Synthesis and flow cytometric evaluation of novel 1,2,3,4-tetrahydroisoquinoline conformationally constrained analogues of nitrobenzylmercaptopurine riboside (NBMPR) designed for probing its conformation when bound to the es nucleoside transporter

Article abstract of DOI:10.1021/jm020405p

Novel regioisomers of conformationally constrained analogues of the potent es nucleoside transporter ligand, nitrobenzylmercaptopurine riboside (NBMPR), designed for probing its bound (bioactive) conformation, were synthesized and evaluated as es transporter ligands by flow cytometry. Purine 6-position 5, 6, 7, or 8-nitro-1,2,3,4-tetrahydroisoquinolylpurine ribosides, in which the nitrobenzyl moiety in NBMPR has been locked into the nitro-1,2,3,4-tetrahydroisoquinoline system, were synthesized by reaction of the appropriate nitro-1,2,3,4-tetrahydroisoquinoline with 6-chloropurine riboside. Flow cytometry was performed using 5-(SAENTA)-X8-fluorescein as the competitive ligand. A high degree of variation in the es transporter binding capacity of the target compounds was observed, with the K_i values ranging from 0.45 nM for the most tightly bound compound (4) to 300 nM for the least tightly bound compound (5). The K_i of NBMPR was 0.70 nM, a little higher than that of compound 4. Compound 4 is the isomer that has the nitro group in the best orientation at the es transporter binding site compared to the other three compounds, 2, 3, and 5.

Full text of DOI:10.1021/jm020405p

J . Med. Chem. 2003, 46, 831-837
831
Syn th esis a n d F low Cytom etr ic Eva lu a tion of Novel
1,2,3,4-Tetr a h yd r oisoqu in olin e Con for m a tion a lly Con str a in ed An a logu es of
Nitr oben zylm er ca p top u r in e Ribosid e (NBMP R) Design ed for P r obin g Its
Con for m a tion Wh en Bou n d to th e es Nu cleosid e Tr a n sp or ter
Zhengxiang Zhu,† J ohn Furr,‡ and J ohn K. Buolamwini†,‡,
*
Department of Pharmaceutical Sciences, College of Pharmacy, 847 Monroe Avenue, Suite 327, University of Tennessee Health
Sciences Center, Memphis, Tennessee 38163 and Department of Medicinal Chemistry, School of Pharmacy,
University of Mississippi, University, Mississippi 38677
Received September 17, 2002
Novel regioisomers of conformationally constrained analogues of the potent es nucleoside
transporter ligand, nitrobenzylmercaptopurine riboside (NBMPR), designed for probing its
bound (bioactive) conformation, were synthesized and evaluated as es transporter ligands by
flow cytometry. Purine 6-position 5, 6, 7, or 8-nitro-1,2,3,4-tetrahydroisoquinolylpurine ribosides,
in which the nitrobenzyl moiety in NBMPR has been locked into the nitro-1,2,3,4-tetrahydro-
isoquinoline system, were synthesized by reaction of the appropriate nitro-1,2,3,4-tetrahydro-
isoquinoline with 6-chloropurine riboside. Flow cytometry was performed using 5-(SAENTA)-
X8-fluorescein as the competitive ligand. A high degree of variation in the es transporter binding
capacity of the target compounds was observed, with the Ki values ranging from 0.45 nM for
the most tightly bound compound (4) to 300 nM for the least tightly bound compound (5). The
Ki of NBMPR was 0.70 nM, a little higher than that of compound 4. Compound 4 is the isomer
that has the nitro group in the best orientation at the es transporter binding site compared to
the other three compounds, 2, 3, and 5.
In tr od u ction
files having to do with toxicity, lack of in vivo efficacy,
and/or lack of selectivity.12 There is therefore a need for
novel inhibitors.
Nucleoside transporters are integral membrane gly-
coproteins, which are responsible for cellular trafficking
of physiological nucleosides and their synthetic ana-
logues.1,2 They are grouped into two categories: (i)
equilibrative transporters (most widely distributed) and
(ii) concentrative (sodium ion-coupled) transporters
(limited to absorptive and secretory tissues and some
cultured cell lines). Two equilibrative transporters with
similar broad substrate specificities have been identified
and designated as es (equilibrative sensitive) and ei
(equilibrative insensitive) on the basis of their sensitiv-
ity or insensitivity to inhibition by S6-(4-nitrobenzyl)-
mercaptopurine riboside (NBMPR, 1), respectively.
Nucleoside transport inhibitors (NTIs) have been shown
to have potential therapeutic applications in heart
disease and stroke as cardioprotective and neuropro-
tective agents, respectively,3-7 in inflammatory disease,8
and in antimetabolite chemotherapy as biological re-
sponse modifiers.9 The es transporter is by far the most
important nucleoside transporter of most mammalian
tissues, especially heart tissue,10,11 and appears to be
the most relevant NT target for therapeutic exploitation.
Several chemical classes have been shown to inhibit the
es nucleoside transporter,12 the most selective of which
are NBMPR and analogues, which inhibit it at low
nanomolar concentrations.1 The therapeutic application
of the available es transporter inhibitors has been
severely hampered by their poor pharmacological pro-
In the absence of a 3D structure of any nucleoside
transporter and for that matter its complex with inhibi-
tors, knowledge of the bioactive conformation of the
most potent and selective inhibitors will be useful for
rational design of new inhibitors.13 We have therefore
embarked on using the conformationally constrained
analogue strategy14 to probe the bioactive conformation
of NBMPR, the prototype es transporter inhibitor (Kd
0.1-1.0 nM), which is at present unknown. Current
structure-activity relationships of es transporter inhibi-
tors12 indicate that for the NBMPR series, the nitroben-
zyl moiety (shown in bold in compounds 1-5) confers
the high-affinity ligand binding capacity. This moiety
is very flexible, with its conformational space involving
torsions about the three bonds, a, b, and c (shown in
compounds 1-5), making that portion of the molecule
the most conformationally variant. In our design, we
envisioned that replacement of the purine 6-position
substituent of NBMPR (1) with nitro-1,2,3,4-tetrahydro-
isoquinolines (2-5) will provide analogues in which the
conformational flexibility of the nitrobenzyl group is
severely restrained. Two of the rotatable bonds (b and
c) in NBMPR have been fixed in the novel tetrahydro-
isoquinoline analogues (2-5). This constrains the high-
affinity-conferring nitrobenzyl group of NBMPR into a
1,2,3,4-tetrahydroisoquinoline ring system. The most
suitable position of the nitro substituent was explored
by varying its position on the aromatic portion of the
tetrahydroisoquinoline ring as shown in compounds
2-5, where the nitro substituent is in the 5-, 6-, 7- or
* To whom correspondence should be addressed. Phone: 901 448-
7533, Fax: 901 448-6828, E-mail: J buolamwini@utmem.edu.
