Constrained NBMPR analogue synthesis, pharmacophore mapping and 3D-QSAR modeling of equilibrative nucleoside transporter 1 (ENT1) inhibitory activity

Article abstract of DOI:10.1016/j.bmc.2008.01.044

Conformationally constrained analogue synthesis was undertaken to aid in pharmacophore mapping and 3D-QSAR analysis of nitrobenzylmercaptopurine riboside (NBMPR) congeners as equilibriative nucleoside transporter 1 (ENT1) inhibitors. In our previous study

Full text of DOI:10.1016/j.bmc.2008.01.044

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 3848–3865
Constrained NBMPR analogue synthesis, pharmacophore
mapping and 3D-QSAR modeling of equilibrative
nucleoside transporter 1 (ENT1) inhibitory activity
Zhengxiang Zhu and John K. Buolamwini*
Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Science Center,
847 Monroe Avenue, Suite 327, Memphis, TN 38163, USA
Received 1 December 2007; revised 20 January 2008; accepted 23 January 2008
Available online 30 January 2008
Abstract—Conformationally constrained analogue synthesis was undertaken to aid in pharmacophore mapping and 3D-QSAR
analysis of nitrobenzylmercaptopurine riboside (NBMPR) congeners as equilibriative nucleoside transporter 1 (ENT1) inhibitors.
In our previous study [J. Med. Chem. 2003, 46, 831–837], novel regioisomeric nitro-1,2,3,4-tetrahydroisoquinoline conformationally
constrained analogues of NBMPR were synthesized and evaluated as ENT1 ligands. 7-NO2-1,2,3,4-Tetrahydroisoquino-2-yl purine
riboside was identified as the analogue with the nitro group in the best orientation at the NBMPR binding site of ENT1. In the
present study, further conformational constraining was introduced by synthesizing 50-O,8-cyclo derivatives. The flow cytometrically
determined binding affinities indicated that the additional 50-O,8-cyclo constraining was unfavorable for binding to the ENT1 trans-
porter. The structure–activity relationship (SAR) acquired was applied to pharmacophore mapping using the PHASE program. The
best pharmacophore hypothesis obtained embodied an anti-conformation with three hydrogen-bond acceptors, one hydrophobic
center, and two aromatic rings involving the 30-OH, 40-oxygen, the NO2 group, the benzyl phenyl and the imidazole and pyrimidine
portions of the purine ring, respectively. A PHASE 3D-QSAR model derived with this pharmacophore yielded an r2 of 0.916 for
four (4) PLS components, and an excellent external test set predictive r2 of 0.78 for 39 compounds. This pharmacophore was used
for molecular alignment in a comparative molecular field analysis (CoMFA) 3D-QSAR study that also afforded a predictive model
with external test set validation predictive r2 of 0.73. Thus, although limited, this study suggests that the bioactive conformation for
NBMPR at the ENT1 transporter could be anti. The study has also suggested an ENT1 inhibitory pharmacophore, and established
a predictive CoMFA 3D-QSAR model that might be useful for novel ENT1 inhibitor discovery and optimization.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
which inhibit it at low nanomolar to subnanomolar con-
centrations.19 Unfortunately, attempts at therapeutic
application of current ENT1 inhibitors have been largely
disappointing due to their poor pharmacological profiles
with regard to toxicity, selectivity, and poor in vivo effi-
cacy.18 Thus, there is a need for novel inhibitors.
Nucleoside transport inhibitors (NTIs) have been shown
to have potential therapeutic applications in heart dis-
ease,2–6 inflammatory disease,7 viral infections,8,9 and
cancer chemotherapy.10–15 The ENT1 transporter, which
is the focus of this research, is the major nucleoside trans-
porter in most mammalian tissues, especially heart tis-
sue,16,17 and appears to be the most relevant NT target
for therapeutic exploitation. Several chemical classes
have been shown to inhibit the ENT1,18 the most potent
and selective of which are NBMPR and its congeners,
Since there are no 3D structures of mammalian nucleo-
side transporters nor their complexes with inhibitors,
knowledge of the 3D pharmacophore of the most potent
and selective inhibitors will be useful for rational design
of new NT inhibitors.20 To that end, the objective of this
study was to continue our probe into the bioactive con-
formation of NBMPR and its analogues as ENT1 nucle-
oside transporter inhibitors through a combination of
conformationally constrained analogue synthesis,1 phar-
macophore mapping, and 3D-QSAR modeling. Current
structure–activity relationship (SAR) studies on ENT1
nucleoside transport inhibitors18 demonstrate that for
Keywords: Equilibrative nucleoside transporter 1 (ENT1); Inhibitor;
Nitrobenzylmercaptopurine riboside (NBMPR); Pharmacophore map-
ping; 3D QSAR; Comparative molecular field analysis; PHASE; Flow
cytometry; Constrained analogues; Synthesis.
*
Corresponding author. Tel.: +1 901 448 7533; fax: +1 901 448
6828; e-mail: jbuolamwini@utmem.edu
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.01.044

Z. Zhu, J. K. Buolamwini / Bioorg. Med. Chem. 16 (2008) 3848–3865
3849
NBMPR analogues, the nitrobenzyl moiety is critical for
high-affinity binding to the transporter. Therefore, in
our previous study, a series of conformationally con-
strained analogues of NBMPR was synthesized by
replacing the purine 6-position nitrobenzyl group with
nitro-1,2,3,4-tetrahydroisoquinolines, thereby locking
two of the rotatable bonds in the flexible nitrobenzyl
moiety into a tetrahydroisoquinoline ring. The most
suitable substitution position of the nitro group was ex-
plored by varying its position on the aromatic ring of the
tetrahydroisoquinoline moiety, as shown in compounds
2–5 (see Fig. 1). The results indicated that compound 4
with the nitro substituent at the 7-position of tetrahy-
droisoquinoline ring most captures the bioactive orien-
tation of the nitrobenzyl moiety of NBMPR.1
However, compounds 2–5, are still flexible; there are
different conformations might greatly influence the bind-
ing affinities of the molecules at the transporter. In con-
tinuation of our probing of the bioactive conformation
of NBMPR in this study, we synthesized another set
of NBMPR analogues with additional conformational
constraining. In this series, the free rotation of the glyco-
0
sidic bond was blocked by forming an O50 –C8 linkage. In
0
doing so, the rotation about the C4 –C5 bond was also
blocked. The most suitable position of the nitro group
was again explored by varying its position on the aro-
matic ring of the tetrahydroisoquinoline moiety, as
shown in Figure 1 (compounds 6–9), where the nitro
substituent is in the 5-, 6-, 7-, or 8-position of the
1,2,3,4-tetrahydroisoquinoline ring, respectively. The
most active NBMPR analogues including the conform-
ationally constrained analogue, compound 4, were used
to develop a common pharmacophore hypothesis using
the PHASE program (Schro¨dinger), which was subse-
quently used for CoMFA 3D-QSAR modeling in at-
tempts to validate the suggested conformation.
10
three major rotatable bonds, the0N9–C glycosidic bond,
0
the C6–N6 bond, and the C4 –C5 bond in the molecules.
For NBMPR analogues and other nucleosides, the base
moiety can adopt two main orientations relative to the
10
sugar moiety about the N9–C glycosidic bond, termed
syn and anti.21 In the syn orientation, the bulky portion
of the base, such as the pyrimidine ring in purine nucleo-
sides or O2 in pyrimidine nucleosides, is orientated over
the sugar ring; and in the anti-conformation it is ori-
ented away from the sugar ring. The intermediate con-
formations between syn and anti are referred to as
high-anti and high-syn, respectively (see Fig. 2). These
2. Chemistry
A limited number of studies have reported the intramo-
lecular cyclization of purine nucleosides. Ikehara et al.
once reported that by activating the 50-OH group with
sodium hydride in dioxane, the resulting nucleophilic –
NO2
NO
NO2
2
N
N
N
N
S
a
b
a
b
c
c
N
N
N
N
N
N
N
N
N
O
N
HO
HO
O
O
O
OH OH
OH OH
OH OH
1, NBMPR
2-5
6-9
Figure 1. Design of conformationally constrained analogues of NBMPR.
10
Figure 2. Different conformations of compound 4 generated by rotation of the N9–C glycosidic bond.

