Synthesis and biochemical evaluation of δ2-isoxazoline derivatives as DNA methyltransferase 1 inhibitors

ARTICLE

pubs.acs.org/jmc

Synthesis and Biochemical Evaluation of Δ²-Isoxazoline Derivatives as

DNA Methyltransferase 1 Inhibitors

Sabrina Castellano,^{†,^}Dirk Kuck,*^{,‡,^}Monica Viviano,^†Jakyung Yoo,^§Fabian Lꢀopez-Vallejo,^§Paola Conti,^||

Lucia Tamborini,^||Andrea Pinto,^||Josꢀe L. Medina-Franco,*^,§and Gianluca Sbardella*^,†

^†Dipartimento di Scienze Farmaceutiche e Biomediche, Epigenetic Med Chem Lab, Universitꢁa degli Studi di Salerno, Via Ponte Don

Melillo, I-84084 Fisciano (SA), Italy

^‡Division of Epigenetics, DKFZ-ZMBH Alliance, German Cancer Research Center, Im Neuenheimer Feld 580, 69120 Heidelberg,

Germany

^§Torrey Pines Institute for Molecular Studies, 11350 SW Village Parkway, Port St. Lucie, Florida 34987, United States

Dipartimento di Scienze Farmaceutiche “Pietro Pratesi”, Universitꢁa degli Studi di Milano, Via Mangiagalli 25, 20133 Milano, Italy

S Supporting Information

b

ABSTRACT: A series of Δ²-isoxazoline constrained analogues

of procaine/procainamide (7aꢀk and 8aꢀk) were prepared

and their inhibitory activity against DNA methyltransferase 1

(DNMT1) was tested. Among them, derivative 7b is far more

potent in vitro (IC₅₀= 150 μM) than other non-nucleoside

inhibitors and also exhibits a strong and dose-dependent

antiproliferative eﬀect against HCT116 human colon carcinoma

cells. The binding mode of 7b with the enzyme was also

investigated by means of a simple competition assay as well as of

docking simulations conducted using the recently published crystallographic structure of human DNMT1. On the basis of the

ﬁndings, we assessed that the mode of inhibition of 7b is consistent with a competition with the cofactor and propose it as a novel

lead compound for the development of non-nucleoside DNMT inhibitors.

’ INTRODUCTION

for the maintenance of CpG methylation patterns in mammals

with hemimethylated CpG dinucleotides serving as preferred

substrates.²³The DNMT3 isoforms (DNMT3A and DNMT3B)

are responsible for de novo methylation during germ cell and

embryonicdevelopment, thus being able to use hemimethylated as

well as unmethylated DNA sequences as substrates. It has been

shown that the inhibition of DNA methyltransferase activity can

lead to demethylation and reactivation of epigenetically silenced

tumor suppressor genes²⁴and, indeed, DNA methylation inhibi-

tors were the ﬁrst successful epigenetic drugs developed and used

as cancer therapeutics.

Epigenetic mechanisms are essential for normal development

and maintenance of tissue-speciﬁc gene expression patterns in

mammals. As a whole, these modiﬁcations create an “epigenetic

landscape”, alteration or disruption of which is a hallmark of

virtually all cases of cancer.^1ꢀ7Both genetic and epigenetic events

regulate the onset of cancer,^8,9but unlike genetic mutations,

epigenetic aberrations are potentially reversible and can be restored

to their normal state.^10,11This makes epigenetic therapy a promis-

ing and valuable approach to chemotherapy as well as chemopre-

vention of cancer.

Two types of DNMT inhibitors have been hitherto described

(Chart 1).^11,25Nucleoside analogues, such as the U.S. Food and

Drug Administration (FDA)-approved 5-azacytidine (5-aza-CR,

Vidaza, Calgene) and 5-aza-2⁰-deoxycytidine (5-aza-CdR, deci-

tabine, Dacogen, Supergen), or 2-pyrimidone-1-β-^D-riboside

(zebularine), or the recently discovered dinucleotide derivative

1 (SGI-110),^26,27exert their eﬀects by incorporation into DNA

inducing substantial DNA demethylation and reactivation of

hypermethylated genes. Yet, they also carry considerable con-

cerns about toxicity. Their use, in fact, is associated with

increased incidence of bone marrow suppression, including

Probably, the most extensively studied epigenetic modiﬁca-

tion in humans is the addition of a methyl group at the carbon-5

position of cytosine residues.¹²DNA methylation in humans

occurs almost exclusively in the context of CpG dinucleotides¹³

clustered in ∼1 kb regions, termed CpG islands.^4,14In addition, it

also occurs at regions of lower CpG density that lie in close

proximity (∼2 kb), termed “CpG island shores”.^15,16

In general, DNA methylation is associated with transcriptional

silencing of genes implicated in the pathogenesis of many

diseases including cancer.^4,8,17ꢀ21Establishment and mainte-

nance of DNA methylation patterns are governed by catalytically

active DNA methyltransferase (DNMT) enzymes.

To date, three²²active DNMTs have been identiﬁed in humans.

DNMT1 is the most abundant among the three and is responsible

Received: August 3, 2011

Published: September 29, 2011

r

2011 American Chemical Society

7663

dx.doi.org/10.1021/jm2010404 J. Med. Chem. 2011, 54, 7663–7677

|

Journal of Medicinal Chemistry

ARTICLE

neutropenia and thrombocytopenia,^11,28ꢀ31even if the toler-

ability of 1 seems to be more favorable.²⁷

hydralazine,^47,48the 4-aminobenzoic acid derivatives procaine and

procainamide,^49ꢀ52as well as other compounds.^53,54

It is worth noting that few eﬀorts have been conducted so far

with non-nucleoside inhibitors to elucidate experimentally the

kinetics and speciﬁc mode of inhibition.

Because of these concerns, the search and the development of

non-nucleoside compounds, which can eﬀectively inhibit DNA

methylation without being incorporated into DNA, is being

actively pursued.^1,11Diﬀerently from nucleoside analogues, non-

nucleoside inhibitors exhibit a wide structural diversity (Chart 2),

as they include dietary polyphenols like (ꢀ)-epigallocatechin-3-

gallate (EGCG),³²curcumin,³³apple polyphenols,³⁴caﬀeic and

chlorogenic acids,³⁵the soy bean isoﬂavone genistein,³⁶the

bisulﬁde bromotyrosine derivatives psammaplins,³⁷the L-trypto-

phan derivative 2 (RG108)^38,39and related maleimide derivatives,⁴⁰

the antibiotic nanaomycin A⁴¹and mithramycin A,⁴²constrained

analogues of S-adenosylhomocysteine (SAH),^43,44the sulfonamide

IM-25,⁴⁵the quinoline derivative SGI-1027,⁴⁶the vasodilatator

Being interested in the development of small-molecule mod-

ulators of epigenetic targets,^{38,39,41,48,53ꢀ72}we focused our atten-

tion on procaine/procainamide as a lead structure for further

modiﬁcation. Originally approved by the FDA as local anesthetic

and antiarrhythmic drug, respectively, procaine and procainamide

have emerged as potential DNA demethylating agents.^45,51It has

been reported that procainamide speciﬁcally inhibits DNMT1

by reducing the aﬃnity with hemimethylated DNA (substrate)

and S-adenosylmethionine (cofactor),^49,52,73thus causing growth

arrest⁵¹and reactivation of tumor suppressor genes in cancer

cells.⁷⁴With the aim to increase potency, according to the frozen

analogue approach, we decided to limit the very high ﬂexibility of

procaine/procainamide scaﬀold by constraining the N-alkylamide

moiety into a 4-substituted- or 5-substituted-oxazoline ring

(derivatives 3,4 and 5,6, Figure 1).⁵⁵Among the synthesized

compounds, unexpectedly, the nitro derivative 5b (Chart 2

Chart 1. Nucleoside Inhibitors of DNMTs

Figure 1. Oxazoline derivatives 3ꢀ6 and Δ²-isoxazoline derivatives 7aꢀ

k and 8aꢀk.

Chart 2. Non-nucleoside Inhibitors of DNMTs

7664

dx.doi.org/10.1021/jm2010404 |J. Med. Chem. 2011, 54, 7663–7677

Journal of Medicinal Chemistry

ARTICLE

Scheme 1. Synthesis of Δ²-Isoxazolines 7aꢀk^a

Scheme 2. Synthesis of Δ²-Isoxazolines 8aꢀk^a

^aReagents and conditions: (a) 3% NaOCl, CH₂Cl₂, 0 ꢀC to room

temperature, 15 min; (b) MsCl, TEA, CH₂Cl₂, 0 ꢀC to room tempera-

ture, 45 min; (c) dimethylamine, THF, 100 ꢀC, sealed tube, 12 h;

(d) TFA/CH₂Cl₂1:3, room temperature, overnight; (e) 1N NaOH,

EtOH, reﬂux, 1 h; (f) zinc powder, acetic acid, room temperature, 1 h.

^aReagents and conditions: (a) R2/R4 ﬂow reactor, EtOAc, K₂CO₃,

80 ꢀC, 10 min; (b) 4N HCl, dioxane, room temperature, 1 h; (c) CH₃I,

K₂CO₃, acetone, room temperature, 12 h; (d) TFA/CH₂Cl₂1:3, room

temperature, overnight; (e) 1N NaOH, EtOH, reﬂux, 1 h; (f) zinc

powder, acetic acid, room temperature,1 h.

and Figure 1) exhibited the highest inhibitory potency against

DNMT1 and, when tested for its eﬀects on the genome methyla-

tion levels in HL60 human myeloid leukemia cells, it revealed a

recognizable demethylation of chromosomal satellite repeats.⁵⁵

To extend structureꢀactivity relationships for this class of

inhibitors, we needed a scaﬀold more stable and versatile than the

oxazoline ring. Therefore, we decided to explore the possibility of

replacing it with the Δ²-isoxazoline (7aꢀk) and to introduce a

new conformational restriction (8aꢀk) as shown in Figure 1.

Herein we report the synthesis of such compounds and their

inhibitory activity toward DNMT1 in both enzymatic and

cellular assays. We also present the results of competition

assays for the most potent enzyme inhibitor. These outcomes

shed light into the binding site of the enzyme and served as basis

to conduct molecular modeling studies of the inhibitor with a

recently published crystallographic structure of human

DNMT1.⁷⁵To the best of our knowledge, this is the ﬁrst

molecular modeling study conducted with the crystallographic

structure of human DNMT1.

Scheme 3. Synthesis of Aldoximes 9aꢀd, 9hꢀj, and 9l and

chlorooximes 12aꢀd, 12hꢀj, and 12l^a

^aReagents and conditions: (a) NH₂OH HCl, Na₂CO₃, H₂O/methanol

3

1:1, room temperature, 3 h; (b) NCS, pyridine, CHCl₃, 40 ꢀC, 0.5ꢀ3 h.

An analogous strategy was exploited to obtain bicyclic-Δ²-

isoxazolines 8 (Scheme 2). As previously reported by us,⁷⁶N-

Boc-protected pyrroline has low reactivity and the yields of 1,3-

dipolar cycloaddition reaction are poor under conventional

reaction conditions, even when the nitrile oxide is slowly

generated from the corresponding stable chloroximes 12. Con-

versely, running the reaction under continuous-ﬂow conditions

resulted in both an increased yield and an acceleration of the

process, straightforwardly generating the N-Boc-protected bicyc-

lic-Δ²-isoxazolines 13.⁷⁶

Removal of the N-Boc group with 4 M HCl solution in

dioxane and subsequent methylation with methyl iodide in

acetone produced derivatives 8aꢀd, 8hꢀj, and 8l. The car-

boxylic acid derivative 8e was obtained by deprotecting the

t-butyl ester 7l with triﬂuoracetic acid at room temperature.

Cleavage of the tosylate group of 8d with NaOH in EtOH

aﬀorded derivative 8g. Finally, reduction with zinc powder in

acetic acid converted the nitro-substituted compounds 8b and 8i

into the corresponding amino derivatives 8f and 8k, respectively.

The substituted oximes 9 were prepared from the correspond-

ing aldehydes under standard conditions. Oximes 9 were then

reacted with NCS in the presence of pyridine to give the

corresponding chlorooximes 12aꢀd, 12hꢀj, and 12l (Scheme 3).

’ CHEMISTRY

The key step for the synthesis of Δ²-isoxazolines 7 was the 1,3-

dipolar cycloaddition of nitrile oxides to allylic alcohol. Nitrile

oxides were formed in situ by the corresponding aldoximes 9

after oxidative chlorination with aqueous NaOCl solution

(common bleach), followed by base-induced dehydrochlorina-

tion of the intermediate hydroxymoyl chlorides (chlorooximes).

The cycloaddition reaction aﬀorded alcohols 10 in good isolated

yields (93ꢀ68%) (Scheme 1).

The nucleophilic displacement of the corresponding mesy-

lates 11 with dimethylamine furnished derivatives 7aꢀd, 7hꢀj,

and 7l. The carboxylic acid derivative 7e was obtained by

deprotecting the t-butyl ester 7l with triﬂuoracetic acid at room

temperature. The phenol derivative 7g was obtained from the

corresponding tosylate 7d after hydrolysis with NaOH. Finally,

reduction with zinc powder in acetic acid converted nitro

derivatives 7b and 7i into the corresponding amino derivatives

7f and 7k, respectively.

7665

dx.doi.org/10.1021/jm2010404 |J. Med. Chem. 2011, 54, 7663–7677

Journal of Medicinal Chemistry

ARTICLE

Table 1. Biochemical DNMT Assay of Compounds 7aꢀk and

8aꢀk against hDNMT1^a,b

compd

ctrl

R

mean (%)^a

SD^b

1.78

100.3

NA^c

0.3

7a

Ph

7b

7c

p-NO₂-Ph

p-CH₃O-Ph

p-TsO-Ph

p-COOH-Ph

p-NH₂-Ph

p-OH-Ph

Bn

0.02

0.09

0.21

0.28

0.37

0.08

0.16

Figure 2. Biochemical DNMT activity assays for compounds 7aꢀk and

8aꢀk against recombinant human DNMT1. SAH was used as reference

drug. All compounds were tested as 2 mM DMSO solutions. The assay

sensitivity and the enzymatic activity required comparably high con-

centrations (500 nM) of enzyme and, consequently, high concentrations

of test compounds. Error bars indicate standard deviations of each

measurement. DMSO (2 mM) alone (ctrl) did not aﬀect the enzymatic

activity of DNMT1. Data are reported as mean ( SD of the relative

enzyme activity in three independent experiments.

