.0 Hz), 7.95 (d, 1H, J = 2.1 Hz), 7.65 (d, 2H, J

= 9.0 Hz), 7.47−7.39 (m, 2H), 6.72 (br t, 1H, J = 4.8 Hz, exchangeable

with deuterium oxide), 3.98 (d, 2H, J = 4.8 Hz), the carboxylic acid

proton could not be detected. ESI-MS m/z: 398 (M + H)⁺. Anal.

(C₁₈H₁₅N₅O₆) C, H, N.

Synthesis of Methyl 5-(2-(3-(4-Cyanophenyl)ureido)acetamido)-

1H-indole-3-carboxylate (4g). Compound 4g was obtained as a white

solid (265 mg, 79%) from compound 12 (300 mg, 0.86 mmol) and

the proper phenyl isocyanate, according to the procedure used for 1k

(vide supra); mp 268−270 °C . (KBr): ν = 3370, 3273, 3113, 2232,

1

1672, 1645, 1591, 1551 cm⁻¹. H NMR (300 MHz, DMSO-d₆): δ

11.86 (s, 1H, exchangeable with deuterium oxide), 10.01 (s, 1H,

exchangeable with deuterium oxide), 9.39 (s, 1H, exchangeable with

deuterium oxide), 8.25 (s, 1H), 8.03 (d, 1H, J = 2.5 Hz), 7.68 (d, 2H, J

= 8.8 Hz), 7.59 (d, 2H, J = 8.8 Hz), 7.45−7.37 (m, 2H), 6.65 (br t, 1H,

J = 5.3 Hz, exchangeable with deuterium oxide), 3.97 (d, 2H, J = 5.3

Hz), 3.79 (s, 3H). ESI-MS m/z: 392 (M + H)⁺, 411 (M + Na)⁺. Anal.

(C₂₀H₁₇N₅O₄) C, H, N.

Synthesis of Methyl 5-(2-(3-(4-(Dimethylamino)phenyl)ureido)-

acetamido)-1H-indole-3-carboxylate (4k). Compound 4k was

obtained as a white solid (228 mg, 65%) from compound 12 (300

mg, 0.86 mmol) and the proper phenyl isocyanate, according to the

procedure used for 1k (vide supra); mp 244−246 °C (decomp).

1

(KBr): ν = 3289, 3118, 2945, 1668, 1630, 1525 cm⁻¹. H NMR (300

MHz, DMSO-d₆): δ 11.85 (s, 1H, exchangeable with deuterium

oxide), 9.94 (s, 1H, exchangeable with deuterium oxide), 8.41 (s, 1H,

exchangeable with deuterium oxide), 8.25 (s, 1H), 8.03 (d, 1H, J = 2.1

Hz), 7.46 (dd, 1H, J = 8.8, 2.1 Hz), 7.40 (d, 1H, J = 8.8 Hz), 7.21 (d,

2H, J = 9.0 Hz), 6.66 (d, 2H, J = 9.0 Hz), 6.25 (br t, 1H, J = 5.4 Hz,

exchangeable with deuterium oxide), 3.92 (d, 2H, J = 5.4 Hz), 3.79 (s,

3H), 2.80 (s, 6H). ESI-MS m/z: 432 (M + Na)⁺. Anal. (C₂₁H₂₃N₅O₄)

C, H, N.

Synthesis of tert-Butyl 5-(2-(3-(4-(Dimethylamino)phenyl)-

ureido)acetamido)-1H-indole-3-carboxylate (4k′). Compound 4k′

was obtained as a white solid (375 mg, 71%) from compound 13 (vide

supra, 600 mg, 1.17 mmol) and the proper phenyl isocyanate,

according to the procedure used for 4a′; mp 225−227 °C. (KBr): ν =

3379, 3325, 2978, 1670, 1641, 1597, 1545 cm⁻¹. ¹H NMR (300 MHz,

DMSO-d₆): δ 11.78 (s, 1H, exchangeable with deuterium oxide), 9.92

(s, 1H, exchangeable with deuterium oxide), 8.50 (s, 1H, exchangeable

with deuterium oxide), 8.32 (s, 1H), 7.90 (d, 1H, J = 2.6 Hz), 7.38−

7.35 (m, 2H), 7.21 (d, 2H, J = 8.7 Hz), 6.67 (d, 2H, J = 8.7 Hz), 6.30

(br t, 1H, J = 5.2 Hz, exchangeable with deuterium oxide), 3.92 (d, 2H,

J = 5.2 Hz), 2.80 (s, 6H), 1.57 (s, 9H). ESI-MS m/z: 452 (M + H)⁺,

474 (M + Na)⁺.

Synthesis of 5-(2-(3-(4-(Dimethylamino)phenyl)ureido)-

acetamido)-1H-indole-3-carboxylic Acid Triﬂuoroacetate (4l). Com-

pound 4l was obtained as a yellow solid (113 mg, 99%) from

compound 4k′ (130 mg, 0.29 mmol), according to the procedure used

for 2f (vide supra); mp 201−204 °C (decomp). IR (KBr): ν = 3356,

3310, 3246, 2960, 1668, 1654, 1597, 1555 cm⁻¹. ¹H NMR (300 MHz,

DMSO-d₆): δ 11.88 (s, 1H, exchangeable with deuterium oxide), 10.02

(s, 1H, exchangeable with deuterium oxide), 9.33 (br s, 1H,

exchangeable with deuterium oxide), 8.26 (s, 1H), 7.96−7.93 (m,

1H), 7.47−7.36 (m, 6H), 6.67 (br t, 1H, J = 4.9 Hz, exchangeable with

deuterium oxide), 3.93 (d, 2H, J = 4.9 Hz), 2.37 (s, 6H), the carboxylic

acid proton and the dimethylammonium salt proton could not be

detected. ESI-MS m/z: 396 (M + H)⁺. Anal. (C₂₂H₂₂F₃N₅O₆) C, H,

N.

Synthesis of tert-Butyl 5-(2-(3-(4-Cyanophenyl)ureido)-

acetamido)-1H-indole-3-carboxylate (4g′). Compound 4g′ was

obtained as a white solid (380 mg, 75%) from compound 13 (vide

supra, 600 mg, 1.17 mmol) and the proper phenyl isocyanate,

according to the procedure used for 4a′; mp 228−230 °C. (KBr): ν =

3348, 3273, 2980, 2224, 1670, 1643, 1589, 1541 cm⁻¹. ¹H NMR (300

MHz, DMSO-d₆): δ 11.76 (s, 1H, exchangeable with deuterium

oxide), 9.96 (s, 1H, exchangeable with deuterium oxide), 9.44 (s, 1H,

exchangeable with deuterium oxide), 8.31 (s, 1H), 7.92−7.90 (m, 1H),

7.97 (d, 2H, J = 8.6 Hz), 7.59 (d, 2H, J = 8.6 Hz), 7.39−7.35 (m, 2H),

6.88 (br t, 1H, J = 4.7 Hz, exchangeable with deuterium oxide), 3.97

(d, 2H, J = 4.7 Hz), 1.56 (s, 9H). ESI-MS m/z: 456 (M + Na)⁺.

