Rapid Synthesis of New DNMT Inhibitors Derivatives of Procainamide

Article abstract of DOI:10.1002/cbic.201100522

DNA methyltransferases (DNMTs) are responsible for DNA methylation, an epigenetic modification involved in gene regulation. Families of conjugates of procainamide, an inhibitor of DNMT1, were conceived and produced by rapid synthetic pathways. Six compoun

Full text of DOI:10.1002/cbic.201100522

DOI: 10.1002/cbic.201100522
Rapid Synthesis of New DNMT Inhibitors Derivatives of
Procainamide
Ludovic Halby,[a, b, c, g] Christine Champion,[a, c] Catherine Sꢀnamaud-Beaufort,[a]
Sophie Ajjan,[a, c] Thierry Drujon,[d] Arumugam Rajavelu,[e] Alexandre Ceccaldi,[a, c]
Renata Jurkowska,[e] Olivier Lequin,[d] William G. Nelson,[f] Alain Guy,[b] Albert Jeltsch,[e]
Dominique Guianvarc’h,[d] Clotilde Ferroud,*[b] and Paola B. Arimondo*[a, g]
DNA methyltransferases (DNMTs) are responsible for DNA
methylation, an epigenetic modification involved in gene regu-
lation. Families of conjugates of procainamide, an inhibitor of
DNMT1, were conceived and produced by rapid synthetic
pathways. Six compounds resulted in potent inhibitors of the
murine catalytic Dnmt3A/3L complex and of human DNMT1, at
least 50 times greater than that of the parent compounds. The
inhibitors showed selectivity for C5 DNA methyltransferases.
The cytotoxicity of the inhibitors was validated on two tumour
cell lines (DU145 and HCT116) and correlated with the DNMT
inhibitory potency. The inhibition potency of procainamide
conjugated to phthalimide through alkyl linkers depended on
the length of the linker; the dodecane linker was the best.
Introduction
Epigenetic modifications are known to control gene expres-
sion. In mammals, methylation of deoxycytidines was shown
to be a major factor in epigenetic regulation and occurs at
CpG sites, which are enriched in so-called CpG islands, often
located in gene promoters.[1–3] Of note, hypermethylation of
the promoters results in gene silencing. The addition of a
methyl group on the carbon-5 position of the cytosine is cata-
lysed by C5 DNA methyltransferases (DNMTs).[4–6] Mammalian
DNMTs are divided in two major families: DNMT1 and DNMT3
(DNMT3A, 3B and 3L), DNMT2 being a tRNA methyltransferase.
With the exception of DNMT3L, which is catalytically inactive,
all share a common catalytic mechanism that uses S-adenosyl-
l-methionine (SAM) as the methyl donor. The epigenetic im-
pairment of information can compromise the normal develop-
ment of an organism and is involved in various diseases, such
as cancer.[7,8] Cancerous cells often present two types of aber-
rant DNA methylation: global hypomethylation leading to ge-
nomic instability and specific hypermethylation of the promot-
ers of certain genes, such as tumour suppressor genes, which
silences them. Interestingly, epigenetic modifications are rever-
sible, and it has been shown that inhibitors of DNMTs are able
to demethylate the promoters and reactivate tumour suppres-
sor genes.[9–11] Therefore DNMT inhibitors offer great promise
for the development of new and more efficient anticancer
strategies.
inhibitor of DNMT to CpG-rich regions, in order to increase its
local concentration at CpG sites. We have already successfully
used this strategy to direct the action of DNA topoisomerase I
and II inhibitors to specific DNA sites.[17–21] Here we synthesised
several conjugates of procainamide (Scheme 1) by developing
a new and rapid synthetic pathway, and found six potent in-
hibitors of Dnmt3A/3L and DNMT1.
[a] L. Halby, Dr. C. Champion, C. Sꢀnamaud-Beaufort, S. Ajjan, Dr. A. Ceccaldi,
Dr. P. B. Arimondo
CNRS-MNHN UMR 7196 INSERM U565
43 rue Cuvier, 75005 Paris (France)
E-mail: paola.arimondo@etac.cnrs.fr
[b] L. Halby, Prof. A. Guy, Prof. C. Ferroud
CNAM UMR 7084 CNRS
2 rue Contꢀ, 75003 Paris (France)
E-mail: clotilde.ferroud@cnam.fr
[c] L. Halby, Dr. C. Champion, S. Ajjan, Dr. A. Ceccaldi
UPMC Universitꢀ Paris 6
75005 Paris (France)
[d] T. Drujon, Prof. O. Lequin, Dr. D. Guianvarc’h
UMR 7203 CNRS-UPMC-ENS and FR2769
4 place Jussieu, 75005 Paris (France)
[e] A. Rajavelu, Dr. R. Jurkowska, Prof. A. Jeltsch
Jacobs University Bremen, School of Engineering and Science
Campus Ring 1, 28759 Bremen (Germany)
Today, two families of DNMT inhibitors are known:[12] nucleo-
side and non-nucleoside analogues. Among the latter, procai-
namide 1, used over the last 30 years as an antiarrhytmic, was
shown to induce significant demethylation of genomic DNA in
human cancer cells, together with its analogue, procaine (an
anaesthetic).[13,14] These compounds are described to bind to
CpG sequences and thereby disturb DNA methylation.[13,15,16] In
this study, we used procainamide as a DNA binder to guide an
[f] Dr. W. G. Nelson
John Hopkins University
1650 Orleans Street, Baltimore, MD 21231 (USA)
[g] L. Halby, Dr. P. B. Arimondo
Present address: CNRS-Pierre Fabre USR3388 ETaC
31035 Toulouse Cedex 01 (France)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201100522.
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157

C. Ferroud, P. B. Arimondo et al.
The ability of compounds 6 and 7 to inhibit human DNMT1
and the murine catalytic Dnmt3A/3L complex was evaluated in
3
a H-methyl group incorporation experiment (Figure 1). DNMT1
was chosen because it has been previously described as a
target of procainamide,[13] while, to our knowledge, no specific
study has been performed on the Dnmt3A/3L complex. Parent
l-RG108 has never been tested on Dnmt3A/3L either, while on
human DNMT1 it showed 34% inhibition at 1 mm.[24] Since
conjugates 6 and 7 showed a moderate increase in activity
compared to parent 1 and l-RG108, a second generation of
derivatives coupled at the carboxylic acid of l-RG108 was pre-
pared. In order to explore different lengths of linkers between
the l-RG108 and procainamide moieties, the latter was at-
tached to a phthalimide by alkyl linkers of 4 to 14 carbon
atoms (Scheme 3). A similar one-pot synthesis was developed:
the nosyl derivative of procainamide 2 was reacted with vari-
ous bromoalkylphthalimides (B1–6) in the presence of K2CO3,
and then treated with thiophenol and water to provide the
corresponding phthalimidoalkyl derivatives of procainamide
(8–13).
Scheme 1. Schematic representation of the conceived conjugates.
Results and Discussion
Synthesis of RG108–procainamide conjugates
Procainamide 1 was conjugated to the known inhibitor l-
RG108 (N-phthalimidoyl protected l-tryptophane).[22–24] The
conjugation was at the carbon-5 position of the indole as, ac-
cording to Schirrmacher et al.,[25] this position could be func-
tionalised without any modification of the activity. We devel-
oped
(Scheme 2). An activation-protection sequence was realised by
reacting the primary amine of with small excess
a rapid pathway requiring few purification steps
1
a
(1.25 equiv) of 2-nitrobenzenesulfonyl chloride (2-nosyl chlo-
ride) to give nosyl derivative 2.[26,27] Compound 3, an OH-deriv-
atized l-RG108, was functionalised with a bromoalkyl linker of
6 or 12 atoms (4 and 5, respectively). An original one-pot reac-
tion was developed: the coupling of 2 to 4 or 5 was per-
formed in Williamson conditions, followed by double deprotec-
tion by thiophenol treatment and addition of water to afford 6
and 7.
