Constrained (l-)-S-adenosyl-l-homocysteine (SAH) analogues as DNA methyltransferase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.03.132

Potent SAH analogues with constrained homocysteine units have been designed and synthesized as inhibitors of human DNMT enzymes. The five membered (2S,4S)-4-mercaptopyrrolidine-2-carboxylic acid, in 1a, was a good replacement for homocysteine, while the corresponding six-member counterpart was less active. Further optimization of 1a, changed the selectivity profile of these inhibitors. A Chloro substituent at the 2-position of 1a, compound 1d, retained potency against DNMT1, while N⁶ alkylation, compound 7a, conserved DNMT3b2 activity. The concomitant substitutions of 1a at both 2- and N⁶ positions reduced activity against both enzymes.

Full text of DOI:10.1016/j.bmcl.2009.03.132

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2742–2746
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Constrained (L-)-S-adenosyl-L-homocysteine (SAH) analogues as DNA
methyltransferase inhibitors
Ljubomir Isakovic a, Oscar M. Saavedra a, David B. Llewellyn a, Stephen Claridge a, Lijie Zhan a,
Naomy Bernstein a, Arkadii Vaisburg a, Nadine Elowe b, Andrea J. Petschner b, Jubrail Rahil b,
Norman Beaulieu c, France Gauthier c, A. Robert MacLeod c, Daniel Delorme a,
Jeffrey M. Besterman c, Amal Wahhab a,
*
a MethylGene Inc., Department of Medicinal Chemistry, 7220 rue Frederick-Banting, Montreal, Quebec, Canada H4S 2A1
b MethylGene Inc., Department of Lead Discovery, 7220 rue Frederick-Banting, Montreal, Quebec, Canada H4S 2A1
c MethylGene Inc., Department of 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:
Potent SAH analogues with constrained homocysteine units have been designed and synthesized as
inhibitors of human DNMT enzymes. The five membered (2S,4S)-4-mercaptopyrrolidine-2-carboxylic
acid, in 1a, was a good replacement for homocysteine, while the corresponding six-member counterpart
was less active. Further optimization of 1a, changed the selectivity profile of these inhibitors. A Chloro
substituent at the 2-position of 1a, compound 1d, retained potency against DNMT1, while N6 alkylation,
compound 7a, conserved DNMT3b2 activity. The concomitant substitutions of 1a at both 2- and N6 posi-
tions reduced activity against both enzymes.
Received 10 February 2009
Revised 23 March 2009
Accepted 25 March 2009
Available online 28 March 2009
Keywords:
Constrained SAH analogues
DNMT1 inhibitors
DNMT3b2 inhibitors
Ó 2009 Elsevier Ltd. All rights reserved.
Abnormalities in human DNA methylation patterns are com-
monly found in human tumors and are implicated in development
and maintenance of human cancers.1a–c DNA hypermethylation in
CpG islands in cancer cells results in alteration of gene expression
patterns and most notably in the loss of expression or silencing of
tumor suppressor genes.2a–c Changes of DNA methylation could be
used for the identification of specific and sensitive tests for the
early detection of some cancers.2d–f DNA methyltransferase 1
(DNMT1) protein is a major contributor to DNMT activity in human
cells and is required to maintain methylation patterns in differen-
tiated cells3a while the de novo DNMTs, DNMT3a, DNMT3b1, and
DNMT3b2 establish DNA methylation during early embryogene-
sis.3b–e A recent review of the enzymatic mechanism of DNMT1
and its implication in the design of novel mechanism-based inhib-
(Vidaza) or degradation (Decitabine) of the active enzymes. Other
small molecule DNMT inhibitors such as procaine and procain-
amide (interrupt binding of DNMT1 to DNA), EGCG, hydralazine,
RG108 and psammaplin A (block the active site of DNMT1 non-
covalently) have been reported.3g,4c More recently Liu et al. have
identified curcumin from virtual screening and docking utilizing
a homology model of DNMT1.4c Curcumin is proposed to work
by blocking the catalytic C1226 of DNMT1 inhibitor enzyme with-
out incorporation into the DNA.
