Dilazep analogues for the study of equilibrative nucleoside transporters 1 and 2 (ENT1 and ENT2)

Article abstract of DOI:10.1016/j.bmcl.2014.10.026

As ENT inhibitors including dilazep have shown efficacy improving oHSV1 targeted oncolytic cancer therapy, a series of dilazep analogues was synthesized and biologically evaluated to examine both ENT1 and ENT2 inhibition. The central diamine core, alkyl chains, ester linkage and substituents on the phenyl ring were all varied. Compounds were screened against ENT1 and ENT2 using a radio-ligand cell-based assay. Dilazep and analogues with minor structural changes are potent and selective ENT1 inhibitors. No selective ENT2 inhibitors were found, although some analogues were more potent against ENT2 than the parent dilazep.

Full text of DOI:10.1016/j.bmcl.2014.10.026

Bioorganic & Medicinal Chemistry Letters 24 (2014) 5801–5804
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Dilazep analogues for the study of equilibrative nucleoside
transporters 1 and 2 (ENT1 and ENT2)
Hilaire Playa a, Timothy A. Lewis b, Amal Ting b, Byung-Chul Suh b, Benito Muñoz b, Robert Matuza c,
Brent J. Passer c, Stuart L. Schreiber b, John K. Buolamwini a, ,
⇑
a Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Science Center, Ste 327 Johnson, 847, Monroe, Memphis, TN 38163, USA
b Center for the Science of Therapeutics, The Broad Institute of MIT and Harvard, 415 Main Street, Cambridge, MA 02142, USA
c Neurosurgery Department, Massachusetts General Hospital, 185 Cambridge Street, Boston, MA 02114, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
As ENT inhibitors including dilazep have shown efficacy improving oHSV1 targeted oncolytic cancer ther-
apy, a series of dilazep analogues was synthesized and biologically evaluated to examine both ENT1 and
ENT2 inhibition. The central diamine core, alkyl chains, ester linkage and substituents on the phenyl ring
were all varied. Compounds were screened against ENT1 and ENT2 using a radio-ligand cell-based assay.
Dilazep and analogues with minor structural changes are potent and selective ENT1 inhibitors. No
selective ENT2 inhibitors were found, although some analogues were more potent against ENT2 than
the parent dilazep.
Received 20 August 2014
Revised 3 October 2014
Accepted 8 October 2014
Available online 28 October 2014
Keywords:
Equilibrative nucleoside transporters (ENTs)
Ó 2014 Elsevier Ltd. All rights reserved.
hENT1
hENT2
rENT2
Dilazep
Nucleoside transporters are trans-membrane proteins that
facilitate the movement of nucleosides, nucleobases, and their ana-
logues across the cell membrane. There are two families of trans-
porters belonging to the solute carrier class of proteins,
concentrative nucleoside transporters (CNTs) and equilibrative
nucleoside transporters (ENTs).1 Both ENTs and CNTs play a vital
role in regulating the levels of nucleosides and nucleobases within
the cell and interstitial space as these highly polar molecules can-
not passively diffuse across cellular membranes.2,3 Substrate spec-
ificity,4 expression levels, and location of expression vary between
the two families of transporters and among the isoforms within
each class,5 enabling the design of potent and selective inhibitors.
Within the equilibrative family, there are four known isoforms in
humans: hENT1, hENT2, hENT3, and hENT4.6 Human ENT3 is an
intracellular transporter and hENT4 has limited expression, func-
tioning as an adenosine transporter only under acidic conditions,7
whereas hENT1 and hENT2 are widely expressed throughout the
body.6
46% related.7 Both hENT1 and hENT2 have broad permeant selec-
tivity, transporting both pyrimidine and purine nucleosides, but
hENT2 transports nucleobases as well and is insensitive to the
nucleoside transport inhibitor S-(4-nitrobenzyl)-6-thioinosine
(NBMPR).6 Likewise, hENT1 and hENT2 possess different Ki values
with regard to dipyridamole and dilazep, two potent nucleoside
transporter inhibitors which are also approved pharmacologic
agents (Fig. 1).8 For each of these inhibitors, hENT1 shows a
significantly greater sensitivity to inhibition.
O
O
MeO
MeO
OMe
OMe
N
N
O
O
OMe
OMe
dilazep (DZ)
O2N
OH
N
N
While hENT1 and hENT2 share greater than 75% sequence
identity to their rodent homologues, the human isoforms are only
HO
N
N
N
OH
N
O
S
N
N
N
N
OH
N
N
OH
HO
⇑
Corresponding author. Tel.: +1 847 578 8341; fax: +1 847 578 6586.
OH
E-mail address: john.buolamwini@rosalindfranklin.edu (J.K. Buolamwini).
Current address: Department of Pharmaceutical Sciences, College of Pharmacy,
Rosalind Franklin University of Medicine and Science, 3333 Green Bay Road,
North Chicago, IL 60064, USA.
dipyramidole (DP)
NBMPR
Figure 1. Inhibitors of ENT1 and ENT2.
http://dx.doi.org/10.1016/j.bmcl.2014.10.026
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

