Click modification in the N 6 region of A3 adenosine receptor-selective carbocyclic nucleosides for dendrimeric tethering that preserves pharmacophore recognition

Article abstract of DOI:10.1021/bc200526c

Adenosine derivatives were modified with alkynyl groups on N⁶ substituents for linkage to carriers using Cu(I)-catalyzed click chemistry. Two parallel series, both containing a rigid North-methanocarba (bicyclo[3.1.0] hexane) ring system in place of ribose, behaved as A₃ adenosine receptor (AR) agonists: (5′-methyluronamides) or partial agonists (4′-truncated). Terminal alkynyl groups on a chain at the 3 position of a N⁶-benzyl group or simply through a N⁶-propargyl group were coupled to azido derivatives, which included both small molecules and G4 (fourth-generation) multivalent poly(amidoamine) (PAMAM) dendrimers, to form 1,2,3-triazolyl linkers. The small molecular triazoles probed the tolerance in A₃AR binding of distal, sterically bulky groups such as 1-adamantyl. Terminal 4-fluoro-3-nitrophenyl groups anticipated nucleophilic substitution for chain extension and ¹⁸F radiolabeling. N⁶-(4-Fluoro-3- nitrophenyl)-triazolylmethyl derivative 32 displayed a K_i of 9.1 nM at A₃AR with ～1000-fold subtype selectivity. Multivalent conjugates additionally containing click-linked water-solubilizing polyethylene glycol groups potently activated A₃AR in the 5′- methyluronamide, but not 4′ truncated series. N⁶-Benzyl nucleoside conjugate 43 (apparent K_i 24 nM) maintained binding affinity of the monomer better than a N⁶-triazolylmethyl derivative. Thus, the N⁶ region of 5′-methyluronamide derivatives, as modeled in receptor docking, is suitable for functionalization and tethering by click chemistry to achieve high A₃AR agonist affinity and enhanced selectivity. This article not subject to U.S. Copyright. Published 2011 by the American Chemical Society.

Full text of DOI:10.1021/bc200526c

Article
pubs.acs.org/bc
Click Modification in the N6 Region of A3 Adenosine Receptor-
Selective Carbocyclic Nucleosides for Dendrimeric Tethering that
Preserves Pharmacophore Recognition
Dilip K. Tosh, Khai Phan, Francesca Deflorian, Qiang Wei, Lena S. Yoo, Zhan-Guo Gao,
and Kenneth A. Jacobson*
Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney
Diseases, National Institutes of Health, Bethesda, Maryland 20892, United States
S
* Supporting Information
ABSTRACT: Adenosine derivatives were modified with
alkynyl groups on N6 substituents for linkage to carriers
using Cu(I)-catalyzed click chemistry. Two parallel series, both
containing a rigid North-methanocarba (bicyclo[3.1.0]hexane)
ring system in place of ribose, behaved as A3 adenosine
receptor (AR) agonists: (5′-methyluronamides) or partial
agonists (4′-truncated). Terminal alkynyl groups on a chain
at the 3 position of a N6-benzyl group or simply through a N6-
propargyl group were coupled to azido derivatives, which
included both small molecules and G4 (fourth-generation)
multivalent poly(amidoamine) (PAMAM) dendrimers, to
form 1,2,3-triazolyl linkers. The small molecular triazoles
probed the tolerance in A3AR binding of distal, sterically bulky
groups such as 1-adamantyl. Terminal 4-fluoro-3-nitrophenyl groups anticipated nucleophilic substitution for chain extension and
18F radiolabeling. N6-(4-Fluoro-3-nitrophenyl)-triazolylmethyl derivative 32 displayed a Ki of 9.1 nM at A3AR with ∼1000-fold
subtype selectivity. Multivalent conjugates additionally containing click-linked water-solubilizing polyethylene glycol groups
potently activated A3AR in the 5′-methyluronamide, but not 4′ truncated series. N6-Benzyl nucleoside conjugate 43 (apparent Ki
24 nM) maintained binding affinity of the monomer better than a N6-triazolylmethyl derivative. Thus, the N6 region of 5′-
methyluronamide derivatives, as modeled in receptor docking, is suitable for functionalization and tethering by click chemistry to
achieve high A3AR agonist affinity and enhanced selectivity.
to full agonist,19 and detailed structure activity relationship
(SAR) studies have already been conducted. A prototypical
A3AR agonist, e.g., ribose 5′-N-methyluronamide Cl-IB-MECA
INTRODUCTION
Selective ligands of the A3 adenosine receptor (AR), a G
■
protein-coupled receptor (GPCR), have antiinflammatory,
anticancer, antiischemic, and myeloprotective activities.1,2
1, is highly receptor subtype-selective and has entered clinical
trials for liver cancer.3 The 4′-thio modification, present in the
A3AR agonists are already in clinical trials for liver cancer,
rheumatoid arthritis, psoriasis, and dry eye disease, with
potent agonist 2, was found to be well-tolerated in receptor
binding.20 Later generation carbocyclic nucleosides containing a
additional envisioned applications for bowel inflammation,
ischemia, and other autoimmune inflammatory disorders.3−7
North (N)-methanocarba (bicyclo[3.1.0]hexane) ring system
in place of the ribose tetrahydrofuryl moiety have led to A3AR
The feasibility of using A3AR antagonists for the treatment of
glaucoma has been demonstrated in animal models.8−10
agonists of high affinity and selectivity. These agonists include
the antiischemic nucleoside MRS3558 3 and its 3-iodo
analogue 4, in which the rigid, fused bicyclic system maintains
a receptor-preferred conformation.21 A parallel approach to
selective binding to the A3AR is based on truncation of the 5′-
uronamide group in ribonucleosides, 4′-thionucleosides, and
carbocyclics, as in 5−8. The effect of truncation is to reduce
efficacy in activation of the receptor, while not precluding
Poly(amidoamine) (PAMAM) dendrimers may serve as
biocompatible carriers for various GPCR drugs, for example,
multivalent nucleoside conjugates for activation of ARs that do
not require drug cleavage.11−13 Such GPCR ligand−dendrimer
(GLiDe) conjugates are sufficiently large to bridge multiple
binding sites present in GPCR dimers and other higher-order
receptor aggregates and have already demonstrated enhanced
pharmacological properties in comparison to the monomeric
ligands.14−18
Received: September 23, 2011
Revised: November 30, 2011
Published: December 17, 2011
Monomeric nucleoside derivatives that bind selectively to the
A3AR have demonstrated a range of efficacies, from antagonist
This article not subject to U.S. Copyright.
Published 2011 by the American Chemical
Society
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Article
selectivity of binding to the A3AR. Thus, truncated oxo 5 and
thio 6 nucleosides are antagonists of the A3AR, and truncated
3-halo substituted 7 and 8 are partial agonists.22−24 Compound
6 lowered intraocular pressure in a mouse model of glaucoma,9
an action characteristic of A3AR antagonists, and compounds 7
and 8 were radiolabeled with 76Br and 125I, respectively, for use
as radioligand probes of the A3AR in vitro and in vivo.25 These
agonists, antagonists, and partial agonists all contained a 3-
substituted benzyl group at the N6 position, which enhanced
A3AR selectivity (Chart 1).
reactions were monitored by thin-layer chromatography
(TLC) using silica gel coated plates with a fluorescence
indicator which were visualized: (a) under UV light, (b) by
dipping in a mixture of anisaldehyde (2.5 mL)/conc. H2SO4 (5
mL)/methanol (425 mL), or (c) by dipping the plate in a
solution of ninhydrin (0.3 g in 100 mL EtOH, containing
AcOH, 1.3 mL) followed by heating. Silica gel column
chromatography was performed with silica gel (SiO2, 200−
400 mesh, 60 Å) using moderate air pressure. Evaporation of
solvents was carried out under reduced pressure at a
temperature below 50 °C. After column chromatography,
appropriate fractions were pooled, evaporated, and dried at
high vacuum for at least 12 h to give the desired products in
Chart 1. Representative Nucleosides That Bind to the A3AR
a
with High Affinity and Selectivity
1
high purity. H NMR ascertained sample purity and spectra
were recorded with a Bruker 400 MHz NMR spectrometer.
