SAR around (l)-S-adenosyl-l-homocysteine, an inhibitor of human DNA methyltransferase (DNMT) enzymes

Article abstract of DOI:10.1016/j.bmcl.2009.03.113

The inhibitory activity of base-modified SAH analogues and the specificity of inhibiting human DNMT1 and DNMT3b2 enzymes was explored. The 6-amino group was essential while the 7-N of the adenine ring of SAH could be replaced by CH- without loss of activi

Full text of DOI:10.1016/j.bmcl.2009.03.113

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2747–2751
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
SAR around (L)-S-adenosyl-L-homocysteine, an inhibitor of human DNA
methyltransferase (DNMT) enzymes
Oscar M. Saavedra a, Ljubomir Isakovic a, David B. Llewellyn a, Lijie Zhan a,
Naomy Bernstein a, Stephen Claridge a, Franck Raeppel a, Arkadii Vaisburg a, Nadine Elowe b,
Andrea J. Petschner b, Jubrail Rahil b, Norman Beaulieu c, A. Robert MacLeod c, Daniel Delorme a,
Jeffrey M. Besterman c, Amal Wahhab a,
*
a MethylGene Inc., Departments of Medicinal Chemistry, 7220 rue Frederick-Banting, Montreal, Quebec, Canada H4S 2A1
b Lead Discovery, 7220 rue Frederick-Banting, Montreal, Quebec, Canada H4S 2A1
c Pharmacology and Cell Biology, 7220 rue Frederick-Banting, Montreal, Quebec, Canada H4S 2A1
a r t i c l e i n f o
a b s t r a c t
Article history:
The inhibitory activity of base-modified SAH analogues and the specificity of inhibiting human DNMT1
and DNMT3b2 enzymes was explored. The 6-amino group was essential while the 7-N of the adenine ring
of SAH could be replaced by CH– without loss of activity against both enzymes. The introduction of small
groups at the 2-position of the adenine moiety favors DNMT1 over DNMT3b2 inhibition whereas alkyl-
ation of the N6-amino moiety favors the inhibition of DNMT3b2 enzyme.
Received 10 February 2009
Revised 23 March 2009
Accepted 25 March 2009
Available online 28 March 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
SAH analogues
DNMT1 inhibitors
DNMT3b2 inhibitors
DNA methylation or the addition of a methyl group at the 50
positions of cytosine residues within a CpG sequence is the main
epigenetic modification in mammals and is responsible for gene
expression in cancer and in normal cells.1 DNA methyltransferase
(DNMT) enzymes catalyze the transfer of a methyl group from
inhibitors are under investigation or are currently in use for the
treatment of cancer. Nucleoside analogs VidazaÒ (5-aza-C), and
DecitabineÒ (5-aza-CdR),10 have recently been approved for
myelodysplastic syndrome while DNMT1 antisense molecules such
as MG98, have demonstrated evidence of clinical activity.11 To ex-
ert their biological effects, 5-aza-C and 5-aza-CdR must be incorpo-
rated into the DNA where they covalently trap the bulky 190-kDa
DNMT enzyme onto the DNA while agents such are MG98 interfere
with the biosynthesis of DNMT1 at the transcriptional level.12a
Other small molecule DNMT inhibitors such as procainamide,
EGCG, RG108, and curcumin12c have been reported.12b
(L
)-S-adenosyl-
the cytosine ring of the DNA.2 The demethylated metabolite,
)-S-adenosyl- -homocysteine ( -SAH) and methylated DNA are
the products of this reaction.3
-SAH binds to DNMTs and inhibits
L
-methionine (SAM), onto the 50 position of
(L
L
L
L
their catalytic reaction and is important for the regulation of bio-
logical transmethylations.4 DNMT1 is required for preservation of
methylation patterns in differentiated cells, while the de novo
DNMTs like DNMT3A and DNMT3B play a role in establishing
methylation during early embryogenesis.5,6 Changes in human
DNA methylation patterns are commonly found in human tumors
and are implicated in development and maintenance of human
cancer.7 The aberrant hypermethylation of the CpG islands in can-
cer cells with concomitant transcriptional inactivation of impor-
tant tumor suppressor genes8 is one of the hallmarks of cancer.