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H. Playa et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5801–5804
Table 1
Scheme 1 Analogues
There are a number of potential therapeutic uses for nucleoside
transporter inhibitors. Studies have shown a correlation between
nucleoside transporter inhibition, specifically hENT1, and reduc-
tion in cellular damage from acute ischemia via effects on tissue
adenosine levels.9,10 Cancer chemotherapy is another area of
potential therapeutic application, especially as a number of current
drugs are transported by nucleoside transporters.11 The current
study was prompted as a result of a collaborative project, which
showed that ENT inhibitors potentiated the activity of oncolytic
herpes simplex I virus (oHSV1) in killing cancer cells.12 Oncolytic
viruses are a treatment that selectively targets cancer cells. Genet-
ically engineered viral vectors spare normal cells, mitigating collat-
eral damage from normal cancer chemotherapy. However, because
oHSV1 has limited replication and spread to neighboring cancer
cells, its potential uses have been limited.13 Prior work showed
that the efficacy of oHSV1 treatment could be improved with the
addition of appropriate pharmaceuticals.14 A high-throughput
screen identified dipyridamole and dilazep, two FDA-approved
drugs that are ENT1 and ENT2 inhibitors (Fig. 1), as efficacious mol-
ecules for increasing the activity of oHSV1.12 The two drugs are
both anti-platelet drugs that act through phosphodiesterase
(PDE) and protein kinase (PK) inhibition. However, experiments
indicated that the mechanism of action for oHSV1 activity
improvement did not involve these mechanisms, but rather
directly involved hENT1 inhibition, as NBMPR (Fig. 1), a known
potent ENT1 inhibitor demonstrated similar results, while PDE
and PK inhibitors did not.13 While both drugs are potent hENT1
inhibitors, at therapeutic levels hENT2 inhibition may occur. To
advance our understanding of how nucleoside transporter inhibi-
tors can improve oHSV1 or other similar therapies, potent selective
inhibitors are needed. As such, dilazep was used as a starting point
to synthesize analogues to explore the structure–activity relation-
ship (SAR) with respect to ENT1 and ENT2 selectivity.
Compound
n
Diamine
DZ
1
2
3
4
5
6
7
8
1
0
2
0
1
2
1
2
1
2
1
2
Homopiperazine
Homopiperazine
Homopiperazine
Piperazine
Piperazine
Piperazine
2,5-Dimethylpiperazine ( )
2,5-Dimethylpiperazine ( )
2,2-Dimethylpiperazine
2,2-Dimethylpiperazine
2,5-Diazabicyclo[2.2.2]octane ( )
2,5-Diazabicyclo[2.2.2]octane ( )
9
10
11
O
O
MeO
MeO
HN
N
O
O
a, b
c
N
O
NH
12
OMe
N
O
O
MeO
MeO
OMe
OMe
O
N
n
OMe
OMe
13
(n = 0)
14 (n = 2)
O
MeO
MeO
O
N
O
d or e
N
12
R
OMe
15 R = Ph
16 R = CH3
A
B
C
Reagents: a) , K2CO3/DMF (93%); b) TFA (93%); c) or , K2CO3/DMF
Dilazep (DZ) analogues were synthesized by varying the sub-
stituents on the phenyl rings, the functional group connecting
them to alkyl linkers of varying length, and the central cyclic dia-
mine. Three bromoalkyl 3,4,5-trimethoxyphenyl esters were trea-
ted with various cyclic diamines to make symmetric compounds
(Scheme 1). Attempts to make the acyclic analogue by treating 1
with N,N0-dimethylethylene diamine were unsuccessful (Table 1).
Unsymmetric analogues were prepared. Alkylation of mono
t-BOC-homopiperazine with bromoester A followed by TFA depro-
tection produced compound 12 which was alkylated with bromo-
ester B or C to give compounds 13 and 14, respectively (Scheme 2).
Compound 12 was acylated to give compounds 15 and 16, respec-
tively. Lower molecular weight analogues were prepared by
treating A and B with either methyl- or benzylhomopiperazine,
methylpiperazine, pyrrolidine, and morpholine (Table 2).
(54% for 13, 50% for 14); d) PhCOCl, Et3N/THF for 15 (63%);
16
Ac2O, pyridine/THF for
(74%)
O
O
MeO
MeO
MeO
MeO
a
NRR
O
Br
O
n
n
n
= 0, 1
OMe
OMe
Reagents: a) excess amine/THF.
Scheme 2. Synthesis of unsymmetrical analogues.
Table 2
Scheme 2 Analogues
Analogues were next prepared removing one, two, or all three
methoxy groups from the phenyl rings, and by adding an electron
withdrawing fluorine substituent (Scheme 3) (Table 3).
The ester groups of dilazep were replaced with an ether,
amide, or heterocycle. 3-Bromo-1-propanol was alkylated with
3,4,5-trimethoxybenzyl chloride and the ether product was treated
with homopiperazine to yield 38 (Scheme 4). Bis-alkylation
of homopiperazine with 3-azido-1-bromopropane followed by
Staudinger reduction gave diamine 39. Treating the diamine with
Compound
n
Amine
17
18
19
20
21
22
23
24
25
26
0
1
0
1
0
1
0
1
0
1
N-Methylhomopiperazine
N-Methylhomopiperazine
N-Benzylhomopiperazine
N-Benzylhomopiperazine
N-Methylpiperazine
N-Methylpiperazine
Pyrrolidine
Pyrrolidine
Morpholine
Morpholine
O
OMe
O
MeO
MeO
OMe
OMe
MeO
MeO
a
O
N
O
Br
3,4,5-trimethoxybenzyl chloride failed to cleanly produce the
desired amino-linked compound, but treatment of the bis-amine
with 3,4,5-trimethoxy benzoyl chloride or acetic anhydride gave
the desired bis-amides 40 and 41, respectively (Scheme 5). Copper
catalyzed cycloaddition of the bis-azide intermediate with 3,4,5-tri-
methoxyphenyl acetylene gave the heterocyclic linked analogue 42.
n
n
N
O
A n = 1
n = 0
C n = 2
n
OMe
O
OMe
B
a) Diamine, K2CO3/DMF
Scheme 1. Synthesis of symmetrical dilazep analogues