Chemical shifts are reported in parts per million (ppm) relative
to tetramethylsilane or using deuterated solvent as the internal
standard (δH: CDCl3 7.26 ppm). For 19F NMR spectra,
CF3CO2H was set as 0 ppm. ESI−high resolution mass
spectroscopic (HRMS) measurements were performed on a
proteomics optimized Q-TOF-2 (Micromass-Waters) using
external calibration with polyalanine. Observed mass accuracies
are those expected on the basis of known performance of the
instrument as well as the trends in masses of standard
compounds observed at intervals during the series of
measurements. Reported masses are observed masses un-
corrected for this time-dependent drift in mass accuracy.
(1S,2R,3S,4R,5S)-4-[2-Chloro-6-{3-(1,6-heptadiynyl)}-9H-
purin-9-yl]-2,3-dihydroxybicyclo[3.1.0]hexane-1-carboxylic
Acid N-Methylamide (9). To a solution of compound 4 (50
mg, 0.09 mmol) in anhydrous DMF (2 mL), PdCl2(PPh3)2
(12.6 mg, 0.01 mmol), CuI (1.7 mg, 0.008 mmol), 1,6-
heptadiyne (0.1 mL, 0.9 mmol), and then triethylamine (0.12
mL, 0.89 mmol) were added and stirred overnight at room
temperature. Solvent was evaporated under vacuum, and the
residue was purified on flash silica gel column chromatography
(CH2Cl2:MeOH = 60:1) to give compound 9 (38 mg, 81%) as
syrup. 1H NMR (CDCl3, 400 MHz) δ 7.8 (s, 1H), 7.42 (s, 1H),
7.24−7.30 (m, 3H), 5.03 (d, J = 6.0 Hz, 1H), 4.84 (s, 1H), 4.79
(br s, 1H), 4.13 (d, J = 6.4 Hz, 1H), 2.94 (s, 3H), 2.55 (t, J =
7.2 Hz, 2H), 2.36−2.41 (m, 2H), 2.18−2.22 (m, 1H), 2.00 (t, J
= 2.8 Hz, 1H), 1.68 (t, J = 9.2 Hz, 1H), 1.85−1.79 (m, 2H),
1.37−1.41 (m, 1H). HRMS calculated for C27H28ClN6O3 (M +
H)+: 519.1911; found 519.1930.
a
Binding Ki values in nM at the human homologue given.
This study applies to multivalent GLiDe conjugates the SAR
developed for monomeric nucleosides as receptor agonists. The
linking step involved click chemistry, which encompasses
various principles of reactivity and has been used extensively
for orthogonal coupling of small molecules to polymers.26,27 In
particular, Cu(I)-catalyzed [3 + 2] cycloaddition reactions that
combine azido and acetylene moieties to form triazole rings are
a convenient and efficient means of linking two molecules.28
Recently, we incorporated dialkynyl groups on an extended
adenine C2 substituent of AR agonists for tethering to
PAMAM dendrimers by Cu(I)-catalyzed click chemistry.29 In
the present series, we have applied this chemistry as a
functionalization approach around the N6 region of adenosine
and its family of A3AR agonists. As with the C2-conjugated
series, the proximal alkyne is designed to promote receptor
recognition, and the distal alkyne may serve as a site for
coupling to macromolecular carriers while maintaining receptor
recognition. Thus, we have used click chemistry to identify
another region on the nucleoside for covalent attachment that
is distal from the pharmacophore and does not interfere with
binding/activation. This region of the nucleosides is predicted
by modeling to extend into the extracellular portion of the
A3AR.23,30
(1S,2R,3S,4R,5S)-4-[2-Chloro-6-{3-(1,7-octadiynyl)}-9H-
purin-9-yl]-2,3-dihydroxybicyclo[3.1.0]hexane-1-carboxylic
Acid N-Methylamide (10). Compound 10 (85%) was
synthesized from compound 4 and 1,7-octadiyne following
1
the same procedure as for compound 2. H NMR (CD3OD,
400 MHz) δ 8.04 (s, 1H), 7.40 (s, 1H), 7.26−7.34 (m, 3H),
5.08 (d, J = 6.8 Hz, 1H), 4.81 (s, 1H), 4.73 (br s, 2H), 4.60 (br
s, 2H), 4.01 (d, J = 6.4 Hz, 1H), 2.87 (s, 3H), 2.43 (t, J = 6.8
Hz, 2H), 2.21−2.26 (m, 2H), 2.05−2.08 (m, 1H), 1.68−1.71
(m, 1H), 1.38−1.40 (m, 1H). HRMS calculated for
C28H30ClN6O3 (M + H)+: 533.2068; found 533.2070.
(1S,2R,3S,4R,5S)-4-[2-Chloro-6-{3-(1,8-nonadiynyl)}-9H-
purin-9-yl]-2,3-dihydroxybicyclo[3.1.0]hexane-1-carboxylic
Acid N-Methylamide (11). Compound 11 (79%) was
synthesized from compound 4 and 1,8-nonadiyne following
the same procedure as for compound 2. 1H NMR (CDCl3, 400
MHz) δ 7.92 (s, 1H), 7.43 (s, 1H), 7.25−7.32 (m, 3H), 5.10
(d, J = 6.6 Hz, 1H), 4.85 (s, 1H), 4.78 (br s, 2H), 4.15 (d, J =
6.3 Hz, 1H), 2.93 (s, 3H), 2.42 (t, J = 6.0 Hz, 2H), 2.22−2.23
EXPERIMENTAL PROCEDURES
■
Chemistry. General Methods. All reagents and solvents
(regular and anhydrous) were of analytical grade and obtained
from commercial suppliers and used without further
purification. Reactions were conducted under an atmosphere
of nitrogen whenever anhydrous solvents were used. All
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Article
(m, 2H), 1.97 (t, J = 2.8 Hz, 1H), 1.71−1.78 (m, 2H), 1.58−
1.61 (m, 6H), 1.48−1.52 (m, 1H). HRMS calculated for
C29H32ClN6O3 (M + H)+: 547.2224; found 547.2239.