3.3

7d

7e

90.6

23.0

20.2

88.0

17.1

14.8

NA^c

28.3

NA^c

34.9

95.6

18.6

NA^c

5.1

7f

7g

7h

7i

p-NO₂-ꢀBn

p-CH₃O-Bn

p-NH₂-Bn

Ph

7j

7k

8a

0.39

’ RESULTS AND DISCUSSION

8b

8c

p-NO₂-Ph

p-CH₃O-Ph

p-TsO-Ph

p-COOH-Ph

p-NH₂-Ph

p-OH-Ph

Bn

0.30

0.39

0.06

Enzymatic Assays. So far, there is no standardized biochem-

ical assay available that delivers reliable data of the enzymatic

activity of DNMTs.⁷⁷As previously reported,^53,55we established

a biochemical in vitro methylation assay using recombinant

DNMT1 isolated and purified from insect cells. The identity

and integrity of recombinant DNMT1 was confirmed by SDS gel

electrophoresis and Western blotting with a DNMT1 specific

antibody (data not shown). Enzymatic activity of purified

DNMT1 was determined by the incorporation of radioactive

labeled methyl groups into hemimethylated oligonucleotide

substrates. Tritium labeled S-adenosylmethionine (SAM) was

used as methyl group donor, and the incorporation of radio-

activity was quantitated by a scintillation counter. The results

showed that the recombinant DNMT1 enzyme has substantial

enzymatic activity (Figure 2).

We performed a preliminary screening of the activity of com-

pounds 7aꢀk and 8aꢀk at the single concentration of 2 mM, using

procaine, 2,^38,39and S-adenosylhomocysteine (SAH) as reference

compounds (2 mM DMSO solution). Procaine (as well as

procainamide) was reported to inhibit DNMT activity at high drug

concentrations. In addition, the assay sensitivity requires compar-

ably high concentrations (500 nM) of enzyme and, consequently,

high concentrations of test compounds. Even if accurate structure

ꢀactivity relationships could not be derived from this assay, a few

empirical considerations could be drawn pointing at the identiﬁca-

tion of potential inhibitors and to rule out inactive compounds.

It is noticeable that while both procaine and 2 were inactive in

this assay, a number of tested compounds show a clear-cut

inhibition of the enzyme activity (Figure 2 and Table 1),

particularly when a nitro- or an amino- group is present as a

substituent of the benzene ring (compounds 7b, 7f, 7i, 7k and 8b,

8f, 8i, 8k). Indeed, at the tested concentration, these compounds

reduced the activity of DNMT1 at a very low level (0.3% in the

case of 7b, Table 1), a particularly signiﬁcant outcome for non-

nucleosidic inhibitors.

8d

8e

8f

0.06

8g

NA^c

84.0

3.8

8h

8i

0.07

0.02

0.05

p-NO₂-Bn

p-CH₃O-Bn

p-NH₂-Bn

8j

1.7

8k

procaine^d

2^d

3.3

NA^c

0.5

SAH^d

0.01

^aThe relative enzymatic activity (in percent) is shown as the mean value

of three measurements. ^bStandard deviation values are indicated in

percentage points. ^cNot active. ^dProcaine, RG108, and SAH (S-

adenosylhomocysteine) were used as reference compounds. All com-

pounds were tested in a concentration of 2 mM against 500 nM of

DNMT1. Compounds with an inhibition greater than 20% were scored

as positive.

case of compounds 7b and 7f, Table 1), but this become less

evident (nearly 2-fold) when the phenyl ring linked to the

heterocycles is replaced by a benzyl moiety (compare the

inhibition elicited by compounds 7b and 7f with those elicited

by 7i and 7k, respectively).

It is noteworthy that quite an opposite trend is observed in

the case of bicyclic derivatives 8. In fact, the nitrophenyl-

substituted pyrrolidinoisoxazoline 8b is 6-fold less active than

its aminophenyl- counterpart (Table 1), whereas the activities

of the benzylic analogues 8i and 8k are comparable. In a

similar way, the introduction of the methoxy group produced

active derivatives only in the case of phenyl-substituted

isoxazolines (compound 7c) and benzyl-substituted pyrroli-

dinoisoxazolines (compound 8j), whereas the corresponding

benzyl- and phenyl- analogues (compounds 7j and 8c, re-

spectively) were inactive.

Similarly to what we previously reported for their oxazolino-

analogues,⁵⁵nitro-substituted isoxazolines derivatives seem to be

more active than their amino- counterparts (nearly 70-fold in the

7666

dx.doi.org/10.1021/jm2010404 |J. Med. Chem. 2011, 54, 7663–7677

Journal of Medicinal Chemistry

ARTICLE

Figure 3. Doseꢀresponse plots for selected compounds 7b, 7f, 7i, 8f, 8i, and 8j against DNMT1. The IC₅₀concentrations of selected compounds were

determined by biochemical DNMT assays under identical conditions (500 nM enzyme, 0.7 μM AdoMet, 400 nM hemimethylated oligo). Each data

point represents the mean ( SD of three measurements, and the data were analyzed by SigmaPlot version 12.0.

Following the above considerations, we then selected deriva-

tives 7b, 7f, 7i, 8f, 8i, and 8j among the most active compounds

and carried out doseꢀresponse assay to generate curves from

which the corresponding IC₅₀values were obtained (Figure 3).

In agreement with results in Table 1, the nitrophenyl isoxazoline

7b showed the highest inhibition of DNMT1 with an IC₅₀value

of 150 μM, whereas its amino- counterpart 7f as well as the

aminophenyl- and the nitrobenzyl-substituted pyrrolidinoisoxa-

zolines 8f and 8i were 2- to 4-fold less potent (IC₅₀values of 270,

570, and 310 μM, respectively). Both the nitrobenzyl isoxazo-

lines 7i and the methoxybenzyl pyrrolidinoisoxazoline 8j were

consistently less active (IC₅₀values of 1600 and 1130 μM,

respectively). SAH was used as a nonspeciﬁc positive control

and conﬁrmed its eﬃcient inhibition of DNMT1 (IC₅₀of 4 μM,

doseꢀresponse curve not shown).

inhibition can be evaluated by holding the inhibitor constant at

its IC₅₀concentration and varying the substrates. The behavior of

the curves obtained under these conditions will point to the

mode of inhibition. For DNMTs, these substrates are the methyl

group donor SAM and the DNA (represented by a short oligo)

and each of them binds to its distinct binding site in the catalytic

domain of DNMT1. Therefore, the concentration of 7b was kept

constant at 150 μM (IC₅₀value, Figure 3), and either the SAM or

the oligo substrate was varied (Figure 4). It can be seen that

increasing the SAM concentration can nearly completely relieve

the enzyme inhibition, whereas this is not affected by increasing

the oligo substrate. According to the model,⁷⁸this is consistent

with an inhibitor that is competitive with respect to SAM and

noncompetitive with respect to the oligo.

Inhibition of Cell Proliferation. Finally, we explored the effect

of compound 7b on the proliferation of HCT116 human colon

carcinoma cell line. To this end, cells were incubated for 72 h with

increasing concentrations (300, 500, and 750 μM) of 7b in

comparison to azacytidine (1 μM) or DMSO alone for control.

The proliferation was assessed by counting viable cells after trypan

blue staining. As shown in Figure 5, in these conditions compound

7b induces a strong and dose-dependent antiproliferative effect,

Competition Assays. As derivative 7b emerged as the most

promising candidate for further development, we decided to gain

a better understanding of the kinetic mechanism(s) for the

observed inhibition of DNMT1. Because mutational analyses

are long-lasting and may affect the enzyme conformation, we

applied a simple method reported by Lai and Wu for assessing

the mode of inhibition.⁷⁸According to this method, the mode of

7667

dx.doi.org/10.1021/jm2010404 |J. Med. Chem. 2011, 54, 7663–7677

Journal of Medicinal Chemistry

ARTICLE

Figure 4. Derivative 7b competes with the SAM binding site. The

catalytic domain of DNMT1 has two binding sites, one for the oligo

substrate and one for the SAM substrate. A modiﬁed biochemical

DNMT activity assay was established to determine the mode of action

of 7b. Competition experiments were performed by increasing the

concentration of the oligo substrate (oligo varied) or, alternatively, the

SAM substrate (SAM varied). The assay was performed under standard

conditions; please note that DNMT1 concentration was always kept

constant (500 nM) as well as the inhibitor (7b) concentration (150 μM).

The initial enzymatic activity under standard conditions in presence of the

inhibitor was deﬁned as 100% inhibition before increasing substrate

concentrations to look for competition events. According to the model

of Lai and Wu (see text), 7b competes with SAM for the cofactor binding

site but not for the oligo-binding site.

Figure 6. (A) Comparison of the binding modes of the R (green) and S

(yellow) conﬁgurations of 7b predicted by Glide. Crystallographic SAH

(gray) is shown as reference. Hydrogen bonds for R-7b are displayed as

magenta dashes. Selected amino acids residues within 4.5 Å of 7b are

shown. Nonpolar hydrogen atoms are omitted for clarity. (B) Two-

dimensional interaction map displaying amino acid residues within 4.5 Å

of R-7b. The ligand proximity contour is depicted with a dotted line. The

ligand solvent exposure is represented with blue circles; larger and

darker circles on ligand atoms indicate more solvent exposure. The

receptor solvent exposure diﬀerences, in the presence and absence of the

ligand, are represented by the size and intensity of the turquoise discs

surrounding the residues; larger and darker discs indicate residues highly

exposed to solvent in the active site when the ligand is absent. Figure

created with the Ligand Interactions application of MOE.

Figure 5. Derivative 7b induces a strong antiproliferative eﬀect after

72 h in HCT116 human colon carcinoma cells compared to DMSO

treated cells. Azacytidine was included as a positive control. Ctrl are

DMSO treated cells. GI₅₀value was determined by SigmaPlot 12.0.

with a GI₅₀value (the concentration of the compound thatinhibits

cell growth by 50%, calculated by SigmaPlot version 12.0, Systat

Software Inc., San Jose, CA) of 360 μM. In the same assay,

derivative 5b does not affect the proliferation rate/doubling time

(not shown).

Molecular Modeling Studies. On the basis of the results of

the competition assay discussed above, the binding mode of 7b

into the binding pocket of the cofactor of human DNMT1 was

analyzed using molecular modeling studies. Molecular docking

and other computational approaches are increasingly being

used to explore the ligand-binding interactions of DNMT in-

hibitors.^79ꢀ81Until now, molecular modeling studies have

been conducted using validated homology models of the methyl-

transferase domain of DNMT1.^79,80,82However, a crystallo-

graphic structure of human DNMT1 has been recently pub-

lished⁷⁵which contains the methyltransferase domain bound

to DNA-containing unmethylated CpG sites. On the basis of the

experimental results of the competition assays, the binding of 7b

into the binding site of the cofactor was further explored using

molecular docking. The docking protocol is presented in the

Experimental Section. Briefly, the R and S configurations of 7b in

neutral and protonated forms (at the amino group) were flexibly

docked into the crystallographic structure of the methyltransfer-

ase domain of human DNMT1 using Glide Extra Precision (XP),

version 5.7.^83,84Before docking 7b, we tested the docking

protocol for its ability to reproduce the experimental binding

mode of the cocrystallized SAH.

To this end, SAH bound to the crystal structure was removed

from the binding pocket and docked back into the cofactor

7668

dx.doi.org/10.1021/jm2010404 |J. Med. Chem. 2011, 54, 7663–7677

Journal of Medicinal Chemistry

ARTICLE

binding site. The root-mean-square deviation between the pre-

dicted conformation and the observed X-ray crystallographic

conformation was 1.5 Å, indicating the capability of the docking

protocol to reproduce the binding mode of SAH.

The calculated docking scores of the R and S conﬁgurations of

7b in protonated form were ꢀ4.0 and ꢀ3.5 kcal/mol, respec-

tively. The docking score of the neutral form of each conﬁgura-

tion was ꢀ3.0 kcal/mol. These results suggest that 7b binds to

the cofactor binding site in the protonated form. The docking

scores of 7b were higher, i.e. less favorable, than the calculated

docking score for SAH, which was ꢀ7.6 kcal/mol. The docking

scores are in agreement with the results of the experimental

DNMT1 inhibition assays, which shows that SAH is a more

potent inhibitor than 7b (see above).

Figure 6a shows a tridimensional binding model of the

protonated R and S conﬁgurations (carbon atoms in green and

yellow, respectively). The structure of cocrystallized SAH is

shown as a reference (carbon atoms in gray).

As shown, Glide XP found a very similar binding mode for

both R and S conﬁgurations. Together with the comparable

docking energies calculated for the two conﬁgurations (see

above), this hints that both enantiomers have similar enzymatic

inhibitory activity. A two-dimensional representation of the

binding mode of R-7b is shown in Figure 6b.

According to the derived docking model, 7b occupies the

binding site of the cofactor which is created by residues from the

motifs IꢀIII and X of the methyltransferase domain.⁸⁵Com-

pound 7b binds in the region where the ^L-homocysteine and

ribose moieties of SAH bind. The Δ²-isoxazoline ring makes

contacts with the side chain of Asn1578 and the backbone of

Phe1145. The phenyl rings makes hydrophobic interactions

mainly with Gly1147, Gly1223, Ala1579, and the backbone

atoms of Ser1146 and Asn1578. The positively charged amino

group forms a hydrogen bond with the side chain of Glu1168,

which is located in motif II. Of note, two oxygen atoms (O2⁰and

O3⁰) of the ribose ring of SAH also form hydrogen bond

interactions with the side chain of Glu1168. Similar hydrogen

bond interactions with the equivalent glutamic acid residue are

observed in other methyltransferases, e.g. Glu40 in the crystal

structure of M.HhaI.⁸⁶

already used as a local anesthetic and an antiarrhythmic drug,

respectively, exhibit a weak DNA demethylating activity (even if at

high drug concentrations) and are “repositionable” as non-nucleo-

side inhibitors. Following on our previous studies that led to the

identiﬁcation of a constrained analogue (5b) of procaine as a lead

compound for the discovery of non-nucleoside inhibitors of

DNMT1, we herein describe the preparation of a series of Δ²-

isoxazoline analogues (7aꢀk and 8aꢀk) and their inhibitory

activity toward DNMT1. While both procaine and 2 (see above)

were inactive in this assay, a number of tested compounds show a

clear inhibition of the enzyme activity. In particular, one of them,

namely derivative 7b, showed the highest inhibition of DNMT1

withanIC₅₀value of 150 μM, noteworthy nearly10-foldimproved

compared to procaine or constrained analogue 5b. Taking advan-

tage of a simple method reported by Lai and Wu,⁷⁸we assessed

that the mode of inhibition of 7b is consistent with a competition

with the cofactor. Of note, these experiments represent one of the

ﬁrst eﬀorts to elucidate experimentally the binding site of a non-

nucleoside inhibitor of DNMT1. On the basis of the experimental

evidence of the competition assays, we conducted molecular

docking of 7b into the binding site of the cofactor in order to

explore its binding orientation and conformation. Docking simula-

tions were conducted with the R and S conﬁgurations using the

recently published crystallographic structure of the methyltrans-

ferase domain of human DNMT1.⁷⁵To our knowledge, this is the

ﬁrst molecular modeling study reported with the crystallographic

structure of human DNMT1. Both enantiomers showed compar-

able binding energy and a very similar binding mode. According to

the bindingmodel, compound 7bbindsinthesamebinding region

of the ^L-homocysteine and ribose moieties of SAH and makes

several hydrogen bonding interactions with amino acid residues

common to SAH. Interestingly, these outcomes seem to exclude a

direct interaction of 7b with DNA, thus diﬀerentiating the

mechanism of inhibition of this compound from those proposed

for procaine⁵¹or procainamide.⁵²As a matter of fact, whereas

relatively high concentrations of these drugs are required for their

inhibition of DNA methylation, derivative 7b is far more potent in

vitro (IC₅₀= 150 μM) and also exhibits a strong and dose-

dependent antiproliferative eﬀect against HCT116 human colon

carcinoma cells. On the basis of these ﬁndings, we propose 7b as a

novel lead compound for the development of non-nucleoside

DNMT inhibitors. As it competes with SAM for the binding site, a

proper derivatization of this scaﬀold could lead to longer “bi-

substrate” inhibitors, able to occupy both DNA and SAM binding

pockets. Indeed, further studies are ongoing to develop analogues

endowed with improved enzyme binding properties and higher

inhibitory potency.