The cofactor (L-)-S-adenosyl-L-methionine (SAM) is the methyl
group donor in the methylation reaction and is required for the
DNA cleavage by most DNMTs. It covalently modifies the DNA at
the carbon-5 of cytosine residues and in the process is converted
to its demethylated metabolite (L-)-S-adenosyl-L-homocysteine
itors was published in 2008 by Svedruzic.3f A second review by Yu
(L
-SAH).3f,g In tissue, the levels of SAM and of SAH are equivalent
ˇ ´
et al., gave an account of the different DNMTs and their biological
functions with the focus on the design of various inhibitors of DNA
hypermethylation as anticancer drugs.3g
(Fig. 1).5a,b SAH can bind to DNMTs and inhibit their catalytic reac-
tion and has an important role in the regulation of biological trans-
methylation.6a–c
The nucleotide analogues 5-azacytidine4a (VidazaÒ) and 5-aza-
20-deoxycytidine4b (Decitabine) are approved anti-cancer drugs
targeting DNA methylation, while a number of other inhibitors
are in different stages of development.3g Such inhibitors, however,
act as mechanism-based (suicide or covalent) inhibitors that are
incorporated into the DNA of the target cell leading to a depletion
O
N
HO
O
NH2
N
X
H2N
N
N
HO
OH
SAM: X= +SMe
SAH: X=S
Sinefungin: X= CH-NH2
* Corresponding author. Tel.: +1 514 337 3333; fax: +1 514 337 0550.
E-mail address: wahhaba@methylgene.com (A. Wahhab).
Figure 1. Structure of SAM, SAH and Sinefungin.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.03.132

L. Isakovic et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2742–2746
2743
Borchardt et al.,7a–f Cohen et al.,8a and others8b–e prepared SAH
analogues as inhibitors for specific non-human DNMTs. More re-
cently, some N6-substituted SAH analogues have been described
as inhibitors of protein arginine methyltransferases.9a,b Nitrogen
analogues of SAM and SAH have been tested against catechol
O-methyltransferase and tRNA methylases.10a–c In addition, Sine-
fungin has been reported to inhibit human DNMT, however, it is
a non-selective inhibitor with potential for toxicity (Fig. 1).11 In
spite of the differences in their sequence identity, the different
classes of DNMTs have a conserved cofactor binding site. There
are numerous published structures of DNMTs in complex with
SAM or SAH, and the position of SAM is relatively similar in each
structure, but the interactions of the methionine moiety are not
conserved between the different classes of DNMTs.8a,12a All the
homocysteine, 2- and N6 positions on binding and selectivity
against DNMT1 and DNMT3b2 enzymes will be shown.
Scheme 1 depicts the general procedure utilized to prepare ana-
logues 1a–k.13a Racemic 1a, 1c, 1d, and 1e have been reported pre-
viously as inhibitors of polyamine biosynthesis.13b,c Alcohol 2a,
(2S,4R)-methyl 1-(tert-butoxycarbonyl)-4-hydroxypyrrolidine-2-
carboxylate, was converted to thioacetate 3a via the mesylate
resulting in inversion of configuration. In situ formation of the thiol
with NaOMe followed by the reaction with either intermediate
4a13d or 4b13e gave the fully protected 5a or 5b, respectively. Ester
hydrolysis of 5a or 5b with KOH (not shown in scheme) followed
by the treatment with a mixture of TFA/H2O resulted in removal
of both the Boc and the acetonide protecting groups to yield the
desired 1a or 1b. Similarly, compound 1c was prepared from alco-
hol 2b, (2S,4S)-methyl 1-(tert-butoxycarbonyl)-4-hydroxypyrrol-
idine-2-carboxylate. Compounds 1d and 1f were prepared from
alcohol 2c, (R)-tert-butyl 3-hydroxypyrrolidine-1-carboxylate, via
the thioacetate 3c. The liberated thiol (not shown in scheme)
was then reacted with 4a or 4b to give acetonides 5d and 5f,
respectively, and the desired 1d and 1f were obtained by deprotec-
tion of the protecting groups. Likewise, compounds 1e and 1g were
prepared starting from alcohols 2d, (S)-tert-butyl 3-hydroxypyrrol-
idine-1-carboxylate, and 2e, (2S,4R)-1-(tert-butoxycarbonyl)-
functional groups of the homocysteine moiety of L-SAH, the termi-
nal amino group, the carboxylic acid group and the sulfur atom, are
involved in binding to the DNMT enzymes.3f,7b,12a SAH analogues
with amino acid modifications tested against the bacterial DNMT
enzymes M.HhaI and M.HaeIII provided further evidence for the
important role of both the amino and carboxylic acid functional
groups for binding to the enzyme.8a
The conformation of the flexible homocysteine moiety in the ac-
tive site of DNMT1 or DNMT3b2 has not been reported. The homo-
cysteine unit could adopt either an extended or folded
conformation, with the extended conformation possibly represent-
ing the catalytically competent conformation.12b,8a We prepared a
large set of compounds focusing on replacements for the homocys-
teine unit to identify analogues with improved drug-like properties
lacking the zwitter-ionic character of SAH. This Letter will describe
replacement of the homocysteine moiety by constrained groups. In
addition, the results of simultaneous modifications of SAH at the
4-hydroxypyrrolidine-2-carboxamide,
correspondingly.