3850
Z. Zhu, J. K. Buolamwini / Bioorg. Med. Chem. 16 (2008) 3848–3865
Oꢀ species would attack the electron-deficient carbon
at position 8 of 8-bromo-20,30-O-isopropylideneadeno-
sine to give 50-O,8-cyclo-20,30-O-isopropylideneadeno-
sine.22 However, when we tried to follow this method
to prepare compound 12 (see Scheme 1), we found
we could not obtain compound 11 by bromination of
compound 10 (which was prepared from compound
4) using bromine in the mixture of dioxane and 10%
Na2HPO4 (1:1) or bromine water in NaOAc buffer as
reported in the literature for the synthesis of 8-bro-
mo-20,30-O-isopropylideneadenosine.23–25 Instead, the
product was identified to be compound 13 when bro-
mine in dioxane/Na2HPO4 (1:1) was used. No products
were obtained when bromine water in NaOAc buffer
was used. An alternative strategy employing direct oxi-
dative cyclization of compound 10 to yield 12, using
lead tetraacetate in dry benzene or N-halosuccinimide
in acetic acid as shown in Scheme 2, which has been
previously used to synthesize 50-O,8-cycloadenosines26
and 50-O,8-cycloguanosines,27 also failed. This led to
the conclusion that the nitrotetrahydroisoquinoline
substituent at the purine C6-position was hindering
the 50-O,C-8-cyclization. Therefore, some other reac-
tion schemes with an appropriate substrate other than
compound 4 were considered. As shown in Scheme 3,
6-chloropurine riboside (14), which does not have a
bulky substituent at the 6-position and is commercially
available, was chosen. 20,30-O-Isopropylidene-6-chloro-
purine riboside (15) was prepared by treating com-
pound 14 with acetone in the presence of a catalytic
amount of hydroperchloric acid (70%). 50-O,8-cyclo-
20,30-O-isopropylidene-6-chloropurine riboside (16)
was successfully prepared by treating compound 15
with N-iodosuccinimide (NIS) in acetic acid. Unfortu-
nately, the deprotection of the 20,30-hydroxyl groups
in compound 16 became another problem. When com-
pound 16 was treated with 0.5 N H2SO4 at 55 ꢁC for
deprotection, a complicated mixture of products was
obtained, with the major product being compound 18
instead of the desired compound 17. No reaction oc-
curred when the temperature was lowered, which led
to our trying several other deprotecting reagents such
as 80% CH3COOH and 80% CF3COOH, but none
was able to selectively remove the isopropylidene group
without cleaving the newly formed 50-O,C-8-cyclo bond
in compound 16. Therefore, other protecting groups
N
N
N
N
NO2
Br2
NO2
NO2
N
N
N
N
N
N
N
N
Br
r.t.
CH3COCH3
N
N
HO
N
HO
HO
O
O O
10
O
O
70% HClO4
Dioxane/10%Na2HPO4
r.t.
o
94%
0-25 C
Br2- H2O
OH OH
4
O O
11
NaOAc buffer
Dioxane
10%Na2HPO4
Br2
30%
N
NO2
N
N
N
N
NO2
HO
HO
N
N
N
O
O
O
N
N
O O
13
O O
12
N
NO2
N
N
O
O
N
N
OHOH
8
Scheme 1.

Z. Zhu, J. K. Buolamwini / Bioorg. Med. Chem. 16 (2008) 3848–3865
3851
N
N
N
N
N
NO2
NO2
NO2
N
N
N
N
N
N
N
N
N
O
CH3COCH3
70% HClO4
Pb(OAc)4
N
HO
HO
O
O
O
Benzene,reflux
0-25oC
94%
O
O
OHOH
O O
NBS or NIS
HOAc, 25oC
4
10
12
N
NO2
N
N
N
O
O
N
OHOH
8
Scheme 2.
Cl
N
Cl
Cl
N
N
N
N
N
N
N
N
O
N
N
HO
N
O
O
O
HO
CH3COCH3
NIS, AcOH
50-60oC 36%
70% HClO4
0-25 C
O O
O O
o
94%
OHOH
14
16
15
0.5N H2SO4
55oC
Cl
N
N
N
N
HO
HO
?
+
O
0.5N H2SO4
55oC
OHOH
18
NO2
Cl
N
NO2
90-93
N
N
N
N
N
N
O
N
N
O
HN
O
O
OHOH
OHOH
17
6-9
Scheme 3.

3852
Z. Zhu, J. K. Buolamwini / Bioorg. Med. Chem. 16 (2008) 3848–3865
Cl
Cl
N
Cl
N
N
N
N
N
N
N
O
N
N
HO
N
O
O
N
HO
ZnCl2, 25oC
p-methoxy-
O
NIS, AcOH
25oC 43%
O
O
19
O
O
20
benzaldehyde
OH OH
81%
14
OMe
OMe
80% TFA
0oC
73%
NO2
Cl
N
NO2
N
N
N
N
N
N
O
O
HN
21-24
N
O
O
N
CaCO3,
reflux
EtOH
47-74%
OH OH
OH OH
17
6-9
Scheme 4.
that can be removed under milder conditions were
sought, and p-anisaldehyde was considered. The final
synthetic methods are shown in Scheme 4, whereby
the 20,30-hydroxyl groups of compound 14 were pro-
tected with p-anisaldehyde catalyzed by zinc chloride
to afford compound 19, which was then subjected to
intramolecular cyclization in the presence of NIS in
acetic acid at ambient temperature to obtain com-
pound 20. The 20,30-O-p-anisylidene group in com-
pound 20 was selectively removed in 80%
trifluoroacetic acid at 0 ꢁC to give the desired com-
pound 17 with the 50-O,8-cyclo bond intact. Coupling
of compound 17 with compounds 21–24 in the pres-
ence of calcium chloride in refluxing ethanol afforded
the target products compounds 6–9, respectively,
according to methods reported in our previous study.1
The compounds were then tested as ligands of the
ENT1 transporter by our reported flow cytometric
method.1,30,31
3. Pharmacophore mapping and 3D-QSAR studies
3.1. Materials and methods
3.1.1. Data sets. Three highly potent and selective ENT1
nucleoside transporter inhibitors, NBMPR, compounds
4 and 25 (see Fig. 3), were chosen as the active com-
pound set for generating common pharmacophore
hypotheses using the PHASE 1.0 program (Schro¨dinger
Inc. San Diego, CA). The training set used for develop-
ing 3D-QSAR models included 77 compounds and com-
prised a series of compounds with a high diversity in
structure and a wide range of ENT1 inhibitory potencies
as reported by Paul et al.28 a series of tetrahydroiso-
quinoline conformationally constrained ENT1 inhibi-
tors reported by us,1 and a series of 50-O,8-cyclo
conformationally constrained ENT1 inhibitors, reported
in this study (see Table 1). The test set included 39
compounds with biological data taken from the
HO
NO2
S
N
N
S
N
NO2
NO2
N
N
N
N
N
N
N
N
N
N
HO
HO
HO
O
O
O
OH OH
OHOH
OHOH
25
1, NBMPR
4
Figure 3. Active compounds used to generate common pharmacophore hypotheses in the PHASE program.