† University of Tennessee Health Sciences Center.
‡ University of Mississippi.
10.1021/jm020405p CCC: $25.00 © 2003 American Chemical Society
Published on Web 01/24/2003

832 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5
Zhu et al.
Sch em e 1a
8-position of the 1,2,3,4-tetrahydrisoquinoline ring,
respectively. In these tetrahydroisoquinoline analogues,
the sulfur atom in NBMPR has of necessity been
replaced by an NH. It is known that this replacement
does not significantly decrease affinity.12
a
Reaction condition: (a) CaCO3/ethanol.
Sch em e 2a
Ch em istr y
All four new aromatic nitro isomers of 6-position nitro-
1,2,3,4-tetrahydoisoquinolinylpurine ribosides NBMPR
analogues were synthesized. Several literature methods
were employed to synthesize the required nitro-1,2,3,4-
tetrahydoisoquinoline (compounds 6-9), which were
subsequently reacted with the commercially available
6-chloropurine riboside (10), according to reaction Scheme
1, to obtain the target compounds. There are few
methods available for the synthesis of tetrahydroiso-
quinolines containing electron-withdrawing groups on
the aromatic ring, which can be problematic.15 The
commercially available 5-nitroisoquinoline (11) was
reduced using sodium borohydride in ethanol to afford
the 5-nitro-1,2,3,4-tetrahydroisoquinoline intermediate
(compound 6) in good yield (90%) according to Scheme
2a.16 The 7-nitro-1,2,3,4-tetrahydroisoquinoline inter-
mediate (compound 8) was prepared in 50% yield by
nitration of the commercially available 1,2,3,4-tetrahy-
droisoquinoline using KNO3 in concentrated sulfuric
acid according to the method of Ajao and Bird17 (see
Scheme 2b). The 6- and 8-nitro-1,2,3,4-tetrahydroiso-
quinoline intermediates 7 and 9 were more difficult to
prepare, but were obtained following reaction Scheme
3 according to the report by Tercel et al.18
a
Reaction conditions: (a) NaBH4/EtOH,
0
°C, (b) KNO3/
concentrated H2SO4, 5 °C.
comparison. Its synthesis was achieved by reacting
compound 12 with compound 10 according to reaction
Scheme 1.
In attempts to find better routes for preparing the
intermediates 7 and 9 because of their low yields, we
tried to prepare compound 9 by the reaction route shown
below (Scheme 4). However, the diazotization and
replacement of the NH2 group in compound 27 to obtain
8-nitroisoquinoline (compound 28), which would have
been subsequently reduced to give the desired compound
9, was not successful. Instead, we obtained 5-hydroxy-
isoquinoline (compound 29), the NMR data of which
compared well with literature values.19 To our knowl-
edge, this is the first report of this kind of a result from
a diazotization in which an NH2 and a NO2 group have
both been replaced in one pot. Another unexpected
result of the reaction is that the diazonium group was
replaced by an OH group instead of a hydrogen atom.
One would have expected a replacement with OH had
the reaction been carried out in the presence of aqueous
HCl, but not after treatment with H3PO2.
The 7-nitro-1,2,3,4-tetrahydroisoquinoline intermedi-
ate (8) used to synthesize compound 4 (see Scheme 2)
was protected by acetylation to give compound 13. The
NO2 group of 13 was reduced to an NH2 group to obtain
compound 14. The NH2 group of 14 was subsequently
protected, and the resulting compound 15 was nitrated
to afford the nitro compounds 16 and 17. Interestingly,
when the amount of KNO3 in the reaction was increased
to improve the yields of compounds 16 and 17, the major
product obtained was the dinitro compound, 22 (50%).
Compounds 16 and 17 were deprotected to give the free
NH2 substituted compounds 18 and 19. It must be
pointed out that the deprotected compounds 18 and 19
were also obtained as minor products during the nitra-
tion of compound 15. This has not previously been noted.
Compounds 18 and 19 were subjected to diazotization
using NaNO2 and HCl and then treatment with H3PO2
to replace the NH2 by H, to obtain compounds 20 and
21. Compounds 20 and 21 were deprotected to obtain
the required intermediates 7 and 9. Compound 23,
which is the analogue of compounds 2-5 without the
nitro substituent, was also synthesized and tested for
SAR
The target constrained NBMPR analogues (2-5) were
tested along with 6-(1,2,3,4-tetrahydroisoquinolinyl)-
purine riboside (23) as es transporter binding ligands
by a facile competitive binding flow cytometric assay
using the K562 chronic myelogenous leukemia cell line.
The high-affinity es transporter fluorescent ligand,
5-(SAENTA)-X8-fluorescein,20 was used as the competi-
tive ligand to be displaced by the test compounds. Flow
cytometry has several advantages over the conventional
radioligand binding assays, in that it eliminates radia-
tion hazards and disposal problems and allows the use
of much fewer cells, as few as 5000 cells compared to 2
million cells per sample for comparable radioligand
assays. As depicted in Figure 1, these novel constrained

Novel 1,2,3,4-Tetrahydroisoquinoline Analogues
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5 833
Sch em e 3a
a
Reaction conditions: (a) (Ac)2O/Et3N, CH2Cl2; (b) NH2NH2‚xH2O/FeCl3‚6H2O/activated carbon/MeOH, reflux; (c) TFAA/TFA/CH2Cl2;
(d) KNO3 (1.2 equiv)/concentrated H2SO4, 4 °C; (e) HCl/MeOH, reflux; (f) (i) NaNO2/HCl/0 °C, (ii) H3PO2; (g) concentrated HCl/MeOH,
reflux; (h) KNO3 (2 equiv)/concentrated H2SO4, 5 °C.