(1S,2R,3S,4R,5S)-4-(2-Chloro-6-(3-(5-(1-(4-fluoro-3-amino-
phenyl)-1H-1,2,3-triazol-4-yl)pent-1-ynyl)benzylamino)-9H-
purin-9-yl)-2,3-dihydroxybicyclo[3.1.0]hexane-1-carboxylic
Acid-N-methylamide (16). Zinc dust (3 mg, 0.045 mmol) was
added to a solution of compound 12 (5 mg, 0.007 mmol) in
acetonitrile (0.5 mL)/acetic acid (0.5 mL) at 0 °C. After
addition, the reaction mixture was brought to room temper-
ature and stirred for 2 h under the same conditions. The
reaction mixture was filtered on a Celite bed, and the filtrate
was evaporated under vacuum. The residue was purified on
flash silica gel column chromatography (CH2Cl2:MeOH =
(1S,2R,3S,4R,5S)-4-(2-Chloro-6-(3-(5-(1-(4-fluoro-3-nitro-
phenyl)-1H-1,2,3-triazol-4-yl)pent-1-ynyl)benzylamino)-9H-
purin-9-yl)-2,3-dihydroxybicyclo[3.1.0]hexane-1-carboxylic
Acid-N-methylamide (12). 4-Fluoro-3-nitro-phenylazide (9.3
mg, 0.051 mmol) and TBTA31 (1 mg, 0.001 mmol) were added
to a solution of compound 9 (19 mg, 0.036 mmol) in t-butanol
(0.6 mL)/CH2Cl2 (0.6 mL)/water (0.6 mL) at room
temperature. Freshly prepared 1 M solution of sodium
ascorbate (36.6 μL) followed by 7.5% solution of copper
sulfate (60.7 μL, 0.018 mmol) was also added into the reaction
mixture and stirred at room temperature overnight. Solvent was
evaporated, and the residue was purified on flash silica gel
column chromatography (CH2Cl2:MeOH = 40:1) to give
1
20:1) to give compound 16 (3 mg, 62%). H NMR (CD3OD,
400 MHz) δ 8.65−8.58 (m, 1H).
(1S,2R,3S,4R,5S)-4-(2-Chloro-6-(3-(7-(1-(4-fluoro-3-amino-
phenyl)-1H-1,2,3-triazol-4-yl)hept-1-ynyl)benzylamino)-9H-
purin-9-yl)-2,3-dihydroxybicyclo[3.1.0]hexane-1-carboxylic
Acid-N-methylamide (17). Compound 17 (64%) was synthe-
sized from compound 14 following the same procedure as for
1
compound 12 (23 mg, 90%) as a syrup. H NMR (CD3OD,
400 MHz) δ 8.53−8.62 (m, 1H), 8.49 (s, 1H), 8.22−8.19 (m,
1H), 8.04 (s, 1H), 7.60−7.67 (m, 2H), 7.39−23 (m, 3H), 5.05
(d, J = 5.6 Hz, 1H), 4.81 (s, 1H), 4.71 (br s, 2H), 4.00 (d, J =
6.8 Hz, 1H), 2.98 (t, J = 7.6 Hz, 2H), 2.86 (s, 3H), 2.53 (t, J =
6.8 Hz, 2H), 2.02−2.08 (m, 3H), 1.82 (t, J = 4.8 Hz, 1H),
1.36−1.39 (m, 1H). HRMS calculated for C33H31ClFN8O3 (M
+ H)+: 701.2151; found 701.2175.
1
compound 16. H NMR (CD3OD, 400 MHz) δ 8.12 (s, 1H),
8.03 (s, 1H), 7.38 (s, 1H), 7.32−7.21 (m, 4H), 7.06−7.02 (m,
1H), 6.90−6.86 (m, 1H), 5.08 (d, J = 7.2 Hz, 1H), 4.80 (s,
1H), 4.70 (br s, 2H), 4.01 (d, J = 6.4 Hz, 1H), 2.87 (s, 3H),
2.80 (t, J = 7.2 Hz, 2H), 2.44 (t, J = 6,8 Hz, 2H), 2.02−2.09 (m,
1H), 1.83−1.78 (m, 2H), 1.68−1.54 (m, 5H), 0.89−0.93 (m,
1H). HRMS calculated for C40H37ClFN8O (M + H)+:
699.2763; found 699.2745.
(1S,2R,3S,4R,5S)-4-(2-Chloro-6-(3-(6-(1-(4-fluoro-3-nitro-
phenyl)-1H-1,2,3-triazol-4-yl)hex-1-ynyl)benzylamino)-9H-
purin-9-yl)-2,3-dihydroxybicyclo[3.1.0]hexane-1-carboxylic
Acid-N-methylamide (13). Compound 13 (94%) was synthe-
sized from compound 10 following the same procedure as for
compound 12. 1H NMR (CD3OD, 400 MHz) δ 8.65−8.58 (m,
1H), 8.45 (s, 1H), 8.23−8.19 (m, 1H), 8.03 (s, 1H), 7.65 (t, J =
8.8 Hz, 1H), 7.41 (s, 1H), 7.33−7.25 (m, 2H), 5.07 (d, J = 6.8
Hz, 1H), 4.80 (s, 1H), 4.72 (br s, 2H), 4.00 (d, J = 6.8 Hz, 1H),
2.87 (s, 3H), 2.51 (t, J = 6.8 Hz, 2H), 2.07−2.04 (m, 1H),
1.97−1.91 (m, 2H), 1.82 (t, J = 5.2 Hz, 2H), 1.74−1.69 (m,
2H), 1.39−1.35 (m, 1H). HRMS calculated for
C34H33ClFN10O5 (M + H)+: 715.2308; found 715.2302.
(1S,2R,3S,4R,5S)-4-(2-Chloro-6-(3-(7-(1-(4-fluoro-3-nitro-
phenyl)-1H-1,2,3-triazol-4-yl)hept-1-ynyl)benzylamino)-9H-
purin-9-yl)-2,3-dihydroxybicyclo[3.1.0]hexane-1-carboxylic
Acid-N-methylamide (14). Compound 14 (96%) was synthe-
sized from compound 11 following the same procedure as for
compound 12. 1H NMR (CD3OD, 400 MHz) δ 8.55−8.59 (m,
1H), 8.41 (s, 1H), 8.16−8.12 (m, 1H), 8.03 (s, 1H), 7.60 (t, J =
9.2 Hz, 1H), 7.36 (s, 1H), 7.30−7.20 (m, 2H), 5.08 (d, J = 6.4
Hz, 1H), 4.80 (s, 1H), 4.69 (br s, 2H), 4.01 (d, J = 6.4 Hz, 1H),
2.87−2.82 (m, 5H), 2.44 (t, J = 6.4 Hz, 2H), 2.08−2.05 (m,
1H), 1.84−1.81 (m, 3H), 1.68−1.58 (m, 4H), 1.39−1.38 (m,
1H). HRMS calculated for C35H35ClFN10O5 (M + H)+:
729.2464; found 729.2463.
(1R,2R,3S,4R,5S)-4-(2-Chloro-6-(3-(hepta-1,6-diynyl)-
benzylamino)-9H-purin-9-yl) 2,3-O-isopropylidine-bicyclo-
[3.1.0]hexane (19). PdCl2(PPh3)2 (29.7 mg, 0.04 mmol), CuI
(8.0 mg, 0.04 mmol), and 1,6-heptadiyne (0.24 mL, 2.11
mmol) followed by triethylamine (60 μL, 0.41 mmol) were
added to a solution of compound 18 (114 mg, 0.211 mmol) in
anhydrous DMF (2 mL), and the reaction mixture was stirred
overnight at room temperature. Solvent was evaporated under
vacuum, and the residue was purified on flash silica gel column
chromatography (hexane:ethyl acetate = 2:1) to give
compound 19 (83 mg, 78%) as foamy syrup. 1H NMR
(CDCl3, 400 MHz) δ 7.72 (s, 1H), 7.42 (s, 1H), 7.28−7.42 (m,
3H), 6.31 (br s, 1H), 5.35 (t, J = 6.0 Hz, 1H), 4.98 (s, 1H), 4.81
(br s, 2H), 4.65 (d, J = 7.6 Hz, 1H), 2.55 (t, J = 7.2 Hz, 2H),
2.38 (dd, J1 = 2.8 Hz, J2 = 3.2 Hz, 2H), 2.08−2.14 (m, 1H),
1.99 (t, J = 2.8 Hz, 1H), 1.82−1.85 (m, 2H), 1.64−1.67 (m,
2H), 1.56 (s, 3H), 1.26 (s, 3H), 0.93−1.00 (m, 1H). HRMS
calculated for C28H29ClN5O2 (M + H)+: 502.2032; found
502.2029.