Synthesis of 5-(2-(3-(4-Cyanophenyl)ureido)acetamido)-1H-in-

dole-3-carboxylic Acid (4h). Compound 4h was obtained as a white

solid (121 mg, 99%) from compound 4g′ (140 mg, 0.32 mmol),

according to the procedure used for 2f (vide supra); mp 238−239 °C

(decomp). IR (KBr): ν = 3344, 3309, 3128, 2926, 2224, 1670, 1664,

1

1595, 1545 cm⁻¹. H NMR (300 MHz, DMSO-d₆): δ 11.75 (s, 1H,

exchangeable with deuterium oxide), 9.99 (s, 1H, exchangeable with

deuterium oxide), 9.40 (s, 1H, exchangeable with deuterium oxide),

8.23 (s, 1H), 7.95 (d, 1H, J = 2.3 Hz), 7.68 (d, 2H, J = 8.5 Hz), 7.58

(d, 2H, J = 8.5 Hz), 7.46−7.38 (m, 2H), 6.65 (br t, 1H, J = 4.9 Hz,

exchangeable with deuterium oxide), 3.95 (d, 2H, J = 4.9 Hz), the

carboxylic acid proton could not be detected. ESI-MS m/z: 376 (M −

H)⁻. Anal. (C₁₉H₁₅N₅O₄) C, H, N.

Synthesis of Methyl 5-(2-(3-(4-Nitrophenyl)ureido)acetamido)-

1H-indole-3-carboxylate (4i). Compound 4i was obtained as a white

solid (244 mg, 69%) from compound 12 (300 mg, 0.86 mmol) and

the proper phenyl isocyanate, according to the procedure used for 1k

(vide supra); mp 288−290 °C (decomp). (KBr): ν = 3364, 3273,

1

3082, 1670, 1645, 1560 cm⁻¹. H NMR (300 MHz, DMSO-d₆): δ

11.85 (s, 1H, exchangeable with deuterium oxide), 10.05 (s, 1H,

exchangeable with deuterium oxide), 9.06 (s, 1H, exchangeable with

deuterium oxide), 8.27 (s, 1H), 8.18 (d, 2H, J = 9.1 Hz), 8.06 (d, 1H, J

K

dx.doi.org/10.1021/jm301097p | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Biochemistry. Plasmids, Antibodies, and Chemicals. All GST

fusion proteins were subcloned into pGEX6P-1 (Amersham

Biosciences), which allows for the induced production of recombinant

proteins fused to the C terminus of glutathione S-transferase. GST-

PABP1,³¹has been previously described. GST-Npl3p was a gift from

Pam Silver (Harvard Medical School, MA, USA) and has been

described previously.³⁷GST-SET7/9 was a gift from Yi Zhang

(Lineberger Comprehensive Cancer Center, Chapel Hill, USA).

GST−G9a was a gift from Yoichi Shinkai (Institute for Virus

Research, Kyoto, Japan).¹²¹Histones H3 and H4 from calf thymus

were purchased from Roche Applied Science. MBP was purchased

from Sigma−Aldrich. The methyl-speciﬁc anti-PABP1 antibody against

the peptide sequence CGAIR*PAAPR*PPFS from PABP1 was raised

in New Zealand white rabbits. The anti-Flag antibody, M2, was

purchased from Sigma−Aldrich. Unless stated otherwise, all

chemicals/reagents were purchased from Sigma-Aldrich.

1k, 3c, 3g, and 4e as well as reference compounds AMI-1 and ellagic

acid were dissolved in DMSO (100%) to obtain 50 mM solutions and

diluted in HBS (10 mM Hepes pH 7.4, 0.15 M NaCl, 0.005% NP40)

with a ﬁnal DMSO concentration of 0.5%. For each sample, the

complete binding study was performed using a six-point concentration

series, typically spanning 0.025−10 μM. Binding experiments were

performed using a ﬂow rate of 50 μL min⁻¹, with 60 s monitoring of

association and 300 s monitoring of dissociation. HBS or 200 μM SAH

in HBS were used as running buﬀer. To evaluate the aﬃnity of H3 or

PABP1(437−488) toward CARM1 in the presence of derivatives 1k,

3c, 3g, and 4e, each substrate was dissolved in HBS at four diﬀerent

concentrations (10 nM, 50 nM, 250 nM, and 1 μM), and ﬁve aliquots

of each sample were dispensed into single-use vials; each derivative

was then added to a complete substrate concentration series to a ﬁnal

concentration of 4 μM, whereas 0.1% DMSO was added to the ﬁfth

series. Binding experiments were performed using a ﬂow rate of 20 μL

min⁻¹, with 60 s monitoring of association and 300 s monitoring of

dissociation, using HBS or 200 μM SAH in HBS as running buﬀer.

Simple interactions were adequately ﬁt to a single-site bimolecular

interaction model (A + B = AB), yielding a single K_D. Sensorgram

elaborations were performed using the BIAevaluation software

provided by GE Healthcare.

Preparation of GST-PRMT Fusion Proteins. Expression of the GST-

PRMT1 fusion protein is described in Supporting Information.

GST-PRMT3 and GST-PRMT4 have been described previously.¹²²

GST-PABP1(437−488) Expression and Puriﬁcation. The construct

for human PABP1 encoding residues 437 to 488, PABP1(437−488),

was generated by subcloning a PCR ampliﬁed fragment from the full

Cellular Methylation Assay. A stable/inducible T-Rex-3XFlag-

PABP1 cell line was established by transfection with pcDNA/FRT/

TO-3XFlag-PAPBP1 and pOG44 plasmids into Flp-in T-Rex HEK293

cells (Invitrogen).

Transfected cells were maintained in Dulbecco’s Modiﬁed Eagle’s

Medium containing fetal bovine serum (10%) supplemented with

blasticidin (10 μg/mL) and hygromycin (100 μg/mL). The resistant

single colony was picked and propagated.

́

length PABP1 gene (a gift from Dr. Bertrand Seraphin at IGBMC)

into the expression vector pGEX-2T (GE Healthcare).

Escherichia coli BL21(DE3) cells transformed with the

PABP1(437−488) plasmid were grown in LB at 37 °C. The GST-

fusion protein expression was induced with 0.2 mM IPTG overnight at

20 °C. Harvested cells were lysed by sonication in 50 mM Tris-HCl,

pH 8.2, 200 mM NaCl. The lysate was clariﬁed by centrifugation at

25000g for 30 min and applied to a 2 mL of Glutathione Sepharose 4B

(GE Healthcare) column. The GST-fusion protein was eluted with

two column volumes of 50 mM reduced glutathione in the lysis buﬀer

and concentrated using an Amicon Ultra with a cutoﬀ of 10 kDa. The

Cells were plated out in 6-well plates at a density of 1 × 10⁵cells/

mL in DMEM media supplemented with 10% FBS. After 12 h, the

media was changed. Cells were either treated with DMSO alone

(negative or background control), tetracyclin alone (positive control),

compound alone, or compound in combination with tetracyclin.

The ﬁnal concentration of compound was 100 μM; the ﬁnal

concentration of tetracyclin was 1 μg/μL. After 24 h, the cells were

harvested and lysed with RIPA buﬀer. The lysates were subsequently

immunoblotted.

Quantiﬁcation of CARM1 Fluorographs. Band intensities were

calculated by staining the bands with Ponceau Red. The stained bands

were then cut out and put in a glass vial each. Then 2 mL of a

scintilization cocktail (CytoScint, MPBio) were added to each vial and

the samples were counted using a Packard 1600 TR liquid

scintilization analyzer. The counts of the excised area were compared

to the DMSO control. This approach was used in Figure 3 and in

Figure S2, Supporting Information.

concentration of 164 μM was measured by UV absorption (ε₂₈₀

=

46370 M⁻¹cm⁻¹), and the protein was stored at −20 °C in 50 mM

Tris pH 8.5, 200 mM NaCl, 0.5 mM dithiothreitol (DTT), 50% (v/v)

glycerol.