The
N-methyl-pyrrolidine analogues 1h and 1i were accessible from
alcohol 2f, (2S,4R)-methyl 4-hydroxy-1-methylpyrrolidine-2-car-
boxylate, and 2g, (R)-1-methylpyrrolidin-3-ol in the order men-
tioned. Esters 1j and 1k were available from intermediates 5a
and 5h, respectively, by treatment with TFA/H2O (Scheme 1).
The synthesis of analogues 7a–d (Scheme 2) with N-6-modifica-
tions began from intermediates 5a, 5b, 5d, and 5f which were
R2
N
R2
N
R2
R2
N
N
NH2
NH2
N
O
N
O
R1
N
N
R1
4a or 4b
b
a
R1
R1
S
S
c then d
or d
N
N
N
N
OH
O
OH
O
HO
2
SAc
1
R3
R3
3
5
1a (2S,4S) R1=CO2H R2= H R3= H
2a (2S,4R) R1= CO2Me R2= Boc
2b (2S,4S) R1= CO2Me R2= Boc
3a (2S,4S) R1= CO2Me R2= Boc
3b (2S,4R) R1= CO2Me R2= Boc
5a (2S,4S) R1= CO2Me R2= Boc R3= H
1b (2 ,4 ) R1=CO H R2= H R3=Cl
5b (2S,4S) R1= CO2Me R2= Boc R3= Cl
5c (2S,4R) R1= CO2Me R2= Boc R3= H
S
S
1c (2S,4R) R1=CO22H R2= H R3= H
R1= H
R1= H
R2= Boc
R2= Boc
3c (3S)
R1= H
R1= H
R2= Boc
R2= Boc
2c (3R)
2d (3S)
R1= H
R1= H
R1= H
R2= H R3= H
R2= H R3= H
R2= H R3=Cl
3d (3R)
5d (3S)
5e (3R)
5f (3S)
R1= H
R1= H
R1= H
R2= Boc R3= H
R2= Boc R3= H
R2= Boc R3= Cl
1d (3S)
1e (3R)
1f (3S)
2e (2 ,4 ) R1= CONH2 R2= Boc
S
R
3e (2 ,4 ) R1= CONH R2= Boc
S
S
2
2f (2S,4R) R1= CO2Me R2= Me
3f (2S,4S) R1= CO2Me R2= Me
3g (4S)
R1= H R2= Me
1g (2 ,4 ) R1=CONH R2= H R3= H
2g (4R)
R1= H
R2= Me
5g (2S,4S) R1= CONH2 R2= Boc R3= H
5h (2S,4S) R1= CO2Me R2= Me R3= H
S
S
2
1h (2S,4S) R1=CO2H R2= Me R3= H
N
R1= H
R2= Me R3= H
5i (3S)
R1= H
R2= Me R3= H
NH2
1i (3S)
O
N
1j (2S,4S) R1=CO2Me R2= H R3= H
1k (2S,4S) R1=CO2Me R2= Me R3= H
Ts
O
N
N
4a R3= H
4b R3= Cl
O
O
R3
Scheme 1. Reagents and conditions: (a) (i) MsCl, pyridine; (ii) KSAc, DMF, 80 °C, 2 h; (b) (i) NaOMe, MeOH, 15 min rt; (ii) intermediate from 3a and 4a to yield 5a; 3a and 4b
to yield 5b; 3b and 4a to yield 5c; 3c and 4a to yield 5d; 3d and 4a to yield 5e; 3c and 4b to yield 5f; 3e and 4a to yield 5g; 3f and 4b to yield 5h; and 3g and 4a to yield 5i;
(c) KOH, H2O, THF; (d) TFA, H2O, rt.