Compounds 9, 12 and 13 were deprotected by using N-
methylhydrazine to give the respective primary amines 14–16,
and coupled to l-RG108 to give compounds 17, 18 and 19, re-
spectively (Scheme 4). In addition, the effect of the configura-
tion of the asymmetric carbon atom on RG108 was investigat-
ed by coupling primary amine 15 to d-RG108 at the carboxylic
acid group to afford 18D. Analogue 20, bearing only the linker
arm of 12 carbon atoms, was also synthesized by reaction of 1-
bromododecane with 2 followed
by treatment with thiophenol.
All
compounds,
l-RG108
amide derivatives (17, 18, and
19) and intermediate phthali-
mides (8 to 13), were tested on
DNMT1 and Dnmt3A/3L activity
at 10 mm (Figure 1). Six com-
pounds showed greatly in-
creased activity compared to the
parent 1 (Table 1). The inhibition
profiles on the two enzymes was
comparable.
Interestingly, in the phthali-
mide family, as the length of the
linker increased the inhibition
activity increased, up to 12
(bearing the 12 carbon atoms
linker). Compounds 12 and 13
proved among the most potent
inhibitors together with the l-
RG108 conjugates 18 and 19
and d-RG108 conjugate 18D, all
bearing long linker arms (Table 1
and Figure S1 in the Supporting
Information). On Dnmt3A/3L, the
l
configuration was slightly
Scheme 2. a) 2-Nitrobenzenesulfonyl chloride, CH2Cl2, 18 h, RT, 55%. b) K2CO3, CH3CN 18 h reflux, 58% for n=4,
61% for n=10. c) K2CO3, KI, CH3CN, 18 h reflux then thiophenol, K2CO3, CH3CN 3 h reflux, then H2O 30 min RT,
68% for n=4, 58% for n=10.
more potent. On DNMT1, 18 and
19 were three- to fourfold less
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ChemBioChem 2012, 13, 157 – 165

Design of Conjugates of Procainamide as DNMT Inhibitors
Scheme 4. a) NH2-NHCH3, MeOH, 18 h, RT, 69% for n=8. b) l-RG108, DCC,
NHS, DMF, 4 h, RT, 58% for n=2 over two steps, 72% for n=8, 46% for
n=10 over the two steps; d-RG108 was used to afford 62% 18D. c) 1-Bro-
mododecane, K2CO3, KI, CH3CN, 18 h, reflux, then thiophenol, 3 h, RT, 94%.
Figure 1. Normalized methylation activity (%) expressed relative to activity
in the absence of inhibitor (“mock”) for A) DNMT1 and B) Dnmt3A/3L in the
presence of compounds (10 mm). Data are meanꢀSE of three experiments.
Study of the mechanism of action
Next we investigated by saturation transfer difference (STD)
NMR spectroscopy experiments which moiety of the conju-
gates interacts with the enzyme. In this context, it has been
recently proposed by modelling that procainamide could inter-
act with DNMT1 as well as the DNA substrate.[31] The STD ex-
periments showed that procainamide did not interact with
DNMT1, Dnmt3A or Dnmt3A/3L, whereas phthalimide and l-
RG108 interacted with the enzymes (Figure 2 and Figure S3).
These results suggest that the procainamide moiety of the
conjugate acts as DNA binder and directs the action of the
phthalimide or l-RG108 on the enzyme. Finally, docking simu-
lations with Dock 6.4 for 12 (Figure 2E and Figure S5), 18 and
20 (data not shown) in the X-ray structure of the catalytic
domain of Dnmt3A (PDB ID:
active than 12. Compound 20, in which the procainamide is at-
tached to the simple dodecane linker, was among the most
potent compounds, while the presence of a primary amine (in
15) decreased the activity, especially on DNMT1 (Figure 1). This
suggests that the presence of a hydrophobic group may be
necessary for the activity. In order to exclude the possibility
that the inhibition was due to aggregation of the molecules
(and thus a non-specific effect),[28] 12, 18 and 20 were tested
in the presence of detergent.[29,30] No significant effect was ob-
served (Figure S2).
2QRV) positioned the phthali-
mide or l-RG108 moiety with
the linker arm in the catalytic
pocket, while the procainamide
moiety protruded outwards
through a cavity. In addition, the
selectivity of the conjugates for
the C5 DNA methyltransferases
was addressed by comparison of
the activity on the murine cata-
Scheme 3. a) K2CO3, KI, CH3CN, 18 h, reflux, then thiophenol, K2CO3, CH3CN, 3 h, reflux, 79% for n=0, 82% for
n=2, 75% for n=4, 83% for n=6, 89% for n=8, 85% for n=10.
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159

C. Ferroud, P. B. Arimondo et al.
Table 1. Biochemical and biological activity of the procainamide conju-
gates. Inhibition (IC50) of murine catalytic complex Dnmt3A/3L and
human DNMT1, and cytotoxicity (TC50) with DU145 and HCT116 cell lines.
Table 2. Methyltransferase selectivity. Inhibition (IC50) of murine catalytic
complex Dnmt3A/3L, EcoDam and G9A.[a]
IC50 [mm]
EcoDam
IC50 [mm][a]
Compounds Dnmt3A/3L DNMT1
TC50 [mm][b]
Cpds
Dnmt3A/3L
G9a
HCT116
DU145
8
11
12
13
410ꢀ240
210ꢀ20
8.5ꢀ4.2
9.2ꢀ3.6
>1000
>1000
1
>500
>500
>500
>300
>250
>1000
>100
>100
>100
>100
>100
430ꢀ44
424ꢀ63
n.d.
700ꢀ68
657ꢀ51
n.d.
n.d.
>1000
200ꢀ40
n.d.
70ꢀ10
l-RG108
phthalimide
160ꢀ50
6
7
8
81ꢀ6
n.d.
n.d.
n.d.
n.d.
[a] Concentration (meanꢀSE of three to five experiments) at which 50%
174ꢀ5
450ꢀ50
185ꢀ20
26ꢀ2
inhibition of the enzyme activity was observed.
180ꢀ10
33ꢀ10
313ꢀ53
72ꢀ13
24ꢀ10
25ꢀ14
9
10
11
12
13
15
17
18
18D
19
20
4.3ꢀ0.8
26ꢀ5
77ꢀ9
10ꢀ5
8.2ꢀ1.9
4.9ꢀ1.3
8.5ꢀ2.1
66ꢀ6
2.1ꢀ0.6
4.8ꢀ2.1
27ꢀ7
8.5ꢀ2.1
tone H3K9 methyltransferase, as described in [32]–[34]. As
shown in Table 2, 12 and 13 were very specific for C5 DNA
methyltransferases. Finally, we determined the cytotoxicity of
the most potent compounds on two tumour cell lines, HCT116
(colon cancer cells) and DU145 (prostate cancer cells). The cy-
totoxicity profile obtained for the two cancer cell lines was
similar to the inhibition profile of the methylation activity
(Table 1 and Figure S4). Again, for the phthalimide family, as
the length of the linker increased the cytotoxicity increased up
to compound 12. Once more, the compounds that showed the
highest cytotoxicity were the phthalimide conjugates 12 and
13, the long amido conjugates of l-RG108 (18 and 19) and of
d-RG108 (18D) and compound 20.
9.7ꢀ2.0
15ꢀ5
5.8ꢀ1.1
43ꢀ0.7
n.d.
89ꢀ5
55ꢀ4
n.d.
4.2ꢀ0.8
10.5ꢀ1.7
9.0ꢀ2.6
3.3ꢀ0.9
21ꢀ9
8.5ꢀ3.1
2.9ꢀ1.3
3.7ꢀ1.8
2.2ꢀ1.2
7.3ꢀ0.5
5.9ꢀ2.6
2.1ꢀ0.6
8ꢀ0.7
21ꢀ2
18ꢀ3
8.2ꢀ0.9
[a] Concentration (meanꢀSE of three to five experiments) at which 50%
inhibition of the enzyme activity was observed. [b] Concentration
(meanꢀSE of three to five experiments) at which 50% inhibition of cellu-
lar proliferation was observed. n.d.=not determined
Conclusion
We have developed a new rapid synthetic pathway to obtain
potent inhibitors of DNMT1 and Dnmt3A/3L, based on the
conjugation of procainamide to l-RG108 or phthalimide. Five
conjugates were at least 50 times more active than their
parent compounds. For the l-RG108 conjugates, the attach-
ment site on l-RG108 was extremely important: conjugation to
the indolo moiety at position 5 resulted in moderately active
compounds, while conjugation at the carboxyl group gave
among the most active compounds. We also showed that the
length of the linker arm plays an important role and that the
12-carbon linker (12 and 18) is the most promising. STD ex-
periments and docking simulations suggest that the procaina-
mide moiety does not interact with the enzyme, in agreement
with the hypothesis of its binding to DNA as a ligand to posi-
tion the phthalimide or l-RG108 to interact with the enzyme.