Protein Methyltransferase Assays. PRMT1, PRMT3, PRMT4/

CARM1, and SET7/9 in Vitro Methylation Assays. In vitro

methylation assays were described in detail previously.⁷⁵Brieﬂy, all

methylation reactions were carried out in the presence of [³H] SAM

(0.42 μM, 79 Ci/mmol, stock solution in diluted HCl/ethanol 9:1, pH

2.0−2.5, GE Healthcare) and PBS (137 mM NaCl, 2.7 mM KCl, 4.3

mM Na₂HPO₄, 1.4 mM KH₂PO₄, pH 7.4). To determine the

speciﬁcity of their inhibitory activity, tested compounds were

incubated with GST-PRMT1 (10 × 10⁻⁸M) and histone H4 (all

1.5 × 10⁻⁶M), GST-PRMT3 (9.0 × 10⁻⁸M) and GAR (4.1 × 10⁻⁷

M), GST-PRMT4 (9.8 × 10⁻⁸M) and histone H3 (all 1.1 × 10⁻⁶M),

and SET 7/9 (1.3 × 10⁻⁷M) and histone H3. Histones were

purchased from Roche. Substrates (0.5 μg, concentration range from

1.1 × 10⁻⁶M to 4.06 × 10⁻⁷M) were incubated with recombinant

enzymes (0.2 μg, concentration range from 1.3 × 10⁻⁷M to 10 × 10⁻⁸

M) in the presence of 0.5 μg of [³H] SAM (0.42 μM) and 50 μM of

compound for 90 min at 30 °C in a ﬁnal volume of 30 μL. Reactions

were run on a 10% SDS-PAGE, transferred to a polyvinylidene ﬂuoride

(PVDF) membrane (Millipore), sprayed with a spray surface

autoradiography enhancer (EN³HANCE, PerkinElmer), and exposed

to ﬁlm overnight. Band intensities were calculated using a Kodak

Image Station 440 and 1D Image Analysis Software (Eastman Kodak

Co.).

Surface Plasmon Resonance Analysis of Binding Interac-

tions. SPR analyses were performed on a Biacore 3000 optical

biosensor equipped with research-grade CM5 sensor chips (Biacore

AB).¹²³Using this platform, one peptide (PABP1(437−488)) or

protein (cal thymus histone H3 or GST-PRMT4 full length) surface,

one BSA surface, and one unmodiﬁed reference surface were prepared

for simultaneous analyses. Proteins (30 μg mL⁻¹in 10 mM sodium

acetate, pH 4.5) were immobilized on individual ﬂow cells of the

sensor chip at a ﬂow rate of 5 mL min⁻¹by using standard amine-

coupling protocols¹²⁴to obtain densities of 12−17 kRU. Compounds

Quantiﬁcation of PABP1 Western Blot. The ﬁlm was scanned

and digitalized. The density of each band was determined using the

Quantity One 1-D analysis software (Bio-Rad) and compared to the

DMSO control. This approach was used in Figure 4.

ASSOCIATED CONTENT

* Supporting Information

■

S

Synthesis of 2,5-disubstituted indole derivatives 1a−j and of

3,5-substituted analogues 2a−h, elemental analysis for all ﬁnal

compounds, preparation of GST-PRMT1 fusion protein,

description of PRMT1 inhibitory assay, additional biological

data. This material is available free of charge via the Internet at

http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author

*Phone: +39-089-96-9770. Fax:+39-089-96-9602. E-mail:

gsbardella@unisa.it.

■

Present Address

^#Celon Pharma Sp. Z.o.o., Laboratory of Medicinal Chemistry,

ulica Ogrodowa 2A, 05−092 Lomianki/Kielpin, Poland.

L

dx.doi.org/10.1021/jm301097p | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(10) Miranda, T. B.; Miranda, M.; Frankel, A.; Clarke, S. PRMT7 Is a

Member of the Protein Arginine Methyltransferase Family with a

Distinct Substrate Specificity. J. Biol. Chem. 2004, 279, 22902−22907.

(11) Kousaka, A.; Mori, Y.; Koyama, Y.; Taneda, T.; Miyata, S.;

Tohyama, M. The distribution and characterization of endogenous

protein arginine N-methyltransferase 8 in mouse CNS. Neuroscience

2009, 163, 1146−1157.

(12) Taneda, T.; Miyata, S.; Kousaka, A.; Inoue, K.; Koyama, Y.;

Mori, Y.; Tohyama, M. Specific regional distribution of protein

arginine methyltransferase 8 (PRMT8) in the mouse brain. Brain Res.

2007, 1155, 1−9.

(13) Selvi, B. R.; Mohankrishna, D. V.; Ostwal, Y. B.; Kundu, T. K.

Small molecule modulators of histone acetylation and methylation: A

disease perspective. Biochim. Biophys. Acta, Gene Regul. Mech. 2010,

1799, 810−828.

(14) Guccione, E.; Bassi, C.; Casadio, F.; Martinato, F.; Cesaroni, M.;

Schuchlautz, H.; Luscher, B.; Amati, B. Methylation of histone H3R2

by PRMT6 and H3K4 by an MLL complex are mutually exclusive.

Nature 2007, 449, 933−937.

(15) Kirmizis, A.; Santos-Rosa, H.; Penkett, C. J.; Singer, M. A.;

Vermeulen, M.; Mann, M.; Bahler, J.; Green, R. D.; Kouzarides, T.

Arginine methylation at histone H3R2 controls deposition of H3K4

trimethylation. Nature 2007, 449, 928−932.

(16) Arrowsmith, C. H.; Bountra, C.; Fish, P. V.; Lee, K.; Schapira,

M. Epigenetic protein families: a new frontier for drug discovery.

Nature Rev. Drug Discovery 2012, 11, 384−400.

(17) Koh, S. S.; Chen, D.; Lee, Y.-H.; Stallcup, M. R. Synergistic

Enhancement of Nuclear Receptor Function by p160 Coactivators and

Two Coactivators with Protein Methyltransferase Activities. J. Biol.

Chem. 2001, 276, 1089−1098.

(18) Wang, H.; Huang, Z.-Q.; Xia, L.; Feng, Q.; Erdjument-Bromage,

H.; Strahl, B. D.; Briggs, S. D.; Allis, C. D.; Wong, J.; Tempst, P.;

Zhang, Y. Methylation of Histone H4 at Arginine 3 Facilitating

Transcriptional Activation by Nuclear Hormone Receptor. Science

2001, 293, 853−857.

(19) Anzick, S. L.; Kononen, J.; Walker, R. L.; Azorsa, D. O.; Tanner,

M. M.; Guan, X.-Y.; Sauter, G.; Kallioniemi, O.-P.; Trent, J. M.;

Meltzer, P. S. AIB1, a Steroid Receptor Coactivator Amplified in

Breast and Ovarian Cancer. Science 1997, 277, 965−968.

(20) Torchia, J.; Rose, D. W.; Inostroza, J.; Kamei, Y.; Westin, S.;

Glass, C. K.; Rosenfeld, M. G. The transcriptional co-activator p/CIP

binds CBP and mediates nuclear-receptor function. Nature 1997, 387,

677−684.