The nitro group of 7b occupies a region similar to the

carboxylate group of SAH making similar interactions with the

binding pocket. Hydrogen bonds between the oxygen atoms of

the nitro group of 7b and the backbone NH of Gly1150 (motif I),

Leu1151, and Val1580 (motif X) were also predicted by Glide

(Figure 6).^75,86

’ CONCLUSION

’ EXPERIMENTAL SECTION

The inactivation of tumor suppressor genes, which often results

from epigenetic silencing associated with DNA hypermethylation,

plays a pivotal role in the development of most forms of human

cancer. Functional knockdown of DNMT1 is reported to reduce

the genomic methylation patterns by approximately 93%.⁸⁷More-

over, complete knock out of DNMT1 led to mitotic catastrophe in

HCT116 cells,⁸⁸indicating the interdependency of genomic

methylation and cell viability but also substantiating the impor-

tance of targeting DNMT1 in cancer chemotherapy. Nucleoside

analogues of cytosine (e.g., 5-azacytidine) eﬀectively inhibit the

activity of DNA methyltransferases, but their high cytotoxicity and

the low therapeutic index make the development of novel non-

nucleoside inhibitors highly desirable. Procaine and procainamide,

Chemistry. All chemicals were purchased from Sigma Aldrich (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 anhy-

drous 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

7669

dx.doi.org/10.1021/jm2010404 |J. Med. Chem. 2011, 54, 7663–7677

Journal of Medicinal Chemistry

ARTICLE

performed on silica gel (silica gel 60, 0.015ꢀ0.040 mm; Merck DC)

5-Hydroxymethyl-3-(4-methoxyphenyl)-4,5-dihydroisoxazole (10c).

1

columns. Melting points were determined on a Stuart SMP30 melting

White solid, 83% yield; mp (EtOAc) 144ꢀ146 ꢀC. H NMR (300

1

point apparatus in open capillary tubes and are uncorrected. H NMR

MHz, CDCl₃): δ 7.60 (d, J = 8.7 Hz, 2H), 6.92 (d, J = 8.7 Hz, 2H),

4.89ꢀ4.79 (m, 1H), 3.89ꢀ3.82 (m, 4H), 3.68 (dd, J = 12.0, 3.7 Hz, 1H),

3.37 (dd, J = 16.8, 10.7 Hz, 1H), 3.25 (dd, J = 16.8, 7.7 Hz, 1H), 1.80 (br s,

1H). ESI-MS m/z: 208 (M + H) ⁺.

5-Hydroxymethyl-3-(4-(tosyloxy)phenyl)-4,5-dihydroisoxazole (10d).

White solid, 83% yield; mp (i-PrOH) 85ꢀ87 ꢀC. ¹H NMR (300 MHz,

CDCl₃): δ7.68 (d, J = 8.0 Hz, 2H), 7.56 (d, J = 8.0 Hz, 2H), 7.30 (d, J = 8.0

Hz, 2H), 7.00 (d, J = 8.0 Hz, 2H), 4.90ꢀ4.79 (m, 1H), 3.84 (dd, J = 12.4,

3.3 Hz, 1H), 3.65 (dd, J = 12.4, 4.4 Hz, 1H), 3.32 (dd, J = 16.8, 11.0 Hz,

1H), 3.22 (dd, J = 16.8, 7.7 Hz, 1H), 2.43 (s, 3H), 2.18 (br s, 1H). ESI-MS

m/z: 348 (M + H) ⁺.

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). When compounds were tested as salts, NMR

data refer to the free base. 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

g95%. 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.

General Procedure for Synthesis of Oximes (Compounds

9aꢀd, 9hꢀj, 9l). To a suspension of the proper aldehyde (30.0 mmol)

and hydroxylamine hydrochloride (2.29 g, 33.0 mmol) in a 1:1 mixture

of H₂O/methanol (40 mL), an aqueous solution of Na₂CO₃(1.59 g,

15.0 mmol, 20 mL) was slowly added. The resulting mixture was stirred

at room temperature for 3 h, and then methanol was evaporated. The

aqueous phase was extracted with Et₂O (3 ꢁ 30 mL). The combined

organic phases were washed with brine (30 mL), dried (Na₂SO₄),

filtered, and concentrated in vacuo to give the title compounds 9, which

were used in the next step without further purification. Yields, physical,

and spectral data of compounds are reported in Supporting Information.

General Procedure for Synthesis of Chlorooximes

(Compounds 12aꢀd, 12hꢀj, 12l). To a solution of the proper

aldoxime (2.75 mmol) in CHCl₃(10 mL), pyridine was added

(20 μL, 0.27 mmol). The reaction mixture was heated at 40 ꢀC, and

N-chlorosuccinimide (405 mg, 3.03 mmol) was added portionwise.

After the reaction was complete (0.5ꢀ3 h, monitored by TLC), the

mixture was diluted with CH₂Cl₂(30 mL) and washed with brine

(3 ꢁ 10 mL). The organic phase was dried (Na₂SO₄), filtered, and

concentrated in vacuo to give the title compounds 12, which were

used in the cycloaddition step without further purification. Yields,

physical, and spectral data of compounds are reported in Support-

ing Information.

3-Benzyl-5-hydroxymethyl-4,5-dihydroisoxazole (10h). Pale-yellow

1

oil, 80% yield. H NMR (300 MHz, CDCl₃): δ 7.40ꢀ7.15 (m, 5H),

4.70ꢀ4.58 (m, 1H), 3.71 (dd, J = 12.1, 3.3 Hz, 1H), 3.67 (s, 2H), 3.51

(dd, J = 12.1, 5.0 Hz, 1H), 2.85 (dd, J = 17.1, 10.5 Hz, 1H), 2.71 (dd, J =

17.1, 7.7 Hz, 1H), 2.10 (br s, 1H). ESI-MS m/z: 192 (M + H) ⁺.

5-Hydroxymethyl-3-(4-nitrobenzyl)-4,5-dihydroisoxazole (10i). Yellow

solid, 93% yield; mp (EtOAc/hexane) 69ꢀ71 ꢀC. ¹H NMR (300 MHz,

CDCl₃): δ 8.20 (d, J = 8.5 Hz, 2H), 7.42 (d, J =8.5 Hz, 2H), 4.74ꢀ4.66 (m,

1H), 3.89ꢀ3.70 (m, 3H), 3.53 (dd, J = 12.2, 4.1 Hz, 1H), 2.89 (dd, J = 17.6,

10.7 Hz, 1H), 2.79 (dd, J = 17.6, 7.5 Hz, 1H), 1.92 (br s, 1H). ESI-MS m/z:

237 (M + H)⁺.

5-Hydroxymethyl-3-(4-methoxybenzyl)-4,5-dihydroisoxazole (10j).

White solid, 78% yield; mp (isopropyl ether) 70ꢀ71 ꢀC. ¹H NMR (300

MHz, CDCl₃): δ 7.11 (d, J = 8.5 Hz, 2H), 6.82 (d, J = 8.5 Hz, 2H),

4.66ꢀ4.54 (m, 1H), 3.76 (s, 3H), 3.66 (dd, J = 12.3, 3.2 Hz, 1H), 3.58 (s,

2H), 3.48 (dd, J = 12.3, 4.6 Hz, 1H), 2.82 (dd, J = 17.3, 11.0 Hz, 1H), 2.68

(dd, J = 17.3, 7.8. Hz, 1H), 2.58 (br s, 1H). ESI-MS m/z: 222 (M + H) ⁺.

3-(4-tert-Butyloxycarbonylphenyl)-5-hydroxymethyl-4,5-dihydroi-

1

soxazole (10l). White solid, 68% yield; mp (i-PrOH) 89ꢀ91 ꢀC. H

NMR (300 MHz, CDCl₃): δ 7.99 (d, J = 8.8 Hz, 2H), 7.69 (d, J = 8.8 Hz,

2H), 4.96ꢀ4.84 (m, 1H), 3.90 (dd, J = 12.4, 3.0 Hz, 1H), 3.70 (dd, J =

12.4, 4.1 Hz, 1H), 3.40 (dd, J = 16.8, 10.7 Hz, 1H), 3.30 (dd, J = 16.8, 8.2

Hz, 1H), 2.20 (br s, 1H), 1.60 (s, 9H). ESI-MS m/z: 278 (M + H) ⁺.

General Procedure for Synthesis of (3-Substituted-4,5-di-

hydroisoxazol-5-yl)methyl Methanesulfonate (Compounds

11aꢀd, 11hꢀj, 11l). To a solution of the proper (3-substituted-4,5-

dihydroisoxazol-5-yl)methanol 10 (1.66 mmol) and triethylamine

(0.28 mL, 2.00 mmol) in dry CH₂Cl₂(20 mL), methanesulfonyl chloride

(0.16 mL, 2.00mmol) wasaddedat 0 ꢀC. Theresulting mixturewas stirred

at room temperature for 45 min, and then the solvent was evaporated. The

residue was taken up with water (20 mL) and extracted with CH₂Cl₂(3 ꢁ

20 mL). The combined organic phases were washed with 1N HCl (2 ꢁ

10 mL), NaHCO₃, saturated aqueous solution (2 ꢁ 10 mL), and brine

(10 mL), dried (Na₂SO₄), filtered, and concentrated in vacuo. Mesylates

11 were obtained in quantitative yields and used without further purifica-

tion in the subsequent reaction. Recrystallization from the suitable solvent

gave an analytical sample.

General Procedure for Synthesis of 3-Substituted-5-hy-

droxymethyl-4,5-dihydroisoxazoles (Compounds 10aꢀd,

10hꢀj, 10l). To a vigorously stirred solution of the proper aldoxime

(7.0 mmol) and allylic alcohol (0.81 mg, 0.95 mL, 14.0 mmol) in

CH₂Cl₂(50 mL) at 0 ꢀC, a solution of common bleach (3% aqueous

solution of NaOCl, 35 mL, 14.0 mmol) was added dropwise, keeping the

temperature below 5 ꢀC. The resulting biphasic mixture was vigorously

stirred at room temperature for 15 min. The aqueous phase was

separated and extracted with CH₂Cl₂(3 ꢁ 20 mL). The combined

organic phases were washed with brine (25 mL), dried (Na₂SO₄),

filtered, and concentrated in vacuo. The residue was purified by silica gel

chromatography (EtOAc/hexane) to give the title compounds 10,

which were recrystallized from the appropriate solvent.

5-Hydroxymethyl-3-phenyl-4,5-dihydroisoxazole Methanesulfonate

1

(11a). White solid; mp (i-PrOH) 109ꢀ111 ꢀC. H NMR (300 MHz,

CDCl₃): δ 7.70ꢀ7.60 (m, 2H), 7.42ꢀ7.38 (m, 3H), 5.06ꢀ4.96 (m, 1H),

4.39 (dd, J = 11.3, 3.9 Hz, 1H), 4.27 (dd, J = 11.3, 5.0 Hz, 1H), 3.45 (dd, J

= 17.0, 11.0 Hz, 1H), 3.30 (dd, J = 17.0, 7.2 Hz, 1H), 3.09 (s, 3H). ESI-MS

m/z: 256 (M + H) ⁺.

5-Hydroxymethyl-3-phenyl-4,5-dihydroisoxazole (10a). White so-

1

lid, 87% yield; mp (EtOAc/hexane) 83ꢀ84 ꢀC. H NMR (300 MHz,

CDCl₃): δ 7.72ꢀ7.60 (m, 2H), 7.45ꢀ7.35 (m, 3H), 4.94ꢀ4.81 (m,

1H), 3.88 (ddd, J = 12.1, 6.3, 3.3 Hz, 1H), 3.68 (ddd, J = 12.1, 6.3, 4.7 Hz,

1H), 3.39 (dd, J = 16.5, 10.4 Hz, 1H), 3.29 (dd, J = 16.5, 8.0 Hz, 1H),

2.09 (t, J = 6.3 Hz, 1H). ESI-MS m/z: 178 (M + H) ⁺.

5-Hydroxymethyl-3-(4-nitrophenyl)-4,5-dihydroisoxazole Methanesul-

fonate (11b). White solid; mp: (MeOH/H₂O) 171ꢀ173 ꢀC. ¹H NMR

(300 MHz, CDCl₃): δ 8.29 (d, J = 8.8 Hz, 2H), 7.84 (d, J = 8.8 Hz, 2H),

5.19ꢀ5.06 (m, 1H), 4.45 (dd, J = 11.3, 4.1 Hz, 1H), 4.39 (dd, J = 11.3, 4.7

Hz, 1H), 3.54 (dd, J = 16.8, 11.0 Hz, 1H), 3.36 (dd, J = 16.8, 7.2 Hz, 1H),

3.09 (s, 3H). ESI-MS m/z: 301 (M + H) ⁺.

5-Hydroxymethyl-3-(4-nitrophenyl)-4,5-dihydroisoxazole (10b).

1

Pale-yellow solid, 82%; mp (methanol) 141ꢀ143 ꢀC. H NMR (300

MHz, CDCl₃): δ 8.30 (d, J = 8.4 Hz, 2H), 7.84 (d, J = 8.4 Hz, 2H),

5.02ꢀ4.92 (m, 1H), 3.96 (ddd, J = 12.1, 4.4, 3.6 Hz, 1H), 3.72 (ddd, J =

12.1, 7.7, 4.4 Hz, 1H), 3.42 (dd, J = 16.5, 10.5, 1H), 3.36 (dd, J = 16.5, 8.5

Hz, 1H), 1.92ꢀ1.84 (m, 1H). ESI-MS m/z: 223 (M + H) ⁺.