Interestingly, 12 and 13 showed very good selectivity for C5
DNA methyltransferases when compared to bacterial DNA
methyltransferase (EcoDam) and mammalian histone G9a
methyltransferase. This observation together with the fact that
the presence of 0.01% Triton X-100 did not affect enzyme in-
hibition indicates that the inhibition is not due to non-specific
aggregation as observed in several screening campaigns.[29,30]
In addition, conjugates 18 and 19 presented a slight selectivity
between Dnmt3A/3L and DNMT1, which can be further im-
proved by substitution, as shown for S-adenosyl-l-homocystein
analogues.[35] Interestingly, the most potent inhibitors, 12, 13,
18, 19 and 20, also showed micromolar cytotoxicity towards
1
Figure 2. 1D H NMR spectra of A) procainamide (500 mm), C) phthalimide
(500 mm), and 1D STD NMR spectra of B) procainamide (500 mm), D) phthali-
mide (500 mm) in the presence of Dnmt3A (8.5 mm). *: contaminant. E) Dock-
ing of 12 in the X-ray crystal structure of Dnmt3A/3L (PDB ID: 2QRV,
chain A).
lytic Dnmt3A/3L complex against bacterial EcoDam N-6 DNA
methyltransferase and the catalytic domain of human G9a his-
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Design of Conjugates of Procainamide as DNMT Inhibitors
J=8.7 Hz, 2Hp8), 7.01 (d, J=8.7 Hz, 2Hp9), 3.44 (m, 2Hp4), 2.94 (m,
6Hp2+p3), 1.10 (t, J=7.2 Hz, 6Hp1); 13C NMR (100 MHz, DMSO): d=
171.5 (Cp6), 153.3 (Cp7), 151.3 (Cn2), 139.4 (Cn1), 138.4 (Cn5), 137.1
(Cn4), 134.9 (Cn3), 133.4 (Cn6), 131.8 (Cp10), 129.2 (Cp8), 124.4 (Cp7),
55.8 (Cp4) 52.0 (Cp3), 40.6 (Cp2), 14.9 (Cp1); HRMS-ESI (m/z) calcd for
C19H24N4O5S: 421.15402 [M+H]+; found: 421.15374.
cancerous cell lines DU145 and HCT116. This is very promising
for their application as antitumor agents.
Experimental Section
Chemicals: General S-adenosyl-l-methionine (SAM) was bought
from Sigma; aliquots were stored at ꢁ808C; procainamide was
from Sigma; oligonucleotides were from Eurogentec (Seraing, Bel-
gium). All chemicals were from Sigma–Aldrich except dibromote-
tradecane, which was from Epsilon Chemie (Brest, France). l-RG108
and d-RG018 were synthesized from l- and d-tryptophane, respec-
tively, according to Schirrmacher et al.[25] Compound 3, an OH-de-
rivatized l-RG108, was obtained as described by Schirrmacher
et al.[25]
(4-Nitrophenyl)methyl-3-{5-[(6-bromohexyl)oxy]-1H-indol-3-yl}-2-
(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)propanoate (4) and (4-ni-
trophenyl)methyl-3-{5-[(6-bromododecyl)oxy]-1H-indol-3-yl}-2-
(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)propanoate (5):
Compound purity was verified by reversed-phase HPLC on an X-
terra C18 MS column (3.9ꢂ100 mm; Waters) with a linear gradient
acetonitrile in 0.1% formic acid (0!95% CH3CN).
Physical measurements: 1H and 13C NMR spectra were acquired
on a Bruker AM400 spectrometer (400 MHz) or on a Bruker Avance
1
II spectrometer equipped with a 13C cryoprobe, at 500 MHz for H
and 125 MHz for 13C; 2D experiments were performed by using
standard Bruker programs. Assignments of the NMR signals are
presented in the Supporting Information.
Dibromoalkyl (1,12-dibromododecane; 400 mg, 1.22 mmol) or 1,6-
dibromohexane (285 mg, 1.22 mmol), K2CO3 (170 mg, 1.22 mmol)
and a catalytic amount of potassium iodide was added to a solu-
tion of 3 (200 mg, 0.41 mmol) in acetone (5 mL). The mixture was
heated at 908C overnight. The mixture was diluted with ethylace-
tate and washed with water. The organic phase was washed with
brine and dried with magnesium sulfate. Solvent was removed and
the residue was purified by flash chromatography on silica gel (cy-
clohexane/ethylacetate, 8:2) to afford 4 (154 mg, yield 58%) and 5
(184 mg, yield 61%) as dark yellow foams.
1
We decided to describe the ABX systems in H NMR spectra as il-
lustrated below. The “external” chemical displacements of the ABX
system (n8 for part B and n1 for part A) will be pointed out, and
the coupling constants Ja and Jb are defined by: Ja = (n1ꢁn2)=
(n7ꢁn8) Jb =(n1ꢁn3)=(n6ꢁn8)
ABX system:
Compound 4: 1H NMR (400 MHz, CDCl3): d=8.10 (dm, J=8.9 Hz,
2Hb4), 7.84 (m, 1Ht1), 7.63 (m, 2Hph3), 7.57 (m, 2Hph4), 7.36 (dm, J=
8.9 Hz, 2Hb3), 7.06 (d, J=8.7 Hz, 1Ht7), 6.32 (m, 2Ht2+t4), 6.71 (dm,
J=8.7 Hz, 1Ht6), 5.25 (m, 3Hb1+t11), 3.85 (m, 2Hl5), 3.66 (d, J=
8.0 Hz, 2Ht10), 3.33 (t, J=6.9 Hz, 2Hl1), 1.76 (m, 2Hl2), 1.70 (m, 2Hl4),
1.22 (m, 4Hl3); 13C NMR (100 MHz, CDCl3): d=168.9 (Cph1), 167.5
(Ct12), 153.6 (Ct5), 147.8 (Cb5), 142.5 (Cb2), 134.2 (Cph4), 131.6 (Cph2),
131.1 (Ct8), 128.3 (Ct2), 127.6 (Ct3), 123.8 (Cb4), 123.5 (Cb3), 123.3
(Cph3), 113.0 (Ct), 111.8 (Ct), 110.5 (Ct9), 101.1 (Ct), 68.7 (Cb1), 66.0 (Cl5),
52.6 (Ct11), 34.1 (Ct10), 32.9 (Cl1), 26.6–25.0 (Cl2+l3+l4); MS-ESI (m/z)
calcd for C32H30N3O7Br: 648.1345–650.1325 [M+H]+; found:
648.1414–650.1386.
n1 and n8 (ABX, 2H; Ja =Hz, Jb =Hz; attribution); NMR spectra of all
tested molecules are available in the Supporting Information.
Chemical synthesis
N-[2-(Diethylamino)ethyl]-4-[(2-nitrobenzene)sulfonamido]benz-
amide (2):
Compound 5: 1H NMR (400 MHz, CDCl3): d=8.09 (dm, J=8.8 Hz,
2Hb4), 7.82 (m, 1Ht1), 7.65 (m, 2Hph3), 7.57 (m, 2Hph4), 7.36 (dm, J=
8.8 Hz, 2Hb3), 7.06 (d, J=8.7 Hz, 1Ht7), 6.90 (m, 2Ht2+t4), 6.69 (dm,
J=8.7 Hz, 1Ht6), 5.23 (m, 3Hb1+t11), 3.83 (m, 2Hl5), 3.65 (d, J=
8.0 Hz, 2Ht10), 3.33 (t, J=6.9 Hz, 2Hl1), 1.77 (m, 2Hl2), 1.69 (m, 2Hl4),
1.42–1.18 (m, 16Hl3); 13C NMR (400 MHz, CDCl3): d=168.9 (Cph1),
167.5 (Ct12), 153.6 (Ct5), 147.8 (Cb5), 142.5 (Cb2), 134.2 (Cph4), 131.6
(Cph2), 131.1 (Ct8), 128.4 (Ct2), 127.6 (Ct3), 123.8 (Cb4), 123.5 (Cb3),
123.3 (Cph3), 113.0 (Ct), 111.8 (Ct), 110.5 (Ct9), 101.1 (Ct), 68.7 (Cb1),
66.0 (Cl5), 52.7 (Ct11), 34.2 (Ct10), 32.9 (Cl1), 29.6–25.0 (Cl2+l3+l4); MS-
ESI (m/z) calcd for C16H20NO2Br: 732.2284–734.2264 [M+H]+; found:
732.2334–734.2354.