(21) Lee, S.-K.; Anzick, S. L.; Choi, J.-E.; Bubendorf, L.; Guan, X.-Y.;

Jung, Y.-K.; Kallioniemi, O. P.; Kononen, J.; Trent, J. M.; Azorsa, D.;

Jhun, B.-H.; Cheong, J. H.; Lee, Y. C.; Meltzer, P. S.; Lee, J. W. A

Nuclear Factor, ASC-2, as a Cancer-Amplified Transcriptional

Coactivator Essential for Ligand-Dependent Transactivation by

Nuclear Receptors in Vivo. J. Biol. Chem. 1999, 274, 34283−34293.

(22) Zhu, Y.; Qi, C.; Jain, S.; Le Beau, M. M.; Espinosa, R.; Atkins, G.

B.; Lazar, M. A.; Yeldandi, A. V.; Rao, M. S.; Reddy, J. K. Amplification

and overexpression of peroxisome proliferator-activated receptor

binding protein (PBP/PPARBP) gene in breast cancer. Proc. Natl.

Acad. Sci. U. S. A. 1999, 96, 10848−10853.

(23) Cheung, N.; Chan, L. C.; Thompson, A.; Cleary, M. L.; So, C.

W. E. Protein arginine-methyltransferase-dependent oncogenesis.

Nature Cell Biol. 2007, 9, 1208−1215.

(24) Scorilas, A.; Black, M. H.; Talieri, M.; Diamandis, E. P. Genomic

Organization, Physical Mapping, and Expression Analysis of the

Human Protein Arginine Methyltransferase 1 Gene. Biochem. Biophys.

Res. Commun. 2000, 278, 349−359.

(25) Goulet, I.; Gauvin, G.; Boisvenue, S.; Cote, J. Alternative

splicing yields protein arginine methyltransferase 1 isoforms with

distinct activity, substrate specificity, and subcellular localization. J.

Biol. Chem. 2007, 282, 33009−33021.

(26) Mathioudaki, K.; Papadokostopoulou, A.; Scorilas, A.;

Xynopoulos, D.; Agnanti, N.; Talieri, M. The PRMT1 gene expression

pattern in colon cancer. Br. J. Cancer 2008, 99, 2094−2099.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

This work was supported by grants from Ministero

dell’Universita

̀

e della Ricerca Scientiﬁca e Tecnologica, PRIN

di Salerno (G.S.), COST Action TD09/

2008 (S.C.), Universita

̀

05 Epigenetics (G.S. and A.M.), and NIEHS Center institu-

tional grants ES007784, ES015188, and DK062248 (M.T.B.).

A.S. was supported by a Fellowship from German Research

Foundation, SP 1262/1-1. C.M. and M.V. were supported by

Universita di Salerno with a postdoctoral research fellowship

̀

and a predoctoral fellowship, respectively. We are indebted to

Dr. Marina Fasolini (Nerviano MS) for helpful discussions.

ABBREVIATIONS USED

■

AIB1, ampliﬁed in breast cancer-1; BL21, Escherichia coli B cells

lacking the Lon protease; BSA, bovine serum albumin;

CARM1, coactivator-associated arginine methyltransferase 1;

CBP, CREB binding protein; CREB, c-AMP response element-

binding; aDMA, asymmetrical dimethylarginine; sDMA, sym-

metrical dimethylarginine; DTT, dithiothreitol; FBXO11, F-

box protein 11; GAR, glycine-rich, arginine rich; GST,

glutathione-S-transferase; HKMT, histone lysine methyltrans-

ferase; hnRNP, heterogeneous nuclear ribonucleoprotein;

HRP, horseradish peroxidase; IPTG, isopropyl-β-^D-1-thioga-

lactopyranoside; LB, lysogeny broth; MMA, monomethylargi-

nine; NF-κB, nuclear factor κB; Npl3p, nuclear shuttling

protein; p300, E1A binding protein 300 kDa; PABP1,

polyadenylate-binding protein 1; PBS, phosphate buﬀer saline;

PRMT, protein arginine methyl transferase; PVDF, polyviny-

lidene ﬂuoride; RmtA, fungal arginine methyltransferase A;

SAM, S-adenosyl methionine; SDS-PAGE, sodium dodecyl

sulfatepolyacrylamide gel electrophoresis; SET, Su(var.) 3−9,

enhancer-of-zeste and trithorax; TBS, Tris buﬀered saline;

TBST, Tris buﬀered saline Tween-20; TCA, trichloroacetic

acid; TFA, triﬂuoroacetic acid

REFERENCES

■

(1) Aletta, J. M.; Hu, J. C.; El-Gewely, M. R. Protein arginine

methylation in health and disease. In Biotechnology Annual Review;

Elsevier: New York, 2008; Vol. 14, pp 203−224.

(2) Bedford, M. T.; Richard, S. Arginine Methylation: An Emerging

Regulator of Protein Function. Mol. Cell 2005, 18, 263−272.

(3) Krause, C. D.; Yang, Z.-H.; Kim, Y.-S.; Lee, J.-H.; Cook, J. R.;

Pestka, S. Protein arginine methyltransferases: evolution and assess-

ment of their pharmacological and therapeutic potential. Pharmacol.

Ther. 2007, 113, 50−87.

(4) Pahlich, S.; Zakaryan, R. P.; Gehring, H. Protein arginine

methylation: cellular functions and methods of analysis. Biochim.

Biophys. Acta, Proteins Proteomics 2006, 1764, 1890−1903.

(5) Bedford, M. T. Arginine methylation at a glance. J. Cell Sci. 2007,

120, 4243−4246.

(6) Smith, B. C.; Denu, J. M. Chemical mechanisms of histone lysine

and arginine modifications. Biochim. Biophys. Acta, Gene Regul. Mech.

2009, 1789, 45−57.

(7) Di Lorenzo, A.; Bedford, M. T. Histone arginine methylation.

FEBS Lett. 2011, 585, 2024−2031.

(8) PRMT1 and other PRMTs except PRMT4 recognize glycine

arginine rich motifs for methylation, whereas PRMT4 recognizes

XXPRXX motif for methylation.

(9) Bedford, M. T.; Clarke, S. G. Protein Arginine Methylation in

Mammals: Who, What, and Why. Mol. Cell 2009, 33, 1−13.

M

dx.doi.org/10.1021/jm301097p | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(27) Papadokostopoulou, A.; Mathioudaki, K.; Scorilas, A.;

Xynopoulos, D.; Ardavanis, A.; Kouroumalis, E.; Talieri, M. Colon

Cancer and Protein Arginine Methyltransferase 1 Gene Expression.

Anticancer Res. 2009, 29, 1361−1366.

(45) Leiper, J.; Nandi, M.; Torondel, B.; Murray-Rust, J.; Malaki, M.;

O’Hara, B.; Rossiter, S.; Anthony, S.; Madhani, M.; Selwood, D.;

Smith, C.; Wojciak-Stothard, B.; Rudiger, A.; Stidwill, R.; McDonald,

N. Q.; Vallance, P. Disruption of methylarginine metabolism impairs

vascular homeostasis. Nature Med. 2007, 13, 198−203.

(46) Chen, X.; Niroomand, F.; Liu, Z.; Zankl, A.; Katus, H. A.; Jahn,

L.; Tiefenbacher, C. P. Expression of nitric oxide related enzymes in

coronary heart disease. Basic Res. Cardiol. 2006, 101, 346−353.

(47) Maas, R. Pharmacotherapies and their influence on asymmetric

dimethylargine (ADMA). Vasc. Med. 2005, 10, S49−S57.

(48) McKinsey, T. A.; Zhang, C. L.; Olson, E. N. Signaling chromatin

to make muscle. Curr. Opin. Cell Biol. 2002, 14, 763−772.