5-Hydroxymethyl-3-(4-methoxyphenyl)-4,5-dihydroisoxazole Metha-

1

nesulfonate (11c). White solid; mp: (EtOAc) 146ꢀ148 ꢀC. H NMR

7670

dx.doi.org/10.1021/jm2010404 |J. Med. Chem. 2011, 54, 7663–7677

Journal of Medicinal Chemistry

ARTICLE

(300 MHz, CDCl₃): δ 7.60 (d, J = 8.8 Hz, 2H), 6.93 (d, J = 8.8 Hz, 2H),

5.04ꢀ4.94 (m, 1H), 4.40 (dd, J = 11.3, 4.1 Hz, 1H), 4.34 (dd, J = 11.3, 5.0

Hz, 1H), 3.85 (s, 3H), 3.48 (dd, J = 16.8, 11.0 Hz, 1H), 3.28 (dd, J = 16.8,

7.2 Hz, 1H), 3.09 (s, 3H). ESI-MS m/z: 286 (M + H) ⁺.

¹H NMR (300 MHz, CDCl₃): δ 7.61 (d, J = 8.8 Hz, 2H), 6.91 (d, J = 8.8

Hz, 2H), 4.93ꢀ4.80 (m, 1H), 3.83 (s, 3H), 3.40 (dd, J = 16.7, 10.5 Hz,

1H), 3.14 (dd, J = 16.7, 7.6 Hz, 1H), 2.63 (dd, J = 12.9, 6.7 Hz, 1H), 2.55

(dd, J = 12.9, 5.6 Hz, 1H), 2.35 (s, 6H). ESI-MS m/z: 235 (M + H) ⁺.

Anal. (C₁₃H₁₈N₂O₂) C, H, N.

5-(N,N-Dimethylaminomethyl)-3-(4-(tosyloxy)phenyl)-4,5-dihydroisox-

azole (7d).White solid, 78% yield; mp (2-propanol) 123ꢀ124 ꢀC. ¹HNMR

(300MHz, CDCl₃):δ7.70 (d, J= 8.2 Hz, 2H), 7.57 (d, J= 8.2 Hz, 2H), 7.31

(d, J = 8.2 Hz, 2H), 7.00 (d, J = 8.2 Hz, 2H), 4.93ꢀ4.82 (m, 1H), 3.32 (dd, J

= 16.8, 10.5 Hz, 1H), 3.12 (dd, J = 16.8, 8.0 Hz, 1H), 2.60 (dd, J = 12.9, 6.3

Hz, 1H), 2.49 (dd, J = 12.9, 6.3 Hz, 1H), 2.43 (s, 3H), 2.31 (s, 6H). ESI-MS

m/z: 375 (M + H) ⁺. Anal. (C₁₉H₂₂N₂O₄S) C, H, N.

3-Benzyl-5-(N,N-dimethyl-aminomethyl)-4,5-dihydroisoxazole Hy-

drochloride (7h). White solid, 76% yield; mp (EtOAc) 80ꢀ82 ꢀC; free

base. ¹H NMR (300 MHz, CDCl₃): δ 7.35ꢀ7.22 (m, 5H), 4.70ꢀ4.60

(m, 1H), 3.68 (s, 2H), 2.86 (dd, J = 17.0, 10.4 Hz, 1H), 2.60 (dd, J =

17.0, 8.0 Hz, 1H), 2.50 (dd, J = 12.8, 6.5 Hz, 1H), 2.33 (dd, J = 12.8,

5.7 Hz, 1H), 2.25 (s, 6H). ESI-MS m/z: 219 (M + H) ⁺. Anal.

5-Hydroxymethyl-3-(4-(tosyloxy)phenyl)-4,5-dihydroisoxazole Metha-

1

nesulfonate (11d). White solid; mp: (i-PrOH) 123ꢀ125 ꢀC. H NMR

(300 MHz, CDCl₃): δ 7.69 (d, J = 8.5 Hz, 2H), 7.57 (d, J = 8.5 Hz, 2H),

7.32 (d, J = 8.5 Hz, 2H), 7.02 (d, J = 8.5 Hz, 2H), 5.07ꢀ4.95 (m, 1H), 4.38

(dd, J = 11.6, 3.9 Hz, 1H), 4.31 (dd, J = 11.6, 3.9 Hz, 1H), 3.44 (dd, J = 17.1,

11.0 Hz, 1H), 3.24 (dd, J = 17.1, 7.1 Hz, 1H), 3.06 (s, 3H), 2.44 (s, 3H).

ESI-MS m/z: 426 (M + H) ⁺.

3-Benzyl-5-hydroxymethyl-4,5-dihydroisoxazole Methanesulfonate

1

(11h). White solid; mp (i-PrOH): 43ꢀ44 ꢀC. H NMR (300 MHz,

CDCl₃): δ 7.38ꢀ7.20 (m, 5H), 4.82ꢀ4.72 (m, 1H), 4.22 (dd, J = 11.3,

3.9 Hz, 1H), 4.15 (dd, J = 11.3, 5.0 Hz, 1H), 3.66 (s, 2H), 2.98 (s, 3H),

2.96 (dd, J = 17.6, 10.9 Hz, 1H), 2.71 (dd, J = 17.6, 7.0 Hz, 1H). ESI-MS

m/z: 270 (M + H) ⁺.

5-Hydroxymethyl-3-(4-nitrobenzyl)-4,5-dihydroisoxazole Methanesul-

fonate (11i). Pale-yellow solid; mp (i-PrOH): 107ꢀ109 ꢀC. ¹HNMR(300

MHz, CDCl₃): δ 8.24 (d, J = 8.8 Hz, 2H), 7.45 (d, J = 8.8 Hz, 2H),

4.92ꢀ4.81 (m, 1H), 4.29 (dd, J = 11.3, 3.8 Hz, 1H), 4.21 (dd, J = 11.3, 4.4

Hz, 1H), 3.83 (s, 2H), 3.08 (s, 3H), 3.02 (dd, J = 17.4, 10.8 Hz, 1H). 2.82

(dd, J = 17.4, 6.8 Hz, 1H). ESI-MS m/z: 315 (M + H) ⁺.

(C₁₃H₁₈N₂O HCl) C, H, N.

3

5-(N,N-Dimethyl-aminomethyl)-3-(4-nitrobenzyl)-4,5-dihydroisox-

azole Hydrochloride (7i). White solid, 79% yield; mp (EtOH/Et₂O)

164ꢀ167 ꢀC; free base. ¹H NMR (300 MHz, CDCl₃): δ 8.20 (d, J = 8.8

Hz, 2H), 7.42 (d, J = 8.8 Hz, 2H), 4.78ꢀ4.66 (m, 1H), 3.78 (s, 2H), 2.89

(dd, J = 17.0, 10.4 Hz, 1H), 2.65 (dd, J = 17.0, 8.0 Hz, 1H), 2.52 (dd, J =

12.9, 6.5 Hz, 1H), 2.40 (dd, J = 12.9, 5.5 Hz, 1H), 2.27 (s, 6H). ESI-MS

5-Hydroxymethyl-3-(4-methoxybenzyl)-4,5-dihydroisoxazole Metha-

1

nesulfonate (11j). White solid; mp (i-PrOH): 104ꢀ105 ꢀC. H NMR

(300 MHz, CDCl₃): δ 7.12 (d, J = 8.7 Hz, 2H), 6.83 (d, J = 8.7 Hz, 2H),

4.83ꢀ4.72 (m, 1H), 4.24 (dd, J = 11.3, 3.9 Hz, 1H), 4.17 (dd, J = 11.3, 5.0

Hz, 1H), 3.79 (s, 3H), 3.62 (s, 2H), 3.03 (s, 3H), 2.96 (dd, J = 17.3, 11.0

Hz, 1H), 2.72 (dd, J = 17.3, 7.1 Hz, 1H). ESI-MS m/z: 300 (M + H) ⁺.

3-(4-tert-Butyloxycarbonylphenyl)-5-hydroxymethyl-4,5-dihydroi-

soxazole Methanesulfonate (11l). White solid; mp (i-PrOH):

m/z: 264 (M + H) ⁺. Anal. (C₁₃H₁₇N₃O₃HCl) C, H, N.

3

5-(N,N-Dimethylaminomethyl)-3-(4-methoxybenzyl)-4,5-dihydroi-

soxazole Hydrochloride (7j). White solid, 79% yield; mp (acetone/Et₂O)

126ꢀ128 ꢀC; free base. ¹H NMR (300 MHz, CDCl₃): δ 7.14 (d, J = 8.5

Hz, 2H), 6.86 (d, J = 8.5 Hz, 2H), 4.69ꢀ4.59 (m, 1H), 3.79 (s, 3H), 3.61

(s, 2H), 2.86 (dd, J = 16.9, 10.3 Hz, 1H), 2.59 (dd, J = 16.9, 8.0 Hz, 1H),

2.49 (dd, J = 12.8, 6.6 Hz, 1H), 2.33 (dd, J = 12.8, 5.7 Hz, 1H), 2.25 (s,

1

128ꢀ129 ꢀC. H NMR (300 MHz, CDCl₃): δ 8.02 (d, J = 8.8 Hz,

2H), 7.68 (d, J = 8.8 Hz, 2H), 5.12ꢀ4.99 (m, 1H), 4.42 (dd, J = 11.7, 4.1

Hz, 1H), 4.35 (dd, J = 11.7, 5.0 Hz, 1H), 3.52 (dd, J = 17.0, 11.1 Hz, 1H),

3.30 (dd, J = 17.0, 7.3 Hz, 1H), 3.08 (s, 3H), 1.60 (s, 9H). ESI-MS m/z:

356 (M + H) ⁺.

6H). ESI-MS m/z: 249 (M + H) ⁺. Anal. (C₁₄H₂₀N₂O₂HCl) C, H, N.

3

5-(N,N-Dimethylaminomethyl)-3-(4-tert-butyloxycarbonylphenyl)-

4,5-dihydroisoxazole (7l). White solid, 81% yield; mp (i-PrOH) 75ꢀ76 ꢀC.

¹H NMR (300 MHz, CDCl₃): δ 8.00 (d, J = 8.8 Hz, 2H), 7.70 (d, J = 8.8

Hz, 2H), 4.98ꢀ4.84 (m, 1H), 3.41 (dd, J = 16.7, 10.6 Hz, 1H), 3.18 (dd, J =

16.7, 8.2 Hz, 1H), 2.63 (dd, J = 12.9, 6.2 Hz, 1H), 2.52 (dd, J = 12.9, 6.2 Hz,

1H), 2.32 (s, 6H), 1.60 (s, 9H). ESI-MS m/z: 305 (M + H) ⁺.

General Procedure for Synthesis of 3-Substituted-5-(N,N-

dimethylaminomethyl)-4,5-dihydroisoxazoles (Compounds

7aꢀd, 7hꢀj, 7l). The proper mesylate (1.30 mmol) was dissolved in a

2 M THF solution of dimethylamine (20 mL, 39.0 mmol) under N₂

atmosphere. The vessel was sealed, and the reaction was stirred at 100 ꢀCfor

12 h and then concentrated in vacuo. The residue was purified by silica gel

chromatography (EtOAc/hexane) to give the title compounds. Com-

pounds 7c,d and 7l were recrystallized from the appropriate solvent.

Compounds 7a,b, and 7hꢀj were dissolved in 4N HCl in dioxane, and

the mixture was stirred at room temperature for 10 min. The solvent was

concentrated in vacuo, and the resulting solid was recrystallized from the

appropriate solvent to yield title compounds as hydrochloride salts.

5-(N,N-Dimethylaminomethyl)-3-phenyl-4,5-dihydroisoxazole Hydro-

chloride (7a). White solid, 79% yield; mp (2-propanol) 188ꢀ190 ꢀC; free

base. ¹H NMR (300 MHz, CDCl₃): δ 7.62ꢀ7.51 (m, 2H), 7.35ꢀ7.23 (m,

3H), 4.86ꢀ4.72 (m, 1H), 3.30 (dd, J = 16.5, 10.5 Hz, 1H), 3.05 (dd, J =

16.5, 8.0 Hz, 1H), 2.53 (dd, J = 12.7, 6.6 Hz, 1H), 2.46 (dd, J = 12.7, 5.2 Hz,

Synthesis of 5-(N,N-Dimethylaminomethyl)-3-(4-hydro-

xyphenyl)-4,5-dihydroisoxazole Hydrochloride (7g). To a solu-

tion of 7d (110 mg, 0.29 mmol) in EtOH (5 mL) an aqueous solution of

1N NaOH (1.45 mL, 1.45 mmol) was added. The mixture was heated at

reflux and stirred for 1 h. The reaction mixture was then cooled to room

temperature, treated with HCl 1 N to pH 9, and concentrated in vacuo.

The residue was treated with water (30 mL) and extracted with EtOAc

(3 ꢁ 30 mL). The combined organic phases were washed with brine

(10 mL), dried (Na₂SO₄), filtered, and concentrated in vacuo to give the

title compound as a free base (61.2 mg, 96%), which was converted in

the corresponding hydrochloride salt following the general procedure

described for compounds 7a,b, and 7hꢀj; mp 232ꢀ235 ꢀC (decomp);

free base. ¹H NMR (300 MHz, DMSO-d₆): δ 9.90 (br s, 1H), 7.47 (d, J =

8.5 Hz, 2H), 6.80 (d, J = 8.5 Hz, 2H), 4.77ꢀ4.66 (m, 1H), 3.37 (dd, J =

16.9, 11.7 Hz, 1H), 3.07 (dd, J = 16.9, 8.0 Hz, 1H), 2.51ꢀ2.39 (m, 2H),

1H), 2.24 (s, 6H). ESI-MS m/z: 205 (M + H) ⁺. Anal. (C₁₂H₁₆N₂O HCl)

3

2.20 (s, 6H). ESI-MS m/z: 221 (M + H)⁺. Anal. (C₁₂H₁₆N₂O₂HCl)

C, H, N.

3

5-(N,N-Dimethylaminomethyl)-3-(4-nitrophenyl)-4,5-dihydroisox-

azole Hydrochloride (7b). White solid, 78% yield; mp (methanol)

262 ꢀC (decomp); free base. ¹H NMR (300 MHz, CDCl₃): δ 8.26 (d, J =

8.7 Hz, 2H), 7.84 (d, J = 8.7 Hz, 2H), 5.06ꢀ4.90 (m, 1H), 3.43 (dd, J =

16.7, 10.6 Hz, 1H), 3.24 (dd, J = 16.7, 8.1 Hz, 1H), 2.66 (dd, J = 12.8, 6.4

Hz, 1H), 2.55 (dd, J = 12.8, 6.0 Hz, 1H), 2.34 (s, 6H). ESI-MS m/z: 250

C, H, N.

Synthesis of 5-(N,N-Dimethylaminomethyl)-3-(4-carboxy-

phenyl)-4,5-dihydroisoxazole (7e). A mixture of TFA and DCM

(1:3, 12 mL) was added to compound 7l (314 mg, 1.03 mmol). The

reaction was stirred at room temperature overnight and then concen-

trated in vacuo to give the title compound as a white solid (256 mg,

99%), which was recrystallized from methanol/H₂O. White solid; mp

249ꢀ251 ꢀC (decomp). ¹H NMR (300 MHz, DMSO-d₆): δ 9.96 (br s,

1H), 8.0 (d, J = 8.2 Hz, 2H), 7.78 (d, J = 8.2 Hz, 2H), 5.25ꢀ5.15 (m,

(M + H) ⁺. Anal. (C₁₂H₁₅N₃O₃HCl) C, H, N.