Sch em e 4a
a
Reaction conditions: (a) TFAA/TFA/CH2Cl2/15 min; (b) KNO3/concentrated H2SO4/0 °C,1 h; (c) concentrated HCl/MeOH/reflux/50
min; (d) (i) NaNO2/HCl/0 °C/2 h, (ii) H3PO2/4 °C/10 h.
Ta ble 1. Flow Cytometrically-Determined Ki Values
compound
Ki (nM)
1 (NBMPR)
2
0.70
250
3
15
4
5
0.45
300
23
150
analogues of NBMPR exhibited a wide range of binding
affinities at the es transporter as measured by their
ability to displace the es nucleoside transporter-specific
ligand,20 shown by the Ki values in Table 1. They range
from a subnanomolar concentration for compound 4 (Ki
0.45 nM) to high nanomolar concentrations for com-
pounds 2 and 5, at least a 500-fold difference. The
results provide some surprises based on the known SAR
of NBMPR analogues. First, one of the new compounds
(4) is as good or even possibly better than the prototype
es transporter inhibitor NBMPR. This is interesting
because, generally, when the sulfur at the 6-position in
NBMPR is replaced by NH as is present in the new
compounds, es transporter binding decreases.12 The
comparable affinities of compound 4 (Ki 0.45 nM) and
F igu r e 1. Equilibrium displacement of SAENTA-fluorescein
ligand by new tetrahydroisoquinoline constrained NBMPR
analogues in K562 cells. Cells were incubated with 25 nM
SAENTA fluorescein in the presence or absence of inhibitor
for 30 min at room temperature and analyzed by flow cytom-
etry (FACSCaliburR). Data were collected on 5000 cells per
sample, and mean channel numbers were used as a measure
of ligand fluorescence output.
NBMPR (Ki 0.70 nM) indicate that conformational
changes at this location in the molecules can make a

834 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5
Zhu et al.
gel (170-400 mesh). Melting points were determined using a
Fisher-J ohns Melting Point Apparatus and are reported
uncorrected. Mass spectra were obtained on a Bruker-HP
Esquire-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.
Gen er a l Meth od for th e P r ep a r a tion of Com p ou n d s
2, 3, 4, 5, 23. A mixture of 6-chloropurine riboside (10, 100
mg, 0.35 mmol), (mono-NO2)-1,2,3,4-tetrahydroisoquinoline
(0.88 mmol), and calcium carbonate (70 mg, 0.70 mmol) in
EtOH (5 mL) was stirred under refluxing for 15 h. The reaction
mixture was filtered, and the filtrate was evaporated in vacuo
at 40 °C. The residue was purified by flash silica gel chroma-
tography followed by recrystallization from MeOH.
significant difference in binding to the es transporter.
Second, we had thought that compound 3 would prob-
ably be the most active of the new analogues since it
has the nitro substituent on the tetrahydroisoquinoline
ring in an equivalent position to the p-nitro substituent
in NBMPR. This is because the known SAR suggests
that para substitution of the nitro group (as in NBMPR)
is the best in terms of es transporter binding, followed
by meta substitution, which in turn is much better than
ortho substitution. As it turned out, the corresponding
para compound 3, is tightly bound (Ki 15 nM), but much
less so than the compound corresponding to the meta
nitro substitution, compound 4 (Ki 0.45 nM). The
compounds corresponding to ortho position substitution,
compounds 2 (Ki 250 nM) and 5 (Ki 300 nM) behaved
as expected, exhibiting activities comparable to the
unsubstituted tetrahydroisoquinoline analogue com-
pound 23 (Ki 150 nM). It appears that compound 3 most
captures the bioactive conformation of the nitrobenzyl
group of NBMPR and should serve as the best template
for rational design of es transporter inhibitors among
these novel NBMPR analogues. These results offer
insights into the bioactive conformation of NBMPR at
the 6-position substituent, which is responsible for high
affinity binding to the es transporter.21 The solid state
conformation of NBMPR has been determined by X-ray
diffraction22 and a solution conformation has also been
determined by NMR.23 The X-ray structure reveals a
syn orientation of the purine system about the glycosidic
bond, whereas the solution conformation reveals the
preponderance of a high-anti orientation of the purine
system about the glycosidic linkage. Conformational
space analysis of the novel conformationally constrained
analogues reported here will shed light on the bioactive
conformation of NBMPR and provide insights for further
es transporter inhibitor design.
The high es transporter binding affinity of compound
4, which is at least equivalent in potency to NBMPR, is
of interest since this compound will be less likely to lose
the nitrobenzyl moiety, and should overcome a major
disadvantage of NBMPR, which has to do with the in
vivo lability of the nitrobenzyl moiety. The nitrobenzyl
group of NBMPR is lost in vivo to form the immuno-
suppressive product mercaptopurine riboside,24 with a
dramatically reduced es transporter affinity (up to a
1000-fold decrease). This lability of the nitrobenzyl
group has actually allowed NBMPR to be used as an
effective photolabeling agent of the es nucleoside
transporter.25-27 This has hampered the clinical ap-
plications of NBMPR. Therefore preclinical development
of the novel compounds 3 and 4, which lock the nitro-
benzyl moiety in place, and are likely to prolong in vivo
activity compared to NBMPR, is warranted. Compounds
3 and 4 are worth further investigation as novel
nucleoside transport inhibitors with potential for clinical
and research applications.
6-{[Mon o- (5, 6, 7, or 8)-NO2-] 1,2,3,4-tetr a h yd r oiso-
qu in o-2-yl}p u r in e Ribosid e (Com p ou n d s 2, 3, 4, 5, 23).