(49) Yildirim, A. O.; Bulau, P.; Zakrzewicz, D.; Kitowska, K. E.;

Weissmann, N.; Grimminger, F.; Morty, R. E.; Eickelberg, O.

Increased Protein Arginine Methylation in Chronic Hypoxia: Role of

Protein Arginine Methyltransferases. Am. J. Respir. Cell Mol. Biol. 2006,

35, 436−443.

(28) Seligson, D. B.; Horvath, S.; Shi, T.; Yu, H.; Tze, S.; Grunstein,

M.; Kurdistani, S. K. Global histone modification patterns predict risk

of prostate cancer recurrence. Nature 2005, 435, 1262−1266.

̂ ́

(29) Cheng, D.; Cote, J.; Shaaban, S.; Bedford, M. T. The Arginine

Methyltransferase CARM1 Regulates the Coupling of Transcription

and mRNA Processing. Mol. Cell 2007, 25, 71−83.

(30) Li, H.; Park, S.; Kilburn, B.; Jelinek, M. A.; Henschen-Edman,

A.; Aswad, D. W.; Stallcup, M. R.; Laird-Offringa, I. A. Lip-

opolysaccharide-Induced Methylation of HuR, an mRNA-Stabilizing

Protein, by CARM1. J. Biol. Chem. 2002, 277, 44623−44630.

(31) Lee, J.; Bedford, M. T. PABP1 identified as an arginine

methyltransferase substrate using high-density protein arrays. EMBO

Rep. 2002, 3, 268−273.

(32) Fujiwara, T.; Mori, Y.; Chu, D. L.; Koyama, Y.; Miyata, S.;

Tanaka, H.; Yachi, K.; Kubo, T.; Yoshikawa, H.; Tohyama, M. CARM1

Regulates Proliferation of PC12 Cells by Methylating HuD. Mol. Cell.

Biol. 2006, 26, 2273−2285.

(33) Jeong, S. J.; Lu, H.; Cho, W. K.; Park, H. U.; Pise-Masison, C.;

Brady, J. N. Coactivator-associated arginine methyltransferase 1

enhances transcriptional activity of the human T-cell lymphotropic

virus type 1 long terminal repeat through direct interaction with Tax. J.

Virol. 2006, 80, 10036−10044.

(34) Hong, H.; Kao, C.; Jeng, M.-H.; Eble, J. N.; Koch, M. O.;

Gardner, T. A.; Zhang, S.; Li, L.; Pan, C.-X.; Hu, Z.; MacLennan, G.

T.; Cheng, L. Aberrant expression of CARM1, a transcriptional

coactivator of androgen receptor, in the development of prostate

carcinoma and androgen-independent status. Cancer 2004, 101, 83−

89.

(50) Hong, E.; Lim, Y.; Lee, E.; Oh, M.; Kwon, D. Tissue-specific and

age-dependent expression of protein arginine methyltransferases

(PRMTs) in male rat tissues. Biogerontology 2012, 13, 329−336.

(51) Ito, T.; Yadav, N.; Lee, J.; Furumatsu, T.; Yamashita, S.;

Yoshida, K.; Taniguchi, N.; Hashimoto, M.; Tsuchiya, M.; Ozaki, T.;

Lotz, M.; Bedford, M.; Asahara, H. Arginine methyltransferase

CARM1/PRMT4 regulates endochondral ossification. BMC Dev.

Biol. 2009, 9, 47.

́

(52) Batut, J.; Duboe, C.; Vandel, L. The Methyltransferases

PRMT4/CARM1 and PRMT5 Control Differentially Myogenesis in

Zebrafish. PLoS One 2011, 6, e25427.

(53) Wang, S.-C. M.; Dowhan, D. H.; Eriksson, N. A.; Muscat, G. E.

O. CARM1/PRMT4 is necessary for the glycogen gene expression

programme in skeletal muscle cells. Biochem. J. 2012, 444, 323−331.

̀

(54) O’Brien, K. B.; Alberich-Jorda, M.; Yadav, N.; Kocher, O.;

(35) Majumder, S.; Liu, Y.; F., O. H., III; Mohler, J. L.; Whang, Y. E.

Involvement of arginine methyltransferase CARM1 in androgen

receptor function and prostate cancer cell viability. Prostate 2006,

66, 1292−1301.

(36) Feng, Q.; Yi, P.; Wong, J.; O’Malley, B. W. Signaling within a

Coactivator Complex: Methylation of SRC-3/AIB1 Is a Molecular

Switch for Complex Disassembly. Mol. Cell. Biol. 2006, 26, 7846−

7857.

DiRuscio, A.; Ebralidze, A.; Levantini, E.; Sng, N. J. L.; Bhasin, M.;

Caron, T.; Kim, D.; Steidl, U.; Huang, G.; Halmos, B.; Rodig, S. J.;

Bedford, M. T.; Tenen, D. G.; Kobayashi, S. CARM1 is required for

proper control of proliferation and differentiation of pulmonary

epithelial cells. Development 2010, 137, 2147−2156.

(55) Tsankova, N.; Renthal, W.; Kumar, A.; Nestler, E. J. Epigenetic

regulation in psychiatric disorders. Nature Rev. Neurosci. 2007, 8, 355−

367.

(37) Frietze, S.; Lupien, M.; Silver, P. A.; Brown, M. CARM1

regulates estrogen-stimulated breast cancer growth through up-

regulation of E2F1. Cancer Res. 2008, 68, 301−306.

(38) Hassa, P. O.; Covic, M.; Bedford, M. T.; Hottiger, M. O. Protein

Arginine Methyltransferase 1 Coactivates NF-κB-Dependent Gene

Expression Synergistically with CARM1 and PARP1. J. Mol. Biol. 2008,

377, 668−678.

(56) Wilson, C. B.; Rowell, E.; Sekimata, M. Epigenetic control of T-

helper-cell differentiation. Nature Rev. Immunol. 2009, 9, 91−105.

(57) Copeland, R. A.; Solomon, M. E.; Richon, V. M. Protein

methyltransferases as a target class for drug discovery. Nature Rev.

Drug Discovery 2009, 8, 724−732.

(58) Cole, P. A. Chemical probes for histone-modifying enzymes.

Nature Chem. Biol. 2008, 4, 590−597.

(39) An, W.; Kim, J.; Roeder, R. G. Ordered cooperative functions of

PRMT1, p300, and CARM1 in transcriptional activation by p53. Cell

2004, 117, 735−748.

(40) Boger, R. H.; Sydow, K.; Borlak, J.; Thum, T.; Lenzen, H.;

Schubert, B.; Tsikas, D.; Bode-Boger, S. M. LDL Cholesterol

Upregulates Synthesis of Asymmetrical Dimethylarginine in Human

Endothelial Cells: Involvement of S-Adenosylmethionine-Dependent

Methyltransferases. Circ. Res. 2000, 87, 99−105.

(41) Tran, C. T. L.; Leiper, J. M.; Vallance, P. The DDAH/ADMA/

NOS pathway. Atherosclerosis Suppl. 2003, 4, 33−40.

(42) Vallance, P.; Leiper, J. Cardiovascular Biology of the

Asymmetric Dimethylarginine:Dimethylarginine Dimethylaminohy-

drolase Pathway. Arterioscler., Thromb., Vasc. Biol. 2004, 24, 1023−

1030.