Z. Zhu, J. K. Buolamwini / Bioorg. Med. Chem. 16 (2008) 3848–3865
Table 1. Structures and inhibitory activities of compounds in the training set
3853
5
8
5
8
6
7
6
7
R
R
N
N
R1
N
N
N
N
N
R1
N
R4
X
N
N
N
N
N
N
N
N
R2
HO
HO
Y
O
O
O
O
z
R2
R3
OH OH
OH OH
OH OH
A
B
C
D
Compound
Type
R
R1
R2
H
R3
R4
X
Y
Z
pIC50
1, NBMPR
2
C
A
A
A
A
B
B
B
B
A
C
C
C
C
C
–S-(4-Nitrobenzyl)
8.07a
5.52a
6.74a
8.26a
5.44a
2.13a
3.61a
6.48a
2.76a
5.74a
7.67a
4.87b
4.97b
3.04b
4.44b
3.59b
6.51b
7.56b
4.49b
4.75b
5.68b
4.86b
5.77b
5.81b
5.20b
4.67b
4.24b
7.48b
5.32b
3.94b
3.64b
3.56b
5.87b
4.04b
5.20b
4.92b
6.34b
5.28b
5.54b
6.09b
3.92b
3.66b
4.28b
4.92b
4.20b
4.87b
3.79b
5.32b
5.94b
8.56b
4.09b
4.2b
5-NO2
6-NO2
7-NO2
8-NO2
5-NO2
6-NO2
7-NO2
8-NO2
H
3
4
5
6
7
8
9
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
–S-(4-Nitrobenzyl)
–NH-n-Amyl
–NH-Benzyl
NH2
H
H
NH-2-Ethoxyethyladenosine
–NH-Furfuryl
H
H
–NH-Isopropyl
–N(CH3)-(4-Nitrobenzyl)
–NH-(4-Nitrobenzyl)
–NH-Phenyl
H
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
D
C
C
C
C
C
C
C
C
D
D
D
C
C
C
C
C
C
C
C
D
H
H
H
–NH-2-Thenyl
–O-Benzyl
H
H
–S-Allyl
–S-Benzyl
H
H
–S-Cyclohexylmethyl
–S-Cyclohexyl
H
H
–S-a,a-Dimethylbenzyl
–S-Ethyl
H
H
–S-(2-Hydroxy-5-nitro-benzyl)
–S-(4-Isopropylbenzyl)
–S- [(2-Methyl-1-naphthyl)-methyl]
–S-Methyl
H
H
H
H
–S-Methyl
–S-Phenylpropyl
–S-Phenyl
H
Tetrahydropyran-2-yl
N
C
N
H
H
–S-2-Methylbenzyl
–S-3-Methylbenzyl
–S-4-Methylbenzyl
–S-Benzyl
H
H
H
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
–S-(3-Bromo-benzyl)
–S-(4-Bromo-benzyl)
–S-Isopropyl
Butyl
Butyl
Butyl
N
N
N
C
C
C
N
N
N
–S-Methyl
–S-(2-Pyridylmethyl)
–S-Butyl
–S-sec-Butyl
–S-(2-Chloro-benzyl)
–S-Ethyl
–S-(2-Fluoro-benzyl)
–S-(4-Fluoro-benzyl)
–S-(2-Hydroxy-5-nitrobenzyl)
–S-Iodo
–S-Propyl
Isobutyl
N
C
N
(continued on next page)

3854
Z. Zhu, J. K. Buolamwini / Bioorg. Med. Chem. 16 (2008) 3848–3865
Table 1 (continued)
Compound
Type
R
R1
R2
R3
R4
X
Y
Z
pIC50
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
C
D
C
D
C
C
C
C
C
C
D
C
C
C
C
C
D
D
D
D
D
D
D
D
D
–S-Isobutyl
–S-Isopropyl
–S-Isopropyl
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
H
5.06b
3.84b
3.72b
4.38b
3.24b
1.94b
3.69b
5.34b
6.75b
4.79b
4.04b
4.49b
4.46b
4.83b
3.35b
4.84b
3.63b
4.00b
3.64b
5.28b
3.94b
4.11b
3.47b
3.71b
3.41b
Propyl
N
N
C
C
N
N
–S-(2-Pyridylmethyl)
–S-(1-Methyl-4-nitroimidazol-5-yl)
–S-(6-Methyl-2-pyridylmethyl)
–S-Methyl
2-Methylbutyl
–S-2-Nitrobenzyl
–S-3-Nitrobenzyl
–S-Phenethyl
–S-(2-Pyridylmethyl)
–S-Propyl
Propyl
N
C
N
–S-(2-Pyridyl-methyl)
–S-(3-Pyridyl-methyl)
–S-(2-Acetophenone)
–S-(40-Chloro-2-acetophenone)
–NH-Isopentyl
–NH-Phenethyl
–NH2
H
H
H
I
C
C
C
C
C
C
C
C
C
N
N
C
C
C
C
C
C
C
N
N
N
N
N
N
N
N
N
H
H
H
–b-D-Ribofuranosyl
–b-D-Ribofuranosyl
–b-D-Ribofuranosyl
–b-D-Ribofuranosyl
–b-D-Ribofuranosyl
–b-D-Ribofuranosyl
–b-D-Ribofuranosyl
–S-Benzyl
–S-Methyl
H
H
Br
I
H
–Chloro
–Methoxy
H
H
H
H
H
–Piperidino
–SH
H
H
a Results taken from Zhu et al.1
b Results taken from Paul et al.28
literature.29–31 All the biological data were normalized to
the flow cytometrically determined data for comparison
using NBMPR as the standard since it was evaluated by
both methods.
charged group (N), positively charged group (P), and
aromatic ring (R). Three highly active compounds,
NBMPR, compounds 4 and 25, were selected for gener-
ating the pharmacophore hypotheses (see Fig. 3). Com-
mon pharmacophore hypotheses were identified using
conformational analysis and a tree-based partitioning
technique. The resulting pharmacophores were then
scored and ranked. Pharmacophores with high-ranking
scores were validated by a partial least square (PLS)
regression-based PHASE 3D-QSAR cross-validation,
and the best pharmacophore hypothesis identified was
further validated by CoMFA 3D-QSAR modeling. All
the molecules used for QSAR studies were aligned to
the pharmacophore hypothesis obtained in PHASE
(see Fig. 5).
3.1.2. Molecular modeling. Three-dimensional structure
building, pharmacophore mapping and CoMFA 3D-
QSAR studies were carried out on a Silicon Graphics
Octane (R12000) workstation with the IRIX 6.5 operat-
ing system running the SYBYL program package, ver-
sion 7.2 (Tripos Associates, St. Louis, MO) and the
PHASE 1.0 program (Schro¨dinger Inc., San Diego,
CA). Molecular energy minimizations were performed
using the Tripos force field with a distance-dependent
dielectric constant and the Powell conjugate gradient
algorithm with an energy change convergence criterion
˚
of 0.001 kcal/mol A. Partial atomic charges were calcu-
3.1.4. Development of a CoMFA 3D-QSAR model. The
PHASE-generated 3D pharmacophore was used as the
alignment template for the CoMFA 3D-QSAR model.
CoMFA descriptors were calculated using a sp3 hybrid-
ized carbon probe atom with a van der Waals radius of
lated using the Gaisteiger–Huckel program in SYBYL.
All the molecules in the present study were aligned to
the best-generated pharmacophore hypothesis (Phar-
m_A) obtained from the PHASE pharmacophore map-
ping exercise.
˚
˚
1.52 A and a charge of +1.0 on a 2 or 1 A spaced 3D cu-
˚
bic lattice with an extension of 4 A units in all directions
3.1.3. Generation of pharmacophore models. PHASE 1.0
implemented in the Maestro 7.0 modeling package
(Schro¨dinger Inc., San Diego, CA) was used to generate
pharmacophore models for ENT1 inhibitors. The 3D
structures of all the molecules used in PHASE were built
in, and imported from SYBYL. Conformers of each
molecule were generated using the MMFF forcefield in
the PHASE program. Pharmacophore feature sites for
the molecules were assigned using a set of features de-
fined in PHASE as hydrogen-bond acceptor (A), hydro-
gen-bond donor (D), hydrophobic group (H), negatively
to encompass the aligned molecules. Steric (Lennard–
Jones 6–12 potential) field energies and electrostatic
(Coulombic potential) fields at each lattice point were
generated and scaled by the CoMFA-STD method in
SYBYL. The SYBYL default energy cut-off of 30 kcal/
mol was used to ensure that there would be no extreme
energy terms to distort the CoMFA models.
In deriving CoMFA 3D-QSAR models PLS regression
analysis was used to correlate the CoMFA descriptors
with biological activities. The CoMFA descriptors were

