Constrained analogues of procaine as novel small molecule inhibitors of DNA methyltransferase-1

Article abstract of DOI:10.1021/jm7015705

Constrained analogues of procaine were synthesized, and their inhibiting activity against DNMT1 was tested. Among them, the most potent compound, derivative 3b, was also able to induce a recognizable demethylation of chromosomal satellite repeats in HL60 human myeloid leukemia cells and thus represents a lead compound for the development of a novel class of non-nucleoside DNMT1 inhibitors.

Full text of DOI:10.1021/jm7015705

J. Med. Chem. 2008, 51, 2321–2325
2321
Constrained Analogues of Procaine as Novel Small Molecule Inhibitors of DNA
Methyltransferase-1
Sabrina Castellano,§ Dirk Kuck,# Marina Sala,§ Ettore Novellino,‡ Frank Lyko,# and Gianluca Sbardella*,§
Dipartimento di Scienze Farmaceutiche, UniVersità degli Studi di Salerno, Via Ponte Don Melillo, 84084 Fisciano (SA), Italy, Dipartimento di
Chimica Farmaceutica e Tossicologica, UniVersità di Napoli “Federico II”, Via D. Montesano 49, 80131 Napoli, Italy, and DiVision of
Epigenetics, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 580, 69120 Heidelberg, Germany
ReceiVed December 14, 2007
Constrained analogues of procaine were synthesized, and their inhibiting activity against DNMT1 was tested.
Among them, the most potent compound, derivative 3b, was also able to induce a recognizable demethylation
of chromosomal satellite repeats in HL60 human myeloid leukemia cells and thus represents a lead compound
for the development of a novel class of non-nucleoside DNMT1 inhibitors.
Chart 1. Nucleoside and Non-Nucleoside Inhibitors of DNMTs
Introduction
Epigenetic alterations are increasingly recognized as valuable
targets for the development of cancer therapies. They not only
occur early in carcinogenesis but also are found in virtually all
cases of cancer.1–4 Importantly, epigenetic alterations do not
involve changes in the DNA sequence and thus are potentially
reversible.2 Of the epigenetic changes seen in cancer, the most
extensively studied is the increased methylation of CpG
dinucleotides clustered in ∼1 kb regions, termed CpG islands.
This change in DNA methylation characteristically results in
the transcriptional silencing of important cancer genes such as
tumor suppressors and caretaker genes.5
Nucleoside analogues like 5-azacytidine, 5-aza-2′-deoxycy-
tidine (decitabine), or 2-pyrimidone-1-ꢀ-D-riboside (zebularine,
Chart 1), though effective in inducing DNA demethylation and
reactivation of hypermethylated genes, carry considerable
concerns about toxicity,6 which is probably associated with the
drugs’ incorporation into DNA. These concerns have strongly
encouraged the search for non-nucleoside inhibitors of DNA
methyltransferases (DNMTsa). However, only a limited number
of them have been described so far (Chart 1), including dietary
polyphenols like (-)-epigallocatechin-3-gallate (EGCG),7 the
bisulfide bromotyrosine derivatives psammaplins,8 the L-tryp-
tophan derivative RG108,9,10 and the 4-aminobenzoic acid
derivatives procaine and procainamide,11–13 originally approved
by the U.S. Food and Drug Administration as local anesthetic
and for the treatment of cardiac arrhythmias, respectively.
Initially proposed as perturbative of the interactions between
DNMTs and CpG-rich sequences, procainamide has been
recently reported to specifically inhibit the maintenance meth-
yltransferase activity of DNMT1 and to demethylate genes
affected by promoter CpG island hypermethylation.14
a lead structure for further modification. Because a high number
of rotatable bonds is usually detrimental for druglikeness,21,22
we decided to partially reduce the very high flexibility of
procaine.
According to the frozen analogue approach,23 we designed
two series of molecules (1–2 and 3–4, Figure 1) where the
N-alkylamide moiety of the lead scaffold was constrained into
a 4-substituted or 5-substituted oxazoline ring. Herein, we report
Being interested in the development of small-molecule
modulators of epigenetic targets,9,10,15–20 we chose procaine as
* To whom correspondence should be addressed. Phone: +39-089-96-
9770. Fax: +39-089-96-9602. E-mail: gsbardella@unisa.it.
§
Università degli Studi di Salerno.