DNMT inhibition is correlated with reactivation of tumor suppres-
sor genes in cancer cells with consequent cancer remission9 mak-
ing it a valuable therapeutic target. Different types of DNMT1
Borchardt et al.13a–f and others14 described the synthesis of a
number of L-SAH analogues having structural modifications of
the amino acid, sugar, and base portions as inhibitors of non-hu-
man DNMTs. More recently, some N6-substituted SAH analogues
have been described as inhibitors of protein arginine methyltrans-
ferases.14f In addition, a number of simplified and less rigid ana-
logues of SAH have been reported, however these compounds,
were devoid of activity against human DNMT1.15 The 20- and 30-hy-
droxy groups of the sugar portion and the 6-amino group of the
adenine moiety of SAH were found to be important structural
requirements for inhibition of DNMTs.16 Some nitrogen analogues
of SAM and SAH have been reported and tested against catechol
O-methyltransferase and tRNA methylases.17 However to the best
of our knowledge, there are no reports on the activity of SAH or
* Corresponding author. Tel.: +1 514 337 3333; fax: +1 514 337 0550.
E-mail address: wahhaba@methylgene.com (A. Wahhab).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.03.113

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O. M. Saavedra et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2747–2751
Table 1
N
DNMT1 and DNMT3b2 enzymatic activity: base modifications, compounds 1-7a
H2N
HO2C
O
NH2
N
S
O
H2N
R
N
N
S
O
HO
OH
HO
OH
M)
OH
1
SAH (racemic)
Compd
R
DNMT 1 IC50
(
l
DNMT 3b2 IC50
0.3
(lM)
N
NH2
Substitutions
Base modifications
1b
2
N
N
N
N
N
6-deamino SAH
6-hydroxy SAH
3-deaza SAH
7-deaza SAH
8-aza SAH
2
3
4
5
6
7
H
N
N
H2N
O
N
R2
S
N
HO2C
N
N
214e
300
>1 mM
45
28
HO
OH
N
R1
1-deaza SAH
N
N
Figure 1. Derivatives of SAH synthesized to probe the binding sites of human
OH
313b
>1 mM
6.8
0.3
3
DNMT1 and DNMT3b2.
N
N
its analogues against human DNMT1 or DNMT3b2 isosymes. In
addition, this is the first account of any inhibitors of the de novo
DNMT3b2 enzyme.
The present paper summarizes the results of our investigation
of modifications of the base, and 2- and N6-substitutions (R1 and
R2, respectively) of SAH (Fig. 1) and the effect on the binding affin-
ity to DNMT1 and DNMT3b2 enzymes.
N
NH2
413b,14i
514e,i
613f
N
N
N
NH2
1.5
N
N
The synthesis of several aza or deaza SAH derivatives was
described previously in the literature. S-nebularinehomocysteine
2,13f S-inosinylhomocysteine 3,13b 3-deaza-SAH 4,13b S-tubercidi-
nylhomocysteine 5,14d and 8-aza-SAH 613f are known compounds
(Table 1). The synthesis of 1-deaza-SAH 7 appeared in the litera-
ture18 however, we prepared it as described in Scheme 1. Commer-
cially available 8 reacted readily with a solution of 9 in methanol
containing 1 equiv of sodium methoxide to give 10. In situ
exchange of the protecting groups afforded the glycoside donor 12.
Pre-activation of 7-nitro-3H-imidazo[4,5-b]pyridine18a and reaction
with 12 and TMSOTf yielded 13. Reduction of the nitro group of 13
and removal of the protecting groups gave 1-deaza-SAH 7.
The 2-substituted analogs listed in Table 2 were prepared as
shown in Schemes 2 and 3.