2-Nitrophenylsufonyl chloride (5 g, 23 mmol) and DMAP (0.25 g,
2 mmol) was added to a solution of procainamide 1 (hydrochloride
salt; 5 g, 18.5 mmol) in dichloromethane (50 mL) and TEA (5 mL).
The mixture was stirred for 18 h at room temperature. The solvent
was removed by vacuum, and the residue was purified by flash
chromatography silica gel with dichloromethane/methanol (9:1+
1.75% ammonia) to give 2 as a pale yellow amorphous solid (hy-
groscopic; 4.3 g, yield 55%). 1H NMR (400 MHz, DMSO): d=8.36
(m, 1Hp11), 7.94 (m, 1Hn3), 7.82 (m, 1Hn6), 7.71 (m, 2Hn4+n5), 7.64 (d,
3-[5-({6-[(4-{[2-(Diethylamino)ethyl]carbamoyl}phenyl)amino]-
hexyl}oxy)-1H-indol-3-yl]-2-(1,3-dioxo-2,3-dihydro-1H-isoindol-2-
yl)propanoic acid (6) and 3-[5-({6-[(4-{[2-(Diethylamino)ethyl]car-
bamoyl}phenyl)amino]dodecyl}oxy)-1H-indol-3-yl]-2-(1,3-dioxo-
2,3-dihydro-1H-isoindol-2-yl)propanoic acid (7):
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161

C. Ferroud, P. B. Arimondo et al.
was purified by flash chromatography on silica gel (cyclohexane/
ethylacetate, 8:2) to afford bromoalkylphthalimides as white amor-
phous solids.
2-(8-Bromooctyl)-2,3-dihydro-1H-isoindole-1,3-dione (B3): n=4;
from 490 mg of 1.8-dibromooctane, B3 was obtained in 54% yield.
1H NMR (400 MHz, CDCl3): d=7.75 (m, 2Hph3), 7.58 (m, 2Hph4), 3.58
(t, J=7.3 Hz, 2Hl5), 3.27 (t, J=6.8 Hz, 2Hl1), 1.76 (m, 2Hl4), 1.58 (m,
2Hl2), 1.39–1.15 (m, 8Hl3); 13C NMR (100 MHz, CDCl3): d=168.4
(Cph1), 133.8 (Cph4), 132.2 (Cph2), 123.1 (Cph3), 38.0 (Cl5), 33.9 (Cl1), 32.7
(Cl4), 28.9–26.7 (Cl2+l3); MS-ESI (m/z) calcd for C16H20NO2Br:
338.0756–340.0735 [M+H]+; found: 338.0805–340.0784.
K2CO3 (160 mg, 1.16 mmol) and the desired l-RG108 derivative 4
(123 mg, 0.19 mmol) or 5 (140 mg, 0.19 mmol) was added to a so-
lution of nosylate 2 (160 mg, 0.38 mmol) in dry acetonitrile (3 mL).
The mixture was refluxed overnight, then thiophenol (50 mL,
0.48 mmol) was added. The reaction mixture was heated at 808C
for 2 h, then water (50 mL) was added, and the mixture was heated
at 808C for 0.5 h. Solvents were removed, and the residue was pu-
rified by preparative HPLC with a C-18 column and a linear gradi-
ent (0–90% acetonitrile with 0.1% TFA) to afford 6 (73 mg, yield
68%) or 7 (83 mg, yield 58%) as dark yellow foams.
2-(8-Bromodecyl)-2,3-dihydro-1H-isoindole-1,3-dione (B4): n=6;
from 540 mg of 1,10-dibromodecane, B4 was obtained in 55%
yield. 1H NMR (400 MHz, CDCl3): d=7.75 (m, 2Hph3), 7.58 (m,
2Hph4), 3.58 (t, J=7.3 Hz, 2Hl5), 3.27 (t, J=6.8 Hz, 2Hl1), 1.76 (m,
2Hl4), 1.58 (m, 2Hl2), 1.39–1.15 (m, 12Hl3); 13C NMR (100 MHz,
DMSO): d=168.4 (Cph1), 133.8 (Cph4), 132.2 (Cph2), 123.1 (Cph3), 38.0
(Cl5), 33.9 (Cl1), 32.8 (Cl4), 29.7–25.6 (Cl2+l3); MS-ESI (m/z) calcd for
C18H24NO2Br: 366.1069–368.1048 [M+H]+; found: 366.1118–
368.1096.
1
2-(8-Bromododecyl)-2,3-dihydro-1H-isoindole-1,3-dione (B5): n=
8; from 590 mg of 1,12-dibromododecane, B5 was obtained in
58% yield. H NMR (400 MHz, CDCl3): d=7.73 (m, 2Hph3), 7.62 (m,
2Hph4), 3.61 (t, J=7.3 Hz, 2Hl5), 3.34 (t, J=6.8 Hz, 2Hl1), 1.72 (m,
2Hl4), 1.61 (m, 2Hl2), 1.33 (m, 16Hl3); 13C NMR (100 MHz, CDCl3):
d=168.4 (Cph1), 133.8 (Cph4), 132.2 (Cph2), 123.1 (Cph3), 38.1 (Cl5), 34.0
(Cl1), 32.8 (Cl4), 29.5–27.0 (Cl2+l3); MS-ESI (m/z) calcd for
C20H28NO2Br: 394.1382–396.1361 [M+H]+; found: 394.1417–
396.1385.
Compound 6: H NMR (400 MHz, DMSO): d=10.55 (s, 1Ht13), 9.28
(sb, 1Ht1), 8.28 (m, 1Hp5), 7.77 (m, 4Hph3+ph4), 7.59 (d, J=8.8 Hz,
2Hp8), 7.07 (d, J=8.7 Hz, 1Ht7), 6.95 (m, 1Ht2 or t4), 6.86 (m, 1Ht2 or t4),
6.54 (m, 3Ht6+p9), 5.02 (t, J=8.3, 1Ht11), 3.77 (m, 2Hl5), 3.52–3.42 (m,
4Ht10+p4), 3.17 (m, 6Hp2+p3), 3.04 (m, 2Hl1), 1.60 (m, 4Hl2+l4), 1.40
(m, 4Hl3), 1.16 (t, J=7.2, 6Hp1); 13C NMR (100 MHz, DMSO): d=
170.3 (Cp6), 167.2 (Ct12), 167.1 (Cph1), 152.3 (Ct5), 151.7 (Cp7), 134.8
(Cph4), 130.9 (Cph2), 130.7 (Ct8), 128.8 (Cp8), 128.6 (Ct2), 127.1 (Ct3),
123.1 (Cph3), 119.7 (Cp10), 112.0 (Ct4 or t6), 111.6 (Cp9), 110.6 (Ct4 or t6),
109.5 (Ct9), 100.5 (Ct7), 67.6 (Cl5), 52.7 (Ct11), 50.2 (Cp3), 46.7 (Cp2), 42.4
(Ct10), 34.3–25.5 (Cl1+l2+l3+l4+p4), 8.4 (Cp1); HRMS-ESI (m/z) calcd for
C38H46O6N5: 668.34426 [M+H]+; found: 668.34475.