Deutsches Krebsforschungszentrum.
Università di Napoli “Federico II”.
#
‡
a Abbreviations: DNMT, DNA methyltransferase; DNMT1, DNA me-
thyltransferase-1; HL60, human myeloid leukemia cell line; SAM, S-
adenosylmethionine; COBRA, combined bisulfite restriction analysis; C1S2,
chromosome 1 satellite 2 repeats; TIMP3, tissue inhibitor of metallopro-
teinase 3; HCT116, human colon carcinoma cell line; RARꢀ2, retinoic acid
receptor ꢀ2; MCF7, human breast adenocarcinoma cell line; Sf9, insect
cell line derived from Spodoptera frugiperda; MOI, multiplicity of infection.
Figure 1. Restricted conformation analogues of procaine.
10.1021/jm7015705 CCC: $40.75
2008 American Chemical Society
Published on Web 03/18/2008

2322 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Brief Articles
Scheme 1a
a Reagents and conditions: (a) NMM, HOBt, DCC, CH2Cl2/DMF 8:1, 0 °C to room temp; (b) Burgess reagent, THF, 80 °C; (c) NaBH4, MeOH, 0 °C;
(d) MsCl, TEA, THF, 0 °C to room temp; (e) diethylamine or pyrrolidine, DMF, 90 °C, sealed tube; (f) Zn, NH4Cl, acetone/water 4:1, room temp.
Scheme 2a
Figure 2. In vitro methylation assay for 1–4 against recombinant
human DNMT1. Procaine was used as reference drug. All compounds
were tested as 2 mM DMSO solutions. Error bars indicate standard
deviations of each measurement. DMSO (2 mM) alone did not affect
the enzymatic activity of DNMT1. Data are reported as the mean (
SD of three independent experiments.
a Reagents and conditions: (a) ZnCl2, chlorobenzene/NMP 95:5, reflux;
(b) MsCl, TEA, THF, 0 °C to room temp; (c) diethylamine or pyrrolidine,
DMF, 90 °C, sealed tube; (d) Zn, NH4Cl, acetone/water 4:1, room temp.
MOI of 5 for 48 h and DNMT1 was purified from cleared lysates
of infected cells by Ni-NTA agarose affinity chromatography
(Supporting Information). The identity and integrity of recom-
binant DNMT1 were confirmed by SDS gel electrophoresis and
Western blotting with a DNMT1 specific antibody (data not
shown). In order to determine the enzymatic activity of purified
DNMT1, we established a biochemical DNA methylation assay.
Methylation rates were measured by the incorporation of
radioactive labeled methyl groups into oligonucleotide sub-
strates. Tritium labeled S-adenosylmethionine (SAM) was used
as methyl group donor. The entire reaction was spotted manually
onto cellulose membranes, washed several times, dried, and after
addition of scintillation cocktail, subjected to a scintillation
counting. The results showed that the recombinant DNMT1
enzyme has substantial enzymatic activity (Figure 2). In
subsequent experiments, this assay was used to test the activity
of derivatives 1–4 (2 mM DMSO solutions). Procaine and
S-adenosylhomocysteine (data not shown) were used as refer-
ences (2 mM DMSO solutions).
the synthesis of such compounds and their inhibitory activity
toward DNA methyltransferases in vitro and in vivo.
Chemistry
The preparation of derivatives 1a,b and 2a,b is reported in
Scheme 1. Peptide coupling of 4-nitrobenzoic acid with (S)-
serine methyl ester hydrochloride gave carboxamide 5, which
underwent a smooth cyclization reaction with Burgess reagent24
to give the oxazoline derivative 6 that was then quantitatively
reduced to the primary alcohol 7.25 The transformation of the
latter into the amine 1a,b was performed by mesylation and
subsequent substitution reaction with diethylamine or pyrroli-
dine, respectively. Finally, reduction of nitro group using zinc
dust and NH4Cl in ethanol26 yielded 2a,b as hydrochloride salts
(Scheme 1).