(43) Leiper, J.; Murray-Rust, J.; McDonald, N.; Vallance, P. S-

Nitrosylation of dimethylarginine dimethylaminohydrolase regulates

enzyme activity: Further interactions between nitric oxide synthase

and dimethylarginine dimethylaminohydrolase. Proc. Natl. Acad. Sci. U.

S. A. 2002, 99, 13527−13532.

(59) Keppler, B. R.; Archer, T. K. Chromatin-modifying enzymes as

therapeutic targets: Part 1. Expert Opin. Ther. Targets 2008, 12, 1301−

1312.

(60) Spannhoff, A.; Sippl, W.; Jung, M. Cancer treatment of the

future: Inhibitors of histone methyltransferases. Int. J. Biochem. Cell

Biol. 2009, 41, 4−11.

(61) Strahl, B. D.; Briggs, S. D.; Brame, C. J.; Caldwell, J. A.; Koh, S.

S.; Ma, H.; Cook, R. G.; Shabanowitz, J.; Hunt, D. F.; Stallcup, M. R.;

Allis, C. D. Methylation of histone H4 at arginine 3 occurs in vivo and

is mediated by the nuclear receptor coactivator PRMT1. Curr. Biol.

2001, 11, 996−1000.

(62) Migliori, V.; Phalke, S.; Bezzi, M.; Guccione, E. Arginine/

lysine−methyl/methyl switches: biochemical role of histone arginine

methylation in transcriptional regulation. Epigenomics 2010, 2, 119−

137.

(63) Kim, D.; Lee, J.; Cheng, D.; Li, J.; Carter, C.; Richie, E.;

Bedford, M. T. Enzymatic Activity Is Required for the in Vivo

Functions of CARM1. J. Biol. Chem. 2010, 285, 1147−1152.

(64) Troffer-Charlier, N.; Cura, V.; Hassenboehler, P.; Moras, D.;

Cavarelli, J. Functional insights from structures of coactivator-

associated arginine methyltransferase 1 domains. EMBO J. 2007, 26,

4391−4401.

(44) Leone, A.; Moncada, S.; Vallance, P.; Calver, A.; Collier, J.

Accumulation of an endogenous inhibitor of nitric oxide synthesis in

chronic renal failure. Lancet 1992, 339, 572−575.

N

dx.doi.org/10.1021/jm301097p | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(65) Zhang, X.; Cheng, X. Structure of the predominant protein

arginine methyltransferase PRMT1 and analysis of its binding to

substrate peptides. Structure 2003, 11, 509−520.

(66) Zhang, X.; Zhou, L.; Cheng, X. Crystal structure of the

conserved core of protein arginine methyltransferase PRMT3. EMBO

J. 2000, 19, 3509−3519.

(67) Sack, J. S.; Thieffine, S.; Bandiera, T.; Fasolini, M.; Duke, G. J.;

Jayaraman, L.; Kish, K. F.; Klei, H. E.; Purandare, A. V.; Rosettani, P.;

Troiani, S.; Xie, D.; Bertrand, J. A. Structural basis for CARM1

inhibition by indole and pyrazole inhibitors. Biochem. J. 2011, 436,

331−339.

(68) Gui, S.; Wooderchak, W. L.; Daly, M. P.; Porter, P. J.; Johnson,

S. J.; Hevel, J. M. Investigation of the Molecular Origins of Protein−

Arginine Methyltransferase I (PRMT1) Product Specificity Reveals a

Role for Two Conserved Methionine Residues. J. Biol. Chem. 2011,

286, 29118−29126.

(69) Yue, W. W.; Hassler, M.; Roe, S. M.; Thompson-Vale, V.; Pearl,

L. H. Insights into histone code syntax from structural and biochemical

studies of CARM1 methyltransferase. EMBO J. 2007, 26, 4402−4412.

(70) Obianyo, O.; Osborne, T. C.; Thompson, P. R. Kinetic

Mechanism of Protein Arginine Methyltransferase 1. Biochemistry

2008, 47, 10420−10427.

(71) Rust, H. L.; Zurita-Lopez, C. I.; Clarke, S.; Thompson, P. R.

Mechanistic Studies on Transcriptional Coactivator Protein Arginine

Methyltransferase 1. Biochemistry 2011, 50, 3332−3345.

(72) Mai, A.; Altucci, L. Epi-drugs to fight cancer: from chemistry to

cancer treatment, the road ahead. Int. J. Biochem. Cell Biol. 2009, 41,

199−213.

(73) Zheng, Y. G.; Wu, J.; Chen, Z.; Goodman, M. Chemical

regulation of epigenetic modifications: opportunities for new cancer

therapy. Med. Res. Rev. 2008, 28, 645−687.

(74) Amur, S. G.; Shanker, G.; Cochran, J. M.; Ved, H. S.; Pieringer,

R. A. Correlation between inhibition of myelin basic protein (arginine)

methyltransferase by sinefungoin and lack of compact myelin

formation in cultures of cerebral cells from embryonic mice. J.

Neurosci. Res. 1986, 16, 367−376.

(75) Cheng, D.; Yadav, N.; King, R. W.; Swanson, M. S.; Weinstein,

E. J.; Bedford, M. T. Small molecule regulators of protein arginine

methyltransferases. J. Biol. Chem. 2004, 279, 23892−23899.

(76) Mai, A.; Cheng, D.; Bedford, M. T.; Valente, S.; Nebbioso, A.;

Perrone, A.; Brosch, G.; Sbardella, G.; De Bellis, F.; Miceli, M.; Altucci,

L. Epigenetic Multiple Ligands: Mixed Histone/Protein Methyltrans-

ferase, Acetyltransferase, and Class III Deacetylase (Sirtuin) Inhibitors.

J. Med. Chem. 2008, 51, 2279−2290.

(77) Mai, A.; Valente, S.; Cheng, D.; Perrone, A.; Ragno, R.; Simeoni,

S.; Sbardella, G.; Brosch, G.; Nebbioso, A.; Conte, M.; Altucci, L.;

Bedford, M. T. Synthesis and Biological Validation of Novel Synthetic

Histone/Protein Methyltransferase Inhibitors. ChemMedChem 2007,

2, 987−991.

(78) Ragno, R.; Simeoni, S.; Castellano, S.; Vicidomini, C.; Mai, A.;

Caroli, A.; Tramontano, A.; Bonaccini, C.; Trojer, P.; Bauer, I.; Brosch,

G.; Sbardella, G. Small Molecule Inhibitors of Histone Arginine

Methyltransferases: Homology Modeling, Molecular Docking, Binding

Mode Analysis, and Biological Evaluations. J. Med. Chem. 2007, 50,

1241−1253.

(82) Huynh, T.; Chen, Z.; Pang, S.; Geng, J.; Bandiera, T.; Bindi, S.;

Vianello, P.; Roletto, F.; Thieffine, S.; Galvani, A.; Vaccaro, W.; Poss,

M. A.; Trainor, G. L.; Lorenzi, M. L.; Gottardis, M.; Jayaraman, L.;

Purandare, A. V. Optimization of Pyrazole Inhibitors of Coactivator

Associated Arginine Methyltransferase 1 (CARM1). Bioorg. Med.

Chem. Lett. 2009, 19, 2924−2927.

(83) Purandare, A. V.; Chen, Z.; Huynh, T.; Pang, S.; Geng, J.;

Vaccaro, W.; Poss, M. A.; Oconnell, J.; Nowak, K.; Jayaraman, L.

Pyrazole inhibitors of coactivator associated arginine methyltransferase

1 (CARM1). Bioorg. Med. Chem. Lett. 2008, 18, 4438−4441.