Com p ou n d 2: yield 65%; mp 159-160 °C; IR: 3438, 1604,
1528, 1352 cm-1 1H NMR (300 MHz, CDCl3) δ 8.15 (1H, s,
;
H-2), 7.86 (1H, d, J ) 7.5 Hz, H-6′′), 7.84 (1H, s, H-8), 7.49
(1H, d, J ) 7.5 Hz, H-8′′), 7.36 (1H, t, J ) 7.8, 8.1 Hz, H-7′′),
6.53 (1H, br d, J ) 9.9 Hz, OH-2′), 5.81 (1H, d, J ) 7.5 Hz,
H-1′), 5.36 (1H, br s, OH-3′), 5.07 (1H, q, J ) 6.9, 5.1 Hz, H-2′),
4.53-4.45 (3H, br m, OH-5′, H-3′, 1′′A), 4.35 (1H, s, H-1′′B),
3.94 (1H, d, J ) 12.9 Hz, H-4′), 3.76 (2H, br m, H-5′), 3.28
(2H, t, J ) 6.0 Hz, H3′′ or H4′′). Anal. (C19H20N6O6) C, H, N.
Com p ou n d 3: yield 66%; mp 127.5-129.5 °C; MS (ESI) m/z
451 (M + Na)+; IR: 3437, 1593, 1523, 1350 cm-1 1H NMR
;
(300 MHz, CD3OD) δ 8.30 (1H, s, H-2), 8.29 (1H, s, H-8), 8.10
(2H, m, H-5′′, 7′′), 7.50 (1H, d, J ) 8.4 Hz, H-8′′), 5.98 (1H, d,
J ) 6.3 Hz, H-1′), 5.50 (2H, br s, OH-2′, 3′), 4.74 (1H,t, J ) 6.0
Hz, OH-5′), 4.58 (3H, br s, H-2′, 3′, 1′′A), 4.32 (1H, q, J ) 5.1,
2.7 Hz, H-4′), 4.17 (1H,d, J ) 2.4 Hz, H-1′′B) 3.89 (1H, q, J )
12.6, 2.4 Hz, H-5′A), 3.74 (1H, q, J ) 9.9, 2.4 Hz, H-5′B), 3.15
(2H, t, J ) 6.0 Hz, H3′′ or H4′′). Anal. (C19H20N6O6‚H2O): C,
H, N.
Com p ou n d 4: yield 61%; mp 172-173 °C; IR: 3422, 1597,
1531, 1346 cm-1;1H NMR (300 MHz, CDCl3) δ 8.18 (1H, s, H-2),
8.08 (1H, s, H-8′′), 8.02 (1H, d, J ) 8.4 Hz, H-6′′), 7.81 (1H, s,
H-8), 7.29 (1H, d, J ) 8.4 Hz, H-5′′), 6.46 (1H, br d, J ) 10.2
Hz, OH-2′), 5.81 (1H, d, J ) 7.5 Hz, H-1′), 5.46 (1H, br s, OH-
3′), 5.07 (1H, q, J ) 6.9, 5.1 Hz, H-2′), 4.60-4.47 (3H, br m,
OH-5′, H-3′, 1′′A), 4.35 (1H, s, H-1′′B), 3.94 (1H, d, J ) 12.3
Hz, H-4′), 3.76 (1H, br t, J ) 12.3, 10.5 Hz, H-5′A), 3.61 (1H,
br d, J ) 3.9 Hz, H-5′B), 3.10 (2H, t, J ) 6.0 Hz, H3′′ or H4′′).
Anal. (C19H20N6O6) C, H, N.
Com p ou n d 5: yield 74%; mp 148-149 °C; MS (ESI) m/z
451 (M + Na)+; IR: 3370, 1590, 1530, 1343 cm-1 1H NMR
;
(300 MHz, CD3COCD3) δ 8.26 (1H, s, H-2), 8.25 (1H, s, H-8),
7.94 (1H, d, J ) 7.5 Hz, H-7′′), 7.60 (1H, d, J ) 7.8 Hz, H-5′′),
7.47 (1H, t, J ) 7.8, 7.8 Hz, H-6′′), 5.95 (1H, d, J ) 6.6 Hz,
H-1′), 5.78 (1H, br s, OH-2′), 5.63 (1H, d, J ) 9.9 Hz, OH-3′),
4.90 (1H, m, H-2′), 4.64 (2H, br m, OH-5′, H-3′), 4.37 (2H, q, J
) 6.0, 3.9 Hz, H-4′, 1′′A), 4.17 (1H, s, H-1′′B), 3.84 (1H, m,
H-5′A), 3.71 (1H, m, H-5′B), 3.21 (2H, t, J ) 6.0 Hz, H3′′ or
H4′′). Anal. (C19H20N6O6) C, H, N.
Com p ou n d 23: yield 95%; mp 157.5-159 °C; IR: 3413,
1600 cm-1; 1H NMR (300 MHz, DMSO-d6) δ 8.45 (1H, s, H-2),
8.29 (1H, s, H-8), 7.20 (4H, m, H-5′′, 6′′, 7′′, 8′′), 5.92 (1H, d, J
) 6.0 Hz, H-1′), 6.47-5.18 (4H, br m, OH-2′, 5′, 3′,H-2′), 4.57
(2H, br m, H-3′, 1′′A), 4.14 (1H, q, J ) 8.1, 5.1 Hz, H-4′), 3.96
(1H, s, H-1′′B), 3.67 (1H, m, H-5′A), 3.56 (1H, m, H-5′B), 3.93
(2H, t, J ) 6.0 Hz, H3′′ or H4′′).
5-Nitr o-1, 2,3,4-tetr a h yd r oisoqu in olin e (6). To a solution
of 11 (500 mg, 2.85 mmol) in acetic acid (50 mL) was added
small portions of NaBH4 until TLC examination of the reaction
mixture indicated the absence of the starting material. The
solution was then poured onto ice, basified with NH3‚H2O, and
then extracted three with CH2Cl2. The organic layer was
washed once with water, dried over sodium sulfate, and
evaporated in vacuo. The residue was purified by silica gel
Exp er im en ta l Section
Ch em istr y. Thin-layer chromatography (TLC) was con-
ducted on silica gel F254 plates (Analtech). Compounds were
visualized by UV light or 5% H2SO4 in EtOH spraying reagent.
