Structure-activity relationships for 4-anilinoquinoline derivatives as inhibitors of the DNA methyltransferase enzyme DNMT1

Article abstract of DOI:10.1016/j.bmc.2013.03.033

A series of 4-anilinoquinoline derivatives related to the known inhibitor SGI-1027, containing side chains of varying pK_a, were prepared by acid-catalysed coupling of the pre-formed side chains with 4-chloroquinolines. The compounds were evaluated for their ability to reduce the level of DNMT1 protein in HCT116 human colon carcinoma cells by Western blotting. With a very strongly basic N-methylpyridinium side chain, only NHCO-linked compounds were effective, whereas less strongly basic ((diaminomethylene)hydrazono)ethyl or 3-methylpyrimidine-2,4-diamine side chains allowed both NHCO- and CONH-linked compounds to show activity. In contrast, the pK_a of the quinoline unit had little apparent influence on activity.

Full text of DOI:10.1016/j.bmc.2013.03.033

Accepted Manuscript
Structure-Activity Relationships for 4-Anilinoquinoline Derivatives as Inhibi‐
tors of the DNA Methyltransferase Enzyme DNMT1
Swarna A. Gamage, Darby G. Brooke, Sanjeev Redkar, Jharna Datta, Samson
T. Jacob, William A. Denny
PII:
S0968-0896(13)00247-2
DOI:
http://dx.doi.org/10.1016/j.bmc.2013.03.033
BMC 10682
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Please cite this article as: Gamage, S.A., Brooke, D.G., Redkar, S., Datta, J., Jacob, S.T., Denny, W.A., Structure-
Activity Relationships for 4-Anilinoquinoline Derivatives as Inhibitors of the DNA Methyltransferase Enzyme
DNMT1, Bioorganic & Medicinal Chemistry (2013), doi: http://dx.doi.org/10.1016/j.bmc.2013.03.033
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Structure-Activity Relationships for 4-Anilinoquinoline Derivatives as
Inhibitors of the DNA Methyltransferase Enzyme DNMT1
Swarna A. Gamage1, Darby G. Brooke1, Sanjeev Redkar,2 Jharna Datta,3,4
Samson T. Jacob,3,4,5 and William A. Denny1,.*
4
Addresses: Auckland Cancer Society Research Centre, School of Medical Sciences, The
2
University of Auckland, Auckland 1142, New Zealand, SuperGen, Inc., Pleasanton, California,
3Department of Molecular and Cellular Biochemistry, 4Comprehensive Cancer Center and
5,Internal Medicine, College of Medicine, The Ohio State University, Columbus, OH 43210
ABSTRACT
A series of 4-anilinoquinoline derivatives related to the known inhibitor SGI-1027, containing
side chains of varying pKa, were prepared by acid-catalysed coupling of the pre-formed side
chains with 4-chloroquinolines. The compounds were evaluated for their ability to reduce the
level of DNMT1 protein in HCT116 human colon carcinoma cells by Western blotting. With
a very strongly basic N-methylpyridinium side chain, only NHCO-linked compounds were
effective, whereas less strongly basic (diaminomethylene)hydrazono)ethyl or 3-
methylpyrimidine-2,4-diamine side chains allowed both NHCO-and CONH-linked
1

compounds to show activity. In contrast, the pKa of the quinoline unit had little apparent
influence on activity.
Graphical Abstract
Keywords: quinoline, anilinoquinoline, DNMT1 inhibitor, synthesis
Abbreviations: DNMT (DNA methyltransferase), DMAP (4-dimethylaminopyridine), DMSO
(dimethylsulfoxide), POCl3 (phosphorous oxychloride)
1. Introduction
Methylation at the C-5 position of cytosine residues is a common occurrence in the
mammalian genome, with about 1% of the bases in the m5C form.1 These methylations are
2