N
N
N
NH2
8
N
N
N
NH2
N
7
45
45
N
a
Values are means of at least two experiments, 20%.
Racemic SAH.
b
alcohol afforded 16. Nucleophilic displacement of tosylate 16 with
the in situ produced thiol from 9, followed by removal of the pro-
tecting groups gave 18.14c
Reaction of intermediate 15 with I2 and nucleophilic substitu-
tion using NH3/isopropanol afforded 19. Intermediate 19 was
converted to 20 following the same reaction sequence as the one
Reaction of 1419 with SnBu3Cl using the conditions described by
Kato et al.20a,b gave 15 which upon electrophilic fluorination using
XeF2/AgTf, nucleophilic substitution employing NH3/isopropanol,
removal of the protecting group and tosylation of the liberated
O
MeO2C
BocHN
O
OMe
S
OMe
OAc
O
b
a
TsO
S
+
O
O
O
O
NHBoc
8
9
10
MeO2C
O
MeO2C
FmocHN
O
d
OMe
c
S
S
11
S
FmocHN
AcO
OAc
O
O
12
N
N
N
O
O
MeO2C
FmocHN
NO2
H2N
HO2C
NH2
N
e
S
N
N
AcO
OAc
HO
OH
13
Scheme 1. Reagents and conditions: (a) 9/NaOMe/MeOH, rt, 30 min then 8, reflux, 3 h; (b) TMSOTf/DCE, rt, 1 h then Fmoc-Cl, rt, 5 h; (c) (i) TFA/water, rt, 30 min; (ii) AcCl/
pyrinine/DCM, rt, 16 h; (d) (i) 7-nitro-3H-imidazo[4,5-b]pyridine/N,O-bistrimethylsilylacetomide/MeCN, rt, 1 h then 2,6-dichloro-9H-purine/TMSOTf, reflux, 30 min; (e) (i)
Fe/HCl/MeOH, reflux, 15 min; (ii) KOH/water/THF, 0 °C, 3 h.

O. M. Saavedra et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2747–2751
2749
Table 2
the tosylate followed by the same reaction sequence as the one
described for the transformation of 16 to 18. Compound 23 was
converted either to the 2-methoxy- or to the 2-methylthio-inter-
mediates, 25a and 25b, respectively, employing sodium methoxide
or sodium methanethiolate, respectively.
DNMT1 and DNMT3b2 enzymatic activity: 2-substituted analogues, compounds 1, 19,
and 22–26a
N
O
H2N
OH
NH2
N
S
O
Compounds 25a and 25b were converted to 26 and 27, respec-
tively, in a manner similar to the synthesis of 18 from 16.
The N6 substituted analogues, compounds 28–34, were
prepared according to the procedure of Lin et al.14f For the concom-
itant substitution of the 2- and N-6 positions, we utilized interme-
diate 12 and reacted it with activated 2,6-dichloro-9H-purine to
afford 35. Displacement of the chloro group of 35 with 2-(biphe-
nyl-4-yl)ethanamine and removal of the protecting groups gave
36 (Scheme 4).
N
N
HO
OH
R
Compd
R
DNMT 1 IC50
(
l
M)
DNMT 32b IC50 (lM)
1
H
F
Cl
I
2.0
2.5
2.9
21.0
6.0
75
0.3
6.8
57.2
256
1.0
NI
1814c
24
20
22
26
27
Me
OMe
SMe
All compounds22a–c were tested against recombinant human
DNMT1 and DNMT3b2 enzymes23 and the results are presented
in Tables 1–3.
216
500
NI: no inhibition at 45
l
M inhibitor concentration.
a
Values are means of at least two experiments, 20%.
We attempted first to explore the importance of the 6-amino
group for inhibition of human DNMT1 and DNMT3b2 enzymes.