1H,13C spectra were recorded on Bruker ARX (300 MHz)
instruments, using CDCl3, CD3OD, (CD3)2SO, or CD3COCD3
as solvents and tetramethylsilane (TMS) as internal standard.
Flash column chromatography was performed on Fisher silica
1
chromatography to give compound 6 (450 mg, yield 90%); H
NMR (300 MHz, CDCl3) δ 7.80 (1H, t, J ) 4.8, 4.5 Hz, H-7),
7.28 (2H, d, J ) 5.7 Hz, H-6,8), 4.11 (2H, s, ArCH2N), 3.17

Novel 1,2,3,4-Tetrahydroisoquinoline Analogues
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5 835
(2H, t, J ) 5.7 Hz, ArCH2CH2N), 3.10 (2H, t, J ) 5.7 Hz,
trated HCl (15 mL) for 50 min. The reaction mixture was
concentrated in vacuo to get rid of MeOH, basified with
ammonium hydroxide, and then extracted with CH2Cl2. The
organic layer was dried over Na2SO4 and evaporated. The
crude product was chromatographed over silica gel to give 1.1
g of compound 18 and 0.5 g of compound 19, 30 and 15% yields,
respectively. Compound 16: MS (ESI) m/z 354 (M + Na)+, m/z
ArCH2CH2N).
7-Nit r o-1, 2,3,4-t et r a h yd r oisoq u in lin e (8). An ice-cold
solution of compound 12 (10.8 g, 80 mmol) in concentrated
sulfuric acid (40 mL) was treated with potassium nitrate (8.8
g, 87 mmol) in small portions, keeping the temperature below
5 °C. The reaction was left overnight at room temperature and
poured onto ice. The resulting solution was basified with NH3‚
H2O, extracted with CH2Cl2. The extract concentrated to
dryness in vacuo. The crude product obtained was converted
to the hydrochloride salt and crystallized from methanol to
give 8.5 g of the hydrochloride (yield 50%), which was basified
to give compound 8. 1H NMR (300 MHz, CDCl3) δ 7.98 (1H, d,
J ) 8.1 Hz, H-6), 7.91 (1H, s, H-8), 7.24 (1H, d, J ) 8.4 Hz,
H-5), 4.10 (2H, s, H-1), 3.17 (2H, t, J ) 6.0 Hz, H-3), 2.89 (2H,
t, J ) 6.0 Hz, H-4).
N-Acetyl-7-n itr o-1, 2,3,4-tetr a h yd r oisoqu in lin e (13). A
mixture of compound 8 (5.4 g, 26 mmol), Et3N (9.8 mL, 70
mmol), and acetic anhydride (5.0 mL, 51 mmol) in CH2Cl2 (70
mL) was stirred at reflux temperature for 1 h. The reaction
mixture was cooled and poured onto ice. The resulting solution
was extracted with CH2Cl2. The organic layer was dried over
sodium sulfate and evaporated in vacuo. The residue was
subjected to by flash silica gel chromatography to obtain 5.3 g
of compound 13 (94% yield). 1H NMR (300 MHz, CDCl3) δ
8.05-8.03 (2H, m, H-6,8), 7.43-7.30 (1H, m, H-5), 4.83 (1.2H,
s, ArCH2N), 4.72 (0.8H, s, ArCH2N), 3.87 (0.8H, t, J ) 5.7 Hz,
ArCH2CH2N), 3.74 (1.2H, t, J ) 5.7 Hz, ArCH2CH2N), 3.02
(1.2H, t, J ) 5.7 Hz, ArCH2CH2N), 2.95 (0.8H, t, J ) 5.7 Hz,
ArCH2CH2N), 2.21 (1.2H, s, COCH3), 2.20 (1.8H, s, COCH3).
1
330 (M - H)-; H NMR (300 MHz, CDCl3), a 3:2 mixture of
amide conformers doubling most signals, δ 11.38 (0.4H, s, NH),
11.32 (0.6H, s, NH), 8.56 (1H, s, H-5), 8.15 (0.4H, s, H-8), 8.13
(0.6H, s, H-8), 4.86 (1.2H, s, ArCH2N), 4.74 (0.8H, s, ArCH2N),
3.88 (0.8H, t, J ) 6.0 Hz, ArCH2CH2N), 3.74 (1.2H, t, J ) 6.0
Hz, ArCH2CH2N), 2.99 (1.2H, t, J ) 6.0 Hz, ArCH2CH2N), 2.93
(0.8H, t, J ) 6.0 Hz, ArCH2CH2N), 2.21 (3H, s, COCH3).