Z. Zhu, J. K. Buolamwini / Bioorg. Med. Chem. 16 (2008) 3848–3865
3855
used as independent variables, and biological activities
Compd 8
Compd 9
Compd 6
Compd 7
1, NBMPR
(pIC50 values) were used as dependent variables. The
predictive value of the models was evaluated first by
leave-one-out (LOO) cross-validation. The cross-vali-
dated coefficient, q2, was calculated using Eq. 1 as
follows:
110
100
90
80
70
60
50
40
30
20
10
0
P
2
ðY predicated ꢀ Y observed
Þ
q2 ¼ 1 ꢀ
ð1Þ
P
2
ðY observed ꢀ Y mean
Þ
where Ypredicted, Yobserved, and Ymean are the predicted,
observed, and mean values of the target property
(pIC50), respectively. (Ypredicted ꢀ Yobserved)2 is the pre-
dictive error sum of squares (PRESS). The number of
components corresponding to the lowest PRESS value
was selected for deriving the final PLS regression models
to minimize the tendency to overfit the data. In addition
to the q2, the number of components, the conventional
correlation coefficient r2 and its standard errors were
also computed. CoMFA coefficient maps were gener-
ated by interpolation of the pair-wise products between
the PLS coefficients and the standard deviations of the
corresponding CoMFA descriptor values. Robustness
of the CoMFA models was checked by group cross-val-
idation and randomization of activity values (Table 8).
-10.2 -9.2
-8.2
-7.2
-6.2
-5.2
-4.2
LogC (M)
Figure 4. Equilibrium displacement of SAENTA-fluorescein by com-
pounds 6–9, the 50-O,8-cyclo highly conformationally constrained
analogues of NBMPR, in K562 cells.
19 nM. The differences emphasize the remarkable regi-
oselectivity of ENT1 among these tetrahydroisoquino-
line NBMPR analogues with respect to the NO2
substituent.28,34 Compared to their less constrained
counterpart compounds 2–5, the binding affinities of
compounds 6–9 were significantly lower (see Table 4,
Ki values increased from the nanomolar range to the
micromolar range except for compound 8). These results
are interesting since compounds 6–9 were obtained by
just forming0a 50-O, 8-linkage to block the free rotation
of the N9–C1 glycosidic bond in compounds 2–5, respec-
tively. The significant loss in binding affinities in going
from compounds 2–5 to compounds 6–9 indicates that
not only is the NBMPR binding site regioselective with
respect to the nitro substituent, but it is also very sensi-
tive to the orientation of the purine ring about the gly-
cosidic linkage. The 50-O,8-cyclo analogues appear not
to present a favorable conformation for binding to
ENT1, supposing other factors are not in play. How-
ever, the fact that compound 8 still exhibited good bind-
ing affinity at the transporter implies that the
conformation of compound 8 does not deviate too far
from the binding mode of NBMPR. These results shed
4. Results and discussion
4.1. ENT1 inhibitory activity of novel 50-O, 8-linkage
constrained nitrotetrahydroisoquinoline NBMPR
analogues
The solid-state conformation of NBMPR has been deter-
mined by X-ray diffraction,32 and a solution conforma-
tion has been proposed using NMR.33 The X-ray
structure reveals the preponderance of a syn orientation
of the purine ring relative to the sugar moiety, whereas
the NMR structure reveals a high-anti orientation. How-
ever, the orientation about the glycosidic bond in the bio-
active conformation remains unknown. The objective of
this study was to probe the bioactive orientation of the
purine ring relative to the sugar moiety about the glyco-
sidic linkage in NBMPR and its analogues when bound
to the ENT1 nucleoside transporter.
The new constrained analogues of NBMPR, compounds
6–9, in which the 50-O, 8-linkage blocks the rotation
0
0
about both the glycosidic bond and C4 –C5 bond, were
synthesized as detailed in the chemistry section and eval-
uated as ENT1 transporter ligands by a flow cytometric
binding assay using the K562 chronic myelogenous leu-
kemia cell line as previously described.1 The concentra-
tion-dependent inhibitory curves are depicted in Figure
4 and Ki values are presented in Table 3. These ana-
logues exhibited a broad range of binding affinities at
the ENT1 nucleoside transporter. Three of them, com-
pounds 6, 7, and 9 exhibited low binding affinities, with
Ki values in the micromolar range, whereas compound
8, the cyclonucleoside analogue corresponding to com-
pound 4, the most tightly bound compound of the pre-
vious tetrahydrisquinoline series,1 bound tightly to the
transporter with a nanomolar Ki value of approximately
more light on the bioactive conformation of NBMPR.
On the other hand, it might be that moving C5 and
0
0
O5 up to join with C8 creates unfavorable interactions
at the transporter that were absent in the non-cyclo
0
compounds 2–5, where the O5 is in a free hydroxyl
group (50-OH) which could participate in both hydro-
gen-bond donor and acceptor interactions. Previous
studies, however, suggest that hydrogen-bond interac-
tions do not appear to be critical at the 50-position,29,35
and thus it might be the loss of flexibility (lower entropy)
imposed by cyclization that accounts for the low
activity.
We continued to carry out pharmacophore mapping
and 3D-QSAR studies to explore the possible bioactive

3856
Z. Zhu, J. K. Buolamwini / Bioorg. Med. Chem. 16 (2008) 3848–3865
Table 2. Structures and inhibitory activities of compounds in the test set
2 3
R
S
4
NHR1
N
6
5
N
N
N
N
N
N
R5
N
HO
O
O
R2
R4 R3
OH OH
A
B
Compound
Type
R
R1
R2
R3
R4
R5
pIC50
94
95
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
2,4,6-Trimethyl
2,4-Dichloro
2-Br
4.48b
7.11b
5.62a
5.82b
5.75a
6.45a
6.55a
6.65b
6.44a
6.48b
6.55a
6.70a
6.61a
8.32b
6.75a
7.09b
6.09b
6.60a
6.80a
7.16a
6.91b
6.21b
3.48b
7.50c
6.37c
6.46c
7.80c
4.33c
5.89c
7.06c
6.01c
5.93c
6.15c
3.94c
6.74c
6.23c
5.38c
6.97c
7.66c
96
97
2-Cl-6-F
2-Cl
2-F
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
2-I
3,4-Dichloro
3-Br
3-CF3
3-Cl
3-F
3-I
3-NO2
4-Br
4-CN
4-COOCH3
4-Cl
4-F
4-I
4-OCF3
4-OCH3
4-tert-Butyl
4-Nitrobenzyl
OH
H
OH
OH
OH
OH
OH
OH
OH
H
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
Cl
2-CH3-3-NO2 Benzyl
2-Cl-4-NO2 Benzyl
4-NO2 Benzyl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
OH
H
2-NO2-4-Cl Benzyl
2-NO2-5-CH3 Benzyl
3-CH3-4-NO2 Benzyl
4-NO2 Benzyl
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
3-NO2-4-CH3 Benzyl
3-NO2-4-Cl Benzyl
3-OCH3 Benzyl
OH
OH
OH
OH
OH
OH
OH
OH
4-Acetamido benzyl
4-Cl Acetamido benzyl
4-NH2 Benzyl
4-NO2 Benzyl
4-NO2 Benzyl
H
a Results taken from the literature.30
b Results taken from the literature.31
c Results taken from the literature.29
conformation(s). Pharmacophore modeling is used to
propose the 3D spatial arrangement of chemical features
that are essential for biological activity of molecules
with respect to a biological target. Three-dimensional
quantitative structure–activity relationship (3D QSAR)
techniques such as comparative molecular field analysis
(CoMFA),36 have been successfully applied in many
aspects regarding drug design and discovery.37 The
CoMFA technique uses both statistical techniques and
molecular graphics to determine correlations between
structural properties of molecules and their biological
activity. The bioactive conformation of each molecule
is chosen and superimposed in a manner supposed to
be the interacting mode with the target receptor. The

Z. Zhu, J. K. Buolamwini / Bioorg. Med. Chem. 16 (2008) 3848–3865
3857
Table 3. Flow cytometrically determined Ki values of 50-O,8-cyclo
compounds
the number of potential pharmacophore hypothesis to
be evaluated. This is a significant advantage over using
only flexible NBMPR analogues. The top five ranking
pharmacophore hypotheses generated were validated
by PHASE 3D-QSAR analysis involving a test set vali-
dation. The resulting best pharmacophore model from
that analysis was further validated by CoMFA 3D-
QSAR modeling.
50-O,8-Cyclo series
Ki (nM)
6
>1000
>1000
18.89
>1000
0.70
7
8
9
NBMPRa
a NBMPR was added as a standard.
For CoMFA 3D-QSAR modeling, an alignment rule for
superposition of the 3D structures of the molecules in a
‘bioactive’ conformation is required. In the absence of a
crystal structure of the target or a complex of target and
ligands, a pharmacophore-based alignment rule is the
accepted norm. Thus we employed the PHASE pharma-
cophore hypothesis generation and validation to obtain
an alignment template for the compounds. All the fea-
tures defined in PHASE: hydrogen-bond acceptor (A),
hydrogen-bond donor (D), hydrophobic group (H), neg-
atively charged group (N), positively charged group (P),
and an aromatic ring (A) were considered for the phar-
macophore generation. All the novel conformationally
constrained analogues of NBMPR synthesized in our
previous study1 and this study were included in the
training set. All the 77 compounds shown in Table 1
were used to develop the PHASE 3D-QSAR models.
All the 39 compounds in Table 2 were used as an exter-
nal test set for PHASE 3D-QSAR model validations for
predictive ability. The top five ranking pharmacophore
hypotheses and their feature compositions are shown
in Table 5. The PLS results of the five PHASE 3D-
QSAR models developed from them and the corre-
sponding prediction results of test set data are listed in
Table 6.
Table 4. Comparison of the Ki values of the two series of conform-
ationally constrained analogues of NBMPRa
Two-way arrows are showing corresponding Kis.
steric and electrostatic fields around the molecules are
then correlated with biological activity. The potent
ENT1 inhibitors, NBMPR, compound 4, and com-
pound 25 (see Fig. 3) were used for pharmacophore gen-
eration with the PHASE program (Schro¨dinger). The
inclusion of compound 4, which is a very potent con-
strained analogue of NBMPR, markedly reduced the
conformational space that the program had to sample,
thereby cutting down on analysis time and reducing
All the top five pharmacophore models comprised six
features (see Table 5). Pharm_A, Pharm_B, and
Pharm_D consisted of three hydrogen-bond acceptors,
one hydrophobic group, and two aromatic rings.
Pharm_C and Pharm_E are composed of two
Table 5. The top five ranking pharmacophore hypotheses generated using PHASEa
Pharmacophore
Pharm_A
Pharm_B
Pharm_C
Pharm_D
Pharm_E
Ranking
Features
Conformation of aligned molecules
1
AAAHRR
anti
2
AAAHRR
anti
3
AADHRR
anti
4
AAAHRRR
anti
5
AADHRR
anti
a The features defined in PHASE include: hydrogen-bond acceptor (A), hydrogen-bond donor (D), hydrophobic group (H), negatively charged group
(N), positively charged group (P), and aromatic ring (R).
Table 6. PLS statistics of PHASE 3D-QSAR models and prediction of test set
Pharmacophore
Pharm_A
Pharm_B
Pharm_C
Pharm_D
Pharm_E
PLS statistics of QSAR model
r2
0.916
0.405
0.886
0.472
137.93
1.081eꢀ32
4
0.901
0.437
0.923
0.388
0.929
0.372
SD
F
193.956
2.034eꢀ37
4
164.534
2.022eꢀ35
4
213.297
9.200eꢀ39
4
233.308
4.852eꢀ40
4
P
PLS components
Results of test set
Predictive r2 for the external test set
0.777
ꢀ0.006
0.125
ꢀ0.184
ꢀ0.742