As it can be seen for compounds 2 and 3 the replacement of the
amino group by a hydrogen or by a hydroxyl group resulted in a
marked reduction in inhibitory activity against both enzymes. This
described for the transformation of 16 to 18. The palladium
catalyzed reaction of 19 with AlMe3 gave 21, which was converted
to 22 as described above (Scheme 2). Compound 2321 was used for
the preparation of compound 24 by transferring the free alcohol to
N
N
N
O
Cl
O
NH2
O
N
Cl
N
N
a
TBDMSO
b,c,d,e
TsO
TBDMSO
TBDMSO
N
N
N
N
N
N
O
O
O
S
O
O
O
F
SnBu3
15
14
16
f
h,c
N
N
N
O
O
NH2
O
MeO2C
BocHN
NH2
NH2
N
N
N
i
TBDMSO
N
N
N
N
N
N
O
O
O
O
O
O
Me
21
F
I
17
19
d,e,f,g
d,e,f,g
g
N
N
N
O
H2N
HO2C
NH2
O
N
HO2C
H2N
O
NH2
HO2C
H2N
NH2
N
N
S
S
S
N
N
N
N
N
N
HO
OH
HO
OH
HO
OH
F
I
Me
22
20
18
Scheme 2. Reagents and conditions: (a) LTMP/THF, ꢀ78 °C, 30 min then SnBu3Cl, ꢀ78 °C, 1 h; (b) 2,6-di-tert-butyl-4-methylpyridine/XeF2/AgTf/DCM, rt, 15 min; (c) NH3/
isopropanol, 2 days, 100 °C, sealed tube; (d) TBAF/THF, 0 °C to rt, 1 h; (e) NaH/THF, 0 °C, 30 min then TsCl, 1 h; (f) 9/NaOMe/MeOH, rt, 30 min then 16, reflux, 1 h; (g) (i) KOH/
THF/water, rt, 1 h, (ii) TFA, rt, 30 min, (iii) TFA/H2O, rt 2 h; (h) I2, THF, 1 h; (i) AlMe3, PdCl2, PPh3, THF, reflux, 2 h.
N
N
O
O
NH2
NH2
N
N
HO
d or e
HO
N
N
N
N
O
O
O
O
Cl
R1
25a, R1= OMe
25b, R1= SMe
23
a,b,c
a,b,c
N
N
O
O
H2N
HO2C
NH2
H2N
HO2C
NH2
N
N
S
S
N
N
N
N
HO
OH
HO
OH
24
R1
Cl
26, R1= OMe
27, R1= SMe
Scheme 3. Reagents and conditions: (a) NaH/THF, 0 °C, 30 min then TsCl, 1 h; (b) 9/NaOMe/MeOH, rt, 30 min then 16, reflux, 1 h; (c) (i) KOH/THF/water, rt, 1 h, (ii) TFA, rt,
30 min, (iii) TFA/H2O, rt 2 h; (d) NaOCH3, DMF, 90 °C, 1 h; (e) NaSCH3, DMF, 80 °C, 1 h.

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O. M. Saavedra et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2747–2751
retained all activity against both enzymes and was as active as race-
N
a
O
MeO2C
Cl
b
mic SAH 1 which indicates that the nitrogen in the 7 position of
adenine is not required for binding to DNMT1 and DNMT3b2
enzymes. This is in agreement with the reported observation that
the N-7 of the adenine moiety of SAH is not important for binding
to other O and N methyltransferases such as indoleethylamine
N-methyltransferase (INMT),14d tRNA methylases(tRNAMT),14d cat-
echol O-methyltransferase (COMT), phenylethanolamine N-methyl-
transferase (PNMT), histamine N methyltransferase (HMT), and
hydroxyindole O-methyltransferase (HIOMT).13b
We explored a variety of substituents at the 2-position of the
adenine ring of SAH, some of which are listed in Table 2. Generally,
DNMT3b2 is less tolerant for substitution at this position than
DNMT1, and none of the analogues prepared was more active than
SAH itself. A 2-methyl group, compound 22, produced a threefold
loss of activity against both enzymes, while halogens led to partial
selectivity for DNMT1 enzyme. Both the 2-fluoro- and 2-chloro
analogues, compounds 18 and 24, respectively, are almost equipo-
tent to SAH against DNMT1 whereas the same compounds caused
22- and 190-fold decrease of inhibitory activity against DNMT3b2.