found predominantly in 5′-m5CpG-3′ sequences, where they are considered to have a key role
in the silencing of genes, especially transposons.2 The major enzyme responsible for the
maintenance of these methylation patterns during replication is the DNA cytosine
methyltransferase DNMT1.3 This is a 1620-residue protein, for which crystal structures of
both the mouse and human forms, complexed with DNA, have been published recently.3
DNA methylation is the cause of a number of inherited disease syndromes, and can also have
a major role in the development of human cancer. It is the most frequent molecular change in
hematopoietic neoplasms,4 and is likely involved in other tumor types; for example, a
percentage of patients with sporadic colorectal cancers show methylation and silencing of the
gene encoding MLH1.5 A recent study of the aberrant promoter methylation profile of tumor
suppressor genes in hematopoietic malignancies showed high frequencies of methylation
compared with healthy controls, suggesting such hypermethylation to be a premalignant
condition.6
The most widely-explored inhibitors of DNMT1 are the suicide inhibitors azacytidine (1;
Vidaza) and decitabine (2; Dacogen). These are antimetabolites that incorporate into DNA in
place of cytosine, and irreversibly trap the enzyme.4,7 Both compounds have been widely
evaluated clinically for the treatment of myelodysplastic syndromes and lymphoproliferative
diseases, which are characterised by a high prevalence of tumor suppressor gene
hypermethylation.8 Both azacytidine and decitabine are approved for the treatment of
myelodysplastic syndrome.4 A phosphorothioate antisense oligodeoxynucleotide (MG98) that
inhibits the translation of the mRNA for DNMT1 is in Phase I trial, given as a continuous 21-
day i.v. infusion every 4 weeks, but did not appear to consistently down-regulate DNMT1
mRNA at doses of up to 240 mg/m2/day.9
3

The proven activity of the azanucleotides has prompted the search for other small-molecule
inhibitors of DNA methylation, but only a few have been described. The polyphenol (−)-
epigallocatechin-3-gallate (3)10 shows demethylating activity in cells, although these
compounds affect a variety of cellular functions, and direct inhibition of DNMT1 has not
been demonstrated. The psammaplin sponge metabolites (e.g., psammaplin G; 4)11 are
reported as potent direct inhibitors of DNA methyltransferases. Another potential mechanism
of inhibition of DNMT1 is through disruption of required enzyme/DNA binding process. The
anaesthetic procaine (5) is known to have demethylating activity in cancer cells, by a
mechanism suggested to involve binding to CpG-rich DNA sequence.12 (Figure 2).
Another well-known class of DNA-binding molecules are those that bind preferentially and
reversibly in the minor groove. Early studies with minor groove binding 4-anilinoquinolinium
bisquaternary salts demonstrated the ability of these compounds to bind selectively to AT-rich
regions of DNA, with a binding site size of 5-6 base pairs, attributed to a combination of
electrostatic, induced dipole-dipole, van der Waal and hydrogen bonding interactions.13 Detailed
NMR14 and X-ray crystallographic15,16 studies show the formation of specific stabilising
hydrogen bonds from the drug amide protons. These compounds have a variety of biological
activities, and were originally developed as anticancer drugs17,18 but their mechanism of action
has not been clearly defined. One member of this class (5; NSC 176319) was considered for
clinical trial, but was too toxic.19 A recent re-investigation of this class focused on replacement
of one or both of the quaternary functions to ameliorate toxicities. The resulting compounds
were shown to have inhibitory effects against DNMT1. One analogue (23; SGI-1027) was
studied in detail20,21 and shown to diminish DNMT1 levels in cells by enhancing its rate of
proteasomal degradation. One analogue (23; SGI-1027) was studied in detail20,21 and shown
4

to diminish levels of DNMT1 (but not DNMT3A or DNMT3B) in cells by enhancing its rate
of proteasomal degradation. It competes competitively with DNMT1 but not with DNA.
Methylation-specific PCR analysis and COBRA of the P16 and TIMP3 genes in the RKO
colon cancer cell line showed20 that these were demethylated following prolonged treatment
with 23. In the present paper we report on the synthesis and structure-activity relationships for
cellular loss of DNMT1 by this class of 4-anilinoquinolines. Due to their similar structures to
that of 23, is is reasonable that they work by a similar mechanism.
2. Results and Discussion
2.1. Chemistry
The pyrimidinyl compounds 6-17 of Table 1 were prepared by acid-catalysed coupling of
appropriate 4-chloroquinolines with the amine side chains 35, 42, 49 or 55. These in turn
were synthesised in a straightforward manner (amide coupling and reduction) from
commercially-available starting materials, with the quaternisation step left as late in the
syntheses as possible (Schemes 1-4). The (diaminomethylene)hydrazono)ethyl compounds
18 and 19 were similarly prepared from 4-chloroquinoline and the amines 62 and 67
respectively (Scheme 5A,B). The corresponding “reverse amides” 20 and 21 were prepared,
in slightly different ways, from the preformed N4-(4-aminophenyl)-6-
dimethylaminoquinoline 68 (Scheme 5C,D). Finally, the phenyl-3-methylpyrimidine-2,4-
diamines 22-28 were prepared likewise from 4-chloroquinolines and the similarly-
constructed amines 78, 83, 86 and 89 (Scheme 6). The 4-chloro-6,7,8-trifluoroquinoline 93
required for the synthesis of 27 was prepared as shown (Scheme 6) from the known ester 90.
5