The enantiomers ent-1a,b and ent-2a,b were prepared analo-
gously starting from (R)-serine methyl ester.
Derivatives 3a,b and 4a,b were obtained by a slight modi-
fication of the general stereospecifical route to 2-oxazolines
previously reported by Serrano et al. (Scheme 2).27 Briefly, a
chlorobenzene solution of 4-nitrobenzonitrile and (R)-3-ami-
nopropane-1,2-diol was refluxed in the presence of a catalytic
amount of zinc chloride and 1-methylpyrrolidin-2-one as
cosolvent to afford the alcohol 9 in 63% yield. The nucleophilic
displacement of the corresponding mesylate 10 with diethyl-
amine and pyrrolidine furnished 3a and 3b, respectively, which
were finally reduced with zinc and NH4Cl in ethanol to give 4a
and 4b as hydrochloride salts (Scheme 2).
Similar to what was previously reported,28 procaine did not
show any activity in this assay. Yet derivatives 1 and 3 exhibited
a weak but distinct inhibition of DNMT1 (Figure 2). In
particular, it is noteworthy that this activity is related to the
presence of the nitro group on the benzene ring (compounds
1a,b, 3a,b, and corresponding enantiomers29), whereas their
anilino counterparts (2a,b, 4a,b, and corresponding enantiomers)
were consistently less active. The replacement of the diethy-
lamino function with a pyrrolidine ring further improved the
inhibitory efficacy of tested compounds (for example, compare
the inhibition of 1a and 3a with the inhibition of their pyrrolidino
counterparts 1b and 3b, respectively). Remarkably, this was
particularly evident in nitro derivatives frozen in the 5-substi-
tuted oxazoline conformation. In fact, the gain in activity is more
marked between 3a and 3b (5-substituted oxazolino derivatives)
than between 1a and 1b (4-substituted equivalents). Finally, it
is also worth mentioning that the inhibitory potencies of 1–4
were always higher than those displayed by their enantiomers
The enantiomers ent-3a,b and ent-4a,b were prepared analo-
gously starting from (S)-3-aminopropane-1,2-diol.
Results and Discussion
In order to establish a biochemical assay for testing the
activity of derivatives 1–4 against DNMT1, we generated
recombinant baculoviruses expressing human DNMT1 with an
N-terminal epitope tag. Sf9 insect cells were infected with a

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2323
particularly sensitive toward demethylation induced by DNMT
inhibitors.32 A weak but detectable demethylation of these
sequences was observed for both procaine and 3b (Figure 3C),
which is consistent with the previously reported slight local
demethylation of the TIMP3 locus in HCT11614 or RARꢀ2
locus in MCF7 cells,12 after treatment with procainamide or
procaine, respectively.
Conclusions
In conclusion, we prepared a series of constrained analogues
of procaine and tested their inhibiting activity against DNMT1.
Among them, derivative 3b was proven the most interesting
compound, exhibiting the highest inhibiting potency, and when
tested for its effects on the genome methylation levels in HL60
human myeloid leukemia cells, it revealed a recognizable
demethylation of chromosomal satellite repeats. These effects
are cell type specific (data not shown), and though weaker than
those obtained with azanucleosides, they are fairly comparable
to other non-nucleoside inhibitors. Importantly, the high cyto-
toxicity and the low therapeutic index of nucleoside analogues
make the development of novel non-nucleoside inhibitors highly
desirable. Thus, derivative 3b might be considered a lead
compound for further studies in this field. Extensive SAR
analysis and molecular modeling studies are ongoing to increase
the knowledge within these series of DNMT1 inhibitors.