(1R,2R,3S,4R,5S)-4-(2-Chloro-6-(3-(nona-1,8-diynyl)-
benzylamino)-9H-purin-9-yl) 2,3-O-isopropylidene-bicyclo-
[3.1.0]hexane (21). Compound 21 (80%) was synthesized
from compound 18 and 1,8-nonadiyne following the same
procedure as for compound 19. 1H NMR (CDCl3, 400 MHz) δ
7.71 (s, 1H), 7.42 (s, 1H), 7.27−7.34 (m, 3H), 6.33(br s, 1H),
5.35 (t, J = 6.4 Hz, 1H), 4.98 (s, 1H), 4.80 (br s, 2H), 4.64 (d, J
= 7.2 Hz, 1H), 2.41 (t, J = 3.6 Hz, 2H), 2.28−2.21 (m, 3H),
2.14−2.08 (m, 1H), 1.97−1.96 (t, J = 2.8 Hz, 1H), 1.54−1.68
(m, 10H), 1.26 (s, 3H), 0.88−0.99 (m, 1H). HRMS calculated
for C30H33ClN5O2 (M + H)+: 530.2323; found 530.2315.
(1R,2R,3S,4R,5S)-4-(2-Chloro-6-(3-(hepta-1,6-diynyl)-
benzylamino)-9H-purin-9-yl) bicyclo[3.1.0]hexane-2,3-diol
(22). A solution of compound 19 (92 mg, 0.18 mmol) in
CH2Cl2 (4 mL) and trifluoromethane sulfonic acid:water (2:1,
3 mL) was stirred at room temperature overnight. Solvent was
evaporated, and the residue was purified on flash silica gel
chromatography (CH2Cl2:MeOH = 70:1) to give compound
(1S,2R,3S,4R,5S)-4-(2-Chloro-6-(3-(6-(1-(adamantyl)-1H-
1,2,3-triazol-4-yl)hex-1-ynyl)benzylamino)-9H-purin-9-yl)-
2,3-dihydroxybicyclo[3.1.0]hexane-1-carboxylic Acid-N-
methylamide (15). Compound 15 (93%) was synthesized
from compound 10 following the same procedure as for
1
compound 12. H NMR (CD3OD, 400 MHz) δ 8.04 (s, 1H),
7.40 (s, 1H), 7.35−7.26 (m, 3H), 5.09 (d, J = 6.8 Hz, 1H), 4.82
(s, 1H), 4.73 (s, 2H), 4.02 (d, J = 6.8 Hz, 1H), 2.87 (s, 1H),
2.44 (t, J = 6.8 Hz, 2H), 2.26−2.21 (m, 3H), 2.17 (m, 8H),
2.08−2.05 (m, 1H), 1.82 (t, J = 4.8 Hz, 1H), 1.70−1.69 (m,
10H), 1.39−1.36 (m, 1H), 0.94−0.88 (m, 1H). HRMS
calculated for C38H45ClN9O3 (M + H)+: 710.3257; found
710.3248.
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1
22 (77 mg, 92%) as a syrup. H NMR (CDCl3, 400 MHz) δ
7.80 (s, 1H), 7.42 (s, 1H), 7.29−7.35 (m, 3H), 6.41 (br s, 1H),
4.87 (s, 1H), 4.81 (br s, 2H), 4.02 (d, J = 6.4 Hz, 1H), 4.00 (s,
1H), 2.69 (d, J = 6.3 Hz, 1H), 2.56 (t, J = 6.8 Hz, 2H), 2.36−
2.41 (m, 2H), 2.03−2.08 (m, 1H), 2.00 (t, J = 2.8 Hz, 1H),
1.85−1.82 (m, 2H), 1.68−1.64 (m, 1H), 1.26−1.30 (m, 2H),
0.90−0.80 (m, 1H). HRMS calculated for C25H25ClN5O2 (M +
H)+: 462.1697; found 462.1696.
purin-9-yl)-bicyclo[3.1.0]hexane-2,3-diol (26). Compound 26
(95%) was synthesized from compound 23 following the same
procedure as for compound 25. 1H NMR (CDCl3, 400 MHz) δ
8.44−8.39 (m, 1H), 8.07−8.11 (m, 1H), 7.83 (s, 1H), 7.77 (s,
1H), 7.51−7.43 (m, 2H), 7.33−7.26 (m, 3H), 6.58 (br s, 1H),
4.87 (s, 1H), 4.79 (br s, 2H), 4.15 (s, 1H), 4.02 (d, J = 6.8 Hz,
1H), 2.89 (t, J = 7.6 Hz, 2H), 2.49 (t, J = 6.8 Hz, 2H), 2.08−
2.04 (m, 1H), 1.96−1.90 (m, 2H), 1.76−1.64 (m, 3H), 1.31−
1.28 (m, 1H), 0.79−0.85 (m, 1H). HRMS calculated for
C32H30ClFN9O4 (M + H)+: 658.2093; found 658.2124.
(1R,2R,3S,4R,5S)-4-(2-Chloro-6-(3-(7-(1-(4-fluoro-3-nitro-
phenyl)-1H-1,2,3-triazol-4-yl)hept-1-ynyl)benzylamino)-9H-
purin-9-yl)bicyclo[3.1.0]hexane-2,3-diol (27). Compound 27
(91%) was synthesized from compound 24 following the same
procedure as for compound 25. 1H NMR (CD3OD, 400 MHz)
δ 8.56−8.54 (m, 1H), 8.40 (s, 1H), 8.13−8.18 (m, 2H), 7.60 (t,
J = 8.8 Hz, 1H), 7.37 (s, 1H), 7.30 (d, J = 6.8 Hz, 1H), 7.24−
7.21 (m, 2H), 4.79 (s, 1H), 4.72−4.69 (m, 3H), 3.90 (d, J = 6.4
Hz, 1H), 2.84 (t, J = 7.2 Hz, 2H), 2.45 (t, J = 6.8 Hz, 2H),
2.01−1.95 (m, 1H), 1.86−1.79 (m, 2H), 1.68−1.58 (m, 5H),
1.33−1.30 (m, 1H), 0.82−0.72 (m, 1H). HRMS calculated for
C33H32ClFN9O4 (M + H)+: 672.2250; found 672.2245.
(1R,2R,3S,4R,5S)-4-(2-Chloro-6-(3-(octa-1,7-diynyl)-
benzylamino)-9H-purin-9-yl) bicyclo[3.1.0]hexane-2,3-diol
(23). PdCl2(PPh3)2 (5.7 mg, 0.008 mmol), CuI (1.0 mg,
0.005 mmol), and 1,7-octadiyne (27 μL, 0.2 mmol) followed by
triethylamine (57 μL, 0.4 mmol) were added to a solution of
compound 18 (20.4 mg, 0.04 mmol) in anhydrous DMF (1.2
mL) and the reaction mixture was stirred overnight at room
temperature. Solvent was evaporated under vacuum, and the
residue was roughly purified on flash silica gel column
chromatography and the obtained product was dissolved in
CH2Cl2 (3 mL) and 2 mL of trifluoromethane sulfonic acid−
water (2:1) were added into it and stirred at room temperature
overnight. Solvent was evaporated, and the residue was purified
on flash silica gel chromatography (CH2Cl2:MeOH = 60:1) to
1
give compound 23 (12.6 mg, 70%) as a syrup. H NMR
(1S,2R,3S,4R,5S)-Ethyl-4-(2-chloro-6-(prop-2-ynylamino)-
9H-purin-9-yl)-2,3-O-isopropylidine-bicyclo[3.1.0]hexane-1-
carboxylate (29). Propargylamine (0.5 mL, 7.5 mmol) and
triethylamine (2.1 mL, 15 mmol) were added to a solution of
compound 28 (620 mg, 0.14 mmol) in anhydrous methanol
(10 mL) and stirred overnight at room temperature. Solvent
was evaporated, and the residue was purified on flash silica gel
column chromatography (hexane:ethyl acetate = 1:1) to give
(CDCl3, 400 MHz) δ 7.80 (s, 1H), 7.42 (s, 1H), 7.35−7.29 (m,
3H), 6.43 (br s, 1H), 4.88 (s, 1H), 4.80 (br s, 2H), 4.03 (d, J =
7.2 Hz, 1H), 3.97 (s, 1H), 2.64 (m, 1H), 2.45 (t, J = 6.4 Hz,
2H), 2.29−2.26 (m, 3H), 2.08−2.06 (m, 1H), 1.98 (t, J = 2.4
Hz, 1H), 1.71−1.66 (m, 3H), 1.28−1.31 (m, 1H), 0.86−0.82
(m, 1H). HRMS calculated for C26H27ClN5O2 (M + H)+:
476.1853; found 476.1869.