2.2. Biology
Data for the synthesised compounds are shown in Table 1. Compounds 6-17 explored the
effects of replacing the quaternary quinolinium unit in 5 and the previously-discussed13
bisquaternary salt compounds with an unquaternised quinoline. The pKa of this unit was
varied by the introduction of amino and dimethylamino substituents at the (resonant) 6-
position, providing a range of values from 7.1-8.2 (calculated using ACD/PhysChem Suite
v12; ACD/Labs, Toronto, Canada). The ability of the compounds to reduce the level of
DNMT1 protein in HCT116 human colon carcinoma cells was measured by Western blotting.
Compounds 6-17 are 4-aminopyridinium monoquaternary salts, divided into four sets of 6-H,
6-NH2 and 6-NMe2 substituted quinolines, in which both the nature of the 4-anilinoquinoline
linker (CONH or NHCO) and the positioning (meta- or para-) of the very strongly basic
(pKA >12) 4-amino-N-methylpyridinium quaternary unit were varied. The results show that
the major determinant for effective inhibition of DNMT1 was the nature of the linker chain;
the CONH-linked compounds 6-11 were broadly ineffective, whereas four out of six of the
corresponding NHCO-linked compounds 12-17 were effective inhibitors at 5 µM
concentration. A secondary influence appeared to be the geometry of the 4-aminopyridinium
linker, with the meta-linked compounds being the more effective. The pKa of the quinoline
had no apparent influence.
Compounds 18-21 are a small subset with a (diaminomethylene)hydrazono)ethyl group in
place of the quinolinium, as a strong (pKa 8.5-9.0) but more delocalised basic group. Where
direct comparisons can be made [18, 19 with quinoliniums 10, 11 (CONH linker) and 20, 21
with quinoliniums 16, 17 (NHCO) linker], the (diaminomethylene)hydrazono)ethyl
compounds have broadly similar (and modest) inhibitory activities to the quinoliniums.
6

Compounds 22-28, bearing a more weakly basic (pKa ~7.5) phenyl-3-methylpyrimidine-2,4-
diamine group, were by far the most active subclass. Compounds 22-25 again varied the
linker (CONH or NHCO) and the positioning (meta- or para-) of the basic group. All were
very effective in inhibition of DNMT1, making it difficult to discern any SAR, but 23 did
appear to be the most active. A more detailed study of 23 (as SGI-1027) has been published
previously.20,21 Finally, three analogues of 23, where the pKa of the quinoline unit was varied
from 2.28 (27) to 7.86 (28) were prepared, to evaluate any effect of quinoline basicity. All
were active, with a slight trend in activity with higher pKa, but all were less effective than the
unsubstituted compound 23.
For a subset of the more active compounds, Western blots showing the reduction in DNMT1
levels in HCT116 human colon carcinoma cells (as both gels and bar graphs) are given in
Figure 1.
3. Conclusions
Apart from the azanucleotide suicide inhibitors such as azacytidine (1) and decitabine (2),
few well-validated inhibitors of DNMT1 have been reported, making the current series of 4-
anilinoquinolines of interest. The limited SAR described above show that very strongly basic
N-methylpyridinium-based side chains can be compatible with good DNMT1 inhibition (e.g.,
compounds 12, 14, 16), but do place a constraint on the type of linker (NHCO versus
CONH), even though the latter are geometrically very similar. Compounds containing the
less strongly basic (diaminomethylene)hydrazono)ethyl group and especially phenyl-3-
7