Boc
N
H
N
N
N
N
N
O
HN
O
N
N
N
R1
b, c, d
a
S
S
R1
5a, b, d, f
or
N
N
N
N
O
Ph
b, d
O
HO
OH
R3
R3
7a R1= CO2H R3= H
7b R1= CO2H R3= Cl
6a R1= CO2Me R3= H
6b R1= CO2Me R3= Cl
7c R1= H
7d R1= H
R3= H
6c R1= H
6d R1= H
R3= H
R3= Cl
R3= Cl
Scheme 2. Reagents and conditions: (a) Intermediates 5a, 5b, 5d, or 5f and azine dihydrochloride, pyridine, reflux, 16 h to give 6a, 6b, 6c and 6d, respectively; (b) 2-
(biphenyl-4-yl)ethanamine, DMF, rt, 48 h; (c) (i) KOH, H2O, THF; (d) TFA, H2O, rt.

2744
L. Isakovic et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2742–2746
converted to triazoles 6a, 6b, 6c, and 6d utilizing azine dihydro-
chloride. Displacement of the triazole group with 2-(biphenyl-4-
yl)ethanamine followed by removal of the protecting groups gave
the desired products 7a–d.13f
All other analogues were prepared in a manner similar to the
above compounds starting form the suitable alcohol.14a The syn-
thesized compounds, 1a–k, 7a–d, 8a–c, 9a–b, 10a–b, 11 and 12,
have the same configuration at the ribose moiety as that of SAH.14b
These analogues were tested against recombinant human enzymes
DNMT1 and DNMT3b2 and their activity is presented in Table 1.15
For constrained SAH analogues incorporating cyclic amines bearing
a carboxylic acid group in place of the homocysteine unit, the ste-
reochemistry at the point of attachment to the sulfur atom and
that at the alpha-amino acid carbon played a role in the observed
activity. Of the five and six membered amino acid analogues (1a,
1c, 8a, 8b, and 8c) both 1a and 8a had reasonable potency against
both enzymes. Remarkably, 1a was almost equipotent to SAH itself
retaining the same level of activity against both enzymes (Table 1).
Compound 1c, the epimer of 1a, was devoid of activity against
DNMT1 and was 270-fold less active against DNMT3b2 indicating
that the inversion of stereochemistry at this center places either
the amine or the carboxylic acid groups in unfavorable position
for interaction with the enzyme. The difference in activity between
Table 1
DNMT1 and DNMT3b2 enzymatic activity of constrained SAH analoguesa
N
Table 2
O
NH2
N
DNMT1 and DNMT3b2 enzymatic activity of cyclic aminesa
R
S
N
N
HO
OH
N
O
NH2
N
R
Compound
R
DNMT1 IC50
0.8
(l
M)
DNMT3b2 IC50
0.2
(lM)
S
N
N
HO
OH
O
HO
L-SAH
H2N
Compound
R
DNMT1 IC50
(l
M)
DNMT3b2 IC50 (lM)
1a
1.1
0.3
HN
HO
O
1a
1.1
0.3
HN
O
HO
1c
8a
HN
HO
>100
54.0
1.5
1d
4.8
44.0
>10
12.0
26.0
32%
51%
16%
100
O
1e
8.0
HN
HN
N
CO2H
1ib
8b
>100
>100
4.0
39
>100
50%
HN
HN
9ab
9bb
10a
10b
18%
0%
HN
CO2H
CO2H
8c
19.0
31.0
HN
HN
1jb
1gb
1h
1kb
O
21.0
O
HN
1%
42%
11
94.0
66.0
O
H2N
NH2
12
13
44
>45
18.0
>100
17.0
41.0
>100
11.0
19%
N
N
H
Me
O
HO
H2N
N
4.5
O
O
14
H2N
a
a
Values are means of at least two experiments, 20%.
Percent inhibition at 45 lM inhibitor concentration.
Values are means of at least two experiments, 20%.