(1R,2R,3S,4R,5S)-4-(2-Chloro-6-(3-(nona-1,8-diynyl)-
benzylamino)-9H-purin-9-yl) bicyclo[3.1.0]hexane-2,3-diol
(24). Compound 24 (89%) was synthesized from compound
1
compound 29 (511 mg, 79%) as a syrup. H NMR (CDCl3,
400 MHz) δ 7.73 (s, 1H), 6.20 (br s, 1H), 5.89 (d, J = 7.0 Hz,
1H), 4.88 (s, 1H), 4.74 (d, J = 7.1 Hz, 1H), 4.47 (br s, 2H),
4.32−4.28 (m, 2H), 4.27−4.13 (m, 1H), 2.32 (s, 1H), 2.24−
2.20 (m, 1H), 1.76−1.72 (m, 1H), 1.68 (s, 3H), 1.57−1.53 (m,
4H), 1.36 (t, J = 7.2 Hz, 3H), 1.30−1.25 (m, 1H). HRMS
calculated for C20H23ClN5O4 (M + H)+: 432.1367; found
432.1362.
(1S,2R,3S,4R,5S)-4-(2-Chloro-6-(prop-2-ynylamino)-9H-
purin-9-yl)-2,3-O-isopropylidine-N-methyl bicyclo[3.1.0]-
hexane-1-carboxamide (30). Methylamine (40% solution, 3
mL) was added to a solution of compound 29 (244 mg, 0.56
mmol) in methanol (9 mL) and stirred for 2 d at room
temperature. Solvent was evaporated under vacuum, and the
residue was purified on flash silica gel column chromatography
(CH2Cl2:MeOH = 70:1) to give compound 30 (148 mg, 63%)
as a syrup. 1H NMR (CD3OD, 400 MHz) δ 8.08 (s, 1H), 5.75
(d, J = 7.2 Hz, 1H), 4.97 (s, 1H), 4.84 (d, J = 6.8 Hz, 1H), 4.35
(br s, 2H), 2.86 (s, 3H), 2.63 (t, J = 2.4 Hz, 1H), 2.15−2.11 (m,
1H), 1.57−1.54 (m, 4H), 1.40 (t, J = 5.2 Hz, 1H), 1.29 (s, 3H).
HRMS calculated for C19H21ClN6O3 (M + H)+: 416.1364;
found 416.1374.
(1S,2R,3S,4R,5S)-4-(2-Chloro-6-(prop-2-ynylamino)-9H-
purin-9-yl)-2,3-dihydroxy-N-methylbicyclo[3.1.0]hexane-1-
carboxamide (31). 10% Trifluoroacetic acid (3 mL) was added
to a solution of compound 30 (70 mg, 0.167 mmol) in
methanol (4 mL) and heated at 70 °C overnight. Solvent was
evaporated under vacuum, and the residue was purified on flash
silica gel column chromatography (CH2Cl2:MeOH = 40:1) to
give compound 31 (55 mg, 87%) as a syrup. 1H NMR
(CD3OD, 400 MHz) δ 8.06 (s, 1H), 5.09 (d, J = 5.6 Hz, 1H),
4.8 (s, 1H), 4.36 (br s, 2H), 4.01 (d, J = 6.4 Hz, 1H), 2.88 (s,
3H), 2.63 (t, J = 2.4 Hz, 1H), 2.08−2.04 (m, 1H), 1.82 (t, J =
1
21 following the same procedure as for compound 22. H
NMR (CD3OD, 400 MHz) δ 8.15 (s, 1H), 7.40 (s, 1H), 7.26−
7.33 (m, 3H), 4.80 (s, 1H), 4.74−4.69 (m, 1H), 4.60 (br s,
2H), 3.92 (d, J = 6.4 Hz, 1H), 2.41 (t, J = 6.4 Hz, 2H), 2.18−
2.19 (m, 3H), 2.01−1.96 (m, 1H), 1.70−1.65 (m, 1H), 1.59−
1.50 (m, 6H), 1.34−1.30 (m, 1H), 0.80−0.74 (m, 1H). HRMS
calculated for C27H29ClN5O2 (M + H)+: 490.2010; found
490.2000.
(1R,2R,3S,4R,5S)-4-(2-Chloro-6-(3-(5-(1-(4-fluoro-3-nitro-
phenyl)-1H-1,2,3-triazol-4-yl)pent-1-ynyl)benzylamino)-9H-
purin-9-yl)-bicyclo[3.1.0]hexane-2,3-diol (25). 4-Fluoro-3-
nitro-phenylazide (7.5 mg, 0.041 mmol) and TBTA (1 mg,
0.001 mmol) were added to a solution of compound 22 (13.5
mg, 0.029 mmol) in CH2Cl2/t-butanol (0.6 mL)/water (0.6
mL) at room temperature. Freshly prepared 1 M solution of
sodium ascorbate (29.2 μL) followed by 7.5% solution of
copper sulfate (48 μL, 0.015 mmol) was also added into the
reaction mixture stirred at room temperature overnight. Solvent
was evaporated, and the residue was purified on flash silica gel
column chromatography (CH2Cl2:MeOH = 45:1) to give
1
compound 25 (17 mg, 91%) as a syrup. H NMR (CD3OD,
400 MHz) δ 861−8.59 (m, 1H), 8.49 (s, 1H), 8.23−8.19 (m,
1H), 8.14 (s, 1H), 7.65 (t, J = 9.2 Hz, 1H), 7.39 (s, 1H), 7.33−
7.24 (m, 3H), 4.79 (s, 1H), 4.72−4.70 (m, 3H), 3.90 (d, J = 6.4
Hz, 1H), 2.99 (t, J = 7.6 Hz, 2H), 2.53 (t, J = 6.8 Hz, 2H),
2.08−1.96 (m, 3H), 1.69−1.65 (m, 1H), 1.33−1.30 (m, 1H),
0.79−0.75 (m, 1H). HRMS calculated for C31H28ClFN9O4 (M
+ H)+: 644.1937; found 644.1962.
(1R,2R,3S,4R,5S)-4-(2-Chloro-6-(3-(6-(1-(4-fluoro-3-nitro-
phenyl)-1H-1,2,3-triazol-4-yl)hex-1-ynyl)benzylamino)-9H-
235
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Article
4.8 Hz, 1H), 1.40−1.37 (m, 1H). HRMS calculated for
C16H18ClN6O3 (M + H)+: 377.1129; found 377.1124.
300 MHz) δ 7.80 (s, 1H), 6.25 (br s, 1H), 5.34 (t, J = 5.7 Hz,
1H), 4.98 (s, 1H), 4.64 (d, J = 7.2 Hz, 1H), 4.48 (s, 2H), 2.03−
2.12 (m, 1H), 1.78 (s, 1H), 1.62−1.67 (m, 1H), 1.54 (s, 3H),
1.25 (s, 3H), 0.88−0.98 (m, 2H). HRMS calculated for
C17H19ClN5O2 (M + H)+: 360.1227; found 360.1218.