Experimental Section
Preparation of (S)-Methyl 2-(4-Nitrophenyl)-4,5-dihydroox-
azole-4-carboxylate (6) and (R)-Methyl 2-(4-Nitrophenyl)-4,5-
dihydrooxazole-4-carboxylate (ent-6). (S)-N-(4-Nitrobenzoyl)-
serine methyl ester (5, 1000.0 mg, 3.728 mmol) was dissolved in
dry THF (14 mL) in a sealed tube, and Burgess reagent [(meth-
oxycarbonylsulfamoyl)triethylammonium hydroxide inner salt]
(1066.2 mg, 4.474 mmol) was added. After the mixture was heated
at 80 °C for 1 h, the solution was evaporated and the crude purified
by column chromatography on silica gel (gradient, hexane/EtOAc,
8:2 to 4:6) to afford pure 6 (727.6 mg, 78%) as a white solid (mp
77–78 °C). (R)-Methyl 2-(4-nitrophenyl)-4,5-dihydrooxazole-4-
carboxylate (ent-6) was prepared under the same reaction conditions,
starting from (R)-N-(4-nitrobenzoyl)serine methyl ester (ent-5). 6:
[R]23 +111.9° (c 3.68, AcOEt). ent-6: [R]23 –110.8° (c 3.92,
Figure 3. Cellular methylation assays. Human myeloid leukemia
(HL60) cells were incubated for 72 h with DMSO (1%), procaine
(0.5 mM), decitabine (0.5 µM), and test compound 3b (0.5 mM).
(A) Genomic cytosine methylation levels were determined by
capillary electrophoretic analysis of isolated genomic DNA as
described in Supporting Information. Methylation levels are shown
relative to DMSO controls. Decitabine treatment induced a strong,
3b a weaker, genomic demethylation. Data are reported as the mean
( SD of three independent experiments. (B) Proliferation assay.
HL60 cells were seeded in duplicate in six-well plates, incubated
for 72 h, and counted for the determination of doubling times. (C)
COBRA assay of chromosomal satellite repeats (C1S2) as described
in Supporting Information. DMSO treated cells revealed methylated
satellites, but procaine as well as 3b induced minor demethylation
of the repetitive elements. Decitabine treatment was included as
positive control and showed the strongest demethylating effect.
D
D
AcOEt). 1H NMR (CDCl3): δ 3.84 (s, 3H), 4.66 (dd, 1H, J ) 10.7,
8.8 Hz), 4.76 (dd, 1H, J ) 8.8, 8.1 Hz), 5.00 (dd, 1H, J ) 10.7,
8.1 Hz), 8.15 (d, 2H, J ) 9.0 Hz), 8.27 (d, 2H, J ) 9.0 Hz). ESI-
MS m/z: 251 (M + H)+. Anal. (C11H10N2O5) C, H, N.
Preparation of (R)-[2-(4-Nitrophenyl)-4,5-dihydrooxazol-4-
yl]methanol (7) and (S)-[2-(4-Nitrophenyl)-4,5-dihydrooxazol-
4-yl]methanol (ent-7). To a solution of (S)-2-(4-nitrophenyl)-4,5-
dihydrooxazole-4-carboxylic acid methyl ester (6, 700.0 mg, 2.798
mmol) in MeOH (100 mL) cooled at 0 °C, NaBH4 was added until
disappearance of the starting material (TLC: silica gel, hexane/
EtOAc 3:7). The reaction mixture was evaporated, taken up with
water (100 mL), and extracted with chloroform (3 × 60 mL). The
organic phase was dried and the solvent evaporated to give pure 7
(608.7 mg, 98%) as a white solid (mp 133–134 °C). (S)-[2-(4-
nitrophenyl)-4,5-dihydrooxazol-4-yl]-methanol (ent-7) was prepared
under the same reaction conditions, starting from (R)-(4-nitrophe-
nyl)-4,5-dihydrooxazole-4-carboxylic acid methyl ester (ent-6). 7:
[R]23 +61.55° (c 1.06, CHCl3). ent-7: [R]23 –62.85° (c 1.07,
CHCl3). H NMR (CDCl3): δ 1.94 (br s, 1H), 3.72 (dd, 1H, J )
11.4, 4.1 Hz), 3.98 (dd, 1H, J ) 11.4, 3.5 Hz), 4.40–4.60 (m, 3H),
7.93 (d, 2H, J ) 8.9 Hz), 8.12 (d, 2H, J ) 8.9 Hz). ESI-MS m/z:
223 (M + H)+.