Percent inhibition at 45 lM inhibitor concentration.
b
b

L. Isakovic et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2742–2746
2745
1a and 8a implies that the distance as well as the configuration
stitution of 1d, compound 1f, resulted in a pronounced reduction
of activity against both DNMT1 and DNMT3b2 enzymes.
plays a role in the observed activity of these inhibitors.16a Com-
pound 8c, the epimer of 8a at the
both enzymes. This is in agreement with the structure of SAH
where only
-SAH is an inhibitor of DNMT enzymes.16b
The methyl ester of our lead 1a, compound 1j, maintained mod-
erate activity against DNMT1, while amide 1g lost activity com-
pletely against DNMT1 and retained slight activity towards
DNMT3b2 (Table 1). Methylation of the amines of 1a and 1j, com-
pounds 1h and 1k, respectively, abrogated DNMT1 activity indicat-
ing that the NH of 1a is engaged in important hydrogen-bonding
interactions in the active site of both enzymes.
Deletion of the carboxylic acid group of 1a, compound 1d, re-
sulted in a fivefold and 60-fold loss of enzymatic activity against
DNMT1 and DNMT3b2, respectively (Table 2). The activity of the
constrained amine 1d against DNMT1 is superior to that of the lin-
ear amines 13 and 14.17 Encouraged by such observation we pre-
pared other cyclic amines, 1e, 1i, 9a and 9b, 10a and 10b, 11 and
12, and tested them in our assays (Table 2). Unfortunately, all were
less active than 1d against both DNMT enzymes. The inferior enzy-
matic activity of 1e versus 1d parallels that of 1c versus. 1a impli-
cating that the (S)-configuration is superior to the (R). Generally,
analogues with a pyrrolidine ring, compounds 1d, 1e, 10a, 10b
and 9, were more active than analogues with other rings such as
azetidine, compound 12, piperidine, compounds 9a and 9b, or 2-
amino-cyclopropyl, compound 11 (Table 2).
In a previous Letter, we have reported that analogues of SAH
bearing a chlorine atom in the 2-position of the purine ring are
more selective towards DNMT1.18 To test whether the same ap-
plies to the constrained analogues of SAH, we prepared compounds
1b and 1f bearing a chlorine atom at the 2-position of the purine
ring. From Table 3, it could be seen that compound 1b retained
all activity against DNMT1, but was sevenfold less active against
DNMT3b2 enzyme as compared to 1a. However, the 2-chloro sub-
a
-carbon was not active against
In addition, we reported that lipophilic groups at the N6 posi-
tion of the purine ring of SAH are tolerated.18 N6 substitution of
the constrained analogue 1a, compound 7a, produced an equipo-
tent inhibitor against DNMT3b2 and a threefold reduction of activ-
ity against DNMT1 (Table 3). On the other hand, N6 substitution of
1d, compound 7c, reduced the activity against both enzymes. Con-
comitant substitution of a 2-chloro and N6 2-(biphenyl-4-yl)ethan-
amine groups on 1a (compound 7b) and 1d (compound 7d) was
not beneficial for enzymatic activity. Compound 7b was slightly
more potent against DNMT1, and 3.5-fold less active against
DNMT3b2, as compared to 1a, while 7d retained activity against
DNMT1 and caused almost twofold reduction of activity against
DNMT3b2 enzyme as compared to 1d.
In conclusion, we have prepared inhibitors of DNMT1 and
DNMT3b2 with constrained homocysteine units that are equipo-
tent to SAH. The five membered (2S,4S)-4-mercaptopyrrolidine-
2-carboxylic acid, in 1a, was a good replacement for homocysteine,
while the corresponding six-membered counterpart was less ac-
tive. Deletion of the carboxylic group, compound 1d, eliminating
the Zwitter-ionic character was less active against both enzymes.
Further optimization of 1a failed to give more potent inhibitors.
2-Chloro substitution of 1a, compound 1b, retained potency
against DNMT1, while N6-substitution, compound 7a, retained
DNMT3b2 activity. The concomitant substitutions of 1a at both
2- and N6 positions lead to a threefold decrease of activity against
both enzymes.
L
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DNMT1
IC50
DNMT3b2
IC50 M)
(
lM)
(l
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Med. Chem. 1975, 18, 300; (d) Borchardt, R. T.; Wu, Y. S. J. Med. Chem. 1976, 19,
197; (e) Borchardt, R. T. Biochem. Pharmacol. 1975, 24, 1542; (f) Borchardt, R. T.;
Huber, J. A.; Wu, Y. S. J. Med. Chem. 1976, 19, 1094.
1a
1b
1d
1f
H
H
H
H
CO2H
CO2H
H
H
Cl
H
1.1
0.82
4.8
0.27
1.9
12.0
>45
H
Cl
36.0
Ph
7ab
7b
7c
CO2H
CO2H
H
H
3.7
2.5
0.26
0.92
Ph
Ph
Ph
Cl
H
15.0
5.0
33.0
8. (a) Cohen, H. M.; Griffiths, A. D.; Tawfik, D. S.; Loakes, D. Org. Biomol. Chem.