H. Playa et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5801–5804
5803
O
O
10 µM Screening of Dilazep Analouges
X
a
O
N
200
150
100
50
O
Br
m
n
n
hENT1
rENT2
N
O
X
X
n
= 0, 1
n
O
m = 1, homopiperazine
m = 0, piperazine
Reagents: a) Diamine, K2CO3/DMF.
Scheme 3. Substitutions on phenyl rings.
Table 3
Scheme 3 Analogues
Compound
n
m
Diamine
X
27
28
29
30
31
32
33
34
35
36
37
1
1
0
1
1
0
0
1
1
1
1
2
1
2
2
1
1
2
1
1
2
1
Homopiperazine
Piperazine
Homopiperazine
Homopiperazine
Piperazine
Piperazine
Homopiperazine
Piperazine
H
H
0
4-OMe
4-OMe
4-OMe
4-OMe
3,5-(OMe)2
3,5-(OMe)2
3,5-(OMe)2
4-F
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17 18 19 20 21
R
P
M
M DP
M DZ
B
µ
µ
N
1
10
M
0 n
10
Figure 2. 10 lM screen of dilazep analogues 1–21. Data presented as average
percent relative uptake to control untreated cells. Error bars are the standard
deviation from the mean.
Piperazine
Homopiperazine
Piperazine
4-F
10 µM Screening of Dilazep Analouges
200
OMe
OMe
b)
a)
Br
N
O
Br
OH
hENT1
rENT2
OMe
150
100
50
MeO
MeO
OMe
OMe
O
N
O
OMe
OMe
38
Reagents: a) 3,4,5-trimethoxybenzyl chloride, K2CO3, THF (15%);
b) homopiperazine, K2CO3, DMF (11%).
Scheme 4. Synthesis of dilazep ether analogue.
0
7
8
0
1
6
1
2
4
R
22 23 24 25 26
2
2
29
3
3
32 33 34 35
3
37 38 39 40
4
P
M
1
M DP
M DZ
B
a
µ
µ
c
N
HN
NH
N3
N
N
N3
H2N
N
N
NH2
10
M
0 n
39
10
b
d or e
Figure 3. 10
lM screen of dilazep analogues 22–42. Data presented as average
percent relative uptake to control untreated cells. Error bars are the standard
deviation from the mean.
N
H
N
N
H
N
N
N
N
Ar
R
N
N
N
R
Ar
N
N
O
O
Table 4
40 R = 3,4,5-(OMe)3Ph
41 R = Ac
42 Ar = 3,4,5-(OMe)3Ph
Selected IC50s with 95% CI of dilazep analogues
Reagents: a) 1-azido-3-bromopropane, K2CO3, THF (55%); b) 3,4,5-trimethoxylphenyl acetylene, Cu,
tBuOH/H2O (97%); c) PPh3, THF/H2O (63%); d) 3,4,5-trimethoxybenzoyl chloride, Et3N, CH2Cl2 (30%);
e) Ac2O, pyridine (9%).
Compound
hENT1 IC50 [nM] (95% CI)
rENT2 IC50 [nM] (95% CI)
DZ
3
4
5
7
17.5 (9.4–33.8)
2.8 (1.6–4.7)
66.1 (35.7–122)
17.7 (12.8–25.7)
3.2 (1.7–6.2)
8800 (2070–37300)
977 (598–1690)
1310 (898–1910)
1080 (643–1800)
Scheme 5. Synthesis of dilazep amide and heterocyclic analogue.
1490 (843–2630)
8
31.0 (13.4–71.7)
4.6 (2.5–8.6)
803 (503–1280)
217 (112–419)
93.9 (27.9–316)
NAa
NA
NA
NA
NA
Compounds were tested in duplicate at 10 lM in a forty-eight
13
15
20
40
well plate cell-based radio-ligand uptake assay, using previously
established transgenic PK15NTD porcine cells expressing individ-
ual recombinant human (h) ENT,13 and H9c2 rat cells expressing
native rENT2, using dilazep (DZ), dipyramidole (DP) and NBMPR
as reference compounds (Figs. 2 and 3). For the rat cells, rENT1
was inhibited with 100 nM NBMPR prior to addition of compounds
to avoid participation of the low level ENT1 expressed by these
cells. Decreased uptake of the 3H[5-]uridine indicated a ‘hit’ and
was confirmed by repeating the assay with seven concentrations
serially diluted over at least four log units in order to establish
dose-response curves (Table 4). These assays were run in triplicate.
a
NA = Insignificant rENT2 inhibitory activity.
Ten compounds were tested in dose-response assays against ENT1,
five of these were also tested for dose-response (Table 4) against
rENT2 based upon their activity at 10 lM.
Dilazep and close analogues are potent hENT1 inhibitors
(IC50 < 100 nM) with little or no activity against rENT2. The central