3

5-(N,N-Dimethylaminomethyl)-3-(4-methoxyphenyl)-4,5-dihydroi-

soxazole (7c). White solid, 75% yield; mp (diisopropylether) 75ꢀ76 ꢀC.

7671

dx.doi.org/10.1021/jm2010404 |J. Med. Chem. 2011, 54, 7663–7677

Journal of Medicinal Chemistry

ARTICLE

1H), 3.80ꢀ3.63 (m, 1H), 3.50ꢀ3.23 (m, 3H), 2.84 (s, 6H). ESI-MS m/

z: 249 (M + H)⁺. Anal. (C₁₃H₁₆N₂O₃) C, H, N.

7.04 (d, J = 7.8 Hz, 2H), 5.34 (dd, J = 9.5, 5.1 Hz, 1H), 4.25ꢀ4.13 (m,

1H), 4.00 (d, J = 12.9 Hz, 1H), 3.85ꢀ3.55 (m, 3H), 2.46 (s, 3H), 1.42 (s,

9H). ESI-MS m/z: 403 (M + H - ^tBu)⁺.

Synthesis of 5-(N,N-Dimethylaminomethyl)-3-(4-ammino-

phenyl)-4,5-dihydroisoxazole Hydrochloride (7f). To an ice

cooled solution of 7b free base (160 mg, 0.64 mmol) in AcOH

(36 mL) zinc powder (336 mg, 5.13 mmol) was added. The resulting

mixture was stirred at room temperature for 1 h, the solid was filtered off,

and the solvent was evaporated. The residue was taken up with EtOAc

(20 mL) and washed with NaHCO₃saturated aqueous solution twice.

The organic phase was dried (Na₂SO₄), filtered, and concentrated in

vacuo. The residue was purified by silica gel chromatography (CH₂Cl₂/

MeOH) to give the title compound (121 mg, 63%), which was converted

in the corresponding hydrochloride salt following the general procedure

described for compounds 7a,b, and 7hꢀj. White solid; mp 268ꢀ270 ꢀC

(decomp); free base. ¹H NMR (300 MHz, CDCl₃): δ 7.47 (d, J = 8.8 Hz,

2H), 6.67 (d, J = 8.8 Hz, 2H), 4.86ꢀ4.76 (m, 1H), 3.87 (br s, 2H); 3.36

(dd, J = 16.5, 10.3 Hz, 1H), 3.11 (dd, J = 16.5, 7.8 Hz, 1H), 2.60 (dd, J =

12.8, 6.4 Hz, 1H), 2.50 (dd, J = 12.8, 6.1 Hz, 1H), 2.32 (s, 6H). ESI-MS

tert-Butyl3-Benzyl-6,6a-dihydro-3aH-pyrrolo[3,4-d]isoxazole-5(4H)-

1

carboxylate (13h)⁷⁶. Pale-yellow oil, 71% yield. H NMR (300 MHz,

CDCl₃): δ 7.21ꢀ7.37 (m, 5H), 5.08 (ddd, J = 9.1, 5.6, 1.5 Hz, 1H),

3.95ꢀ3.80 (m, 3H), 3.62ꢀ3.50 (m, 2H), 3.45 (dd, J = 13.5, 5.6 Hz, 1H),

3.29 (dd, J = 11.7, 8.5Hz, 1H), 1.45 (s, 9H). ESI-MSm/z: 325 (M + Na)⁺.

tert-Butyl 3-(4-Nitrobenzyl)-6,6a-dihydro-3aH-pyrrolo[3,4-d]iso-

xazole-5(4H)-carboxylate (13i)⁷⁶. Yellow solid, 67% yield; mp

(EtOAc/hexane) 108ꢀ110 ꢀC. ¹H NMR (300 MHz, CDCl₃): δ

8.20 (d, J = 8.5 Hz, 2H), 7.45 (d, J = 8.5 Hz, 2H), 5.12 (dd, J = 8.2, 4.8

Hz, 1H), 4.00ꢀ3.80 (m, 2H), 3.75ꢀ3.55 (m, 3H), 3.45 (dd, J = 12.6,

4.8 Hz, 1H), 3.32 (dd, J = 11.8, 8.2 Hz, 1H), 1.42 (s, 9H). ESI-MS

t

m/z: 292 (M + H ꢀ Bu)⁺.

tert-Butyl 3-(4-Methoxybenzyl)-6,6a-dihydro-3aH-pyrrolo[3,4-d]iso-

xazole-5(4H)-carboxylate (13j)⁷⁶. Pale-yellow oil, 64% yield. ¹H NMR

(300 MHz, CDCl₃): δ 7.15 (d, J = 8.5 Hz, 2H), 6.88 (d, J = 8.5 Hz, 2H),

5.07 (ddd, J = 9.1, 5.8, 1.6 Hz, 1H), 3.80 (s, 3H), 3.85ꢀ3.75 (m, 2H),

3.60ꢀ3.40 (m, 4H), 3.30 (dd, J = 11.8, 8.5 Hz, 1H), 1.45 (s, 9H). ESI-MS

m/z: 355 (M + Na)⁺.

m/z: 220 (M + H)⁺. Anal. (C₁₂H₁₇N₃O HCl) C, H, N.

3

5-(N,N-Dimethylaminomethyl)-3-(4-aminobenzyl)-4,5-di-

hydroisoxazole Hydrochloride (7k). Reduction of compound 7i

(154 mg, 0.58 mmol) according to the procedure used for 7f yielded the

free amine (102 mg, 75%), which was converted to the hydrochloride

salt following the general procedure described for compounds 7a,b, and

7hꢀj. White solid; mp (EtOH/Et₂O) 211ꢀ212 ꢀC (decomp); free

base. ¹H NMR (300 MHz, CDCl₃): δ 6.99 (d, J = 8.2 Hz, 2H), 6.62 (d,

J = 8.2 Hz, 2H), 4.79ꢀ4.46 (m, 1H), 3.75 (br s, 2H), 3.54 (s, 2H), 2.86

(dd, J = 17.0, 10.3 Hz, 1H), 2.58 (dd, J = 17.0, 10.3 Hz, 1H), 2.50 (dd, J =

12.8, 7.0 Hz, 1H), 2.35 (dd, J = 12.8, 5.5 Hz, 1H), 2.26 (s, 6H). ESI-MS

tert-Butyl 3-(4-(tert-Butoxycarbonyl)phenyl)-6,6a-dihydro-3aH-

pyrrolo[3,4-d]isoxazole-5(4H)-carboxylate (13l). White solid, 69%

yield; mp (EtOAc/hexane) 143ꢀ145 ꢀC. ¹H NMR (300 MHz, CDCl₃):

δ 8.02 (d, J = 8.2 Hz, 2H), 7.67 (d, J = 8.2 Hz, 2H), 5.34 (dd, J = 9.4, 5.6

Hz, 1H), 4.27ꢀ4.18 (m, 1 H), 4.00 (d, J = 12.9 Hz, 1H), 3.80ꢀ3.58 (m,

3H), 1.65 (s, 9H), 1.40 (s, 9H). ESI-MS m/z: 389 (M + H)⁺.

General Procedure for the Synthesis of 5-Methyl-3-sub-

stituted-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazoles

(Compounds 8aꢀd, 8hꢀj, 8l). A solution of the proper N-Boc-

protected derivative 13 (1.00 mmol) in 4 M HCl solution in dioxane

(5.0 mL, 20.0 mmol) was stirred for 1 h and then concentrated in vacuo.

The residue was treated with water (10 mL) and washed with CH₂Cl₂

twice. The aqueous phase was basified with NaHCO₃and extracted with

EtOAc (3 ꢁ 30 mL). The combined organic phases were washed with

brine (10 mL), dried (Na₂SO₄), filtered, and concentrated in vacuo to

afford the crude, unprotected derivative, which was used for the next step

without further purification.

To a solution of the proper unprotected 4,5,6,6a-tetrahydro-3aH-

pyrrolo[3,4-d]isoxazole (0.80 mmol) in acetone (8 mL), K₂CO₃

(442 mg, 3.20 mmol) and methyl iodide (49 μL, 0.80 mmol) were

added. The resulting mixture was stirred at room temperature for

12 h, the solid was ﬁltered oﬀ, and the solvent was evaporated. The

residue was puriﬁed by silica gel chromatography (CH₂Cl₂/MeOH)

to give the title compounds. Derivatives 8aꢀd were recrystallized

from the appropriate solvent. Compounds 8h and 8i were converted

in the corresponding hydrochloride salts following the general

procedure described for compounds 7a,b and 7hꢀj. Compound 8j

(53.0 mg, 0.21 mmol) was dissolved in EtOH (3 mL), and a solution

of oxalic acid (30.0 mg, 0.24 mmol) in EtOH (1 mL) was added. The

mixture was stirred at room temperature for 10 min, and the resulting

solid was recrystallized from EtOH/Et₂O to yield title compounds as

oxalate salt.

m/z: 234 (M + H)⁺. Anal. (C₁₃H₁₉N₃O HCl) C, H, N.

3

General Procedure for the Synthesis of tert-Butyl 3-Sub-

stituted-6,6a-dihydro-3aH-pyrrolo[3,4-d]isoxazole-5(4H)-car-

boxylates (Compounds 13aꢀd, 13hꢀj, 13l). A 0.25 M solution of

N-Boc-3-pyrroline 9 (169 mg, 1.00 mmol) in EtOAc (4 mL) and a 0.37 M

solution of the proper chloroxime 12 (1.50 mmol) in EtOAc (4 mL) were

prepared. The tworeactant streamswere mixedusing a simpleT-piece and

delivered to an Omnifit glass column (6.6 mm id by 100 mm length) filled

with K₂CO₃(540 mg, 4.00 mmol) heated at 90 ꢀC at a total flow rate of

0.1 mL min^ꢀ1, equivalent to a residence time of about 10 min. A 100 psi

backpressure regulator was applied to the system. The solvent was

evaporated, and the product was purified by silica gel column chroma-

tography (hexane/EtOAc 7:3) to give the title compound 13.

tert-Butyl 3-Phenyl-6,6a-dihydro-3aH-pyrrolo[3,4-d]isoxazole-5(4H)-

carboxylate (13a)⁷⁶. White solid, 69% yield; mp (EtOAc/hexane)

1

146ꢀ147 ꢀC (decomp). H NMR (300 MHz, CDCl₃): δ 7.65ꢀ7.55

(m, 2H), 7.45ꢀ7.35 (m, 3H), 5.28 (ddd, J = 9.3, 5.5, 1.1 Hz, 1H),

4.25ꢀ4.15 (m, 1H), 3.90 (d, J = 12.6 Hz, 1H), 3.51ꢀ3.70 (m, 3H), 1.40

t

(s, 9H). ESI-MS m/z: 233 (M + H ꢀ Bu)⁺.

tert-Butyl 3-(4-Nitrophenyl)-6,6a-dihydro-3aH-pyrrolo[3,4-d]isoxazole-5

(4H)-carboxylate (13b)⁷⁶.Pale-yellow solid, 60% yield; mp (EtOAc/hexane)

189ꢀ190 ꢀC(decomp).¹H NMR (300 MHz, CDCl₃):δ8.28 (d, J=8.8Hz,

2H), 7.80 (d, J = 8.8 Hz, 2H), 5.40 (dd, J = 8.3, 4.1 Hz, 1H), 4.30ꢀ4.20 (m,

1H), 4.03 (d, J = 13.2 Hz, 1H), 3.85ꢀ3.55 (m, 3H), 1.42 (s, 9H). ESI-MS

t

m/z: 278 (M + H ꢀ Bu)⁺.

5-Methyl-3-phenyl-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole

(8a). White solid, 48% yield; mp (EtOAc/hexane) 119ꢀ121 ꢀC

tert-Butyl 3-(4-Methoxyphenyl)-6,6a-dihydro-3aH-pyrrolo[3,4-d]

isoxazole-5(4H)-carboxylate (13c)⁷⁶. White solid, 73% yield; mp

(EtOAc/hexane) 137ꢀ138 ꢀC. ¹H NMR (300 MHz, CDCl₃): δ 7.56

(d, J = 8.8 Hz, 2H), 6.93 (d, J = 8.8 Hz, 2H), 5.26 (dd, J = 9.3, 5.2 Hz,

1H), 4.25ꢀ4.15 (m, 1H), 3.95 (d, J = 12.6 Hz, 1H), 3.83 (s, 3H),

1

(decomp). H NMR (300 MHz, CDCl₃): δ 7.68 (m, 2H), 7.40 (m,

3H), 5.22 (dd, J = 9.1, 4.5 Hz, 1H), 4.21ꢀ4.16 (m, J = 9.1, 7.4, 1.7 Hz,

1H), 3.26 (d, J = 11.0 Hz, 1H), 3.09 (d, J = 9.6 Hz, 1H), 2.51 (dd, J = 9.6,

7.4 Hz, 1H), 2.45 (dd, J = 11.0, 4.5 Hz, 1H), 2.33 (s, 3H). ESI-MS m/z:

203 (M + H)⁺. Anal. (C₁₂H₁₄N₂O) C, H, N.

5-Methyl-3-(4-nitrophenyl)-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]iso-

xazole (8b). White solid, 38% yield; mp (EtOAc/hexane) 161ꢀ162 ꢀC. ¹H

NMR (300 MHz, CDCl₃): δ 8.27 (d, J = 7.7 Hz, 2H), 7.85 (d, J = 7.7 Hz,

2H), 5.33 (dd, J = 4.3, 9.5, 1H), 4.23ꢀ4.17 (m, 1H), 3.33 (d, J = 11.2 Hz,

t

3.72ꢀ3.58 (m, 3H), 1.40 (s, 9H). ESI-MS m/z: 263 (M + H ꢀ Bu)⁺.

tert-Butyl 3-(4-(Tosyloxy)phenyl)-6,6a-dihydro-3aH-pyrrolo[3,4-d]

isoxazole-5(4H)-carboxylate (13d)⁷⁶. Pale-yellow solid, 58% yield;

1

mp (EtOAc/hexane) 189ꢀ190 ꢀC. H NMR (300 MHz, CDCl₃): δ

7.72 (d, J = 8.2 Hz, 2H), 7.56 (d, J = 8.2 Hz, 2H), 7.33 (d, J = 7.8 Hz, 2H),

7672

dx.doi.org/10.1021/jm2010404 |J. Med. Chem. 2011, 54, 7663–7677

Journal of Medicinal Chemistry

ARTICLE

1H), 3.08 (d, J = 9.7, 1H), 2.66ꢀ2.43 (m, 2H), 2.37 (s, 3H). ESI-MS m/z:

248 (M + H)⁺. Anal. (C₁₂H₁₃N₃O₃) C, H, N.