methylpyrimidine-2,4-diamine side chains allowed both types of linker to to be permissive. In
contrast, the pKa of the quinoline unit had little apparent influence on activity. A previous20,21
detailed study of the mechanism of action of 23 suggested that it can restore the normal
function of tumor suppressors by the induction of accelerated proteasomal degradation of
DNMT1 protein. It is likely that the inhibitory analogues discussed here function in the same
way. Other studies on 23 suggest it has a broadly similar mode of action to that of 5-
azacytidine,22,23 including being less potent to cancer cells harboring DNMT3B gene
amplification,24 and a recent computational study25 discussed the development of novel
DNMT1 inhibitors based on 23.
4. Materials and Methods
4.1. Chemistry. Intermediates and final products were analysed by reverse-phase HPLC
(Alltima C18 5 µm column, 150 × 3.2 mm; Alltech Associated, Inc., Deerfield, IL) using an
Agilent HP1100 equipped with a diode-array detector. Mobile phases were gradients of 80%
CH3CN/20% H2O (v/v) in 45 mM ammonium formate at pH 3.5 and 0.5 mL/min. Purity was
determined by monitoring at 330 50 nm and was >95%. Final product purity was also
assessed by combustion analysis carried out in the Campbell Microanalytical Laboratory,
University of Otago, Dunedin, New Zealand. Melting points were determined on an
Electrothermal 2300 Melting Point Apparatus. NMR spectra were obtained on a Bruker
Avance 400 spectrometer at 400 MHz for 1H and 100 MHz for 13C spectra.
The details of the preparation and chemical characterisation of all the compounds of Table 1
are provided in Supplementary Data.
8

4.2. Biology
4.2.1. Inhibition of DNMT1 in HCT116 cells: The human colon carcinoma cell line
HCT116 was obtained from ATCC and cultured in MEM according to supplier’s protocol.
Exponentially growing cells were treated with different drugs or DMSO (vehicle) for 24h.
Cells were harvested and whole extracts were subjected to Western blot analysis. Whole cell
extracts were immunoblotted with anti-DNMT1 or anti-GAPDH antibodies. The signal was
developed with ECLTM (GE Healthcare) after incubation with appropriate secondary
antibodies.
Acknowledgements
The authors thank Supergen Inc. and Cancer Society Auckland for support.
Supplementary Data
Supplementary data associated with this article can be found, in the online version, at doi
*****.
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13

Table 1. Structures and biological activities of 4-anilinoquinoline DNMT1 inhibitors
b
No
R
X
link Y
pKaa
TD50
(µM)
12.5
25
Dmnt1c
(% loss)
quin sidech
6
7
8
9
H
H
CONH
CONH
CONH
CONH
m
p
A
A
A
A
A
A
A
A
A
A
A
A
B
B
B
7.05
7.05
7.75
7.76
7.86
7.86
7.41
7.41
8.11
8.12
8.22
8.23
7.03
7.02
7.38
>12
>12
>12
>12
>12
>12
>12
>12
>12
>12
>12
>12
8.73
8.99
8.56
0
0
6-NH2
6-NH2
m
p
12.5
50
2
0
10 6-NMe2 CONH
11 6-NMe2 CONH
m
p
25
0
50
16
80
0
12
13
H
H
NHCO
NHCO
NHCO
NHCO
m
p
25
12.5
25
14 6-NH2
15 6-NH2
m
p
80
0
50
16 6-NMe2 NHCO
17 6-NMe2 NHCO
18 6-NMe2 CONH
19 6-NMe2 CONH
20 6-NMe2 NHCO
m
p
25
75
45
0
50
m
p
25
25
30
40
m
25
14

21 6-NMe2 NHCO
p
m
p
B
C
C
C
C
C
C
C
7.35
7.04
7.05
7.40
7.40
6.30
2.28
7.86
8.45
7.64
7.68
7.63
7.58
7.68
7.64
7.64
25
25
25
25
25
25
25
25
34
75
97
85
75
30
18
43
22
23
24
25
H
H
H
H
CONH
CONH
NHCO
NHCO
CONH
m
p
26 6-Cl
p
27 6,7,8-triF CONH
28 6-NMe2 CONH
p
p
Footnotes for Table 1
apKa of the quinoline or the side chain basic centres, calculated by ACD/PhysChem Suite v12
(average of tautomers); bTD50 : approximate drug concentration at which cells are 50%
viable; cDNMT1: the % loss of DNMT1 protein compared to untreated control, in HCT116
human colon carcinoma cells, measured by Western blotting, at a compound concentration of
5 µM.
15