Compound 17: MS (ESI) m/z 354 (M + Na)+, m/z 330 (M -
H)-;1H NMR (300 MHz, CDCl3), a 3:2 mixture of amide
conformers doubling most signals, δ 9.64 (1H, br s, NH), 8.21
(0.4H, d, J ) 8.7 Hz, H-5), 8.18 (0.6H, d, J ) 8.7 Hz, H-5),
7.46 (0.4H, d, J ) 8.7 Hz, H-6), 7.43 (0.6H, d, J ) 8.7 Hz, H-6),
4.89 (1.2H, s, ArCH2N), 4.75 (0.8H, s, ArCH2N), 3.86 (0.8H, t,
J ) 6.0 Hz, ArCH2CH2N), 3.78 (1.2H, t, J ) 6.0 Hz, ArCH2-
CH2N), 3.01 (1.2H, t, J ) 6.0 Hz, ArCH2CH2N), 2.96 (0.8H, t,
J ) 6.0 Hz, ArCH2CH2N), 2.06 (3H, s, COCH3). Compound
18: MS (ESI) m/z 258 (M + Na)+; 1H NMR (300 MHz, CDCl3),
a 2:1 mixture of amide conformers doubling most signals, δ
7.94 (1H, s, H-5), 6.60 (1H, s, H-8), 5.96 (2H, br s, NH2), 4.68
(1.3H, s, ArCH2N), 4.56 (0.7H, s, ArCH2N), 3.80 (0.7H, t, J )
6.0 Hz, ArCH2CH2N), 3.67 (1.3H, t, J ) 6.0 Hz, ArCH2CH2N),
2.85 (1.3H, t, J ) 6.0 Hz, ArCH2CH2N), 2.79 (0.7H, t, J ) 6.0
Hz, ArCH2CH2N), 2.18 (3H, s, COCH3); 13C NMR δ 169.9 and
169.7 (COCH3), 143.6 and 143.2, 142.5 and 141.5, 131.5 and
131.3, 123.4 and 123.5 (C-6, 7,9,10), 126.1 and 125.5, 116.1
and 115.5 (C-5,8), 48.1, 44.4, 44.1, 39.9 (C-1,3), 28.5 and 27.5
(C-4), 22.1 and 21.8 (COCH3). Compound 19: MS (ESI) m/z
258 (M + Na)+, m/z 234 (M - H)-; 1H NMR (300 MHz, CDCl3),
a 3:2 mixture of amide conformers doubling most signals, δ
7.10 (0.6H, d, J ) 8.4 Hz, H-5), 7.05 (0.4H, d, J ) 8.7 Hz, H-5),
6.73 (0.6H, d, J ) 8.4 Hz, H-6), 6.68 (0.4H, d, J ) 8.4 Hz, H-6),
5.52 (1.2H, s, NH2), 5.34 (0.8H, s, NH2), 4.93 (0.8H, s,
ArCH2N), 4.81 (1.2H, s, ArCH2N), 3.79 (1.2H, t, J ) 6.0 Hz,
ArCH2CH2N), 3.68 (0.8H, t, J ) 6.0 Hz, ArCH2CH2N), 2.83
(0.8H, t, J ) 6.0 Hz, ArCH2CH2N), 2.77 (1.2H, t, J ) 6.0 Hz,
ArCH2CH2N), 2.16 (3H, s, COCH3). 13C NMR δ 169.5 and 169.2
(COCH3), 142.9 and 142.4, 131.0 and 130.1, 125.7, 124.3 (C-7,
8,9,10), 135.4 and 134.5, 117.7 and 117.2 (C-5, 6), 47.3, 43.7,
42.9, 39.1 (C-1, 3), 29.0 and 28.1 (C-4), 22.1 and 21.6 (COCH3).
N-Acetyl-7-a m in o-1, 2,3,4-tetr a h yd r oisoqu in olin e (14).
A mixture of compound 13 (5.1 g, 23 mmol), activated carbon
(4.2 g), ferric chloride hexahydrate (2.1 g, 7.6 mmol), and
methanol (140 mL) was stirred under reflux for 20 min. To
the boiling mixture was added hydrazine hydrate (8.5 g, 265
mmol) dropwise, the mixture was refluxed for an additional 4
h, cooled, and filtered, and the residue was washed with
methanol. The filtrate was concentrated in vacuo and the
residue recrystallized from EtOAc to give compound 14 (3.8
1
g, 85% yield). H NMR [300 MHz, (CD3)2SO], a 3:2 mixture of
amide conformers doubling most signals, δ 6.80 (1H, d, J )
8.1 Hz, H-5), 6.40 (1H, d, J ) 7.8 Hz, H-6), 6.33 (1H, s, H-7),
4.89 (2H, br s, NH2), 4.46 (0.8H, s, ArCH2N), 4.41 (1.2H, s,
ArCH2N), 3.57 (2H, t, J ) 5.7 Hz, ArCH2CH2N), 2.66 (1.2H, t,
J ) 6.0 Hz, ArCH2CH2N), 2.55 (0.8H, t, J ) 6.0 Hz, ArCH2-
CH2N), 2.06 (1.2H, s, COCH3), 2.05 (1.8H, s COCH3).
N-Acetyl-7- tr iflu or oa ceta m id o-1, 2,3,4-tetr a h yd r oiso-
qu in olin e (15). A solution of compound 14 (3.0 g, 17 mmol),
trifluoroacetic acid (2 mL, 28 mmol), and trifluoroacetic
anhydride (4 mL, 28 mmol) in CH2Cl2 was refluxed for 15 min
and cooled, and the mixture was poured onto ice and extracted
with CH2Cl2. The extract was dried over sodium sulfate and
concentrated in vacuo. The crude product was chromato-
graphed over silica gel to give 4.5 g of compound 15 (94% yield).
N-Acetyl-6 (8)-n itr o-1, 2,3,4-tetr ah ydr oisoqu in olin e (20,
21). To a stirred solution of compound 18 (19) (290 mg, 1.23
mmol) in HCl (6 M, 4 mL) was added dropwise a solution of
NaNO2 (110 mg, 1.6 mmol) in water (0.5 mL) at 0 °C. After
the mixture was stirred at 0 °C for 2 h, hypophosphorous acid
(50% aqueous solution, 1.4 mL) was added dropwise, and the
mixture was stirred at 40 °C for 10 h. The reaction mixture
was then poured into water and extracted with methylene
chloride. The organic layer was dried over NaSO4 and evapo-
rated. The crude product was purified by silica gel chroma-
tography to afford compound 20 (21). Compound 20 (176 mg,
65%): MS (ESI) m/z 243 (M + Na)+, m/z 219 (M - H)-; 1H
NMR (300 MHz, CDCl3), a 2:1 mixture of amide conformers
doubling most signals, δ 8.08-8.03 (2H, m, H-5, 7), 7.31 (1H,
d, J ) 8.7 Hz, H-8), 4.83 (1.3H, s, ArCH2N), 4.72 (0.7H, s,
ArCH2N), 3.87 (0.7H, t, J ) 6.0 Hz, ArCH2CH2N), 3.74 (1.3H,
t, J ) 6.0 Hz, ArCH2CH2N), 3.02 (1.3H, t, J ) 5.7 Hz, ArCH2-
CH2N), 2.96 (0.7H, t, J ) 5.7 Hz, ArCH2CH2N), 2.21 (3H, s,
COCH3). Compound 21 (233 mg, 86%): MS (ESI) m/z 243 (M
+ Na)+; 1H NMR (300 MHz, CDCl3), a 3:2 mixture of amide
conformers doubling most signals, δ 7.97 (0.6H, d, J ) 10.2
Hz, H-7), 7.94 (0.4H, d, J ) 10.2 Hz, H-7), 7.49-7.32 (2H, m,
H-6, 5), 5.09 (0.8H, s, ArCH2N), 4.99 (1.2H, s, ArCH2N), 3.86
(1.2H, t, J ) 6.0 Hz, ArCH2CH2N), 3.75 (0.8H, t, J ) 6.0 Hz,
ArCH2CH2N), 3.03 (0.8H, t, J ) 6.0 Hz, ArCH2CH2N), 2.98
(1.2H, t, J ) 6.0 Hz, ArCH2CH2N), 2.21 (1.8H, s, COCH3), 2.19
(1.2H, s, COCH3).