5-Methyl-3-(4-methoxyphenyl)-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,

CDCl₃): δ 7.47 (d, J = 8.5 Hz, 2H), 6.67 (d, J = 8.5 Hz, 2H), 5.15 (dd,

J = 9.3, 4.4 Hz, 1H), 4.15ꢀ4.08 (m, 1H), 3.87 (br s, 2H), 3.22 (d, J = 10.7

Hz, 1H), 3.06 (d, J = 9.6 Hz, 1H), 2.55ꢀ2.40 (m, 2H), 2.32 (s, 3H). ESI-

4-d]isoxazole (8c). White solid, 33% yield; mp (EtOAc/hexane)

MS m/z: 218 (M + H)⁺. Anal. (C₁₂H₁₅N₃O HCl) C, H, N.

3

1

158ꢀ160 ꢀC. H NMR (300 MHz, CDCl₃): δ 7.61 (d, J = 8.8 Hz,

3-(4-Aminobenzyl)-5-methyl-4,5,6,6a-tetrahydro-3aH-

pyrrolo[3,4-d]isoxazole oxalate (8k). Reduction of compound

8i (140 mg, 0.54 mmol) according to the procedure used for 7f

yielded the free amine (103 mg, 83%), which was converted to the

oxalate salt following the general procedure described for compound

8j. White solid; mp (EtOH/Et₂O) 164ꢀ165 ꢀC; free base. ¹H NMR

(300 MHz, CDCl₃): δ 7.02 (d, J = 8.5 Hz, 2H), 6.64 (d, J = 8.5 Hz,

2H), 4.96 (dd, J = 9.3, 4.6 Hz, 1H), 3.77 (d, J = 15.1 Hz, 1H), 3.64 (br

s, 2H), 3.49 (dd, 9.3, 7.5 Hz, 1H), 3.38 (d, J = 15.1 Hz, 1H), 3.19 (d,

J = 10.9 Hz, 1H), 2.94 (d, J = 9.7 Hz, 1H), 2.31 (s, 3H), 2.31ꢀ2.25

(m, 1H), 2.18 (dd, J = 9.7, 7.5 Hz, 1H). ESI-MS m/z: 232 (M + H)⁺.

2H), 6.91 (d, J = 8.8 Hz, 2H), 5.19 (dd, J = 10.0, 4.6 Hz, 1H), 4.17ꢀ4.12

(m, 1H), 3.84 (s, 3H), 3.25 (d, J = 10.9 Hz, 1H), 3.07 (d, J = 9.0, 1H),

2.59ꢀ2.40 (m, 2H), 2.32 (s, 3H). ESI-MS m/z: 233 (M + H)⁺. Anal.

(C₁₃H₁₆N₂O₂) C, H, N.

5-Methyl-3-(4-(tosyloxy)phenyl)-4,5,6,6a-tetrahydro-3aH-pyrrolo-

[3,4-d]isoxazole (8d). White solid, 32% yield; mp (EtOAc/hexane)

122ꢀ123 ꢀC. ¹H NMR (300 MHz, CDCl₃): δ 7.70 (d, J = 8.5 Hz, 2H),

7.59 (d, J = 8.5 Hz, 2H), 7.32 (d, J = 8.5 Hz, 2H), 7.00 (d, J = 8.5 Hz,

2H), 5.22 (dd, J = 9.4, 4.1 Hz, 1H), 4.14ꢀ4.08 (m, 1H), 3.28 (d, J = 11.9

Hz, 1H), 3.07 (d, J = 9.4, 1H), 2.49ꢀ2.38 (m, 5H), 2.33 (s, 3H). ESI-

MS m/z: 373 (M + H)⁺. Anal. (C₁₉H₂₀N₂O₄S) C, H, N.

Anal. (C₁₃H₁₇N₃O H₂CO₄) C, H, N.

3

3-Benzyl-5-methyl-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole

Hydrochloride (8h). White solid, 31% yield; mp (EtOH/Et₂O) 122ꢀ

123 ꢀC; free base. ¹H NMR (300 MHz, CDCl₃): δ 7.37ꢀ7.16 (m, 5H),

4.97 (dd, J = 4.6, 9.3 Hz, 1H), 3.89 (d, J = 15.1 Hz, 1H), 3.50 (d, J = 15.1

Hz, 1H), 3.55ꢀ3.42 (m, 1H), 3.20 (d, J = 10.9 Hz, 1H), 2.96 (d, J = 9.4

Hz, 1H), 2.31 (s, 3H), 2.26 (dd, J = 10.9, 4.6 Hz, 1H), 2.17 (dd, J = 9.4,

3-(4-Carboxyphenyl)-5-methyl-4,5,6,6a-tetrahydro-3aH-

pyrrolo[3,4-d]isoxazole (8e). Deprotection of compound 8l

(200 mg, 0.66 mmol) according to the procedure used for 7e yielded

the title compounds (162 mg, 99%). White solid; mp (methanol/H₂O)

255ꢀ257 ꢀC dec. ¹H NMR (300 MHz, DMSO-d₆): δ 10.20 (br s, 1H),

8.02 (d, J = 8.5 Hz, 2 H), 7.84 (d, J = 8.5 Hz, 2H), 5.50 (dd, J = 9.6, 4.4

Hz, 1H), 4.82ꢀ4.70 (m, 1H), 3.98ꢀ3.80 (m, 1H), 3.78ꢀ3.60 (m, 1H),

3.58ꢀ3.25 (m, 2H), 2.78 (s, 3H). ESI-MS m/z: 247 (M + H)⁺. Anal.

(C₁₃H₁₄N₂O₃) C, H, N.

7.3 Hz, 1H). ESI-MS m/z: 217 (M + H)⁺. Anal. (C₁₃H₁₆N₂O HCl)

3

C, H, N.

5-Methyl-3-(4-nitrobenzyl)-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]

isoxazole Hydrochloride (8i). Pale-yellow solid, 40% yield; mp (EtOH/

Et₂O) 239ꢀ240 ꢀC (decomp); free base. ¹H NMR (300 MHz, CDCl₃):

δ 8.20 (d, J = 8.5 Hz, 2H), 7.45 (d, J = 8.5 Hz, 2H), 5.03 (dd, J = 9.3, 4.6

Hz, 1H), 3.93 (d, J = 15.5 Hz, 1H), 3.64 (d, J = 15.5 Hz, 1H), 3.55ꢀ3.45

(m, 1H), 3.23 (d, J = 10.9 Hz, 1H), 2.94 (d, J = 9.7 Hz, 1H), 2.31 (s, 3H),

2.27 (dd, J = 10.9, 4.6 Hz, 1H), 2.20 (dd, J = 9.7, 7.2 Hz, 1H). ESI-MS

DNMT1 Expression, Purification, and Assay. Cloning and

Purification of Recombinant DNMT1. DNMT1 was produced and

purified as described before.⁵⁵Briefly, proteins were expressed in Sf9

insect cells (derived from Spodoptera frugiperda) and purified by affinity

chromatography and gel filtration. The protein concentration of purified

DNMT was determined by Bradford assay and verified by using

Coomassie blue stained SDS/polyacrylamide gels and suitable molec-

ular mass markers of known concentration. Protein identity was con-

firmed by immunoblotting using a DNMT1-specific antibody (Santa

Cruz) according to the manufacturer’s protocol.

m/z: 262 (M + H)⁺. Anal. (C₁₃H₁₅N₃O₃HCl) C, H, N.

3

3-(4-Methoxybenzyl)-5-methyl-4,5,6,6a-tetrahydro-3aH-pyrrolo-

[3,4-d]isoxazole Oxalate (8j). White solid, 34% yield; mp (Et₂O/

EtOH) 138ꢀ140 ꢀC; free base. ¹H NMR (300 MHz, CDCl₃): δ 7.14

(d, J = 8.4 Hz, 2H), 6.83 (d, J = 8.4 Hz, 2H), 4.94 (dd, J = 9.2, 4.6 Hz,

1H), 3.86ꢀ3.75 (m, 1H), 3.77 (s, 3H), 3.53ꢀ3.39 (m, 2H), 3.17 (d,

J = 10.9 Hz, 1H), 2.93 (d, J = 9.4 Hz, 1H), 2.29 (s, 3H), 2.23 (dd, J =

10.9, 4.6 Hz, 1H), 2.14 (dd, J = 9.4, 7.4 Hz, 1H). ESI-MS m/z: 247 (M

Biochemical DNMT Activity Assay and DoseꢀResponse Assays. DNA

methylation assays were carried out in total reaction volume of 25 μL

containing 0.4 μM hemimethylated or unmethylated oligonucleotide

substrate purchased from MWG (upper strand: 5⁰-GATCGCXGATG-

CGXGAATXGCGATXGATGCGAT-3⁰, X = 5mC for hemimethylated

or X = C for unmethylated substrate, and lower strand: 5⁰-ATCGC-

ATCGATCGCGATTCGCGCATCGGCGATC-3⁰), purified DNMT

(500 nM) in reaction buffer (100 mM KCl, 10 mM TrisCl pH 7.5, 1 mM

EDTA), and BSA (1 mg/mL). All reactions were carried out at 37 ꢀC in

the presence of 0.7 μM [methyl-³H] AdoMet (2.6 TBq/mmol,

PerkinElmer). After 3 h, the reaction was stopped by adding 10 μL of

20% SDS and spotting of the whole volume onto DE81 cellulose paper.

Filters were baked at 80 ꢀC for 2 h and washed three times with cold 0.2 M

NH₄HCO₃, three times with distilled water, and once with 100% ethanol.

After drying, filters were transferred into Mini-Poly Q vial (PerkinElmer)

and 5 mL of Ultima Gold LSC Cocktail was added per vial. Analysis was

done in a scintillation counter, each measurement was repeated once.

Competition Assay. For the determination of the mode of action of

7b in the catalytic domain of DNMT1, we used the model of Lai and

Wu⁷⁸as recommended by the NIH Chemical Genomics Center

(http://www.ncgc.nih.gov/guidance) and adapted it to our biochemical

DNMT activity assay. The conditions of the biochemical DNMT

activity assay (see above) were slightly modified for the competition

experiments: the inhibitor concentration was held constant at 150 μM,

the reaction time at 37 ꢀC was decreased to 1 h. The concentrations of

either SAM substrate or oligo substrate were increased to test for

competition with 7b, which should result in a decreasing inhibition.

Cell Culture. HCT116 cells were obtained directly from the American

Type Culture Collection (ATCC, www.atcc.org) and passaged in our

+ H)⁺. Anal. (C₁₄H₁₈N₂O₂H₂CO₄) C, H, N.

3

5-Methyl-3-(4-(tert-butoxycarbonyl)phenyl)-4,5,6,6a-tetrahydro-

1

3aH-pyrrolo[3,4-d]isoxazole (8l). Colorless oil, 45% yield. H NMR

(300 MHz, CDCl₃): δ 8.00 (d, J = 8.8 Hz, 2H), 7.70 (d, J = 8.8 Hz, 2H),

5.25 (dd, J = 9.6, 4.4 Hz, 1H), 4.22ꢀ4.15 (m, 1H), 3.28 (d, J = 11.0 Hz,

1H), 3.06 (d, J = 9.3 Hz, 1H), 2.51 (dd, J = 9.3, 7.4 Hz, 1H), 2.45 (dd, J =

11.0, 4.4 Hz, 1H), 2.32 (s, 3 H), 1.58 (s, 9 H). ESI-MS m/z: 303 (M +

H)⁺.

3-(4-Hydroxyphenyl)-5-methyl-4,5,6,6a-tetrahydro-3aH-

pyrrolo[3,4-d]isoxazole (8g). Deprotection of compound 8d

(70.0 mg, 0.19 mmol) according to the procedure used for 7g yielded

the title compound as a white solid (40.5 mg, 95%), which was

1

recrystallized from EtOAc/hexane; mp 126ꢀ127 ꢀC. H NMR (300

MHz, DMSO-d₆): δ 9.87 (s, 1H), 7.46 (d, J = 8.5 Hz, 2H), 6.79 (d, J =

8.5 Hz, 2H), 5.05 (dd, J = 9.4, 4.3 Hz, 1H), 4.25ꢀ4.20 (m, 1H), 3.01 (d,

J = 10.8 Hz, 1H), 2.85 (d, J = 9.5 Hz, 1H), 2.33 (dd, J = 9.5, 7.4 Hz, 1H),

2.25 (dd, J = 10.8, 4.3 Hz, 1H), 2.15 (s, 3H). ESI-MS m/z: 219 (M +

H)⁺. Anal. (C₁₂H₁₄N₂O₂) C, H, N.

3-(4-Aminophenyl)-5-methyl-4,5,6,6a-tetrahydro-3aH-

pyrrolo[3,4-d]isoxazole Hydrochloride (8f). Reduction of

compound 8b (135 mg, 0.55 mmol) according to the procedure

used for 7l yielded the free amine (101 mg, 85%), which was

converted to the hydrochloride salt following the general procedure

described for compounds 7a,b, and 7hꢀj. White solid; mp (EtOH/

Et₂O) 211ꢀ212 ꢀC (decomp); free base. ¹H NMR (300 MHz,

7673

dx.doi.org/10.1021/jm2010404 |J. Med. Chem. 2011, 54, 7663–7677

Journal of Medicinal Chemistry

ARTICLE

laboratory for fewer than 6 months after resuscitation. HCT116 were

cultured in DMEM/Ham’s F12 (BIOCHROM) supplemented with 10%

FCS (Invitrogen). Cellproliferation and viability was assessedbycounting

trypan blue stained cells by TC10 automated cell counter (Bio-Rad).

Compounds. SAH, 2 and procaine were purchased by Sigma-Aldrich,

and both were dissolved in water to 50 mM stocks and stored in aliquots

at ꢀ80 ꢀC.

’ REFERENCES

(1) Sharma, S.; Kelly, T. K.; Jones, P. A. Epigenetics in cancer.

Carcinogenesis 2010, 31, 27–36.

(2) Jones, P. A.; Baylin, S. B. The Epigenomics of Cancer. Cell 2007,

128, 683–692.

(3) Esteller, M. Epigenetics in Cancer. N. Engl. J. Med. 2008, 358,

1148–1159.

(4) Portela, A.; Esteller, M. Epigenetic modiﬁcations and human

disease. Nature Biotechnol. 2010, 28, 1057–1068.

(5) Bernstein, B. E.; Meissner, A.; Lander, E. S. The Mammalian

Epigenome. Cell 2007, 128, 669–681.

(6) Kouzarides, T. Chromatin Modiﬁcations and Their Function.

Cell 2007, 128, 693–705.

(7) Suzuki, M. M.; Bird, A. DNA methylation landscapes: provoca-

tive insights from epigenomics. Nature Rev. Genet. 2008, 9, 465–476.

(8) Feinberg, A. P.; Ohlsson, R.; Henikoﬀ, S. The epigenetic

progenitor origin of human cancer. Nature Rev. Genet. 2006, 7, 21–33.

(9) Jones, P. A.; Martienssen, R. A Blueprint for a Human Epigen-

ome Project: The AACR Human Epigenome Workshop. Cancer Res.

2005, 65, 11241–11246.