Preparation of (S)-(2-(4-Nitrophenyl)-4,5-dihydrooxazol-4-
yl)methyl Methanesulfonate (8) and (R)-(2-(4-Nitrophenyl)-4,5-
dihydrooxazol-4-yl)methyl Methanesulfonate (ent-8). To a so-
lution of (R)-[2-(4-nitrophenyl)-4,5-dihydrooxazol-4-yl]methanol (7,
370.0 mg, 1.66 mmol) and triethylamine (0.278 mL, 2.00 mmol)
ent-1–4, with the sole exception of 1a, which was slightly less
active than ent-1a.
As 3b showed the strongest inhibitory effects among tested
derivatives, we also explored its effect on the genomic DNA
methylation level of HL60 human myeloid leukemia cells. Cells
were incubated for 72 h with 0.5 mM drug solutions in DMSO,
and genomic cytosine methylation levels were quantitatively
determined by capillary electrophoresis.30 The proliferation of
HL60 cells (population doubling time) was also assessed.
The results demonstrated a slight decrease of the global
methylation level after 3b treatment compared to procaine
(Figure 3A). More pronounced demethylation was observed with
5-aza-2′-deoxycytidine (Figure 3A), but this effect was ac-
companied by substantial cytotoxicity (Figure 3B). However,
3b did not reveal any detectable cytotoxic effects on HL60 cells
(Figure 3B), thus encouraging the further development of this
scaffold. We also used combined bisulfite restriction analysis
(COBRA)31 to analyze the methylation status of chromosomal
satellite repeats. This assay has been demonstrated to be
D
D
1

2324 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Brief Articles
in dry THF (20 mL) methanesulfonyl chloride (0.155 mL, 2.00
mmol) was added at 0 °C. The resulting mixture was 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
chloroform (3 × 40 mL). The organic phase was dried and the
solvent evaporated to give mesylate 8 (502.5 mg, 98%), which was
used without further purification in the subsequent reaction. (R)-
(2-(4-Nitrophenyl)-4,5-dihydrooxazol-4-yl)methyl methanesulfonate
(ent-8) was prepared under the same reaction conditions, starting
from ent-7.
General Procedure for the Synthesis of (2-(4-Nitrophenyl)-
4,5-dihydrooxazol-4-yl)methanamines 1a,b and ent-1a,b. Ex-
ample: (R)-N-Ethyl-N-((2-(4-nitrophenyl)-4,5-dihydrooxazol-4-
yl)methyl)ethanamine(1a). (S)-(2-(4-Nitrophenyl)-4,5-dihydrooxazol-
4-yl)methyl methanesulfonate (8, 342.3 mg, 1.14 mmol) was
dissolved in dry DMF (14 mL) in a sealed tube, and diethylamine
(1.50 mL, 34.2 mmol) was added. After being heated at 90 °C
overnight, the mixture was evaporated, taken up with water (20
mL), and extracted with ethyl acetate (3 × 50 mL). The organic
layer was dried and evaporated. The crude was purified by column
chromatography on silica gel (gradient, chloroform/methanol, 98:2
to 90:10) to afford pure 1a (253.8 mg, 90%) as a yellow solid (mp
79–80 °C). [R]23D +21.05° (c 3.80, CHCl3). 1H NMR (CDCl3): δ
1.02 (t, 6H, J ) 7.1), 2.46–2.70 (m, 5H), 2.82 (dd, 1H, J ) 12.7,
4.5 Hz), 4.26–4.32 (m, 1H), 4.45–4.53 (m, 2H), 8.11 (d, 2H, J )
8.9 Hz), 8.23 (d, 2H, J ) 8.9 Hz). ESI-MS m/z: 278 (M + H)+.
Anal. (C14H19N3O3) C, H, N. (2-(4-Nitrophenyl)-4,5-dihydrooxazol-
4-yl)methanamines 1b and ent-1a,b were prepared under the same
reaction conditions, starting from mesylates 8 or ent-8 and the
proper amine.