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H. Playa et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5801–5804
due solely to ENT1 and not ENT2 inhibition.12 Further studies with
oHSV1 and the compounds described here support this hypothesis,
results of these studies will be reported shortly. Other chemical
scaffolds will need to be explored to discover potent and selective
inhibitors of ENT2 to ascertain more about the role and importance
of this transporter. oHSV therapy of cancer has now advanced to
phase 3 clinical trials.15 There is an urgent need to evaluate and
utilize therapeutics that will enhance oHSV efficacy. Further stud-
ies of dilazep analogues should be considered in appropriate ani-
mal cancer models with the aim of progressing to clinical trials.
O
Ar
O
O
N
N
O
N
O
Ar
Ar
O
Ar
O
N
5
O
4
O
Ar
O
O
N
N
O
N
O
Ar
Ar
O
Ar
O
N
7 (+)
N
O
8
3
Ar
O
O
Ar
O
N
N
Ar
O
O
N
O
Ar
O
O
13
Acknowledgements
Figure 4. Structures of hENT1 active compounds (Ar = 3,4,5-(OMe)3Ph).
This work was funded in part by the NIH-MLPCN program (1
U54 HG005032-1 awarded to S.L.S.) as well as NIH RO1GM10405
(J.K.B.) and Grant RO1CA102139 (R.L.M.).
Uptake inhibition of rat and human ENT2
150
hENT2
rENT2
Supplementary data
Supplementary data (chemistry experimental procedures and
characterization of all compounds plus biological assay details)
associated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.bmcl.2014.10.026.
100
50
0
References and notes
7
8
5
9
1
7
0
3
2
3
Z
2
4
40
2
19
D
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homopiperazine ring can be replaced with a piperazine, or methyl-
substitued piperazine. The alkyl chains can be extended or short-
ened by one carbon with little change in potency. When the ester
bonds of DZ were replaced with an ether or heterocycle, hENT1
activity was diminished, but activity was observed with the corre-
sponding amide (40). Compounds with the 3,4,5-(OMe)3 substi-
tuted phenyl rings displayed activity (Fig. 4), though two of these
groups are not required based upon the activities of 15 and 20.
No potent rENT2 inhibitors were found, though compounds 3
and 5 have IC50s ca. 1 lM. Selected compounds demonstrating
activity against rENT2 were cross-screened against hENT2
(Fig. 5). Only minor differences in activities were observed
between the two assays with the compounds tested.
15. Kaufman, H. L.; Bines, S. D. Future Oncol. 2010, 6, 941.
Given these results, the activity previously reported on the effi-
cacy of oHSV1 treatment in the presence of dilazep is most likely