However, a 2-iodo group lead to loss of activity against both en-
zymes. It is noteworthy that a 2-fluoro or 2-chloro-group conferred
selectivity amongst human DNMT enzymes and this could be a
beneficial tool for the preparation of more selective inhibitors. All
other substituents such as a methoxy or a thiomethyl group, com-
pounds 26, and 27, respectively, produced inactive compounds
(Table 2).
N
12
S
O
N
FmocHN
N
AcO
OAc
Cl
35
N
H
N
H2N
HO2C
N
S
Ph
N
N
HO
OH
Cl
36
Scheme 4. Reagents and conditions: (a) 12/N,O-bistrimethylsilylacetomide/MeCN,
rt, 1 h then 2,6-dichloro-9H-purine/TMSOTf, reflux, 30 min; (b) (i) 3-phenylpropan-
1-amine/DCE, rt, 16 h; (ii) KOH/water/THF, rt, 16 h.
Table 3
DNMT1 and DNMT3b2 enzymatic activity: N6 alkylation compounds 28–36a
N
O
H2N
OH
NHR
N
S
O
N
N
HO
OH
R1
Compd
R
R1 DNMT 1 IC50
(l
M) DNMT 3b2 IC50
0.3
(lM)
1
H
H
2.0
We also investigated the tolerance of substitution at the N6
position of the adenine ring. A phenyl and a benzyl group resulted
in a marked decrease of activity against DNMT1, while the more
flexible lipophilic moeities such as 3-phenylpropanyl and p-phe-
nyl-phenethyl groups produced compounds, 31 and 32, respec-
tively, with moderate activity. The DNMT3b2 enzyme seems to
be more tolerant for substitution at this position however the best
compounds, 31 and 32, are twofold less active than SAH itself. The
concomitant 2- and N6 substitution, compound 36, resulted in
slight decrease of activity against DNMT1 and a substantial loss
of activity against DNMT3b2, as compared to 32.
In conclusion, some differences in binding requirements of base
modified SAH analogues have been observed between human
DNMT1 and DNMT3b2 enzymes. The 6-amino group is essential
while the 7-N of the adenine ring of SAH can be replaced without
loss of activity against both enzymes. The introduction of small
groups at the 2-position of the adenine moiety favors DNMT1 over
DNMT3b2 whereas alkylation of the N6-amino moiety favors the
inhibition of DNMT3b2 enzyme. The study, however, failed to iden-
tify modifications capable of increasing inhibitory activity of SAH
against either enzyme.
2814f
2914f
30
H
154
2
H
H
H
H
H
H
61
27
7.4
5.4
22
18
1
2
31
0.6
0.6
3.6
0.9
Ph
32
33
N
OMe
34
References and notes
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29
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loss of activity while S-tubercidinylhomocysteine (7-deaza SAH) 5

O. M. Saavedra et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2747–2751
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A
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sepharose FF and Hitrap Heparin columns (Amersham Biosciences. The
ˇ
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2008, 16, 2276; (i) Serafinowski, P. Synthesis 1985, 926.
DNMT3b2 enzyme was purified on
a Hitrap SP-sepharose column and
15. Botta, M.; Saladino, R.; Pedraly-Noy, G.; Nicoletti, R. Med. Chem. Res. 1994, 4,
323.
underwent buffer exchange, using PD-10 column (Amersham Biosciences).