3858
Z. Zhu, J. K. Buolamwini / Bioorg. Med. Chem. 16 (2008) 3848–3865
Figure 5. The best-generated pharmacophore model (Pharm_A) obtained from PHASE. Pharmacophore features are red vectors for hydrogen-bond
acceptors (A), orange rings for aromatic groups (R), and green balls for hydrophobic functions (H). NBMPR, compounds 4 and 25 were aligned to
the pharmacophore. For the molecules, blue indicates nitrogen, red indicates oxygen, yellow refers to sulfur, gray indicates carbon, and white
indicates hydrogen.
hydrogen-bond acceptors, one hydrogen-bond donor,
one hydrophobic group, and two aromatic rings. All
the five top pharmacophores considered had an anti-
conformation. These pharmacophore hypotheses affor-
ded PHASE 3D-QSAR models with good PLS statistics
results (especially with regard to conventional r2). How-
ever, only one of them, Pharm_A, performed excellently
on prediction of the external test set (see Table 6). The
Pharm_A pharmacophore produced a PHASE 3D-
QSAR model with r2 of 0.916, four (4) PLS components,
and an excellent external test set predictive r2 of 0.777.
The large value of F indicates a statistically significant
regression model, which is supported by the small value
of the variance ratio (P), an indication of a high degree
of confidence (PHASE user manual). This pharmaco-
phore was chosen for further QSAR analysis. Figure 5
Figure 6. Pharmacophore feature mapping onto compounds 4 and 8 and their conformational comparison. Pharmacophore features are red vectors
for hydrogen-bond acceptors (A), orange rings for aromatic groups (R), and green balls for hydrophobic functions (H). NBMPR, compounds 4 and
25 were aligned to the pharmacophore. Molecules are blue for nitrogen, red for oxygen, yellow for sulfur, gray for carbon, and white for hydrogen.

Z. Zhu, J. K. Buolamwini / Bioorg. Med. Chem. 16 (2008) 3848–3865
3859
shows Pharm_A mapped onto the structures of
NBMPR, compounds 4 and 25. The three hydrogen-
bond acceptor features mapped onto the 30-OH, the ri-
bose ring oxygen, and the nitro group. The 30-OH has
been identified as an important structural feature for
interaction with nucleoside transporters.18,38–40 The
hydrophobic function is mapped onto the N6-benzyl
ring, and the two aromatic groups are mapped onto
the purine system. The importance of hydrophobicity
of the purine 6-position substituent was highlighted in
the study of Paul et al.28
onto all the chemical features encoded in the pharmaco-
phore, which is consistent with the results obtained from
the flow cytometric assay showing that compound 8 was
able to bind well to the ENT1 transporter with a Ki val-
ues in the nanomolar range (Ki = 18.89 nM, see Table
3). Figure 7 shows that compounds 6, 7, and 9 are only
partly mapped onto the pharmacophore with two or
more encoded features being unmatched, which is con-
sistent with the biological results as well (Ki values of
compounds 6, 7, and 9 are all in the micromolar range,
see Table 3). These findings suggest that the anti-confor-
mation might be the optimal conformation for NBMPR
and its analogues in binding to ENT1. Other factors
may also contribute to or be responsible for the low
Compound 4 adopts an anti-conformation, whereas
compound 8 adopts a high-anti-conformation due to
the restriction imposed by the 50-O, 8-linkage (see
Fig. 6). However, such a conformational shift is not
large enough to prohibit compound 8 from mapping
activity of these compounds such as unfavorable inter-
actions of the region around the C8–O5 cyclic linkage.
The reduced flexibility (lower entropy), possible atom
0
Figure 7. Pharmacophore feature mapping onto compounds 6, 7, and 9. Pharmacophore features are red vectors for hydrogen-bond acceptors (A),
orange rings for aromatic groups (R), and green balls for hydrophobic functions (H). NBMPR, compounds 4 and 25 were aligned to the
pharmacophore. For the molecules, blue indicates nitrogen, red indicates oxygen, yellow refers to sulfur, gray indicates carbon, and white indicates
hydrogen.
Figure 8. Superimposition of training set (A) and test set (B) aligned to the pharmacophore model, Pharm_A, imported from PHASE.

3860
Z. Zhu, J. K. Buolamwini / Bioorg. Med. Chem. 16 (2008) 3848–3865
Table 9. Residuals of the predictions of the test set by the CoMFA
3D-QSAR model
bumping (unfavorable van der Waals), charge and/or
hydrophobic/hydrophilic mismatch could also be the
cause of the observed low binding affinity.
Compound
Actual pIC50
Residual
94
95
4.48
7.11
5.62
5.82
5.75
6.45
6.55
6.65
6.44
6.48
6.55
6.70
6.61
8.32
6.75
7.09
6.09
6.60
6.80
7.16
6.91
6.21
3.48
7.50
6.37
6.46
7.80
4.33
5.89
7.06
6.01
5.93
6.15
3.94
6.74
6.23
5.38
6.97
7.66
ꢀ0.9
0.67
0.02
To further validate the Pharm_A pharmacophore
hypothesis, a Pharm_A-dependent alignment of the
training and test set compounds was exported to the
SYBYL program and used for CoMFA 3D-QSAR
modeling. The high sensitivity of CoMFA to molecular
alignment led to elimination of compounds as outliers.
All the conformationally constrained analogues of
NBMPR that we have synthesized, compounds 2–5,1
compound 26, and compounds 6–9 were accommodated
in the training set (see Fig. 8) for the alignments of train-
ing set and test set compounds used for the CoMFA 3D-
QSAR derivation. The CoMFA model was developed
with 57 compounds in the training set and afforded an
r2 of 0.894 and q2 value of 0.591 (PLS statistics results
of CoMFA model are shown in Table 7). To make sure
96
97
0.14
0.01
98
99
0.75
0.77
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
ꢀ0.34
0.22
0.06
ꢀ0.01
0.36
0.53
0.29
0.36
ꢀ0.17
ꢀ0.77
0.03
0.3
0.91
Table 7. PLS statistics of CoMFA 3D-QSAR model
0.93
0.25
PLS statistics
CoMFA
q2
0.591
0.894
0.472
188.072
5
ꢀ1.41
0.04
0.68
r2
s
ꢀ0.29
ꢀ0.12
ꢀ0.51
0.97
F
PLS components
Contribution
Steric
0.373
0.627
0.04
0.13
Electrostatic
0.17
ꢀ0.38
ꢀ0.25
0.89
Table 8. Results of group cross-validation and randomization exercise
for the CoMFA 3D-QSAR model
0.02
0.49
Exercise
PLS statistics
CoMFA
Group validation (20 runs)
Randomization (20 runs)
Average q2 (SD)
Rand q2
0.572 (0.032)
0.0005
0.12
0.27
9
9
8
7
6
5
4
A
B
8
7
6
5
4
3
2
1
3
3
4
5
6
7
8
9
1
2
3
4
5
6
7
8
9
Actual pIC
Actual pIC
50
50
Figure 9. Curves for training set (A) and test set (B) activity predictions by the CoMFA 3D-QSAR model.