2005, 3, 152. and references therein; (b) Botta, M.; Saladino, R.; Pedraly-Noy,
G.; Nicoletti, R. Med. Chem. Res. 1994, 4, 323; (c) Borchardt, T.; Wu, Y. S.; Huber,
J. A. J. Med. Chem. 1976, 19, 1104; (d) Houston, M. D.; Matuszewska, B.;
Borchardt, R. T. J. Med. Chem. 1985, 28, 478; (e) Crooks, P. A.; Tribe, M. J.; Pinney,
R. J. J. Pharm. Pharmacol. 1984, 36, 85.
7d
H
Cl
27.0
a
Values are means of at least two experiments, 20%.
b
N-6-Substituted SAH (2-amino-4-(((2S,3S,4R,5R)-5-(6-(2-(biphenyl-4-yl)ethyl-
amino)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methylthio)butanoic
acid) IC DNMT1 = 5.4 M; DNMT3b2 = 0.6
M.17
9. (a) Lin, Q.; Jiang, F.; Shultz, P. G.; Gray, N. S. J. Am. Chem. Soc. 2001, 123, 11608;
(b) Hirano, T.; Hoshino, K.; Iwanami, N.; Kagechika, H. In Poster, 236th National
l
l
50

2746
L. Isakovic et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2742–2746
Meeting of the American Chemical Society; Philadelphia, PA, August 17–21, 2008;
MEDI 118.
10. (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.
1H), 4.06 (m, 1H), 3.94 (m, 1H), 3.65 (m, 3H), (3H assumed under H2O at 4.87),
2.8–3.0 (m, 5H), 1.68 (m, 1H). MS: calcd 576; found 577 (MH+); 7b: 1H NMR
(DMSO-d6) d 8.49 (s, 1H), 7.6–7.7 (m, 4H), 7.1–7.5 (5H), 5.85 (d, 1H, 5.9 Hz),
4.66 (m, 1H), 4.13 (m, 1H), 4.04 (m, 1H), 3.3–3.8 (m, 7H), 2.98 (m, 4H), 1.76 (m,
1H). MS: calcd 610; found 611 (MH+).
11. Barbés, C.; Sánchez, J.; Yebra, M. J.; Robert-Geró, M.; Hardisson, C. FEMS
Microbiol. Lett. 1990, 69, 239.
15. Enzymes and biological assays: The full length cDNAs of DNMT1 (Swissprot
accession number P26358) and of DNMT3b2 (Swissprot accession number
Q9UBC3-2) were cloned in the pBlueBac4.5 vector (Invitrogen) These
constructs were used to generate recombinant baculoviruses using the Bac-
12. (a) Shieh, F.-K.; Reich, N. O. J. Mol. Biol. 2007, 373, 1157; (b) Liebert, K.; Horton,
J. R.; Chahar, S.; Orwick, M.; Cheng, X.; Jeltsch, A. J. Biol. Chem. 2007, 282, 22848.
13. (a) Experimental details are in MethylGene patent application: Wahhab, A.;
Besterman, J.; Delorme, D.; Isakovic, L.; Llewellyn, D.; Rahil, J.; Saavedra, O.;
Deziel, R., International Patent Application WO 2006/078752, 2006.; (b)
Douglas, K. A.; Zormeier, M. M.; Marcolina, L. M.; Woster, P. M. Bioorg Med.
Chem. Lett. 1991, 1, 267; (c) Guo, J. Q.; Wu, Y. Q.; Farmer, W. L.; Douglas, K. A.;
Woster, P. M. Bioorg Med. Chem. Lett. 1993, 3, 147; (d) Baddiley, J.; Jamieson, G.
A. J. Chem. Soc. 1954, 4280; (e) Montgomery, J. A.; Shortnacy, A. T.; Thomas, H. J.
J. Med. Chem. 1974, 17, 1197; (f) Samano, V.; Miles, R. W.; Robins, M. J. J. Am.
Chem. Soc. 1994, 116, 9331.