(1R,2R,3S,4R,5S)-4-(2-Chloro-6-(prop-2-ynylamino)-9H-
purin-9-yl) bicyclo[3.1.0]hexane-2,3-diol (38). A solution of
compound 37 (40 mg, 0.11 mmol) in methanol (3 mL) and
10% trifluoromethane sulfonic acid−water (3 mL) was heated
at 70 °C overnight. Solvent was evaporated, and the residue was
purified on flash silica gel chromatography (CH2Cl2:MeOH =
50:1) to give compound 38 (33 mg, 93%) as a syrup. 1H NMR
(CD3OD, 300 MHz) δ 8.15 (s, 1H), 4.79 (s, 1H), 4.70 (t, J =
6.0 Hz, 1H), 4.36 (s, 2H), 3.89 (d, J = 6.6 Hz, 1H), 2.62 (s,
1H), 1.95−2.01 (m, 1H), 1.62 −1.68 (m, 1H), 1.28−1.32 (m,
1H), 0.72−0.79 (m, 1H). HRMS calculated for C14H15ClN5O2
(M + H)+: 320.0914; found 320.0898.
(1S,2R,3S,4R,5S)-4-(2-Chloro-6-((1-(4-fluoro-3-nitrophen-
yl)-1H-1,2,3-triazol-4-yl)methylamino)-9H-purin-9-yl)-2,3-di-
hydroxy-N-methylbicyclo[3.1.0]hexane-1-carboxamide (32).
4-Fluoro-3-nitro-phenylazide (9.7 mg, 0.053 mmol) and TBTA
(1 mg, 0.001 mmol) were added to a solution of compound 31
(14.4 mg, 0.038 mmol) in t-butanol (0.8 mL)/water (0.8 mL)
at room temperature. Freshly prepared 1 M solution of sodium
ascorbate (38.2 μL) followed by 7.5% solution of copper sulfate
(63 μL, 0.019 mmol) was also added into the reaction mixture
stirred at room temperature overnight. Solvent was evaporated,
and the residue was purified on flash silica gel column
chromatography (CH2Cl2:MeOH = 30:1) to give compound
1
32 (19 mg, 92%) as a syrup. H NMR (CD3OD, 400 MHz) δ
8.62−8.61 (m, 2H), 8.27−8.24 (m, 1H), 8.06 (s, 1H), 7.69−
7.64 (m, 1H), 5.08 (d, J = 6.8 Hz, 1H), 4.94 (br s, 2H), 4.81 (s,
1H), 4.01 (d, J = 6.0 Hz, 1H), 2.87 (s, 3H), 2.07−2.04 (m,1H),
1.82 (t, J = 4.8 Hz, 1H), 1.39−1.37 (m, 1H). HRMS calculated
for C22H21ClFN10O5 (M + H)+: 559.1369; found 559.1346.
(1S,2R,3S,4R,5S)-4-(2-Chloro-6-((1-(4-fluoro-3-amino)-1H-
1,2,3-triazol-4-yl) methylamino)-9H-purin-9-yl)-2,3-dihy-
droxy-N-methylbicyclo[3.1.0]hexane-1-carboxamide (33).
Compound 33 (73%) was synthesized from compound 32
(1R,2R,3S,4R,5S)-4-(2-Chloro-6-((1-(4-fluoro-3-nitrophen-
yl)-1H-1,2,3-triazol-4-yl)methylamino)-9H-purin-9-yl)bicyclo-
[3.1.0]hexane-2,3-diol (39). 4-Fluoro-3-nitro-phenylazide
(9.88 mg, 0.054 mmol) and TBTA (1 mg, 0.001 mmol) were
added to a solution of compound 38 (12.4 mg, 0.038 mmol) in
t-butanol (0.6 mL)/water (0.6 mL) at room temperature. A
freshly prepared 1 M solution of sodium ascorbate (38.7 μL)
followed by 7.5% solution of copper sulfate (51.6 μL, 0.015
mmol) were also added to the reaction mixture, which was then
stirred at room temperature overnight. Solvent was evaporated,
and the residue was purified on flash silica gel column
chromatography (CH2Cl2:MeOH = 30:1) to give compound
1
following the same procedure as for compound 16. H NMR
(CD3OD, 400 MHz) d 8.32 (s, 1H), 8.06 (s, 1H), 7.27−7.25
(m, 1H), 7.13−7.08 (m, 1H), 6.99−6.95 (m, 1H), 5.08 (d, J =
7.2 Hz, 1H), 4.82 (s, 2H), 4.00 (d, J = 6.8 Hz, 1H), 2.87 (s,
3H), 2.07−2.04 (m,1H), 1.82 (t, J = 4.8 Hz, 1H), 1.40−1.38
+
1
(m, 1H). HRMS calculated for C22H23ClFN10O3 (M + H) :
39 (18 mg, 96%) as a syrup. H NMR (CD3OD, 300 MHz) δ
529.1627; found 529.1616.
8.60−8.64 (m, 2H), 8.22−8.27 (m, 1H), 8.15 (s, 1H), 7.62−
7.68 (m, 1H), 4.79 (s, 1H), 4.70 (t, J = 5.1 Hz, 1H), 4.37 (s,
2H), 3.88 (d, J = 6.6 Hz, 1H), 2.62 (t, J = 2.4 Hz, 1H), 1.95−
1.99 (m, 1H), 1.62−1.68 (m, 1H), 1.26−1.32 (m, 1H), 0.72−
0.79 (m, 1H). HRMS calculated for C20H18ClFN9O4 (M +
H)+: 502.1154; found 502.1150.
(1R,2R,3S,4R,5S)-4-(2-Chloro-6-(1-adamantyl-1H-1,2,3-tri-
azol-4-yl)methylamino)-9H-purin-9-yl)bicyclo[3.1.0]hexane-
2,3-diol (40). Compound 40 (92%) was prepared from
compound 38 following the same method for compound 37.
1H NMR (CD3OD, 300 MHz) δ 8.13 (s, 1H), 8.02 (s, 1H),
4.81−4.84 (m, 2H), 4.79 (s, 1H), 4.70 (t, J = 6.0 Hz, 1H), 3.89
(d, J = 6.0 Hz, 1H), 2.24 (s, 10H), 1.95−1.97 (m, 1H), 1.82 (s,
6H), 1.63−1.66 (m, 1H), 1.28−1.32 (m, 1H), 0.73−0.77 (m,
1H). HRMS calculated for C24H30ClN8O2 (M + H)+:
497.2180; found 497.2164.
Dendrimer Conjugates. Synthetic procedures for each of
three dendrimer conjugates are provided below. After reaction
in DMSO as specified, each conjugate was separated from small
molecular weight impuries by extensive dialysis for 2 d and then
lyophilized. We utilized Spectra/Por dialysis tubing, MWCO
3500, diameter 11.5 mm (Spectrum Laboratories, Rancho
Dominguez, CA, USA). Typically, 10−15 mg of dendrimer
from the reaction mixture was to a volume of 5−7 mL with
DMSO, transferred to the tubing, and placed in stirring water in
a 1.5 L beaker. The water was changed every 2 h, 5× daily, for 3
d. Then, after lyophilization each dendrimer conjugate was
redissolved in DMSO (∼1 mL per 2 mg) and treated with an
equal volume of aqueous sodium−EDTA solution (0.5 M) to
remove the residual copper by complexation. The mixture was
stirred overnight at room temperature followed by extensive
dialysis for 2 d as before and lyophilization to give the desired
dendrimer conjugate.