Z. Zhu, J. K. Buolamwini / Bioorg. Med. Chem. 16 (2008) 3848–3865
3861
that these results were not obtained by chance, PLS runs
with scrambled (randomized) pIC50 values and group
cross-validations were performed (see Table 8). In the
randomization control exercise, the majority of the anal-
yses afforded very poor q2 values, indicating that the
CoMFA model generated with the actual data did not
arise fortuitously. The deviations in the q2 values result-
ing from the group cross-validations were minimal, sug-
gesting the model is robust. The model was further
validated by using it to predict the ENT1 inhibitory
activities of an external test set of 39 compounds, and
it showed a strong predictive ability, with a predictive
r2 of 0.73 (see Fig. 9). Residuals of the predictions of
the test set by the CoMFA 3D-QSAR model are shown
in Table 9. The PLS coefficient contour maps depict re-
gions around the molecules in 3D space where changes
in physicochemical features increase or decrease potency
as predicted (see Fig. 10). The green colored areas de-
note regions that prefer sterically bulky groups in the
CoMFA model, whereas yellow colored regions are ste-
rically restricted. The blue contours indicate regions that
favor electropositive substituents, whereas red contours
mark regions favoring electronegative groups. As shown
in Figure 10, a red region near the nitro group of
NBMPR shows that an electronegative substituent at
this site increases potency. This is consistent with the
pharmacophore hypothesis (Pharm_A) in which the ni-
tro group at this position is an hydrogen-bonding accep-
tor, and SAR studies that show high potencies for
NBMPR, 27, and 34 compared to their respective coun-
terparts without nitro substitution, compounds 39, 54,
and 29 (see Table 1). The green contour near the para
position of the 6-benzyl ring indicates that generally a
sterically bulky substituent is favored at this site; how-
ever, a yellow region is on the other side of the 6-benzyl
group indicating that there is a limit as to the degree of
bulkiness of the substituent at this region. The yellow
contour near the ortho position of 6-benzyl group shows
that the steric bulk around this region is disfavored. The
blue contour near the purine ring shows that an electron
deficient system is favored at this site. The pharmaco-
phore map (Figs. 5 and 6) and CoMFA contour map
(Fig. 10) complement each other with the former
appearing to capture more specific features than the lat-
ter. The results obtained from the CoMFA study further
validated the Pharm_A hypothesis, suggesting that the
anti-conformation might be a possible bioactive confor-
mation of NBMPR and its analogues.
5. Conclusion
In our continuing efforts to probe the bioactive confor-
mation of NBMPR and its analogues at the ENT1
transporter, we carried out the synthesis and flow cyto-
metric evaluation of new, more conformationally con-
strained analogues, combined them with our
previously synthesized constrained analogues, as well
as a diverse literature reported NBMPR congeners to
derive a 3D pharmacophore model of a bioactive con-
formation. CoMFA 3D-QSAR modeling was utilized
in validation of this pharmacophore, which consists of
three hydrogen-bond acceptors, one hydrophobic
group, and two aromatic rings, generated using the
PHASE program. The use of a substantial number of
compounds in both the training and the external test sets
Figure 10. PLS coefficient SD steric and electrostatic contour maps of CoMFA 3D-QSAR model. Red, negative electrostatic potential enhances
*
potency; blue, positive electrostatic potential favors activity; green, bulky substituents enhance potency; yellow, bulky substituents are unfavorable to
activity.

3862
Z. Zhu, J. K. Buolamwini / Bioorg. Med. Chem. 16 (2008) 3848–3865
in the validation process, leads us to believe that the
anti-conformation indicated by this pharmacophore
model, is a possible bioactive conformation of NBMPR
at the ENT1 nucleoside transporter. This remains to be
tested by experimental structural biology studies. The
established CoMFA 3D-QSAR model of high predictive
ability and robustness should be useful for the design
and optimization of new ENT1 inhibitors.
(426.39): C, 53.52%; H, 4.26%; N, 19.71%. Found: C,
53.18%; H, 4.24%; N, 19.31%.
6.2.1.2. Compound 7. Yield 74%; mp 179–180 ꢁC; MS
1
m/z 427 (M++H); H NMR (300 MHz, (CD3)2SO), d
8.29 (1H, s, H-2), 8.09 (1H, s, H-500), 8.05 (1H, d,
J = 4.4 Hz, H-700), 7.56 (1H, d, J = 8.1 Hz, H-800), 6.05
(1H, s, H-10), 5.64 (1H, br s, OH-20, disappears upon
D2O exchange), 5.33 (3H, br s, H-100, OH-30, simplifies
on D2O exchange), 4.63 (1H, m, H-20), 4.58 (1H, br
m, H-50A), 4.45 (1H, m, H-30), 4.36 (2H, br s, H-300),
4.25 (1H, d, J = 5.1, H-50B), 4.13 (1H, d, J = 12.6 Hz,
H-40), 3.06 (2H, t, J = 5.4 Hz, H-400), HPLC 20.8 min
(97%).
6. Experimental
6.1. Chemistry
Thin-layer chromatography (TLC) was conducted on
silica gel F254 plates (Analtech). Compounds were visu-
alized by UV light or 5% H2SO4 in EtOH spraying re-
6.2.1.3. Compound 8. Yield 55%; mp 188–190 ꢁC; MS
1
m/z 449 (M++Na); H NMR (300 MHz, (CD3)2SO), d
1
agent. H, 13C spectra were recorded on Bruker ARX
8.31 (1H, s, H-2), 8.20 (1H, s, H-800), 8.07 (1H, d,
J = 7.5 Hz, H-600), 7.49 (1H, t, J = 7.8 Hz, H-500), 6.07
(1H, s, H-10), 5.65 (1H, d, J = 6.6, OH-20, disappears upon
D2O exchange), 5.38 (1H, d, J = 5.4 Hz, OH-30, simplifies
upon D2O exchange), 5.33 (2H, br s, H-100), 4.66 (1H, d,
J = 2.1, H-20), 4.59 (1H, br d, H-50A), 4.47 (1H, t,
J = 5.4 Hz, H-30), 4.43 (2H, br s, H-300), 4.25 (1H, t,
J = 6.6, H-50B), 4.13 (1H, d, J = 12.6 Hz, H-40), 3.05
(2H, t, J = 5.7 Hz, H-400), HPLC 23.9 min (95%).
(300 MHz) instruments, using CDCl3, CD3OD,
(CD3)2SO or CD3COCD3 as solvents and tetramethyl-
silane (TMS) as internal standard. Column chromatog-
raphy was performed on Fisher silica gel (170–400
mesh). Melting points were determined using a Fisher–
Johns Melting Point Apparatus and are reported uncor-
rected. Mass spectra were obtained on a Bruker-HP Es-
quire-LC mass spectrometer, and IR spectra in KBr
with a Perkin-Elmer (System 2000 FT-IR) spectrometer.
All solvents and reagents were bought from Aldrich and
used without further purification except drying when
necessary. Purity of compounds 7, 8, and 9 was checked
6.2.1.4. Compound 9. Yield 47%; mp 171–172 ꢁC; MS
1
m/z 449 (M++Na); H NMR (300 MHz, (CD3)2SO), d
8.28 (1H, s, H-2), 7.94 (1H, d, J = 8.1 Hz, H-700), 7.58
(1H, d, J = 6.9 Hz, H-500), 7.46 (1H, t, J = 7.8 Hz, H-
600), 6.03 (1H, s, H-10), 5.62 (1H, d, J = 6.6, OH-20, dis-
appears on D2O exchange), 5.55 (2H, br s H-100), 5.36
(1H, d, J = 5.4 Hz, OH-30, disappears upon D2O ex-
change), 4.63 (1H, d, J = 2.4, H-20), 4.58 (1H, br d, H-
50A), 4.44 (1H, t, J = 5.7 Hz, H-30), 4.37 (2H, br s, H-
300); 4.22 (1H, t, J = 6.3, H-50B), 4.12 (1H, d,
J = 12.9 Hz, H-40), 3.04 (2H, t, J = 5.7 Hz, H-400), HPLC
21.1 min (99%).
with
a
Hewlett–Packard 1100 HPLC apparatus
equipped with a Platinum EPS 100A 5 lm C18 analyti-
cal column (150 · 4.6 mm, Phenomenex, Torrance, CA)
in a linear gradient solvent system, H2O/CH3CN from
100/0 to 20/80 in 30 min; the flow rate was 1 ml/min.
Peaks were detected by UV absorption with a diode ar-
ray detector.
6.2. General method for the preparation of compounds 6–9
A mixture of 50-O,8-cyclo-6-chloropurine riboside (17,
100 mg, 0.35 mmol), Mono-(5, 6, 7, or 8)-NO2-1,2,3,4-
tetrahydroisoquinoline (21, 22, 23, or 24, 157 mg,
0.88 mmol), and calcium carbonate (70 mg, 0.7 mmol)
in ethanol (5 ml) was stirred under reflux for 15 h. The
reaction mixture was filtered, and the filtrate was evap-
orated in vacuo at 40 ꢁC. The residue was purified by
flash silica gel chromatography followed by recrystalli-
zation from methanol.
6.3. 6-(700-NO2-1,2,3,4-Tetrahydroisoquino-2-yl)-20,30-O-
p-isopropylidene purine riboside (10)
To a suspension of 6-(700-NO2-1,2,3,4-tetrahydroisoqui-
no-2-yl) purine riboside (4, 1.11 g, 2.6 mmol) in 30 ml
of acetone was added 0.4 ml of 70% HClO4 at 0 ꢁC.
The reaction mixture was stirred overnight at room tem-
perature, after which the reaction mixture was neutral-
ized with NH3ÆH2O and evaporated to dryness under
aspirator pressure at 40 ꢁC. The crude product was
recrystallized from ethanol to give 1.14 g of 6-(700-NO2-
1,2,3,4-tetrahydroisoquino-2-yl)-20,30-O-p-isopropylidene
purine riboside (10) (94% yield). Mp 88–90 ꢁC; MS m/z
6.2.1. 6-{[Mono-(500,600,700, or 800)-NO2-]-1,2,3,4-tetrahydro-
isoquino-2-yl}-50-O, 8-cyclo-purine riboside (6–9)
6.2.1.1. Compound 6. Yield 74%; mp 250–252 ꢁC; MS
1
1
m/z 427 (M++H); H NMR (300 MHz, (CD3)2SO), d
469 (M++H), H NMR (300 MHz, CDCl3), d 8.36 (1H,
8.29 (1H, s, H-2), 7.87 (1H, d, J = 8.1 Hz, H-600),7.67
(1H, d, J = 7.5 Hz, H-800), 7.48 (1H, t, J = 7.8 Hz, H-
700), 6.04 (1H, s, H-10), 5.63 (1H, d, J = 6.6, OH-20, dis-
appears on D2O exchange), 5.36 (1H, d, J = 5.4 Hz, OH-
30, disappears on D2O exchange), 5.32 (2H, br s, H-100),
4.63 (1H, d, J = 2.1, H-20), 4.58 (1H, br d, H-50A), 4.45
(1H, t, J = 5.4 Hz, H-30), 4.36 (2H, br s, H-300), 4.25 (1H,
t, J = 6.6, H-50B), 4.13 (1H, d, J = 12.6 Hz, H-40), 3.10
(2H, t, J = 5.7 Hz, H-400). Anal. Calcd for C19H18N6O6
s, H-2), 8.16 (1H, s, H-800), 8.08 (1H, d, J = 7.8 Hz, H-
600), 7.35 (1H, t, J = 7.8 Hz, H-500), 6.70 (1H, d,
J = 6.0 Hz, OH-50, disappeared upon D2O exchange),
5.85 (1H, d, J = 5.4 H-10), 5.52 (2H, br s, H-100), 5.25
(1H, t, J = 5.4, H-20), 5.14 (1H, t, J = 5.4 Hz, H-30),
4.58 (3H, br d, H-300, H-50A), 4.0 (1H, d, J = 12.6 Hz,
H-40), 3.80 (1H, t, J = 6.6, H-50B), 3.12 (2H, t,
J = 5.7 Hz, H-400), 1.66 (3H, s, CH3 of isopropylidene
group), 1.38 (3H, s, CH3 of isopropylidene group).