Typically purities of DNMT1 and DNMT3b2 enzyme preparations were above
95% and 70%, respectively. Purified enzyme stocks were frozen at ꢀ80 °C in
aliquots prior to use in enzymatic assays. Assay, DNMT1: the enzyme/Oligo
16. (a) Borchardt, T.; Wu, Y. S.; Huber, J. A. J. Med. Chem. 1976, 19, 1104; (b)
Houston, M. D.; Matuszewska, B.; Borchardt, R. T. J. Med. Chem. 1985, 28, 478;
(c) Crooks, P. A.; Tribe, M. J.; Pinney, R. J. J. Pharm. Pharmacol. 1984, 36, 85.
17. (a) Chang, C.-D.; Coward, J. K. J. Med. Chem. 1976, 19, 684; (b) Minnick, A. A.;
Kenyon, G. J. Org. Chem. 1988, 53, 4952; (c) Thompson, M.; Makhalfia, A.;
Hornby, D. P.; Blackburn, G. M. J. Org. Chem. 1999, 64, 7467.
18. (a) Cristalli, G.; Franchetti, P.; Grifantini, M.; Vittori, S.; Bordoni, T.; Geroni, C. J.
Med. Chem. 1987, 30, 1686; (b) Lim, J.; Winkler, W. C.; Nakamura, S.; Scott, V.;
Breaker, R. R. Angew. Chem., Int. Ed. 2006, 45, 964.
mixture (10 ll) is added to the inhibitor (3 ll) in a round bottom 96-well plate
and the mixture is pre-incubated for 10 min at 37 °C. Final enzyme
concentration is 25 nM and the hemi-methylated oligonucleotide (MYG167:
ATC GCA TCG ATC GCG ATT CGC GCA TCG GCGATC; MYG166: GAT XGC XGA
TGX GXG AAT XGX GAT XGA TGX GAT (X: 5-methylcytosine, the two oligos are
hybridized to form a duplex) at a final concentration of 2.6
[methyl-3H] mixture (10
l, 20% hot and the remaining is cold SAM. SAM was
0.55 mCi/ml, with a specific activity of 55–85 Ci/mmol) was then added for a
final concentration of 3
M. All solutions are diluted with water and a 10ꢁ
assay buffer (50 mM Tris–HCl pH 7.6, 5% Glycerol, 1 mM EDTA, 100 g/ml BSA,
1 mM DTT). The final reaction was incubated for 15 min at 37 °C and the
reaction was stopped with 150 l of a solution of SAH, and harvested onto a
lM. The SAM
l
19. Camp, D.; Li, Y.; McClaskey, A.; Moni, R. W.; Quinn, R. J. Bioorg. Med. Chem. Lett.
1998, 8, 695.
l
20. (a) Kato, K.; Hayakawa, H.; Tanaka, H.; Kumamoto, H.; Miyasaka, T.
Tetrahedron. Lett. 1995, 36, 6507; (b) Kato, K.; Hayakawa, H.; Tanaka, H.;
Kumamoto, H.; Shindoh, S.; Miyasaka, T. J. Org. Chem. 1997, 62, 6833.
21. Castro-Pichel, J.; Garcia-Lopez, M. T.; De Las Heras, F. G. Tetrahedron 1987, 383.
22. (a) Experimental details are in MethylGene patent, Amal Wahhab, Jeffrey
Besterman, Daniel Delorme, Ljubomir Isakovic, David Llewellyn, Jubrail Rahil,
Oscar Saavedra, Robert Deziel, International Patent Application WO 2006/
078752, 2006; (b) All compounds were >95% pure and were routinly
characterized by 1H NMR and MS, and in some instances by 13C NMR and
l
l
DEAE filtermat (Wallac Cat# 1450-522) using a Tomtec Cell harvester. The
Filtermat was washed using a cold 20 mM solution of NH4HCO3. The filtermat
was dried on a hot plate (set at a low temperature) and MeltiLexTM scintillant
(Wallac Cat#1450-441) was melted over the filtermat. The filtermat was read
using a Wallac top-count beta-counter. The assay procedure for DNMT3b2 is
exactly as DNMT except final enzyme concentration was 188 nM.