( 1 S , 2 R , 3 S , 4 R , 5 S ) - 4 - ( 2 - C h l o r o - 6 - ( ( 1 - ( 4 - ( 3 -
fluoropropylcarbamoyl)phenyl)-1H-1,2,3-triazol-4-yl)-
methylamino)-9H-purin-9-yl)-2,3-dihydroxy-N-
methylbicyclo[3.1.0] hexane-1-carboxamide (34). Com-
pound 34 (89%) was synthesized from compound 31 following
1
the same procedure as for compound 32. H NMR (CD3OD,
400 MHz) δ 8.56 (s, 1H), 8.06 (s, 1H), 8.02 (d, J = 8.8 Hz,
2H), 7.96 (d, J = 8.8 Hz, 2H), 5.08 (d, J = 6.8 Hz, 2H), 4.95 (br
s, 2H), 4.82 (s, 1H), 4.61 (t, J = 5.6 Hz, 1H), 4.51 (t, J = 5.6
Hz, 1H), 4.00 (d, J = 6.8 Hz, 1H), 3.54 (t, J = 6.8 Hz, 2H), 2.87
(s, 3H), 2.08−1.96 (m, 3H), 1.82 (t, J = 4.8 Hz, 1H), 1.40−
1.36 (m, 1H). HRMS calculated for C26H29ClFN10O4 (M + H)
+: 599.2046; found 599.2030.
(1S,2R,3S,4R,5S)-4-(2-Chloro-6-((1-(adamantyl)-1H-1,2,3-
triazol-4-yl)methylamino)-9H-purin-9-yl)-2,3-dihydroxy-N-
methylbicyclo[3.1.0]hexane-1-carboxamide (35). Compound
35 (95%) was synthesized from compound 31 following the
1
same procedure as for compound 32. H NMR (CD3OD, 400
MHz) δ 8.04 (s, 1H), 8.03 (s, 1H), 5.08 (d, J = 6.8 Hz, 1H),
4.82 (s, 2H), 4.01 (d, J = 6.4 Hz, 1H), 2.87 (s, 3H), 2.25 (s,
9H), 2.07−2.04 (m, 1H), 1.84−1.81 (m, 7H), 1.40−1.36 (m,
1H). HRMS calculated for C26H33ClN9O3 (M + H)+:
554.2395; found 554.2383.
(1R,2R,3S,4R,5S)-4-(2-Chloro-6-(prop-2-ynylamino)-9H-
purin-9-yl) 2,3-O-isopropylidene-bicyclo[3.1.0]hexane (37).
Propargylamine (47 μL, 0.7 mmol) and triethylamine (0.28
mL, 1.9 mmol) were added to a solution of compound 36 (50
mg, 0.14 mmol) in anhydrous methanol (2 mL) and stirred
overnight at room temperature. Solvent was evaporated, and
the residue was purified on flash silica gel column
chromatography (hexane:ethyl acetate = 1:1) to give
1
compound 37 (45 mg, 85%) as a syrup. H NMR (CD3OD,
236
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Bioconjugate Chemistry
Article
For multiple steps of ultrafiltration and biological assays,
dilute solutions (1 μM) of the dendrimer conjugates were
prepared in 50 mM Tris-HCl (pH 7.5) and 10 mM MgCl2
buffer. Wash buffer solutions whose concentrations of Tris-
HCl, MgCl2, and DMSO match those of the solutions of
dendrimer conjugates were also prepared for rinsing the
centrifugal filters prior to ultrafiltration. Centrifugal filters
(Milipore Ltd., Amicon Ultra-0.5, 10 000 MW Cutoff) were
rinsed with 400 μL of wash buffer for 5 min at 14 000 × g, after
which the filters were placed into new microcentrifuge tubes.
Ultrafiltration of the solutions of dendrimer conjugates was
then performed by adding 400 μL of each solution to a filter,
spinning the filters for 15 min at 14 000 × g, transferring the
filtrate to a microcentrifuge tube for analysis in a radioligand
binding assay and resuspending the material remaining in each
filter in 400 μL of buffer. This process was repeated 8 times to
ensure the complete removal of contaminants. After the eighth
spin, the remaining material was collected by inverting the
filters, placing them into new microcentrifuge tubes, and
spinning the filters for 2 min at 1000 × g. Once collected, the
remaining material for each dendrimer sample (approximately
20 μL) was resuspended in 400 μL of 50 mM Tris-HCl (pH
7.5) and 10 mM MgCl2 buffer. Finally, each of the fractions
collected during the rinse and ultrafiltration steps and the
resuspended material retained by the filter were tested in a
radioligand binding assay using A3 expressing CHO cell derived
membranes to determine binding activity. This confirmed that
no small molecular impurities remained following multiple
ultrafiltration steps.
G4 PAMAM, Conjugated with Compound 9 and PEG (43).
Freshly prepared aqueous sodium ascorbate (1 M, 22 μL) was
added to a mixture of PEG-azide dendrimer 42 (15 mg, 0.25
μmol, prepared as described32) and compound 9 (5.7 mg,
10.98 μmol) in a mixture of DMSO (0.7 mL) and water (0.7
mL), followed by addition of 7.5% aqueous cupric sulfate (146
μL, 43.93 μmol). The reaction mixture was stirred at room
temperature overnight, and the product was purified by dialysis
and EDTA treatment, as described above. The mixture was
lyophilized to give compound 41 (14 mg, 72%) as a colorless
foamy solid. 1H NMR (DMSO, 400 MHz) δ 8.88 (s), 8.10 (s),
7.82 (s), 7.56 (s), 7.37 (s), 7.29 (br s), 5.42 (s), 4.93 (s), 4.82
(m), 4.66 (m), 4.35 (s), 4.13 (s), 3.32 (s), 3.10 (br s), 2.87 (br
s), 2.33 (s), 2.17 (br s), 1.73 (br s), 1.60 (br s), 1.29 (br s). ESI-
MS: calcd. 77 501; found 77 512.
(CD3OD, 400 MHz) δ 8.56 (br s, 1H), 8.10−7.94 (br m), 7.56
(br s), 5.42 (br s), 4.94 (s), 4.67 (br m), 4.35 (br s), 4.12 (br s),
3.50 (br s), 3.2 (s), 2.8 (br s), 2.31 (br s), 1.82 (br s), 1.59 (br
s), 1.29 (br s). ESI-MS: calcd. 71 511; found 71 413.
Pharmacology. Cell Culture and Membrane Prepara-
tion. Chinese hamster ovary (CHO) cells stably expressing
either the recombinant hA1 or hA3AR and human embryonic
kidney (HEK) 293 cells stably expressing the human (h)
A2AAR were cultured in DMEM and F12 (1:1), supplemented
with 10% fetal bovine serum, 100 units/mL penicillin, 100 μg/
mL streptomycin, and 2 μmol/mL glutamine. In addition, we
added 800 μg/mL Geneticin to the hA2AAR media and 500 μg/
mL Hygromycin B to the hA1AR, hA2BAR, and hA3AR media.
After harvesting the cells, we centrifuged them at 250 × g for 5
min at 4 °C. The pellet was resuspended in 50 mM Tris-HCl
buffer (pH 7.5), containing 10 mM MgCl2. The suspension was
homogenized with an electric homogenizer for 10 s and was
then recentrifuged at 20 000 × g for 30 min at 4 °C. The
resultant pellet was homogenized again, resuspended in the
buffer mentioned above in the presence of 3 U/mL adenosine
deaminase, finally pipetted into 1 mL vials, and this crude
membrane preparation was stored at −80 °C until binding
experiments were conducted. The concentration of protein was
determined using a BCA Protein Assay Kit from Pierce
Biotechnology (Rockford, IL).33
Radioligand Binding Studies. Radioligand binding assays at
A1, A2A, and A3ARs were performed according to the
procedures described previously.34−36 Each tube in the binding
assay contained 100 μL of a crude membrane preparation in
suspension (20 μg of protein), 50 μL of a stock solution of
agonist radioligand, and 50 μL of increasing concentrations of
the test ligands in Tris-HCl buffer (50 mM, pH 7.5) containing
10 mM MgCl2. Nonspecific binding was determined using a
final concentration of 10 μM 5′-N-ethylcarboxamidoadenosine
(NECA), a nonspecific agonist, diluted with the buffer.