Z. Zhu, J. K. Buolamwini / Bioorg. Med. Chem. 16 (2008) 3848–3865
3863
6.4. 6-(700-NO2-1,2,3,4-Tetrahydroisoquino-2-yl)-20,30-O-
p-isopropylidene-8-OH purine riboside (13)
(1H, s, H-10), 5.08 (1H, d, J = 5.4, H-40), 4.70 (2H, m,
H-50), 4.52 (1H, d, J = 6.0 Hz, H-30), 4.24 (1H, d,
J = 6.0 Hz, H-20), 2.12 (3H, s, CH3 of isopropylidene
group), 1.32 (3H, s, CH3 of isopropylidene group).
6-(700-NO2-1,2,3,4-Tetrahydroisoquino-2-yl)-20,30-O-p-iso-
propylidene purine riboside (10, 500 mg, 1.07 mmol)
was dissolved in a mixture of dioxane and 10% disodium
hydrogen phosphate buffer (32 ml, 1:1, v/v), then bro-
mine (204.7 mg, 1.28 mmol) was added while stirring.
The reaction mixture was stirred for 20 h at ambient
temperature, followed by the addition of 2 N NaHSO3
to reduce the excess bromine. The solution was extracted
with CH2Cl2. The combined organic layer was washed
with 2 N NaHSO3, dried over Na2SO4, and evaporated
to give sticky residue, which was purified on preparative
TLC plates using MeOH/EtOAc mixture (1:20 v/v) as
the solvent system to afford 13 (156 mg, 30% yield).
Mp 228–230 ꢁC; MS m/z 484 (M++H); 1H NMR
(300 MHz, CDCl3), d 8.98 (1H, s, H-2), 8.58 (2H, br s,
H-800, OH-8, simplifies on D2O exchange), 8.38 (1H, d,
J = 7.8 Hz, H-600), 7.54 (1H, t, J = 7.8 Hz, H-500), 6.32
(1H, s, H-10), 5.49 (1H, d, J = 5.4, H-20), 5.12 (1H, m,
H-30), 4.45 (4H, m, H-100, 300), 3.65 (1H, t, J = 6.6, H-
50A), 3.45 (1H, m, H-40), 3.30 (3H, m, H-400, H-50B),
1.40 (3H, s, CH3 of isopropylidene group), 1.25 (3H, s,
CH3 of isopropylidene group).
6.7. 20,30-O-Isopropylidene-8-hydroxy-6-chloropurine
riboside (18)
50-O,8-cyclo-20,30-O-Isopropylidene-6-chloropurine ribo-
side (16, 100 mg, 0.31 mmol) was suspended in 0.5 N
H2SO4 (4 ml) and stirred at 55 ꢁC for 15 h. The reaction
mixture was neutralized with NH3ÆH2O and evaporated
to dryness in vacuo. The residue was subjected to chroma-
tography on preparative TLC plates using a mixture of
MeOH/EtOAC (1:20) as the solvent system. The purified
product was characterized as 8-hydroxy-20,30-O-isopropyl-
idene-6-chloropurine riboside (18) (23.3 mg, 25% yield).
MS (ESI) m/z 325 (M++Na); 1H NMR (300 MHz,
(CD3)2SO), d 12.31 (1H, br s, OH-8), 8.48 (1H, s, H-2),
5.73 (1H, d, J = 6.4 Hz, H-10), 5.31 (1H, d, J = 6.6, OH-
20), 5.12 (1H, d, J = 5.7 Hz, OH-30), 4.89 (1H, q,
J = 10.8 Hz, H-20), 4.76 (1H, t, J = 6.6 Hz, OH-50), 4.21
(1H, t, J = 6.0 Hz, H-30), 3.86 (1H, t, J = 6.0 Hz, H-40),
3.62 (1H, m, H-50A), 3.48 (1H, m, H-50B).
6.8. 20,30-O-p-Anisylidene-6-chloropurine riboside (19)
6.5. 20,30-O-Isopropylidene-6-chloropurine riboside (15)
A suspension of zinc chloride (2.65 g, 19.4 mmol) in p-
methoxybenzaldehyde (10 ml) was stirred for 30 min at
30–40 ꢁC, and then 6-chloropurine riboside (14) (1 g,
3.5 mmol) was added. The mixture was stirred at room
temperature for 5 days. The semi-solid product was
poured on ice and extracted with CHCl3 (3· 40 ml)
and the combined organic extracts were washed with
water, dried over anhydrous Na2SO4, and evaporated.
The residue was chromatographed on silica gel. Eluate
of 60% ethyl acetate in hexane was collected and evapo-
rated to give compound 19 (1.2 g, 81%). Mp 128–
130 ꢁC; MS (ESI) m/z 427 (M++Na); 1H NMR
(300 MHz, (CD3)2SO), a 2:1 mixture of stereoisomers
doubling most signals, d 8.92 (1H, s, H-8), 8.84 (1H, s,
H-2), 7.45 (2H, d, J = 9.0 Hz, ortho-H of p-anisylidene
ring), 6.88 (2H, d, J = 9.0 Hz, para-H of p-anisylidene
ring), 6.45 (1H, d, J = 3.0 Hz, H-10), 6.21 (1H, s, p-anisy-
lidene CH), 5.52 (1H, m, H-20), 5.15 (1H, t, J = 6.0 Hz,
OH-50), 5.12 (1H, m, H-30), 4.38 (1H, q, J = 6.0 Hz, H-
40), 3.78 (3H, s, OCH3), 3.63 (2H, m, H-50). Anal. Calcd
for C18H17ClN4O5 (404.81): C, 53.41%; H, 4.23%; Cl,
8.76%, N, 13.84%. Found: C, 53.27%; H, 4.23%; Cl,
8.64%, N, 13.76%.
A suspension of 6-chloro purine riboside (14, 1.0 g,
3.5 mmol) in 42 ml of acetone was stirred overnight with
0.6 ml of 70% HClO4 at room temperature. The reaction
mixture was neutralized with NH3ÆH2O, after which the
mixture was evaporated to dryness under aspirator pres-
sure at 40 ꢁC. The crude product was recrystallized from
ethanol to give 1.1 g of 6-(700-NO2-1,2,3,4-tetra-
hydroisoquino-2-yl)-20,30-O-p-isopropylidene
purine
riboside (15) (94% yield). Mp 160–161 ꢁC; MS m/z 449
1
(M++Na); H NMR (300 MHz, CDCl3), d 8.80 (1H, s,
H-8), 8.26 (1H, s, H-2), 5.98 (1H, d, J = 5.4 H-10),
5.22 (1H, t, J = 5.4, H-20), 5.14 (1H, t, J = 5.4 Hz, H-
30), 4.96 (1H, dd, J = 6.0, 2.1 Hz, OH-50, disappeared
upon D2O exchange), 4.58 (1H, br s, H-40), 4.0 (1H, d,
J = 7.8 Hz, H-50A), 3.84 (1H, t, J = 6.6, H-50B), 1.68
(3H, s, CH3 of isopropylidene group), 1.42 (3H, s,
CH3 of isopropylidene group).
6.6. 50-O,8-cyclo-20,30-O-Isopropylidene-6-chloropurine
riboside (16)
A
mixture of 20,30-O-isopropylidene-6-chloropurine
riboside (15) (960 mg, 2.94 mmol) and N-iodosuccini-
mide (NIS) (2.01 g, 8.8 mmol) in acetic acid (29 ml)
was stirred at 50–60 ꢁC for 3 days. Acetic acid was re-
moved in vacuo. The concentrated reaction mixture
was neutralized with NH3.H2O and extracted with ethyl
acetate (3· 30 ml). The organic layer was separated and
washed with water (3· 10 ml), dried over anhydrous
Na2SO4, and evaporated. The residue was subjected to
chromatography on silica gel eluting with Hexane/
EtOAc (3:2) to provide 344 mg of 50-O,8-cyclo-20,30-O-
p-anisylidene-6-chloropurine riboside (16) as crystals
(36%). Mp 224–226 ꢁC; MS (ESI) m/z 347 (M++Na);
1H NMR (300 MHz, CDCl3), d 8.62 (1H, s, H-2), 6.44
6.9. 50-O,8-cyclo-20,30-O-p-Anisylidene-6-chloropurine
riboside (20)
A mixture of 20,30-O-p-anisylidene-6-chloropurine ribo-
side (19) (1.0 g, 2.48 mmol) and N-iodosuccinimide
(NIS) (1.6 g, 7.4 mmol) in acetic acid (24 ml) was stirred
at ambient temperature for 3 days. Acetic acid was re-
moved in vacuo. The concentrated reaction mixture
was neutralized with NH3ÆH2O and extracted with ethyl
acetate (3· 30 ml). The organic layer was separated and
washed with water, dried over anhydrous Na2SO4, and
evaporated. The residue was subjected to chromatogra-