Molecular Modeling. To conduct the docking studies, the protonated

and neutral states of the R and S enantiomers of 7b were prepared using

Molecular Operating Environment (MOE), version 2008.10.⁸⁹The

crystal structure of human DNMT1 bound to SAH was retrieved from

the Protein Data Bank (PDB entry 3PTA).⁷⁵Docking was performed

with the methyltransferase domain using the Glide (Grid-Based Ligand

Docking with Energetics) program, version 5.7.^83,84The Protein Pre-

paration Wizard module of Maestro was used to prepare the protein⁹⁰as

we reported previously for other systems.⁹¹For docking, the scoring

grids were centered on the crystal structure of SAH using the default

bounding sizes, with an inner box of 10 Å on each side and an outer box

of 25.4 Å on each side. Flexible docking with default parameters was

used. Glide XP (Extra Precision) was employed for all docking

calculations.⁸⁴The best docked poses were selected as the ones with

the lowest GlideScore; the more negative the Glide Score, the more

favorable the binding.

(10) Bird, A. DNA methylation patterns and epigenetic memory.

Genes Dev. 2002, 16, 6–21.

(11) Yoo, C. B.; Jones, P. A. Epigenetic therapy of cancer: past,

present and future. Nature Rev. Drug Discovery 2006, 5, 37–50.

(12) In addition to 5-methylcytosines, 5-hydroxymethyl-2⁰-deoxy-

cytidine has also been observed, although it seems not to be present in

cancer cell lines. These new DNA modiﬁcations need to be further

studied to determine their implications for normal and diseased

epigenetic regulation. See: Kriaucionis, S.; Heintz, N. The Nuclear

DNA Base 5-Hydroxymethylcytosine Is Present in Purkinje Neurons

and the Brain. Science 2009, 324, 929–930.

(13) Non-CG methylation has recently been described in humans at

CHG and CHH sites (where H is A, C or T). CHG and CHH

methylation has been found in stem cells and the levels of non-CpG

methylation decrease during diﬀerentiation and are restored in induced

pluripotent stem cells, suggesting a key role in origin and maintenance of

pluripotent state. Mechanisms of non-CpG methylation remain

unclear. See: Lister, R.; Pelizzola, M.; Dowen, R. H.; Hawkins, R. D.;

Hon, G.; Tonti-Filippini, J.; Nery, J. R.; Lee, L.; Ye, Z.; Ngo, Q. M.;

Edsall, L.; Antosiewicz-Bourget, J.; Stewart, R.; Ruotti, V.; Millar, A. H.;

Thomson, J. A.; Ren, B.; Ecker, J. R. Human DNA methylomes at base

resolution show widespread epigenomic diﬀerences. Nature 2009,

462, 315–322. Laurent, L.; Wong, E.; Li, G.; Huynh, T.; Tsirigos, A.;

Ong, C. T.; Low, H. M.; Kin Sung, K. W.; Rigoutsos, I.; Loring, J.; Wei,

C.-L. Dynamic changes in the human methylome during diﬀerentiation.

Genome Res. 2010, 20, 320–321.

’ ASSOCIATED CONTENT

S

Supporting Information. Elemental analysis for com-

b

pounds 7aꢀk and 8aꢀk, characterization data of compounds

9aꢀd, 9hꢀj, 9l, 12aꢀd, 12hꢀj, and 12l. This material is

available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION

Corresponding Author

*For D.K.: phone, +49-6221-42-3800; E-mail, d.kuck@dkfz.de.

For J.L.M-F.: phone/fax, +1-772-345-4685; E-mail, jmedina@

tpims.org. For G.S.: phone, +39-089-96-9770; fax, +39-089-96-

9602; E-mail: gsbardella@unisa.it.

Author Contributions

^{^}These two authors contributed equally to this work.

(14) Esteller, M. Epigenetics in evolution and disease. Lancet 2008,

372, S90–S96.

’ ACKNOWLEDGMENT

(15) Doi, A.; Park, I.-H.; Wen, B.; Murakami, P.; Aryee, M. J.;

Irizarry, R.; Herb, B.; Ladd-Acosta, C.; Rho, J.; Loewer, S.; Miller, J.;

Schlaeger, T.; Daley, G. Q.; Feinberg, A. P. Diﬀerential methylation of

tissue- and cancer-speciﬁc CpG island shores distinguishes human

induced pluripotent stem cells, embryonic stem cells and ﬁbroblasts.

Nature Genet. 2009, 41, 1350–1353.

(16) Irizarry, R. A.; Ladd-Acosta, C.; Wen, B.; Wu, Z.; Montano, C.;

Onyango, P.; Cui, H.; Gabo, K.; Rongione, M.; Webster, M.; Ji, H.;

Potash, J. B.; Sabunciyan, S.; Feinberg, A. P. The human colon cancer

methylome shows similar hypo- and hypermethylation at conserved

tissue-speciﬁc CpG island shores. Nature Genet. 2009, 41, 178–186.

(17) Balch, C.; Nephew, K. P.; Huang, T. H. M.; Bapat, S. A.

Epigenetic “bivalently marked” process of cancer stem cell-driven

tumorigenesis. Bioessays 2007, 29, 842–845.

This work was supported by grants from Ministero

dell’Universitꢁa e della Ricerca Scientiﬁca e Tecnologica—PRIN

2008 (S.C.), Universitꢁa di Salerno (G.S.), COST Action TD09/

05 Epigenetics (G.S.), Universitꢁa degli Studi di Milano (P.C., A.

P., L.T.), the State of Florida (J.L.M.-F.), and the Menopause &

Women’s Health Research Center (J.L.M.-F.). D.K. thanks Sara

Myers for technical assistance with the DNMT1 activity assay. M.

V. was supported by Universitꢁa di Salerno with predoctoral

fellowships.

’ ABBREVIATIONS USED

DMEM, Dulbecco’s Modiﬁed Eagle’s Medium; DNMT, DNA

methyltransferase; FCS, fetal calf serum; HCT116, human colon

carcinoma cell line; SAH, S-adenosyl-^L-homocysteine; SAM,

S-adenosyl methionine; Sf9, insect cell line derived from Spodop-

tera frugiperda; MOI, multiplicity of infection

(18) Hewagama, A.; Richardson, B. The genetics and epigenetics of

autoimmune diseases. J. Autoimmun. 2009, 33, 3–11.

(19) Liu, L.; van Groen, T.; Kadish, I.; Tollefsbol, T. O. DNA

methylation impacts on learning and memory in aging. Neurobiol. Aging

2009, 30, 549–560.

7674

dx.doi.org/10.1021/jm2010404 |J. Med. Chem. 2011, 54, 7663–7677

Journal of Medicinal Chemistry

ARTICLE

(20) Ohm, J. E.; McGarvey, K. M.; Yu, X.; Cheng, L.; Schuebel, K. E.;

Cope, L.; Mohammad, H. P.; Chen, W.; Daniel, V. C.; Yu, W.; Berman,

D. M.; Jenuwein, T.; Pruitt, K.; Sharkis, S. J.; Watkins, D. N.; Herman,

J. G.; Baylin, S. B. A stem cell-like chromatin pattern may predispose

tumor suppressor genes to DNA hypermethylation and heritable silenc-

ing. Nature Genet. 2007, 39, 237–242.

Deacetylase and DNA Methyltransferase. J. Org. Chem. 2003, 68,

3866–3873.

(38) Brueckner, B.; Garcia Boy, R.; Siedlecki, P.; Musch, T.; Kliem,

H. C.; Zielenkiewicz, P.; Suhai, S.; Wiessler, M.; Lyko, F. Epigenetic

Reactivation of Tumor Suppressor Genes by a Novel Small-Molecule

Inhibitor of Human DNA Methyltransferases. Cancer Res. 2005, 65,

6305–6311.

(21) Richardson, B. Primer: epigenetics of autoimmunity. Nature

Clin. Pract. Rheum. 2007, 3, 521–527.

(39) Siedlecki, P.; Garcia Boy, R.; Musch, T.; Brueckner, B.; Suhai,

S.; Lyko, F.; Zielenkiewicz, P. Discovery of Two Novel, Small-Molecule

Inhibitors of DNA Methylation. J. Med. Chem. 2006, 49, 678–683.

(40) Suzuki, T.; Tanaka, R.; Hamada, S.; Nakagawa, H.; Miyata, N.

Design, synthesis, inhibitory activity, and binding mode study of novel DNA

methyltransferase 1 inhibitors. Bioorg. Med. Chem. Lett. 2010, 20, 1124–1127.

(41) Kuck, D.; Caulﬁeld, T.; Lyko, F.; Medina-Franco, J. L. Nanao-

mycin A Selectively Inhibits DNMT3B and Reactivates Silenced Tumor

Suppressor Genes in Human Cancer Cells. Mol. Cancer Ther. 2010,

9, 3015–3023.

(22) A fourth enzyme previously known as DNMT2 has strong

sequence similarities with 5-methylcytosine methyltransferases but does

not methylate DNA and was shown to methylate position 38 in aspartic

acid transfer RNA. To better reﬂect its biological function, the name for

this enzyme has been changed to tRNA aspartic acid methyltransferase 1

(TRDMT1). See: Goll, M. G.; Kirpekar, F.; Maggert, K. A.; Yoder, J. A.;

Hsieh, C.-L.; Zhang, X.; Golic, K. G.; Jacobsen, S. E.; Bestor, T. H.

Methylation of tRNAAsp by the DNA Methyltransferase Homolog

Dnmt2. Science 2006, 311, 395–398.

(42) Lin, R.-K.; Hsu, C.-H.; Wang, Y.-C. Mithramycin A inhibits

DNA methyltransferase and metastasis potential of lung cancer cells.

Anti-Cancer Drugs 2007, 18, 1157–1164.

(43) Isakovic, L.; Saavedra, O. M.; Llewellyn, D. B.; Claridge, S.;

Zhan, L.; Bernstein, N.; Vaisburg, A.; Elowe, N.; Petschner, A. J.; Rahil,

J.; Beaulieu, N.; Gauthier, F.; MacLeod, A. R.; Delorme, D.; Besterman,

J. M.; Wahhab, A. Constrained (^L-)-S-adenosyl-L-homocysteine (SAH)

analogues as DNA methyltransferase inhibitors. Bioorg. Med. Chem. Lett.

2009, 19, 2742–2746.

(44) Saavedra, O. M.; Isakovic, L.; Llewellyn, D. B.; Zhan, L.;

Bernstein, N.; Claridge, S.; Raeppel, F.; Vaisburg, A.; Elowe, N.;

Petschner, A. J.; Rahil, J.; Beaulieu, N.; MacLeod, A. R.; Delorme, D.;

Besterman, J. M.; Wahhab, A. SAR around (^L)-S-adenosyl-L-homocys-

teine, an inhibitor of human DNA methyltransferase (DNMT) enzymes.

Bioorg. Med. Chem. Lett. 2009, 19, 2747–2751.

(45) Lin, Y.-S.; Shaw, A.; Wang, S.-G.; Hsu, C.-C.; Teng, I.-W.;

Tseng, M.-J.; Huang, T.; Chen, C.-S.; Leu, Y.-W.; Hsiao, S.-H. Identi-

ﬁcation of novel DNA methylation inhibitors via a two-component

reporter gene system. J. Biomed. Sci. 2011, 18, 3.

(23) Goll, M. G.; Bestor, T. H. Eukaryotic cytosine methyltrans-

ferases. Annu. Rev. Biochem. 2005, 74, 481–514.

(24) Egger, G.; Liang, G.; Aparicio, A.; Jones, P. A. Epigenetics in

human disease and prospects for epigenetic therapy. Nature 2004,

429, 457–463.

(25) 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.

(26) Yoo, C. B.; Jeong, S.; Egger, G.; Liang, G.; Phiasivongsa, P.;

Tang, C.; Redkar, S.; Jones, P. A. Delivery of 5-Aza-2⁰-Deoxycytidine to

Cells Using Oligodeoxynucleotides. Cancer Res. 2007, 67, 6400–6408.

(27) Chuang, J. C.; Warner, S. L.; Vollmer, D.; Vankayalapati, H.;

Redkar, S.; Bearss, D. J.; Qiu, X.; Yoo, C. B.; Jones, P. A. S110, a 5-Aza-2⁰-

Deoxycytidine-Containing Dinucleotide, Is an Eﬀective DNA Methyla-

tion Inhibitor In Vivo and Can Reduce Tumor Growth. Mol. Cancer

Ther. 2010, 9, 1443–1450.

(28) Brueckner, B.; Lyko, F. DNA methyltransferase inhibitors: old

and new drugs for an epigenetic cancer therapy. Trends Pharmacol. Sci.

2004, 25, 551–554.

(46) Datta, J.; Ghoshal, K.; Denny, W. A.; Gamage, S. A.; Brooke,

D. G.; Phiasivongsa, P.; Redkar, S.; Jacob, S. T. A New Class of

Quinoline-Based DNA Hypomethylating Agents Reactivates Tumor

Suppressor Genes by Blocking DNA Methyltransferase 1 Activity and

Inducing Its Degradation. Cancer Res. 2009, 69, 4277–4285.

(47) Cornacchia, E.; Golbus, J.; Maybaum, J.; Strahler, J.; Hanash, S.;

Richardson, B. Hydralazine and procainamide inhibit T cell DNA

methylation and induce autoreactivity. J. Immunol. 1988, 140, 2197–2200.

(48) Singh, N.; Due~nas-Gonzꢀalez, A.; Lyko, F.; Medina-Franco, J. L.

Molecular Modeling and Molecular Dynamics Studies of Hydralazine with

Human DNA Methyltransferase 1. ChemMedChem 2009, 4, 792–799.

(49) Lin, X.; Asgari, K.; Putzi, M. J.; Gage, W. R.; Yu, X.; Cornblatt,

B. S.; Kumar, A.; Piantadosi, S.; DeWeese, T. L.; De Marzo, A. M.;

Nelson, W. G. Reversal of GSTP1 CpG Island Hypermethylation and

Reactivation of π-Class Glutathione S-Transferase (GSTP1) Expression

in Human Prostate Cancer Cells by Treatment with Procainamide.

Cancer Res. 2001, 61, 8611–8616.

(50) Scheinbart, L. S.; Johnson, M. A.; Gross, L. A.; Edelstein, S. R.;

Richardson, B. C. Procainamide inhibits DNA methyltransferase in a

human T cell line. J. Rheumatol. 1991, 18, 530–534.

(51) Villar-Garea, A.; Fraga, M. F.; Espada, J.; Esteller, M. Procaine Is

a DNA-Demethylating Agent with Growth-Inhibitory Eﬀects in Human

Cancer Cells. Cancer Res. 2003, 63, 4984–4989.

(52) Lee, B. H.; Yegnasubramanian, S.; Lin, X.; Nelson, W. G.

Procainamide Is a Speciﬁc Inhibitor of DNA Methyltransferase 1.

J. Biol. Chem. 2005, 280, 40749–40756.

(53) Kuck, D.; Singh, N.; Lyko, F.; Medina-Franco, J. L. Novel and

selective DNA methyltransferase inhibitors: docking-based virtual screen-

ing and experimental evaluation. Bioorg. Med. Chem. 2010, 18, 822–829.