TM
N-Blue DNA according to the manufacturer’s instructions (Invitrogen) Nuclear
extracts were prepared from High Five insect cells infected with the
recombinant baculoviruses. The DNMT1 enzyme was purified on sequential
Q-sepharose FF and Hitrap Heparin columns (Amersham Biosciences. The
DNMT3b2 enzyme was purified on
a Hitrap SP-sepharose column and
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
14. (a) The piperidine type analogues, compounds 8a, 8b and 8c (Table 1) were
prepared in a manner similar to that for 1a, starting from alcohols (2S,4R)-
methyl 1-(tert-butoxycarbonyl)-4-hydroxypiperidine-2-carboxylate, (2S,4S)-
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
methyl
1-(tert-butoxycarbonyl)-4-hydroxypiperidine-2-carboxylate,
and
(2R,4R)-methyl 1-(tert-butoxycarbonyl)-4-hydroxypiperidine-2-carboxylate,
respectively, while compounds 9a and 9b were easily reached from alcohols
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 10X
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
(R)-tert-butyl
3-hydroxypiperidine-1-carboxylate
and
tert-butyl
4-
l
hydroxypiperidine-1-carboxylate, respectively. The 2-methylpyrrolidine
analogues 10a and 10b were made starting from (R)-tert-butyl 2-
l
(hydroxymethyl)pyrrolidine-1-carboxylate
and
(S)-tert-butyl
2-
l
(hydroxymethyl)pyrrolidine-1-carboxylate in the order mentioned. The 1,2-
cyclopentyl analogue 11 was prepared from tert-butyl (1S,2S)-2-
hydroxycyclopentylcarbamate and the azetidine analogue 12, was prepared
from tert-butyl 3-hydroxyazetidine-1-carboxylate following the same
chemistry described for 1a.; (b) All compounds were >95% pure and were
routinely characterized by 1H NMR and in some instances by 13C NMR and
NOE. Characterization of 1b: 1H NMR (D2O) d (ppm) 7.88 (s, 1H), 5.61 (d, 1H,
J = 4.5 Hz, 1H), 4.46 (dd, J = 4.5 Hz, J = 5 Hz, 1H), 4.14 (dd, J = 5 Hz, J = 5.3 Hz,
1H), 4.05 (m, 2H), 3.45 (m, 2H), 3.16 (m, 1H), 2.88 (dd, J = 14 Hz, J = 5.1 Hz, 1H),
2.78 (dd, J = 14 Hz, J = 6.1 Hz, 1H), 2.57 (m, 1H). MS: calcd 430.08 (100%),
432.08 (37%); found 431.3 (100%), 433.3 (17%) (MH+); 1f: 1H NMR (D2O) d
(ppm) 8.26 (br, 1H), 8.08 (s, 1H), 5.8 (d, 1H, J = 4.7 Hz, 1H), 4.7 (dd, J = 4.7 Hz,
J = 5.1 Hz, 1H), 4.27 (dd, J = 5.1Hz, J = 5.1 Hz, 1H), 4.16 (m, 1H), 3.48 (m, 1H),
3.33 (m, 1H), 3.33 (m, 2H), 3.21 (m, 1H), 3.02 (dd, J = 4.9 Hz, J = 12.1 Hz, 1H),
2.26 (dd, J = 4.5 Hz, J = 14.1 Hz, 1H), 2.89 (dd, J = 7 Hz, J = 14.1 Hz, 1H), 2.20 (m,
1H), 1.82 (m, 1H). MS: calcd 386.09 (100%), 388.09 (37%); found 387.2 (100%),
389.1 (40%) (MH+); 7a: 1H NMR (DMSO-d6) d 8.26 (s, 1H), 8.18 (s, 2H), 7.86 (br
s, 1H), 7.54 (m, 4H), 7.35 (m, 2H), 7.24 (m, 3H), 5.82 (d, 1H, J = 5.5 Hz), 4.64 (m,
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.
16. (a) The difference in the observed activity between 1a and 8a could be due to
steric factors which could interfere with the ability to form effiencent H-
bonding interactions with the active site of the enzyme.; (b) Kumar, R.;
Srivastava, R.; Singh, R. K.; Surolia, A.; Rao, D. N. Bioorg. Med. Chem. 2008, 16,
2276.
17. Jamieson, G. A. J. Org. Chem. 1963, 2397.
18. 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, B.;
MacLeod, A.R.; Delorme, D.; Besterman, J. F.; Wahhab, A. Biorg. Med. Chem. Lett.
2009, 19, 2747.