(84) Therrien, E.; Larouche, G.; Manku, S.; Allan, M.; Nguyen, N.;

Styhler, S.; Robert, M.-F.; Goulet, A.-C.; Besterman, J. M.; Nguyen, H.;

Wahhab, A. 1,2-Diamines as inhibitors of co-activator associated

arginine methyltransferase 1 (CARM1). Bioorg. Med. Chem. Lett. 2009,

19, 6725−6732.

(85) Bonham, K.; Hemmers, S.; Lim, Y.-H.; Hill, D. M.; Finn, M. G.;

Mowen, K. A. Effects of a novel arginine methyltransferase inhibitor on

T-helper cell cytokine production. FEBS J. 2010, 277, 2096−2108.

(86) Dowden, J.; Hong, W.; Parry, R. V.; Pike, R. A.; Ward, S. G.

Toward the development of potent and selective bisubstrate inhibitors

of protein arginine methyltransferases. Bioorg. Med. Chem. Lett. 2010,

20, 2103−2105.

(87) Feng, Y.; Li, M.; Wang, B.; Zheng, Y. G. Discovery and

Mechanistic Study of a Class of Protein Arginine Methylation

Inhibitors. J. Med. Chem. 2010, 53, 6028−6039.

(88) Obianyo, O.; Causey, C. P.; Osborne, T. C.; Jones, J. E.; Lee, Y.-

H.; Stallcup, M. R.; Thompson, P. R. A Chloroacetamidine-Based

Inactivator of Protein Arginine Methyltransferase 1: Design, Synthesis,

and In Vitro and In Vivo Evaluation. ChemBioChem 2010, 11, 1219−

1223.

(89) Osborne, T.; Weller Roska, R. L.; Rajski, S. R.; Thompson, P. R.

In Situ Generation of a Bisubstrate Analogue for Protein Arginine

Methyltransferase 1. J. Am. Chem. Soc. 2008, 130, 4574−4575.

(90) Selvi, B. R.; Batta, K.; Kishore, A. H.; Mantelingu, K.; Varier, R.

A.; Balasubramanyam, K.; Pradhan, S. K.; Dasgupta, D.; Sriram, S.;

Agrawal, S.; Kundu, T. K. Identification of a Novel Inhibitor of

Coactivator-Associated Arginine Methyltransferase 1 (CARM1)-

mediated Methylation of Histone H3 Arg-17. J. Biol. Chem. 2010,

285, 7143−7152.

(91) Wan, H.; Huynh, T.; Pang, S.; Geng, J.; Vaccaro, W.; Poss, M.

A.; Trainor, G. L.; Lorenzi, M. V.; Gottardis, M.; Jayaraman, L.;

Purandare, A. V. Benzo[d]imidazole Inhibitors of Coactivator

Associated Arginine Methyltransferase 1 (CARM1)Hit to Lead

Studies. Bioorg. Med. Chem. Lett. 2009, 19, 5063−5066.

(92) Castellano, S.; Milite, C.; Ragno, R.; Simeoni, S.; Mai, A.;

Limongelli, V.; Novellino, E.; Bauer, I.; Brosch, G.; Spannhoff, A.;

Cheng, D.; Bedford, M. T.; Sbardella, G. Design, Synthesis and

Biological Evaluation of Carboxy Analogues of Arginine Methyl-

transferase Inhibitor 1 (AMI-1). ChemMedChem 2010, 5, 398−414.

(93) Dillon, M. B. C.; Bachovchin, D. A.; Brown, S. J.; Finn, M. G.;

Rosen, H.; Cravatt, B. F.; Mowen, K. A. Novel Inhibitors for PRMT1

Discovered by High-Throughput Screening Using Activity-Based

Fluorescence Polarization. ACS Chem. Biol. 2012, 7, 1198−204.

(94) Obianyo, O.; Causey, C. P.; Jones, J. E.; Thompson, P. R.

Activity-Based Protein Profiling of Protein Arginine Methyltransferase

1. ACS Chem. Biol. 2011, 6, 1127−1135.

(79) Spannhoff, A.; Heinke, R.; Bauer, I.; Trojer, P.; Metzger, E.;

Gust, R.; Schule, R.; Brosch, G.; Sippl, W.; Jung, M. Target-Based

Approach to Inhibitors of Histone Arginine Methyltransferases. J. Med.

Chem. 2007, 50, 2319−2325.

(95) Hart, P. T.; Lakowski, T. M.; Thomas, D.; Frankel, A.; Martin,

N. I. Peptidic Partial Bisubstrates as Inhibitors of the Protein Arginine

N-Methyltransferases. ChemBioChem 2011, 12, 1427−1432.

(96) Dowden, J.; Pike, R. A.; Parry, R. V.; Hong, W.; Muhsen, U. A.;

Ward, S. G. Small molecule inhibitors that discriminate between

protein arginine N-methyltransferases PRMT1 and CARM1. Org.

Biomol. Chem. 2011, 9, 7814−7821.

(97) Cheng, D.; Valente, S.; Castellano, S.; Sbardella, G.; Di Santo,

R.; Costi, R.; Bedford, M. T.; Mai, A. Novel 3,5-Bis-

(bromohydroxybenzylidene)piperidin-4-ones as Coactivator-Associ-

ated Arginine Methyltransferase 1 Inhibitors: Enzyme Selectivity and

Cellular Activity. J. Med. Chem. 2011, 54, 4928−4932.

(80) Spannhoff, A.; Machmur, R.; Heinke, R.; Trojer, P.; Bauer, I.;

Brosch, G.; Schule, R.; Hanefeld, W.; Sippl, W.; Jung, M. A novel

̈

arginine methyltransferase inhibitor with cellular activity. Bioorg. Med.

Chem. Lett. 2007, 17, 4150−4153.

(81) Allan, M.; Manku, S.; Therrien, E.; Nguyen, N.; Styhler, S.;

Robert, M.-F.; Goulet, A.-C.; Petschner, A. J.; Rahil, G.; Robert

́

MacLeod, A.; Deziel, R.; Besterman, J. M.; Nguyen, H.; Wahhab, A. N-

Benzyl-1-heteroaryl-3-(trifluoromethyl)-1H-pyrazole-5-carboxamides

as inhibitors of co-activator associated arginine methyltransferase 1

(CARM1). Bioorg. Med. Chem. Lett. 2009, 19, 1218−1223.

O

dx.doi.org/10.1021/jm301097p | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(98) Cheng, D.; Bedford, M. T. Xenoestrogens Regulate the Activity

of Arginine Methyltransferases. ChemBioChem 2011, 12, 323−329.

(99) Bissinger, E.-M.; Heinke, R.; Spannhoff, A.; Eberlin, A.; Metzger,

(115) Lakowski, T. M.; Zurita-Lopez, C.; Clarke, S. G.; Frankel, A.

Approaches to measuring the activities of protein arginine N-

methyltransferases. Anal. Biochem. 2010, 397, 1−11.

(116) Lakowski, T. M.; Frankel, A. Sources of S-adenosyl-l-

homocysteine background in measuring protein arginine N-methyl-

transferase activity using tandem mass spectrometry. Anal. Biochem.

2010, 396, 158−160.

(117) Yadav, N.; Lee, J.; Kim, J.; Shen, J.; Hu, M. C.; Aldaz, C. M.;

Bedford, M. T. Specific protein methylation defects and gene

expression perturbations in coactivator-associated arginine methyl-

transferase 1-deficient mice. Proc. Natl. Acad. Sci. U. S. A. 2003, 100,

6464−6468.