Figure 1. Depletion of DMNT1 protein in HCT116 cells by quinazolines
HCT116 cells were treated with different concentrations (2.5, 5.0 and 10.0 μM) of the
indicated compounds for 24 hours. Whole cell extracts were subjected to Western blot
analysis with anti-DNMT1 antibody, to measure the degree of depletion of DNMT1 protein.
16

Figure 2
2
2
+
2
+
2
2
17

Scheme 1
Reagents and Conditions: (i) TsOH/180 °C; (ii) H2/Pd/C/MeOH; (iii) 4-nitrobenzoyl
chloride/pyridine/dioxane; (iv) DMF/MeOTs/20 °C, then ion- exchange; (v) Fe
dust/EtOH/H2O; (vi) 4-chloroquinolines/MeOH/cat. HCl/reflux.
18

Scheme 2
2
+
-
2
2
2
+
-
2
2
2
+
-
2
Reagents and conditions: As for Scheme 1
19

Scheme 3
Reagents and conditions: (i) CDI/DMF/55 °C 1 h, then 4-nitroaniline/DMAP/150 °C; (ii) p-
TsOH; (iii) pyridylpyridiniumchloride/ 180 °C; (iv) DMF/MeOTs; (v) Fe
dust/EtOH/H2O/H+/then ion exchange; (vi) 4-chloroquinolines/EtOH/H2O/H+/reflux.
20

Scheme 4
Reagents and conditions: (i) TsOH/180 oC; (ii) SOCl2, then 4-nitroaniline/pyridine; (iii)
EDMF/MeOTs/20 oC, then ion exchange; (iv) Fe dust/EtOH/trace HCl; (v) 4-
chloroquinolines, EtOH, H2O, H+, reflux.
21

Scheme 5
A
O
O N
2
2
CH
iii
3
2
i
57
2
2
+
NH
N
2
H N
2
59:
R=NO
61
62
: R = NO
2
2
ii
iv
ii
compound
18
NH
60:
2
R=NH
2
: R = NH
2
2
of Table 1
.H SO
2
4
58
Me
O
Me
NH
N
N
B
2
O
NH
N
iii
i
2
CH
N
NH
3
58
+
2
N
H
NH
2
O N
2
R
.HCl
.2HCl
iv
63
R
64
65
66
67
: R = NO
: R = NO
2
2
ii
ii
compound
: R = NH
: R = NH
2
2
19
of Table 1
H
NH
2
N
Me
C
O
O
HN
HN
HO C
2
COCH
3
Me N
2
Me N
2
v
-
Cl
vi
compound
+
N
H
N
+
H
+
20
70
of Table 1
-
69
Cl
68
O
Me
.HCl
NH
D
NH
N
2
CH
i
viii
21
compound
N
3
+
58
of Table 1
2
HO C
2
HO C
2
72
71
Reagents and conditions: (i) MeOH, c.HCl, reflux, 14 h; (ii) Fe, EtOH, H2O, cat. c.HCl,
reflux, 14 h; (iii) 4-nitrobenzoyl chloride, pyridine, dioxane, reflux, 2 h; (iv) 4-chloro-
6(dimethylaminoquinoline, EtOH, H20, cHCl, reflux; (v) EDCI, DMAP, DMF, 20°C; (vi)
MeOH, HCl, reflux; (vii) aminoguanidine bicarbonate , c.HCl, MeOH, reflux, 2 h; 68, EDCI,
DMAP, DMF.
Scheme 6
22

Reagents and conditions: (i) 2-Ethoxyethanol, c.HCl, reflux; (ii) Fe, 2:1 EtOH/H2O, 2% v/v
AcOH, reflux, 1.5 h; (iii) 4-nitrobenzoyl chloride, pyridine, dioxane, 50 °C, 5 d; (iv) Fe, 2:1
EtOH/H2O, c.HCl; (v) 4-chloroquinoline, EtOH, H2O, H+, reflux; (vi) H2, 10%Pd-C, MeOH;
(vii) 1M NaOH/reflux; (viii) Ph2O/reflux; (ix) POCl3/reflux
23

Graphical Abstract
+
2
2
2
2
2
24