1
H NMR (300 MHz, (CD3)2SO), a 3:2 mixture of amide
conformers doubling most signals, δ 11.24 (0.8H, s, NH), 11.21
(1.2H, s, NH), 7.55-7.40 (2H, m, H5,8), 7.22 (0.6H,s, H-6), 7.20
(0.4H, s, H-6), 4.64 (0.8H, s, ArCH2N), 4.57 (1.2H, s, ArCH2N),
3.65 (2H, t, J ) 6.3 Hz, ArCH2CH2N), 2.84 (1.2H, t, J ) 6.0
Hz, ArCH2CH2N), 2.73 (0.8H, t, J ) 6.0 Hz, ArCH2CH2N), 2.06
(1.2H, s, COCH3), 2.05 (1.8H,s COCH3).
N-Acetyl-7-tr iflu or oa ceta m id o-6 (8)-n itr o-1, 2,3,4-tet-
r a h yd r oisoqu in olin e (16, 17) a n d N-Acetyl-7-a m in o-6 (8)-
n itr o-1, 2,3,4-tetr a h yd r oisoqu in olin e (18, 19). Compound
15 (4.5 g, 16 mol) was dissolved in 30 mL of ice-cold concen-
trated H2SO4. To this solution was added powdered potassium
nitrate (2 g, 20 mmol) portionwise, keeping the temperature
around 4 °C. The mixture was stirred at 0 °C overnight and
then poured onto ice. The resulting solution was basified with
ammonium hydroxide and extracted with CH2Cl2. The organic
layer was dried over sodium sulfate and concentrated to
dryness. The product was a mixture of compounds 16 and 17
(as determined by 1H NMR). Hydrolysis of compounds 16 and
17 was achieved by refluxing in MeOH (60 mL) and concen-

836 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5
Zhu et al.
6(8)-Nit r o-1, 2,3,4-t et r a h yd r oisoq u in olin e (7 a n d 9).
Hydrolysis of 20 (21) (190 mg, 0.86 mmol) in MeOH (6 mL)
and concentrated hydrochloride acid (3 mL) at refluxing
temperature for 10 h afforded 6(and 8)-nitro-1,2,3,4-tetrahy-
droisoquinolines (100 mg, 66%), which were separated by flash
silica gel chromatography. 6-NO2-1,2,3,4-tetrahydroisoquino-
line (20): MS (ESI) m/z 179 (M + H)+; 1H NMR (300 MHz,
CDCl3) δ 7.99-7.96 (2H, m, H-5, 7), 7.16 (1H, d, J ) 9.0 Hz,
H-8), 4.10 (2H,s, ArCH2N), 3.17 (2H, t, J ) 6.0 Hz, ArCH2-
CH2N), 2.90 (2H, t, J ) 6.0 Hz, ArCH2CH2N). 8-NO2-1,2,3,4-
tetrahydroisoquinoline (21): MS (ESI) m/z 179 (M + H)+; 1H
NMR (300 MHz, CD3OD) δ 7.80 (1H, d, J ) 8.1 Hz, H-7), 7.44
(1H, d, J ) 7.5 Hz, H-5), 7.33 (1H, t, J ) 8.1, 7.8 Hz, H-6),
4.16 (2H, s, ArCH2N), 3.07 (2H, t, J ) 6.0 Hz, ArCH2CH2N),
2.92 (2H, t, J ) 6.0 Hz, ArCH2CH2N).
N-Acet yl-6,8-d in it r o-7-t r iflu or oa cet a m id o-1,2,3,4-t et -
r a h yd r oisoqu in olin e (22). Compound 15 (426 mg, 1.49
mmol) was dissolved in 5 mL was ice-cold concentrated H2-
SO4. To this solution was added powdered potassium nitrate
(301.3 mg, 2.98 mmol) portionwise, keeping the temperature
under 5 °C. The mixture was stirred at 4 °C for a further 4 h
and then poured onto ice. The resulting solution was basified
with ammonium hydroxide and extracted with CH2Cl2. The
organic layer was dried over sodium sulfate and concentrated
to dryness. The crude product was chromatographed over silica
gel to give 280 mg of N-acetyl-6,8-dinitro-7-trifluoroacetamido-
1,2,3,4-tetrahydroisoquinoline (50%). MS (ESI) m/z 399 (M +
Na)+, m/z 375 (M - H)-; IR: 3448, 1640, 1647, 1270, 1171
cm-1; 1H NMR (300 MHz, DMSO-d6), a 3:1 mixture of amide
conformers doubling some signals, δ 7.74 (1H, s, H-5), 4.48
(2H, s, ArCH2N), 3.70 (2H, t, J ) 5.7 Hz, ArCH2CH2N), 2.90
(1.5H, t, J ) 5.7 Hz, ArCH2CH2N), 2.78 (0.5H, t, J ) 5.7 Hz,
ArCH2CH2N), 2.07 (2.3H, s, COCH3), 2.07 (0.7H, s, COCH3).