(54) Medina-Franco, J.; Lꢀopez-Vallejo, F.; Kuck, D.; Lyko, F.

Natural products as DNA methyltransferase inhibitors: a computer-

aided discovery approach. Mol. Diversity 2011, 15, 293–304.

(29) Brueckner, B.; Kuck, D.; Lyko, F. DNA Methyltransferase

Inhibitors for Cancer Therapy. Cancer J. 2007, 13, 17–22.

(30) Lyko, F.; Brown, R. DNA Methyltransferase Inhibitors and the

Development of Epigenetic Cancer Therapies. J. Natl. Cancer Inst. 2005,

97, 1498–1506.

(31) Stresemann, C.; Lyko, F. Modes of action of the DNA

methyltransferase inhibitors azacytidine and decitabine. Int. J. Cancer

2008, 123, 8–13.

(32) Fang, M. Z.; Wang, Y.; Ai, N.; Hou, Z.; Sun, Y.; Lu, H.; Welsh,

W.; Yang, C. S. Tea Polyphenol (ꢀ)-Epigallocatechin-3-Gallate Inhibits

DNA Methyltransferase and Reactivates Methylation-Silenced Genes in

Cancer Cell Lines. Cancer Res. 2003, 63, 7563–7570.

(33) Liu, Z.; Xie, Z.; Jones, W.; Pavlovicz, R. E.; Liu, S.; Yu, J.; Li, P.-

k.; Lin, J.; Fuchs, J. R.; Marcucci, G.; Li, C.; Chan, K. K. Curcumin is a

potent DNA hypomethylation agent. Bioorg. Med. Chem. Lett. 2009,

19, 706–709.

(34) Fini, L.; Selgrad, M.; Fogliano, V.; Graziani, G.; Romano, M.;

Hotchkiss, E.; Daoud, Y. A.; De Vol, E. B.; Boland, C. R.; Ricciardiello, L.

Annurca Apple Polyphenols Have Potent Demethylating Activity and

Can Reactivate Silenced Tumor Suppressor Genes in Colorectal Cancer

Cells. J. Nutr. 2007, 137, 2622–2628.

(35) Lee, W. J.; Zhu, B. T. Inhibition of DNA methylation by caﬀeic

acid and chlorogenic acid, two common catechol-containing coﬀee

polyphenols. Carcinogenesis 2006, 27, 269–277.

(36) Fang, M. Z.; Chen, D.; Sun, Y.; Jin, Z.; Christman, J. K.; Yang,

C. S. Reversal of Hypermethylation and Reactivation of p16INK4a,

RARβ, and MGMT Genes by Genistein and Other Isoﬂavones from

Soy. Clin. Cancer Res. 2005, 11, 7033–7041.

(37) Pina, I. C.; Gautschi, J. T.; Wang, G.-Y.-S.; Sanders, M. L.;

Schmitz, F. J.; France, D.; Cornell-Kennon, S.; Sambucetti, L. C.;

Remiszewski, S. W.; Perez, L. B.; Bair, K. W.; Crews, P. Psammaplins

from the Sponge Pseudoceratina purpurea: Inhibition of Both Histone

7675

dx.doi.org/10.1021/jm2010404 |J. Med. Chem. 2011, 54, 7663–7677

Journal of Medicinal Chemistry

ARTICLE

(55) Castellano, S.; Kuck, D.; Sala, M.; Novellino, E.; Lyko, F.;

Sbardella, G. Constrained analogues of procaine as novel small molecule

inhibitors of DNA methyltransferase-1. J. Med. Chem. 2008, 51,

2321–2325.

(56) Massa, S.; Mai, A.; Sbardella, G.; Esposito, M.; Ragno, R.; Loidl,

P.; Brosch, G. 3-(4-Aroyl-1H-pyrrol-2-yl)-N-hydroxy-2-propenamides,

a New Class of Synthetic Histone Deacetylase Inhibitors. J. Med. Chem.

2001, 44, 2069–2072.

of Cell Diﬀerentiation and Proliferation. ChemMedChem 2006, 1,

1073–1080.

(70) Bartolini, S.; Mai, A.; Artico, M.; Paesano, N.; Rotili, D.; Spadafora,

C.; Sbardella, G. 6-[1-(2,6-Diﬂuorophenyl)ethyl]pyrimidinones Antago-

nize Cell Proliferation and Induce Cell Diﬀerentiation by Inhibiting

(a Nontelomeric) Endogenous Reverse Transcriptase. J. Med. Chem.

2005, 48, 6776–6778.

(71) Ornaghi, P.; Rotili, D.; Sbardella, G.; Mai, A.; Filetici, P. A novel

Gcn5p inhibitor represses cell growth, gene transcription and histone

acetylation in budding yeast. Biochem. Pharmacol. 2005, 70, 911–917.

(72) Mai, A.; Massa, S.; Ragno, R.; Esposito, M.; Sbardella, G.;

Nocca, G.; Scatena, R.; Jesacher, F.; Loidl, P.; Brosch, G. Binding Mode

Analysis of 3-(4-Benzoyl-1-methyl-1H-2-pyrrolyl)-N-hydroxy-2-prope-

namide: A New Synthetic Histone Deacetylase Inhibitor Inducing

Histone Hyperacetylation, Growth Inhibition, and Terminal Cell Dif-

ferentiation. J. Med. Chem. 2002, 45, 1778–1784.

(73) Lu, Q.; Wu, A.; Richardson, B. C. Demethylation of the same

promoter sequence increases CD70 expression in lupus T cells and T cells

treated with lupus-inducing drugs. J. Immunol. 2005, 174, 6212–6219.

(74) Segura-Pacheco, B.; Trejo-Becerril, C.; Perez-Cardenas, E.;

Taja-Chayeb, L.; Mariscal, I.; Chavez, A.; Acu~na, C.; Salazar, A. M.;

Lizano, M.; Duen~as-Gonzalez, A. Reactivation of tumor suppressor

genes by the cardiovascular drugs hydralazine and procainamide and

their potential use in cancer therapy. Clin. Cancer Res. 2003, 9,

1596–1603.

(75) Song, J.; Rechkoblit, O.; Bestor, T. H.; Patel, D. J. Structure of

DNMT1ꢀDNA Complex Reveals a Role for Autoinhibition in Main-

tenance DNA Methylation. Science 2011, 331, 1036–1040.

(76) Castellano, S.; Tamborini, L.; Viviano, M.; Pinto, A.; Sbardella,

G.; Conti, P. Synthesis of 3-Aryl/benzyl-4,5,6,6a-tetrahydro-3aH-

pyrrolo[3,4-d]isoxazole Derivatives: A Comparison between Conven-

tional, Microwave-Assisted and Flow-Based Methodologies. J. Org.

Chem. 2010, 75, 7439–7442.

(77) Recently, a ﬂuorescence-based assay using a short DNA duplex

immobilized on 96-well plates was employed to screen ﬂavones and

ﬂavanones for the inhibition of murine catalytic Dnmt3a/3L.Ceccaldi,

A.; Rajavelu, A.; Champion, C.; Rampon, C.; Jurkowska, R.; Jankevicius,

G.; Sꢀenamaud-Beaufort, C.; Ponger, L.; Gagey, N.; Dali Ali, H.; Tost, J.;

Vriz, S.; Ros, S.; Dauzonne, D.; Jeltsch, A.; Guianvarc’h, D.; Arimondo,

P. B. C5-DNA Methyltransferase Inhibitors: From Screening to

Eﬀects on Zebraﬁsh Embryo Development. ChemBioChem 2011, 12,

1337–1345.

(78) Lai, C.-J.; Wu, J. C. A Simple Kinetic Method for Rapid

Mechanistic Analysis of Reversible Enzyme Inhibitors. Assay Drug Dev.

Technol. 2003, 1, 527–535.

(79) Yoo, J.; Medina-Franco, J. Homology modeling, docking and

structure-based pharmacophore of inhibitors of DNA methyltransferase.

J. Comput.-Aided Mol. Des. 2011, 25, 555–567.

(57) 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.

(58) Piaz, F.; Vassallo, A.; Rubio, O.; Castellano, S.; Sbardella, G.; De

Tommasi, N. Chemical biology of histone acetyltransferase natural

compounds modulators. Mol. Diversity 2011, 15, 401–416.

(59) 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.

(60) 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 Methyltransfer-

ase Inhibitor 1 (AMI-1). ChemMedChem 2010, 5, 398–414.

(61) Sbardella, G.; Castellano, S.; Vicidomini, C.; Rotili, D.; Nebbioso,

A.; Miceli, M.; Altucci, L.; Mai, A. Identiﬁcation of long chain

alkylidenemalonates as novel small molecule modulators of histone

acetyltransferases. Bioorg. Med. Chem. Lett. 2008, 18, 2788–2792.

(62) Mai, A.; Rotili, D.; Tarantino, D.; Nebbioso, A.; Castellano, S.;

Sbardella, G.; Tini, M.; Altucci, L. Identiﬁcation of 4-hydroxyquinolines

inhibitors of p300/CBP histone acetyltransferases. Bioorg. Med. Chem.

Lett. 2009, 19, 1132–1135.

(63) Spannhoﬀ, A.; Kim, Y. K.; Raynal, N. J. M.; Gharibyan, V.; Su,

M.-B.; Zhou, Y.-Y.; Li, J.; Castellano, S.; Sbardella, G.; Issa, J.-P. J.;

Bedford, M. T. Histone deacetylase inhibitor activity in royal jelly might

facilitate caste switching in bees. EMBO Rep. 2011, 12, 238–243.

(64) Colussi, C.; Rosati, J.; Straino, S.; Spallotta, F.; Berni, R.; Stilli,

D.; Rossi, S.; Musso, E.; Macchi, E.; Mai, A.; Sbardella, G.; Castellano, S.;

Chimenti, C.; Frustaci, A.; Nebbioso, A.; Altucci, L.; Capogrossi, M. C.;

Gaetano, C. Nε-Lysine acetylation determines dissociation from GAP

junctions and lateralization of connexin 43 in normal and dystrophic

heart. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 2795–2800.

(65) 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 Methyltransfer-

ase, Acetyltransferase, and Class III Deacetylase (Sirtuin) Inhibitors.

J. Med. Chem. 2008, 51, 2279–2290.

(80) Medina-Franco, J. L.; Caulﬁeld, T. Advances in the computa-

tional development of DNA methyltransferase inhibitors. Drug Discovery

Today 2011, 16, 418–425.

(81) Yoo, J.; Medina-Franco, J. Trimethylaurintricarboxylic acid

inhibits human DNA methyltransferase 1: insights from enzymatic

and molecular modeling studies. J. Mol. Model. 2011, DOI: 10.1007/

s00894-011-1191-4. Published Online: July 29, 2011.

(82) Siedlecki, P.; Boy, R. G.; Comagic, S.; Schirrmacher, R.; Wiessler,

M.; Zielenkiewicz, P.; Suhai, S.; Lyko, F. Establishment and functional

validation of a structural homology model for human DNA methyltrans-

ferase 1. Biochem. Biophys. Res. Commun. 2003, 306, 558–563.

(83) Glide, version 5.7; Schr€odinger, LLC: New York, NY, 2011.

(84) Friesner, R. A.; Murphy, R. B.; Repasky, M. P.; Frye, L. L.;

Greenwood, J. R.; Halgren, T. A.; Sanschagrin, P. C.; Mainz, D. T. Extra

Precision Glide: Docking and Scoring Incorporating a Model of

Hydrophobic Enclosure for ProteinꢀLigand Complexes. J. Med. Chem.

2006, 49, 6177–6196.

(66) 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. ChemMed-

Chem 2007, 2, 987–991.

(67) 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.

(68) Mai, A.; Rotili, D.; Tarantino, D.; Ornaghi, P.; Tosi, F.;

Vicidomini, C.; Sbardella, G.; Nebbioso, A.; Miceli, M.; Altucci, L.;

Filetici, P. Small-Molecule Inhibitors of Histone Acetyltransferase

Activity: Identiﬁcation and Biological Properties. J. Med. Chem. 2006,

49, 6897–6907.

(69) Sbardella, G.; Bartolini, S.; Castellano, S.; Artico, M.; Paesano,

N.; Rotili, D.; Spadafora, C.; Mai, A. 6-Alkylthio-4-[1-(2,6-diﬂuo-

rophenyl)alkyl]-1H-[1,3,5]triazin-2-ones (ADATs): Novel Regulators

(85) Jeltsch, A. Beyond Watson and Crick: DNA Methylation and

Molecular Enzymology of DNA Methyltransferases. ChemBioChem

2002, 3, 274–293.

7676

dx.doi.org/10.1021/jm2010404 |J. Med. Chem. 2011, 54, 7663–7677

Journal of Medicinal Chemistry

ARTICLE

(86) Klimasauskas, S.; Kumar, S.; Roberts, R. J.; Cheng, X. Hhal

methyltransferase ﬂips its target base out of the DNA helix. Cell 1994,

76, 357–369.

(87) Rhee, I.; Bachman, K. E.; Park, B. H.; Jair, K.-W.; Yen, R.-W. C.;

Schuebel, K. E.; Cui, H.; Feinberg, A. P.; Lengauer, C.; Kinzler, K. W.;

Baylin, S. B.; Vogelstein, B. DNMT1 and DNMT3b cooperate to silence

genes in human cancer cells. Nature 2002, 416, 552–556.

(88) Chen, T.; Hevi, S.; Gay, F.; Tsujimoto, N.; He, T.; Zhang, B.;

Ueda, Y.; Li, E. Complete inactivation of DNMT1 leads to mitotic

catastrophe in human cancer cells. Nature Genet. 2007, 39, 391–396.

(89) Molecular Operating Environment (MOE), version 2008.10;

Chemical Computing Group Inc.: Montreal, Quebec, Canada, 2008.

(90) Schr€odinger Suite 2011 Protein Preparation Wizard, Epik version

2.2, Impact version 5.7, Prime version 3.0; Schr€odinger, LLC: New York,

NY, 2011.

(91) Hernꢀandez-Campos, A.; Velꢀazquez-Martínez, I.; Castillo, R.;

Lꢀopez-Vallejo, F.; Jia, P.; Yu, Y.; Giulianotti, M. A.; Medina-Franco, J. L.

Docking of Protein Kinase B Inhibitors: Implications in the Structure-

Based Optimization of a Novel Scaﬀold. Chem. Biol. Drug Des. 2010, 76,

269–276.

7677

dx.doi.org/10.1021/jm2010404 |J. Med. Chem. 2011, 54, 7663–7677

Article Doi

Synthesis and biochemical evaluation of δ2-isoxazoline derivatives as DNA methyltransferase 1 inhibitors

DOI: 10.1021/jm2010404

Source and publish data:

Authors:

Article abstract of DOI:10.1021/jm2010404

Full text of DOI:10.1021/jm2010404

Products guided by the article

R&D Labs maybe for 1338732-62-9

Relevant to this article

Hot Product