1
2-(8-Bromotetradecyl)-2,3-dihydro-1H-isoindole-1,3-dione (B6):
n=10; from 640 mg of 1,14-dibromotetradecane, B6 was obtained
in 51% yield. 1H NMR (400 MHz, CDCl3): d=7.73 (m, 2Hph3), 7.62
(m, 2Hph4), 3.60 (t, J=7.3 Hz, 2Hl5), 3.35 (t, J=6.8 Hz, 2Hl1), 1.74 (m,
2Hl4), 1.61 (m, 2Hl2), 1.40–1.10 (m, 20Hl3); 13C NMR (100 MHz,
CDCl3): d=168.5 (Cph1), 133.8 (Cph4), 132.1 (Cph2), 123.1 (Cph3), 38.1
(Cl5), 34.1 (Cl1), 32.9 (Cl4), 29.6–26.8 (Cl2+l3); MS-ESI (m/z) calcd for
C22H32NO2Br: 422.1695–424.1674 [M+H]+; found: 422.1720–
424.1691.
1
Compound 7: H NMR (400 MHz, DMSO): d=10.55 (s, 1Ht13), 9.33
(sb, 1Ht1), 8.28 (m, 1Hp5), 7.77 (m, 4Hph3+ph4), 7.58 (d, J=8.8 Hz,
2Hp8), 7.07 (d, J=8.7 Hz, 1Ht7), 6.95 (m, 1Ht2 or t4), 6.85 (m, 1Ht2 or t4),
6.55 (dd, J=2.3; 8.7 Hz, 1Ht6), 6.51 (d, J=8.7 Hz, 2Hp9), 5.03 (t, J=
8.4 Hz, 1Ht11), 3.79–3.71 (m, 2Hl5), 3.48 (m, 4Ht10+p4), 3.16 (m, 6Hp2+
p3), 2.99 (t, J=8.4 Hz, 2Hl1), 1.62–1.49 (m, 4Hl2+l4), 1.4–1.1 (m,
22Hl3+p1); 13C NMR (100 MHz, DMSO): d=170.1 (Cp6), 167.0 (Ct12),
166.9 (Cph1), 152.1 (Ct5), 151.5 (Cp7), 134.6 (Cph4), 130.9 (Cph2), 130.7
(Ct8), 128.8 (Cp8), 128.6 (Ct2), 127.1 (Ct3), 123.1 (Cph3), 119.5 (Cp10),
111.8 (Ct4 or t6), 111.4 (Cp9), 110.4 (Ct4 or t6), 109.3 (Ct9), 100.3 (Ct7), 67.5
(Cl5), 52.5 (Ct11), 50.0 (Cp3), 46.5 (Cp2), 34.1–21.9 (Cl1+l2+l3+l4+t10+p4),
8.2 (Cp1); HRMS-ESI (m/z) calcd for C44H58O6N5 [M+H]+: 752.43816;
found: 752.43892.
Synthesis of phthalimido derivatives of procainamide 8 to 13:
K2CO3 (160 mg, 1.16 mmol) and the desired bromoalkylpthtalimide
2-(8-Bromobutyl)-2,3-dihydro-1H-isoindole-1,3-dione (B1) and 2-(8-
Bromohexyl)-2,3-dihydro-1H-isoindole-1,3-dione (B2) were bought
from Sigma–Aldrich.
(0.19 mmol) was added to a solution of the nosylate 2 (160 mg,
0.38 mmol) in dry acetonitrile (3 mL). The mixture was refluxed
overnight, then thiophenol (50 mL, 0.48 mmol) was added, and the
solution was stirred at room temperature for 3 h. Solvent was re-
moved and the residue was purified by flash chromatography on
silica gel with dichloromethane/methanol (98:2+1.75% ammonia)
as eluent.
Synthesis of bromoalkylphthalimides (B3–B6):
N-[2-(Diethylamino)ethyl]-4-{[10-(1,3-dioxo-2,3-dihydro-1H-iso-
indol-2-yl)butyl]amino}benzamide (8): n=0; from 54 mg of N-(4-
bromobutyl)phthalimide, 8 was obtained as a yellow oil with a
yield of 79%. 1H NMR (400 MHz, CDCl3): d=7.77 (m, 2Hph3), 7.64
(m, 2Hph4), 7.54 (dm, J=8.7 Hz, 2Hp8), 6.71 (m, 1Hp5), 6.48 (dm, J=
8.7 Hz, 2Hp9), 4.02 (m, 1Hp11), 3.66 (t, J=7.2 Hz, 2Hl5), 3.38 (dd, J=
5.4, 11.4 Hz, 2Hp4), 3.13 (m, 2Hl1), 2.56 (t, J=6.1 Hz, 2Hp3), 2.49 (q,
Potassium phthalimide (2.5 mmol) was added to a solution of di-
bromoalkyl (1.8 mmol) in DMF (5 mL), and the mixture was stirred
overnight at room temperature, then diluted with ethylacetate and
washed with water. The organic phase was washed with brine and
dried on magnesium sulfate. Solvent was removed, and the residue
162
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ChemBioChem 2012, 13, 157 – 165

Design of Conjugates of Procainamide as DNMT Inhibitors
J=7.2 Hz, 4Hp2), 1.65–1.60 (m, 2Hl4), 1.58–1.53 (m, 2Hl2), 0.97 (t, J=
7.3 Hz, 6Hp1); 13C NMR (100 MHz, CDCl3): d=168.5 (Cph1), 167.2
(Cp6), 150.7 (Cp7), 134.0 (Cph4), 132.0 (Cph2), 128.6 (Cp8), 123.3 (Cph3),
122.9 (Cp10), 111.7 (Cp9), 51.5 (Cp3); 46.8 (Cp2), 43.0 (Cl1), 37.6 (Cl5),
37.1 (Cp4), 26.5; 26.2 (Cl2+l4), 12.0 (Cp1); HRMS-ESI (m/z) calcd for
C25H33O3N4: 437.25472 [M+H]+; found: 437.25487.
N-[2-(Diethylamino)ethyl]-4-{[10-(1,3-dioxo-2,3-dihydro-1H-iso-
indol-2-yl)tetradecyl]amino}benzamide (13): n=10; from 80 mg
of N-(14-bromotetradecyl)phthalimide, 13 was obtained as a pale
yellow solid with a yield of 85%. 1H NMR (400 MHz, CDCl3): d=
7.73 (m, 2Hph3), 7.61 (m, 4Hph4+p8), 7.12 (m, 1Hp5), 6.48 (d, J=
8.8 Hz, 2Hp9), 4.15 (m, 1Hp11), 3.58 (t, J=7.1 Hz, 2Hl5), 3.46 (dd, J=
5.6, 11.3 Hz, 2Hp4), 3.03 (dd, J=6.6, 12.5 Hz, 2Hl1), 2.74 (t, J=5.8 Hz,
2Hp3), 2.65 (q, J=7.2 Hz, 4Hp2), 1.57–1.48 (m, 4Hl2+l4), 1.33–1.10 (m,
20Hl3), 1.04 (t, J=7.1 Hz, 6Hp1); 13C NMR (100 MHz, CDCl3): d=
168.4 (Cph1), 167.9 (Cp6), 151.3 (Cp7), 133.8 (Cph4), 132.1 (Cph2), 128.5
(Cp8), 123.1 (Cph3), 121.6 (Cp10), 111.5 (Cp9), 52.2 (Cp3); 47.3 (Cp2), 43.5
(Cl1), 38.0 (Cl5), 36.8 (Cp4), 29.5–26.8 (Cl2+l3+l4), 11.3 (Cp1); HRMS-ESI
(m/z) calcd for C35H53O3N4: 577.41122 [M+H]+; found: 577.41085.