for 2 h, hypophosphorous acid (50% aqueous solution, 0.3 mL)
was added dropwise, and the mixture was stirred at 4 °C for
10 h. The reaction mixture was poured into water and
extracted with CH2Cl2. The organic layer was dried over
NaSO4 and evaporated. The crude product was chromato-
graphed over silica gel to obtain compound 29 (15 mg, 49%);
MS (ESI) m/z 146 (M + H)+; 1H NMR (300 MHz, DMSO-d6), δ
10.69 (1H, s, OH), 9.45 (1H, s, ArCH2N), 8.44 (1H, d, J ) 9.0
Hz, H-3), 7.70 (1H, d, J ) 5.7 Hz, H-4), 7.56 (1H, t, J ) 7.8,
7.8 Hz, H-7), 7.34 (1H, d, J ) 8.1 Hz, H-8). 6.97 (1H, d, J )
7.8 Hz, H-6). Literature:19 1H NMR (300 MHz, CDCl3) δ 9.22
(1H, s, ArCH2N), 8.51 (1H, d, J ) 5.9 Hz, H-3), 8.05 (1H, d, J
) 5.9 Hz, H-4), 7.46 (2H, m, H-7 and H-8), 7.07 (1H, d, J )
7.4 Hz, H-6).
Biologica l Testin g. The compounds were tested to deter-
mine their es nucleoside transporter binding ability by a flow
cytometric assay.20 Human leukemia K562 cells growing in
RPMI 1640 medium were washed once and resuspended at
1.6 × 106 cells/mL in phosphate-buffered saline at pH 7.4, and
incubated with 5-(SAENTA)-X8-fluorescein (25 nM) in the
presence or absence of varying concentrations of test com-
pounds at room temperature for 45 min. Flow cytometric
measurements of cell-associated fluorescence were then per-
formed with a FACSCalibur (Becton Dickinson, San J ose, CA)
equipped with a 15 mW-argon laser (Molecular Resources Flow
Cytometry Facility, University of Tennessee Health Sciences
Center). In each assay, 5000 cells were analyzed from suspen-
sions of 4 × 105 cells/mL. The units of fluorescence were
arbitrary channel numbers. Percentage (%) of control (i.e., es
transporter-specific fluorescence in the presence of SAENTA-
fluor without test compounds) was calculated for each sample
by the equation below (eq 1).
5- Tr iflu or oa ceta m id oisoqu in olin e (25). A solution of 24
(291.3 mg, 2 mmol), trifluoroacetic acid (0.7 mL, 9.8 mmol)
and trifluoroacetic anhydride (1.4 mL, 9.8 mmol) in CH2Cl2 (6
mL) was refluxed for 15 min. The reaction mixture was cooled,
poured onto ice, and neutralized with ammonium hydroxide
to obtain a light yellow solid, which was filtered and dried to
afford compound 25 (432.3 mg, 90% yield). 1H NMR (300 MHz,
CDCl3) δ 9.37 (1H, s, H-1), 8.69 (1H, d, J ) 6.0 Hz, H-3), 8.22
(1H, d, J ) 8.4 Hz, H-8), 8.00 (1H, d, J ) 6.0 Hz, H-4), 7.72
(1H, t, J ) 8.1, 8.4 Hz, H-7), 7.61 (1H, d, J ) 8.1 Hz, H-6).
5-Tr iflu or oa ceta m id o-8-n itr oisoqu in olin e (26). Com-
pound 25 (300 mg, 1.3 mmol) was dissolved in 3.5 mL of ice-
cold concentrated H2SO4. To this solution was added powdered
potassium nitrate (200 mg, 2.0 mmol) portionwise, keeping the
temperature around 0 °C. The mixture was stirred at 3 °C for
further 1 h and then poured onto ice. The resulting solution
was basified with ammonium hydroxide and extracted with
CH2Cl2. The organic layer was dried over sodium sulfate and
concentrated to dryness. The crude product was chromato-
graphed over silica gel to give 214 mg of compound 26 (60%).
MS (ESI) m/z 286 (M + H)+; IR: 3422, 1740, 1562, 1523, 1312,
1340 cm-1; 1H NMR (300 MHz, DMSO-d6) δ 11.96 (1H, s, NH),
9.86 (1H, s, H-1), 8.80 (1H, d, J ) 6.0 Hz, H-3), 8.55 (1H, d, J
) 8.1 Hz, H-7), 8.10 (1H, d, J ) 8.4 Hz, H-6), 8.00 (1H, d, J )
5.7 Hz, H-4).
5-Am in o-8-n itr oisoqu in olin e (27). Compound 26 (100 mg,
0.35 mmol) was hydrolyzed in MeOH (2.5 mL) and concen-
trated HCl (0.6 mL) at refluxing temperature for 50 min. The
reaction mixture was evaporated in vacuo, basified with
ammonium hydroxide, and extracted with CH2Cl2. The organic
layer was dried over Na2SO4 and evaporated; the crude product
was chromatographed over silica gel to give 57 mg of compound
27 (85%). MS (ESI) m/z 190 (M + H)+; IR: 3328, 1650, 1558,
1276 cm-1; 1H NMR (300 MHz, DMSO-d6) δ 10.16 (1H, s, H-1),
8.60 (1H, d, J ) 5.7 Hz, H-3), 8.45 (1H, d, J ) 8.7 Hz, H-7),
8.17 (1H, d, J ) 5.7 Hz, H-4), 7.82 (2H, s, NH2), 6.83 (1H, d,
J ) 9.0 Hz, H-6).
(SFs) × 100
% control )
(1)
(SFf)
where SFs is the es transporter-specific fluorescence of test
samples, and SFf is the es transporter-specific fluorescence of
the SAENTA-fluor ligand standard in mean channel numbers.
The results were fed into the PRISM program (GraphPad,
San Diego, CA) to derive concentration-dependent curves, as
shown in Figure 1. From these curves, the IC50 values were
obtained and used to calculate inhibition constants (Ki) values
from eq 2
Ki ) IC50/(1 + [L]/KL)
(2)
where [L] and KL are the concentration and the Kd value of
the SAENTA-fluor, respectively.
The Ki values were used to compare the abilities of the new
compounds to displace the es transporter-specific ligand (5-
(SAENTA)-X8-fluorescein,20 and for that matter their affinity
for the es transporter.
Ack n ow led gm en t . Financial support from the
American Heart Association Southeast Affiliate Grant
No. 9950872V is hereby acknowledged. We thank Ms.
Yu-Ping Xiao for providing technical support and per-
forming the flow cytometry experiments.
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