3864
Z. Zhu, J. K. Buolamwini / Bioorg. Med. Chem. 16 (2008) 3848–3865
phy on SiO2 and eluted with Hexane/EtOAc mixture
(1:1) to provide 429 mg of 50-O,8-cyclo-20,30-O-p-anisy-
lidene-6-chloropurine riboside (20) as a crystal (yield
43%). mp 215–216 ꢁC; MS (ESI) m/z 425 (M++Na);
1H NMR (300 MHz,CDCl3), d 8.71 (1H, s, H-2), 7.40
(2H, d, J = 9.0 Hz, ortho-H of p-anisylidene ring), 6.94
(2H, d, J = 9.0 Hz, para-H of p-anisylidene ring), 6.67
(1H, s, H-10), 6.19 (1H, s, p-anisylidene CH), 5.25 (1H,
d, J = 6.0, H-20), 4.92 (2H, q, J = 9.0 Hz, H-50), 4.69
(1H, q, J = 9.0 Hz, H-30), 4.31 (1H, d, J = 12.0 Hz, H-
40), 3.83 (3H, s, OCH3). Anal. Calcd for C18H15ClN4O5
(402.80): C, 53.67%; H, 3.75%; N, 13.91%. Found: C,
53.23%; H, 3.77%; N, 13.68%.
fluorescence of the SAENTA-fluorescein ligand stan-
dard in mean channel numbers. Inhibition constants
were calculated from the IC50 values Ki values using
the following equation:
Ki ¼ IC50=ð1 þ ½Lꢂ=KLÞ
ð3Þ
where [L] and KL are the concentration and the Kd value
of the SAENTA-fluorescence, respectively. The results
were fed into the PRISM program (GraphPad, San Die-
go, CA) to derive concentration-dependent curves, as
shown in Figure 3. From these curves, the IC50 values
were obtained and used to calculate Ki values, which
were used to compare the abilities of compounds to
displace the ENT1 (es) transporter-specific ligand,
5- (SAENTA)-X8-fluorescein,35 and for that matter
their affinity for the transporter.
6.10. 50-O,8-cyclo-6-Chloropurine riboside (17)
50-O,8-cyclo-20, 30-O-p-anisylidene-6-chloropurine ribo-
side (20) (320 mg, 0.8 mmol) was suspended in 80% tri-
fluoroacetic acid (4 ml) and stirred in an ice bath for
4 h. The reaction mixture was neutralized with NH3Æ-
H2O and evaporated in vacuo to remove water. The
resulting residue was dissolved in ethyl acetate and sub-
jected to chromatography on silica gel using a mixture
of Hexane/EtOAc (1:4) as eluant to give 166 mg of 50-
O,8-cyclo-6-chloropurine riboside (17) as crystals
(73%). Mp 212–213 ꢁC; MS (ESI) m/z 307 (M++Na);
1H NMR (300 MHz, (CD3)2SO), d 8.71 (1H, s, H-2),
6.12 (1H, s, H-10), 5.65 (1H, d, J = 6.6, OH-20), 5.43
(1H, d, J = 5.7 Hz, OH-30), 4.74 (1H, q, J = 10.8 Hz,
H-20), 4.64 (1H, br s, H-50A), 4.46 (1H, t, J = 6.0 Hz,
H-30), 4.33 (1H, br d, J = 6.0 Hz, H-50B), 4.29 (1H, br
d, J = 4.8 Hz, H-40). Anal. Calcd for C10H9ClN4O4
(284.66): C, 42.19%; H, 3.19%; Cl, 12.45%; N, 19.68%.
Found: C, 42.25%; H, 3.30%; Cl, 12.09; N, 19.33%.
Acknowledgment
Financial support from the National Heart Lung and
Blood Institute, Grant No. K01-HL067479 awarded to
J.K.B. is gratefully acknowledged.
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ð2Þ
ðSFf Þ
where SFs is the ENT1 transporter-specific fluorescence
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