N-[2-(Diethylamino)ethyl]-4-{[10-(1,3-dioxo-2,3-dihydro-1H-iso-
indol-2-yl)hexyl]amino}benzamide (9): n=2; from 59 mg of N-(6-
bromohexyl)phthalimide, 9 was obtained as a pale yellow oil with
1
a yield of 82%. H NMR (400 MHz, CDCl3): d=7.75 (m, 2Hph3), 7.62
(m, 2Hph4), 7.52 (dm, J=8.7 Hz, 2Hp8), 6.68 (m, 1Hp5), 6.44 (dm, J=
8.7 Hz, 2Hp9), 3.98 (m, 1Hp11), 3.61 (t, J=7.2 Hz, 2Hl5), 3.37 (dd, J=
5.4, 11.4 Hz, 2Hp4), 3.04 (m, 2Hl1), 2.55 (t, J=6.1 Hz, 2Hp3), 2.47 (q,
J=7.2 Hz, 4Hp2), 1.62–1.50 (m, 4Hl2+l4), 1.47–1.25 (m, 4Hl3), 0.95 (t,
J=7.3 Hz, 6Hp1); 13C NMR (100 MHz, CDCl3): d=168.5 (Cph1), 167.3
(Cp6), 150.9 (Cp7), 133.9 (Cph4), 132.1 (Cph2), 128.6 (Cp8), 123.2 (Cph3),
122.7 (Cp10), 111.6 (Cp9), 51.5 (Cp3); 46.8 (Cp2), 43.3 (Cl1), 37.8 (Cl5),
37.1 (Cp4), 29.1, 28.5, 26.5 (Cl2+l3+l4), 12.0 (Cp1); HRMS-ESI (m/z) calcd
for C27H37O3N4: 465.28602 [M+H]+; found: 465.28588.
4-[(6-Aminohexyl)amino]-N-[2-(diethylamino)ethyl]benzamide
(14), 4-[(6-Aminododecyl)amino]-N-[2-(diethylamino)ethyl]benza-
mide (15), and 4-[(6-Aminotetradecyl)amino]-N-[2-(diethylami-
no)ethyl]benzamide (16): Methylhydrazine (100 mL) was added to
a solution of 9 (56 mg, 0.12 mmol) or 12 (72 mg, 0.14 mmol) or 13
(57 mg, 0.10 mmol) in ethanol (1 mL). The mixture was stirred at
room temperature overnight. The solvent was removed, toluene
was added and coevaporated until methylhydrazine was complete-
ly removed. The residue of the reaction with 12 was purified by
flash chromatography silica gel with dichloromethane/methanol
(97:3+1.75% ammonia) to give 15 (41 mg, 69%) as pale yellow
foams. Compounds 14 and 16, residues of deprotections of 9 and
13, were used in the following step without further purification.
N-[2-(Diethylamino)ethyl]-4-{[10-(1,3-dioxo-2,3-dihydro-1H-iso-
indol-2-yl)octyl]amino}benzamide (10): n=4; from 64 mg of N-(8-
bromooctyl)phthalimide, 10 was obtained as a yellow foam with a
yield of 75%. 1H NMR (400 MHz, CDCl3): d=7.75 (m, 2Hph3), 7.62
(m, 2Hph4), 7.54 (dm, J=8.7 Hz, 2Hp8), 6.68 (m, 1Hp5), 6.47 (dm, J=
8.7 Hz, 2Hp9), 3.95 (m, 1Hp11), 3.60 (t, J=7.2 Hz, 2Hl5), 3.38 (dd, J=
5.5, 11.5 Hz, 2Hp4), 3.04 (m, 2Hl1), 2.56 (t, J=6.1 Hz, 2Hp3), 2.48 (q,
J=7.3 Hz, 4Hp2), 1.61–1.48 (m, 4Hl2+l4), 1.35–1.20 (m, 8Hl3), 0.95 (t,
J=7.3 Hz, 6Hp1); 13C NMR (100 MHz, CDCl3): d=168.5 (Cph1), 167.3
(Cp6), 151.0 (Cp7), 133.9 (Cph4), 132.2 (Cph2), 128.6 (Cp8), 123.1 (Cph3),
122.7 (Cp10), 111.6 (Cp9), 51.6 (Cp3); 46.8 (Cp2), 43.5 (Cl1), 38.0 (Cl5),
37.1 (Cp4), 29.3–26.7 (Cl2+l3+l4), 12.0 (Cp1). HRMS-ESI (m/z) calcd for
C29H41O3N4 [M+H]+: 493.31732; found: 493.31695.
Compound 14: MS-ESI (m/z) calcd for C19H34ON4: 335.281 [M+H]+;
found: 335.298.
Compound 15: 1H NMR (400 MHz, CDCl3): d=7.53 (d, J=8.7 Hz,
2Hp8), 6.69 (m, 1Hp5), 6.50 (d, J=8.7 Hz, 2Hp9), 4.01 (m, 1Hp11), 3.41
(m, 2Hp4), 2.61–2.43 (m, 8Hp2+l1+p3), 3.03 (m, 2HNH ), 1.53 (m, 2Hl2),
2
1.45–1.10 (m, 24Hl3+l4+l5), 0.96 (t, J=7.1 Hz, 6Hp1); 13C NMR
(100 MHz, CDCl3): d=167.2 (Cp6), 150.9 (Cp7), 128.6 (Cp8), 122.6
(Cp10), 111.6 (Cp9), 51.5 (Cp3); 47.0 (Cp2), 46.8 (Cl1), 37.1 (Cp4), 31.9 (Cl2),
29.5–27.1 (Cl2+l3), 22.7 (Cl4), 14.1 (Cl5), 12.0 (Cp1); MS-ESI (m/z) calcd
for C25H47ON4: 419.374 [M+H]+; found: 419.402.
N-[2-(Diethylamino)ethyl]-4-{[10-(1,3-dioxo-2,3-dihydro-1H-iso-
indol-2-yl)decyl]amino}benzamide (11): n=6; from 70 mg of N-
(10-bromodecyl)phthalimide, 11 was obtained as a pale yellow
1
solid with a yield of 83%. H NMR (400 MHz, CDCl3): d=7.76 (m,
2Hph3), 7.63 (m, 2Hph4), 7.55 (d, J=8.7 Hz, 2Hp8), 6.70 (m, 1Hp5), 6.49
(d, J=8.8 Hz, 2Hp9), 3.95 (m, 1Hp11), 3.60 (t, J=7.2 Hz, 2Hl5), 3.38
(dd, J=5.6, 11.3 Hz, 2Hp4), 3.05 (dd, J=6.8, 12.3 Hz, 2Hl1), 2.55 (t,
J=5.9 Hz, 2Hp3), 2.47 (q, J=7.1 Hz, 4Hp2), 1.64–1.49 (m, 4Hl2+l4),
1.26–1.15 (m, 12Hl3), 0.95 (t, J=7.1 Hz, 6Hp1); 13C NMR (100 MHz,
CDCl3): d=168.5 (Cph1), 167.3 (Cp6), 151.0 (Cp7), 133.9 (Cph4), 132.2
(Cph2), 128.6 (Cp8), 123.1 (Cph3), 122.6 (Cp10), 111.6 (Cp9), 51.5 (Cp3);
46.8 (Cp2), 43.5 (Cl1), 38.0 (Cl5), 37.1 (Cp4), 29.4–26.8 (Cl2+l3+l4), 12.0
(Cp1); HRMS-ESI (m/z) calcd for C31H45O3N4: 521.34862 [M+H]+;
found: 521.34819.
Compound 16: MS-ESI (m/z) calcd for C27H51ON4: 447.41 [M+H]+;
found: 447.41.