General Procedure for the Synthesis of 4-Substituted-4-(4,5-
dihydrooxazol-2-yl)anilines 2a,b and ent-2a,b. Example: (R)-4-
(4-(Diethylaminomethyl)-4,5-dihydrooxazol-2-yl)aniline (2a). A
solution of (R)-N-ethyl-N-((2-(4-nitrophenyl)-4,5-dihydrooxazol-4-
yl)methyl)ethanamine (1a, 250.0 mg, 0.9 mmol) in acetone/water
4:1 (10 mL) was treated with zinc dust (471.5 mg, 7.2 mmol) and
ammonium chloride (385.5 mg, 7.2 mmol), and the resulting
suspension was stirred for 45 min at 20 °C. The reaction mixture
was then filtered, and the filtrate was evaporated. The crude was
purified by column chromatography on basic alumina, eluting with
chloroform to afford the pure hydrochloride of 2a (250.0 mg, 97%)
as a hygroscopic yellow solid foam. [R]23D +1.62° (c 3.08, MeOH).
1H NMR (DMSO-d6): δ 0.94 (t, 6H, J ) 7.0) 2.29–2.65 (m, 6H),
4.00–4.05 (m, 1H), 4.21–4.39 (m, 2H), 5.66 (s, 2H), 6.54 (d, 2H,
J ) 8.3 Hz), 7.51 (d, 2H, J ) 8.3 Hz). ESI-MS m/z: 248 (M +
H)+. Anal. (C14H21N3O·HCl) C, H, N. 4-(4,5-Dihydrooxazol-2-
yl)anilines 2b and ent-2a,b were prepared under the same reaction
conditions, starting from the corresponding nitro derivative 1b or
ent-1a,b.
The (2-(4-nitrophenyl)-4,5-dihydrooxazol-5-yl)methanamines
3a,b and ent-3a,b were prepared via the mesylates 10 according
to the same procedure described above for 1a.
The 5-substituted-4-(4,5-dihydrooxazol-2-yl)anilines 4a,b and
ent-4a,b were prepared by reduction of the corresponding nitro
derivatives 3a,b or ent-3a,b according to the same procedure
described above for 2a.
Acknowledgment. This work was partially supported by
grants from Regione Campania 2003, LR 5/02, Ministero
dell’Università e della Ricerca Scientifica e Tecnologica (PRIN
2005), Università di Salerno, Italy (G.S.), and the Helmholtz
Association of National Research Centers (Ideenwettbewerb
Gesundheit) and the Fonds der Chemischen Industrie (F.L.).
Supporting Information Available: Experimental chemical and
biological procedures and characterization data for 1-4 and for
intermediates 5–10, as well as for the corresponding enantiomers.
This material is available free of charge via the Internet at http://
pubs.acs.org.
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Preparation of (R)-(2-(4-Nitrophenyl)-4,5-dihydrooxazol-5-
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5-yl)methanol (ent-9). Zinc chloride (460.0 mg, 50 mol %) was
melted under vacuum in a two-necked flask, and chlorobenzene
(15 mL) was added under nitrogen at room temperature, followed
by 4-nitrobenzonitrile (1000 mg, 6.75 mmol), (R)-3-aminopropane-
1,2-diol (738 mg, 8.10 mmol), and 1-methyl-2-pyrrolidinone (0.5
mL). The resulting yellow solution was heated at reflux for 24 h.
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residue dissolved in dichloromethane (30 mL). The solution was
washed with saturated aqueous sodium hydrogen carbonate (2 ×
10 mL), dried, and concentrated under vacuum. The crude was
purified by column chromatography on silica gel (gradient,
chloroform/methanol, 98:2 to 90:10) to afford pure 9 (944.8 mg,
63%) as a yellow solid (mp 129–131 °C). ent-9 was prepared under
the same reaction conditions, starting from (S)-3-aminopropane-
1,2-diol. 9: [R]23 +5.39° (c 5.560, CHCl3). ent-9: [R]23 –5.77°
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D
D
1
(c 5.195, CHCl3). H NMR (CDCl3): δ 2.65 (br s, 1H), 3.75 (dd,
1H, J ) 12.3, 5.4 Hz), 3.82–3.95 (m, 2H), 4.12 (dd, 1H, J ) 15.2,
10.0 Hz), 4.82–4.90 (m, 1H), 8.06 (d, 2H, J ) 9.0 Hz), 8.20 (d,
2H, J ) 9.0 Hz). ESI-MS m/z: 223 (M + H)+. Anal. (C10H10N2O4)
C, H, N.

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