(118) Monod, J.; Wyman, J.; Changeux, J.-P. On the nature of

allosteric transitions: a plausible model. J. Mol. Biol. 1965, 12, 88−118.

(119) Zorn, J. A.; Wells, J. A. Turning enzymes ON with small

molecules. Nature Chem. Biol. 2010, 6, 179−188.

(120) A few PRMT1 activators, named AMAs, were previously

reported by some of us. See: Cheng, D.; Yadav, N.; King, R. W.;

Swanson, M. S.; Weinstein, E. J.; Bedford, M. T. Small molecule

regulators of protein arginine methyltransferases. J. Biol. Chem. 2004,

279, 23892−23899 (reference 75)..

(121) Tachibana, M.; Sugimoto, K.; Fukushima, T.; Shinkai, Y. SET

Domain-containing Protein, G9a, Is a Novel Lysine-preferring

Mammalian Histone Methyltransferase with Hyperactivity and Specific

Selectivity to Lysines 9 and 27 of Histone H3. J. Biol. Chem. 2001, 276,

25309−25317.

E.; Cura, V.; Hassenboehler, P.; Cavarelli, J.; Schule, R.; Bedford, M.

̈

T.; Sippl, W.; Jung, M. Acyl derivatives of p-aminosulfonamides and

dapsone as new inhibitors of the arginine methyltransferase hPRMT1.

Bioorg. Med. Chem. 2011, 19, 3717−3731.

(100) Ragno, R.; Mai, A.; Simeoni, S.; Caroli, A.; Valente, S.; Perrone,

A.; Castellano, S.; Sbardella, G. In Small Molecule Inhibitors of Histone

Arginine Methyltransferases: Updated Structure-Based 3-D QSAR Models

with Improved Robustness and Predictive Ability, Frontiers in CNS and

Oncology Medicinal Chemistry, ACS-EFMC, Siena, Italy, October 7−

9, 2007, COMC-010.

(101) Somei, M.; Yamada, F. Simple indole alkaloids and those with a

non-rearranged monoterpenoid unit. Nat. Prod. Rep. 2005, 22, 73−

103.

(102) Narayana, B.; Ashalatha, B. V.; Vijaya Raj, K. K.; Fernandes, J.;

Sarojini, B. K. Synthesis of some new biologically active 1,3,4-

oxadiazolyl nitroindoles and a modified Fischer indole synthesis of

ethyl nitro indole-2-carboxylates. Bioorg. Med. Chem. 2005, 13, 4638−

4644.

(103) Pierson, P. D.; Fettes, A.; Freichel, C.; Gatti-McArthur, S.;

Hertel, C.; Huwyler, J. R.; Mohr, P.; Nakagawa, T.; Nettekoven, M.;

Plancher, J.-M.; Raab, S.; Richter, H.; Roche, O.; Rodríguez Sarmiento,

R. M. A.; Schmitt, M.; Schuler, F.; Takahashi, T.; Taylor, S.; Ullmer,

C.; Wiegand, R. 5-Hydroxyindole-2-carboxylic Acid Amides: Novel

Histamine-3 Receptor Inverse Agonists for the Treatment of Obesity.

J. Med. Chem. 2009, 52, 3855−3868.

(122) Frankel, A.; Yadav, N.; Lee, J.; Branscombe, T. L.; Clarke, S.;

Bedford, M. T. The Novel Human Protein Arginine N-Methyl-

transferase PRMT6 Is a Nuclear Enzyme Displaying Unique Substrate

Specificity. J. Biol. Chem. 2002, 277, 3537−3543.

(104) Vicker, N.; Su, X.; Ganeshapillai, D.; Purohit, A.; Reed, M. J.;

Potter, B. V. L. Preparation of arylsulfonylbenzazoles as inhibitors of

11-beta-hydroxy steroid dehydrogenase type 1 and type 2. Patent

s2003-GB4590 2004037251, 20031023, 2003.

(105) Too, K.; Brown, D. M.; Holliger, P.; Loakes, D. Effect of a

hydrogen bonding carboxamide group on universal bases. Collect.

Czech. Chem. Commun. 2006, 71, 899−911.

(123) Cooper, M. Label-free screening of biomolecular interactions.

Anal. Bioanal. Chem. 2003, 377, 834−842.

(124) Johnsson, B.; Lofas, S.; Lindquist, G. Immobilization of

̈

̊

proteins to a carboxymethyldextran-modified gold surface for

biospecific interaction analysis in surface plasmon resonance sensors.

Anal. Biochem. 1991, 198, 268−277.

(106) McBride, A. E.; Cook, J. T.; Stemmler, E. A.; Rutledge, K. L.;

McGrath, K. A.; Rubens, J. A. Arginine Methylation of Yeast mRNA-

Binding Protein Npl3 Directly Affects Its Function, Nuclear Export,

and Intranuclear Protein Interactions. J. Biol. Chem. 2005, 280,

30888−30898.

(107) Gary, J. D.; Lin, W.-J.; Yang, M. C.; Herschman, H. R.; Clarke,

S. The Predominant Protein−Arginine Methyltransferase from

Saccharomyces cerevisiae. J. Biol. Chem. 1996, 271, 12585−12594.

(108) Henry, M. F.; Silver, P. A. A novel methyltransferase (Hmt1p)

modifies poly(A)+-RNA-binding proteins. Mol. Cell. Biol. 1996, 16,

3668−3678.

(109) For these compounds, we were not able to obtain reproducible

results due to solubility issues.

(110) Dal Piaz, F.; Tosco, A.; Eletto, D.; Piccinelli, A. L.; Moltedo,

O.; Franceschelli, S.; Sbardella, G.; Remondelli, P.; Rastrelli, L.; Vesci,

L.; Pisano, C.; De Tommasi, N. The Identification of a Novel Natural

Activator of p300 Histone Acetyltranferase Provides New Insights into

the Modulation Mechanism of this Enzyme. ChemBioChem 2010, 11,

818−827.

(111) Milite, C.; Castellano, S.; Benedetti, R.; Tosco, A.; Ciliberti, C.;

Vicidomini, C.; Boully, L.; Franci, G.; Altucci, L.; Mai, A.; Sbardella, G.

Modulation of the activity of histone acetyltransferases by long chain

alkylidenemalonates (LoCAMs). Bioorg. Med. Chem. 2011, 19, 3690−

3701.

(112) One RU represents the binding of 1 pg of protein per square

mm.

(113) Coan, K. E. D.; Shoichet, B. K. Stoichiometry and Physical

Chemistry of Promiscuous Aggregate-Based Inhibitors. J. Am. Chem.

Soc. 2008, 130, 9606−9612.

(114) Giannetti, A. M.; Koch, B. D.; Browner, M. F. Surface Plasmon

Resonance Based Assay for the Detection and Characterization of

Promiscuous Inhibitors. J. Med. Chem. 2008, 51, 574−580.

P

dx.doi.org/10.1021/jm301097p | J. Med. Chem. XXXX, XXX, XXX−XXX

Article Doi

Identification of small-molecule enhancers of arginine methylation catalyzed by coactivator-associated arginine methyltransferase 1

DOI: 10.1021/jm301097p

Source and publish data:

Authors:

Article abstract of DOI:10.1021/jm301097p

Full text of DOI:10.1021/jm301097p

Products guided by the article

R&D Labs maybe for 1408083-10-2

Relevant to this article

Hot Product