N-[2-(Diethylamino)ethyl]-4-({6-[2-(1,3-dioxo-2,3-dihydro-1H-iso-
indol-2-yl)-3-(1H-indol-3-yl)propanamido]hexyl}amino)benza-
mide (17), N-[2-(Diethylamino)ethyl]-4-({6-[2-(1,3-dioxo-2,3-dihy-
dro-1H-isoindol-2-yl)-3-(1H-indol-3-yl)propanamido]dodecyl}ami-
no)benzamide (18 and 18D) and N-[2-(Diethylamino)ethyl]-4-({6-
[2-(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)-3-(1H-indol-3-yl)pro-
panamido]tetradecyl}amino)benzamide (19):
N-[2-(Diethylamino)ethyl]-4-{[10-(1,3-dioxo-2,3-dihydro-1H-iso-
indol-2-yl)dodecyl]amino}benzamide (12): n=8; from 75 mg of
N-(12-bromododecyl)phthalimide, 12 was obtained as
a pale
yellow solid with a yield of 89%. 1H NMR (400 MHz, CDCl3): d=
7.74 (m, 2Hph3), 7.62 (m, 2Hph4), 7.54 (d, J=8.7 Hz, 2Hp8), 6.66 (m,
1Hp5), 6.48 (d, J=8.7 Hz, 2Hp9), 3.97 (m, 1Hp11), 3.59 (t, J=7.2 Hz,
2Hl5), 3.38 (dd, J=5.6, 11.5 Hz, 2Hp4), 3.05 (m, 2Hl1), 2.56 (t, J=
6.0 Hz, 2Hp3), 2.47 (q, J=7.1 Hz, 4Hp2), 1.60–1.50 (m, 4Hl2+l4), 1.28–
1.10 (m, 16Hl3), 0.96 (t, J=7.1, 6Hp1); 13C NMR (100 MHz, CDCl3):
d=168.4 (Cph1), 167.2 (Cp6), 151.0 (Cp7), 133.8 (Cph4), 132.2 (Cph2),
128.5 (Cp8), 123.1 (Cph3), 122.6 (Cp10), 111.6 (Cp9), 51.5 (Cp3); 46.8 (Cp2),
43.5 (Cl1), 38.0 (Cl5), 37.2 (Cp4), 29.5–27.0 (Cl2+l3+l4), 12.0 (Cp1); HRMS-
ESI (m/z) calcd for C33H49O3N4: 549.37992 [M+H]+; found:
549.37938.
NHS (N-hydroxysuccinimide; 41 mg, 0.36 mmol), DCC (N,N’-dicyclo-
hexylcarbodiimide; 50 mg, 0.24 mmol) and DIPEA (N,N-diisopropy-
lethylamine; 40 mL, 0.40 mmol) were added to a solution of l-
RG108 (80 mg, 0.24 mmol) in DMF (500 mL). The mixture was stirred
ChemBioChem 2012, 13, 157 – 165
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163

C. Ferroud, P. B. Arimondo et al.
at room temperature for 2 h then filtered to removed DCU (dicy-
clohexylurea), and then 14 (crude product from 0.05 mmol of 9) or
15 (41 mg, 0.1 mmol) or 16 (crude product from 0.1 mmol of 13)
was added and the mixture was stirred at room for 2 h. The sol-
vent was removed, and the residue was purified by flash chroma-
tography on silica gel with dichloromethane/methanol (99:1+
0.5% ammonia) as eluent to give 17 (from 14) as a pale yellow
solid (52 mg, yield 58% after two steps from 9), 18 (from 15) as a
pale yellow solid (52 mg; yield 72%, [a]2D0>: ꢁ75.5 (c=1, MeOH)) or
19 (from 16) as a pale yellow solid (35 mg, yield 48% over two
steps from 13). The same procedure with d-RG108 as starting ma-
teriel was applied to 15 give 18D in 62% yield ([a]2D0 =+73.3 (c=1,
MeOH)).
(m, 1Hp11), 3.79–3.10 (ABX, Ja =7.1 Hz, Jb =15.0 Hz, 2Ht10), 3.46 (dd,
J=5.6, 11.6 Hz, 2Hp4), 3.22 (dd, J=7.1 Hz, 12.9 Hz, 2Hl5), 3.14 (dd,
J=7.0, 12.6 Hz, 2Hl1), 2.623 (t, J=6.1 Hz, 2Hp3), 2.56 (q, J=7.1 Hz,
4Hp2), 1.62 (m, 2Hl4), 1.46–1.10 (m, 22Hl3+l2), 1.04 (t, J=7.1 Hz,
6Hp1); 13C NMR (100 MHz, CDCl3): d=168.7 (Ct12), 168.3 (Cph1), 167.4
(Cp6), 151.0 (Cp7), 136.3 (Cph2), 134.2 (Cph4), 131.8 (Ct8), 128.7 (Cp8),
127.1 (Ct3), 123.64 (Cph3), 122.8 (Ct2), 122.4 (Cp10), 122.2 (Ct5 or t6),
119.8 (Ct5 or t6), 118.6 (Ct7), 111.7 (Cp9), 111.4 (Ct9), 111.3 (Ct4), 55.2
(Ct11), 51.6 (Cp3); 46.9 (Cp2), 43.7 (Cl1), 40.0 (Cl5), 37.3 (Cp4), 29.7–25.5
(Cl2+l3+l4+t10), 12.0 (Cp1); HRMS-ESI (m/z) calcd for C46H63O4N6:
763.49053 [M+H]+; found: 763.49146. calcd for [M+Na]+:
795.51675; found: 795.51693.
N-[2-(Diethylamino)ethyl]-4-[(2-ethyldecyl)amino]benzamide
1
(20):
Compound 17: H NMR (400 MHz, CDCl3): d=8.69 (sb, 1Ht1), 7.70–
7.51 (m, 7Hph4+t7+p8+ph3), 7.21 (m, 1Ht4), 7.05–6.94 (m, 4Ht2+t5+t6+
p5), 6.47 (dm, J=8.8 Hz, 2Hp9), 6.30 (m, 1Ht13), 5.16 (t, J=7.1 Hz,
1Ht11), 4.18 (m, 1Hp11), 3.75–3.49 (m, 4Ht10+p4), 3.13–2.54 (m, 10Hl5+
l1+p3+p2), 1.45 (m, 2Hl4), 1.33 (m, 2Hl2), 1.24–1.05 (m, 8Hp1+l3);
13C NMR (100 MHz, CDCl3): d=168.7 (Ct12), 168.2 (Cph1), 167.4 (Cp6),
151.0 (Cp7), 136.3 (Cph2), 134.1 (Cph4), 131.6 (Ct8), 128.6 (Cp8), 126.9
(Ct3), 123.4 (Cph3), 122.9 (Ct2), 122.5 (Cp10), 122.2 (Ct5 or t6), 119.6
(Ct5 or t6), 118.6 (Ct7), 111.6 (Cp9), 111.3 (Ct9), 111.0 (Ct4), 55.0 (Ct11), 51.5
(Cp3); 46.8 (Cp2), 43.5 (Cl1), 39.9 (Cl5), 37.1 (Cp4), 29.5–25.5 (Cl2+l3+l4+
t10), 12.0 (Cp1); HRMS-ESI (m/z) calcd for C38H47O4N6: 651.3653
[M+H]+; found: 651.36598.
K2CO3 (160 mg, 1.16 mmol) and 1-bromododecane (47 mg,
0.19 mmol) were added to a solution of nosylate 2 (160 mg,
0.38 mmol) in dry acetonitrile (3 mL). The mixture was refluxed
overnight, then thiophenol (50 mL, 0.48 mmol) was added, and the
reaction mixture was stirred at room temperature for 3 h. Solvent
was removed and the residue was purified by flash chromatogra-
phy on silica gel with dichloromethane/methanol (98:2+1.75%
ammonia) to afford 20 (72 mg; 94%) as a white amorphous solid.
1H NMR (400 MHz, CDCl3): d=7.56 (dm, J=8.7 Hz, 2Hp8), 6.70 (m,
1Hp5), 6.44 (dm, J=8.7 Hz, 2Hp9), 3.93 (m, 1Hp11), 3.37 (dd, J=5.5,
11.6 Hz, 2Hp4), 3.05 (dd, J=7.04, 12.7 Hz, 2Hl1), 2.56 (t, J=5.7 Hz,
2Hp3), 2.49 (q, J=7.1 Hz, 4Hp2), 1.58–1.50 (m, 2Hl2), 1.30–1.18 (m,
16Hl3), 0.96 (t, J=7.1 Hz, 6Hp1), 0.81 (t, J=7.1 Hz, 3Hl5); 13C NMR
(100 MHz, CDCl3): d=167.3 (Cp6), 151.0 (Cp7), 128.6 (Cp8), 122.6
(Cp10), 111.6 (Cp9), 51.5 (Cp3); 46.8 (Cp2), 43.5 (Cl1), 37.1 (Cp4), 31.9 (Cl2),
29.5–27.1 (Cl2+l3), 22.7 (Cl4), 14.1 (Cl5), 12.0 (Cp1); HRMS-ESI (m/z)
calcd for C25H46ON3: 404.36354 [M+H]+; found: 404.36492.
1
Compound 18: H NMR (500 MHz, CDCl3): d=8.31 (sb, 1Ht1), 7.77
(m, 2Hph3), 7.67–7.59 (m, 5Hph4+p8+t7), 7.27 (d, J=8.0 Hz, 1Ht4), 7.
14 (t, J=7.5 Hz, 2Ht5 or t6), 7. 08 (t, J=7.5 Hz, 2Ht5 or t6), 7.02 (d, J=
2.0 Hz, 1Ht2), 6.74 (m, 1Hp5), 6.54 (dm, J=9.0 Hz, 2Hp9), 6.25 (m,
1Ht13), 5.23 (dd, J=7.0, 9.1 Hz, 1Ht11), 4.00 (t, J=5.0 Hz 1Hp11),
3.80–3.63 (ABX, Ja =15.0 Hz, Jb =7.0 Hz, 2Ht10), 3.45 (dd, J=5.6,
11.6 Hz, 2Hp4), 3.22 (dd, J=7.0 Hz, 12.9 Hz, 2Hl5), 3.14 (dd, J=7.0,
12.6 Hz, 2Hl1), 2.61 (t, J=6.5 Hz, 2Hp3), 2.55 (q, J=7.0 Hz, 4Hp2),
1.62 (m, 2Hl4), 1.43–1.10 (m, 18Hl3+l2), 0.95 (t, J=7.0 Hz, 6Hp1);
13C NMR (100 MHz, CDCl3): d=168.6 (Ct12), 168.2 (Cph1), 167.3 (Cp6),
150.9 (Cp7), 136.2 (Cph2), 134.1 (Cph4), 131.6 (Ct8), 128.5 (Cp8), 126.9
(Ct3), 123.4 (Cph3), 122.7 (Ct2), 122.5 (Cp10), 122.2 (Ct5 or t6), 119.6
(Ct5 or t6), 118.6 (Ct7), 111.6 (Cp9), 111.2 (Ct9), 111.1 (Ct4), 55.0 (Ct11), 51.4
(Cp3); 46.7 (Cp2), 43.5 (Cl1), 39.8 (Cl5), 37.1 (Cp4), 29.7–25.4 (Cl2+l3+l4+
t10), 12.0 (Cp1); HRMS-ESI (m/z) calcd for C44H59O4N6: 735.45923
[M+H]+; found: 735.45973.
Biological activity: C5 DNA methyltransferase activity assay: The
C-terminal catalytic domain of murine Dnmt3a (aa. 623–908) and
the C-terminal domain of Dnmt3L (aa. 208–421), obtained as de-
scribed in [36], were preincubated together (0.6 and 1 mm, respec-
tively) for 20–30 min. at room temperature. Tested compounds
were added, together with [3H]methyl S-adenosyl-l-methionine
(SAM; 280 nm, 0.15 mCi) and unmethylated duplex (5’-GATCG
CCGAT GCGCG AATCG CGATC GATGC GAT-3’/5’-ATCGC ATCGA
TCGCG ATTCG CGCAT CGGCG ATC-3’, 0.2 mm) for 1 h at 378C in re-
action buffer (50 mL, HEPES (20 mm, pH 7.2), KCl (50 mm), EDTA
(1 mm), BSA (25 mgmLꢁ1)). The human DNMT1 was produced in
H19 cells by the bacculovirus system as described in ref. [13]. The
enzyme (20 nm) was incubated in reaction buffer (50 mL, HEPES
(20 mm, pH 7.2), KCl (50 mm), EDTA (1 mm), BSA (25 mgmLꢁ1)) in
the presence of the same hemimethylated DNA duplex (5’-GAT-
MeCG CMeCGAT GMeCGMeCG AATMeCGMe CGATMeC GATGMeC
GAT-3’/5’-ATCGC ATCGA TCGCG ATTCG CGCAT CGGCG ATC-3’,
0.2 mm) and the tested compounds with [3H]methyl SAM (280 nm,
0.15 mCi) and unlabeled SAM (1.28 mm) for 2 h at 378C. After incu-
bation, we removed the unincorporated SAM and the enzymes
with P-30 Tris Micro Bio-Spin Biospin chromatography columns
(Bio-rad), and determined incorporation of radioactivity by liquid
scintillation counting (Wallac Microbeta 1450 Trilux, PerkinElmer).
Background levels were determined in samples lacking the DNA
substrate. The results were plotted as relative methylation activity
1
Compound 18D: H NMR (500 MHz, CDCl3): d=8.31 (sb, 1Ht1), 7.76
(m, 2Hph3), 7.68–7.60 (m, 5Hph4+p8+t7), 7.29 (d, J=8.0 Hz, 1Ht4), 7.
14 (t, J=7.5 Hz, 2Ht5 or t6), 7.08 (t, J=7.5 Hz, 2Ht5 or t6), 7.02 (d, J=
2.0 Hz, 1Ht2), 6.74 (m, 1Hp5), 6.55 (dm, J=9.0 Hz, 2Hp9), 6.25 (m,
1Ht13), 5.23 (dd, J=7.0 Hz, 9.1 Hz, 1Ht11), 3.99 (t, J=5.0 Hz, 1Hp11),
3.80–3.63 (ABX, Ja =7.0 Hz, Jb =15.0 Hz, 2Ht10), 3.44 (dd, J=5.6,
11.6 Hz, 2Hp4), 3.22 (dd, J=7.0 Hz, 12.9 Hz, 2Hl5), 3.13 (dd, J=7.0,
12.6 Hz, 2Hl1), 2.61 (t, J=6.5 Hz, 2Hp3), 2.55 (q, J=7.0 Hz, 4Hp2),
1.61 (m, 2Hl4), 1.41–1.10 (m, 18Hl3+l2), 1.03 (t, J=7.0 Hz, 6Hp1);
13C NMR (100 MHz, CDCl3): d=168.6 (Ct12), 168.2 (Cph1), 167.3 (Cp6),
150.9 (Cp7), 136.2 (Cph2), 134.1 (Cph4), 131.6 (Ct8), 128.5 (Cp8), 126.9
(Ct3), 123.4 (Cph3), 122.7 (Ct2), 122.5 (Cp10), 122.2 (Ct5 or t6), 119.6
(Ct5 or t6), 118.6 (Ct7), 111.6 (Cp9), 111.2 (Ct9), 111.1 (Ct4), 55.0 (Ct11), 51.4
(Cp3); 46.7 (Cp2), 43.5 (Cl1), 39.8 (Cl5), 37.1 (Cp4), 29.7–25.4 (Cl2+l3+l4+
t10), 12.0 (Cp1); HRMS-ESI (m/z) calcd for C44H59O4N6 [M+H]+:
735.45923; found: 735.45954.
1
Compound 19: H NMR (500 MHz, CDCl3): d=8.19 (sb, 1Ht1), 7.76
(m, 2Hph3), 7.67–7.60 (m, 5Hph4+t7+p8), 7.29 (s, 1Ht4), 7.14–7.08 (m,
2Ht5+t6), 7.03 (d, J=2.2 Hz, 1Ht2), 6.70 (m, 1Hp5), 6.55 (dm, J=
8.8 Hz, 2Hp9), 6.21 (m, 1Ht13), 5.23 (dd, J=7.0 Hz, 9.1 Hz, 1Ht11), 3.96
164
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ChemBioChem 2012, 13, 157 – 165

Design of Conjugates of Procainamide as DNMT Inhibitors
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1
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1
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Cell lines: DU145, a human prostate cancer cell line, and HCT116,
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Acknowledgements
This work was supported by ANR-06 JCJCJ-0080 and by DFG
grant JE 252/6 to A.J. A.C. was the recipient of an ARC fellowship
and CC of an INSERM-CNRS BDI. The authors thank Lionel
Dubost and Dr. Arul Marie of the MNHN Mass Spectroscopy Ser-
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CNRS FRE 3206 for use of the microbeta counter, and Dr. Ahmed
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Keywords: cancer
·
conjugation
·
DNA methylation
·
inhibitors · rapid synthesis
[1] R. S. Illingworth, A. P. Bird, FEBS Lett. 2009, 583, 1713–1720.
Received: August 16, 2011
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23, 781–783.
Published online on December 14, 2011
ChemBioChem 2012, 13, 157 – 165
ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
165