The mixtures were incubated at 25 °C for 60 min. Binding
reactions were terminated by filtration through Whatman GF/
B filters under a reduced pressure using a MT-24 cell harvester
(Brandell, Gaithersburg, MD). Filters were washed three times
with 5 mL of 50 mM ice-cold Tris-HCl buffer (pH 7.5). The
radioactive agonists [3H]R-N6-(phenylisopropyl)adenosine (R-
PIA) 47 and [3H]2-[p-(2-carboxyethyl)phenyl-ethylamino]-5′-
N-ethylcarboxamidoadenosine (CGS21680) 48 were used for
the A1 and A2AAR assays, respectively, while [125I]N6-(4-amino-
3-iodobenzyl)adenosine-5′-N-methyluronamide (I-AB-MECA)
49 was used for the A3AR assay. Filters for A1 and A2AAR
binding were placed in scintillation vials containing 5 mL of
Hydrofluor scintillation buffer and counted using a PerkinElm-
er Tricarb 2810TR Liquid Scintillation Analyzer. Filters for
A3AR binding were counted using a PerkinElmer Cobra II γ-
counter.
G4 PAMAM, Conjugated with Compound 23 (45). Freshly
prepared aqueous sodium ascorbate (1 M, 90 μL) was added to
a mixture of azido dendrimer 44 (5.6 mg, 0.35 μmol, prepared
as described29) and compound 23 (10.7 mg, 22.4 μmol) in a
mixture of DMSO (0.6 mL) and water (0.6 mL), followed by
addition of 7.5% aqueous cupric sulfate (150 μL, 44.8 μmol).
The reaction mixture was stirred at room temperature
overnight, and the product was purified purified by dialysis
and EDTA treatment, as described above. The mixture was
lyophilized to give compound 45 (12.9 mg, 79%) as a white
foamy solid. 1H NMR (DMSO, 400 MHz) δ 8.86 (s), 8.24 (s),
7.25−7.36 (m), 5.13 (d, J = 4.4 Hz), 4.63 (br s), 4.54 (d, J = 7.2
Hz), 3.79 (br s), 2.76 (s), 2.67 (s), 2.43 (t, J = 6.4 Hz), 2.33 (br
s), 2.21 (br s), 1.81 (br s), 1.59 (br s), 1.51 (br s), 1.23 (s), 1.11
(d, J = 3.6 Hz), 0.62 (br s). ESI-MS: calcd. 46 343; found 46
024.
Cyclic AMP Accumulation Assay. Intracellular cyclic
AMP levels were measured with a competitive protein binding
method.37 CHO cells that expressed the recombinant hA3AR
were harvested by trypsinization. After centrifugation and
resuspending in medium, cells were planted in 24-well plates in
1.0 mL medium. After 24 h, the medium was removed and cells
were washed three times with 1 mL DMEM, containing 50 mM
HEPES, pH 7.4. Cells were then treated with the agonist
NECA (10 μM) and/or test compound (1 or 10 μM) in the
presence of rolipram (10 μM) and adenosine deaminase (3
units/mL). After 30 min, forskolin (10 μM) was added to the
medium, and incubation was continued for an additional 15
G4 PAMAM, Conjugated with Compound 31 and PEG
(46). Compound 46 (63%) was prepared from compound 31
1
following the same method as for compound 43. H NMR
237
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Table 1. Potency of a series of (N)-methanocarba adenosine derivatives at three subtypes of human ARs
a
All experiments were done on CHO or HEK293 (A2A only) cells stably expressing one of three subtypes of hARs. The binding affinity for A1, A2A,
and A3ARs was expressed as Ki values (n = 3−5) and was determined by using agonist radioligands ([3H]R-PIA, 47; [3H]CGS21680, 48; or [125I]I-
AB-MECA, 49; respectively), unless noted. A percent in parentheses refers to inhibition of radioligand binding at 10 μM, unless noted. Ki app values
238
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Table 1. continued
b
c
are provided for the dendrimer derivatives 43 and 46. Values from Tchilibon et al.21 and Tosh et al.23 4, MRS1898; 8, MRS5127; 16, MRS5362;
32, MRS5415; 33, MRS5519; 43, MRS5351; 46, MRS5439. Control dendrimer structures given in Scheme 5; binding data for 41 as reported.32
Nucleoside dendrimer conjugate structures given in Scheme 5. Percent inhibition of radioligand binding at 1 μM.
d
e
f
Scheme 1. Synthesis of 5′-N-Methyluronamide (N)-Methanocarba Adenosine Derivatives Linked through a N6-Benzyl Group as
Model Compounds
min. The reaction was terminated by removing the supernatant,
and cells were lysed upon the addition of 200 μL of 0.1 M ice-
cold HCl. The cell lysate was resuspended and stored at −20
°C. For determination of cyclic AMP production, a cyclic AMP
kit (Sigma, St. Louis, MO) was used.
Data Analysis. Binding and functional parameters were
calculated using Prism 5.0 software (GraphPAD, San Diego,
CA, USA). IC50 values obtained from competition curves were
converted to Ki values using the Cheng-Prusoff equation.38
A2AAR X-ray structure39 shows four disulfide bridges present in
the EL region of this receptor, but only one is relevant to the
A3AR, i.e., a bond between Cys77 (TM3) and Cys166 (EL2)
that is conserved among family A GPCRs. The conformational
differences in the EL region between A2AAR with its four
disulfides and A3AR with a single disulfide and their effects on
ligand recognition are the subject of ongoing studies. The top-
ranked A3AR model built using the Homology Model tool of
Molecular Operating Environment53 was used for the docking of
nucleosides.
Data were expressed as mean
standard error. The
concentrations of the dendrimer-ligand complexes are meas-
ured by the concentration of the dendrimer, not the ligand.
Therefore, binding Ki values of the dendrimer derivative are
expressed as Ki app values.
Molecular Modeling. Homology Model and Ligand
Construction. Detailed modeling procedures and a Scheme
(S2) are in the Supporting Information. Briefly, the sequence of
the human A3AR was mapped onto the crystal structure of the
A2AAR (minus the T4-lysozyme insertion) in complex with the
agonist UK-43209739 (PDB ID 3QAK)40 as a structural
template for the homology modeling of the A3AR. The UK-
The structure of conjugate 46 was imported as a mol file into
MOE, where it was subjected to energy minimization using the
CHARMM27 forcefield and subjected to 230 ns of Molecular
Dynamics (MD) simulations in a solvated system using
Desmond54 as implemented in the software from Schrodinger
̈
(Cambridge, MA). The final configuration of the dendrimer−
PEG conjugate 46 from the simulation in water was then used
for the building of the complex A3AR-dendrimer conjugate.
Molecular Docking. A portion of 46 containing the N6-
triazolylmethyl (N)-methanocarba nucleoside moiety and part
of the dendrimer chain (Figure S1) was minimized with the
239
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Scheme 2. Synthesis of 4′-Truncated (N)-Methanocarba Adenosine Derivatives Linked through a N6-Benzyl Group as Model
Compounds
Scheme 3. Synthesis of 5′-N-Methyluronamide (N)-Methanocarba Adenosine Derivatives Linked through a N6-Triazolylmethyl
Group as Model Compounds
Polak-Ribiere conjugate gradient (PRCG) minimization and
used as starting point for the docking study. The flexible
docking of the ligand in the A3AR model was performed by
means of Glide and MacroModel in the software from
RESULTS
■
Chemical approaches to couple ring-constrained nucleoside
monomers by click chemistry through their N6 position to
PAMAM dendrimers were explored. The effects of these
structural changes on receptor recognition were examined in
radioligand binding assays and functional assays of cyclic AMP.
Furthermore, the binding of a representative dendrimer
conjugate was visualized and specific amino acid interaction
predicted with molecular modeling using an A3AR homology
model based on the recently reported X-ray structures of an
agonist-bound A2AAR.39
Chemical Synthesis. We prepared various (N)-methano-
carba adenosine derivatives (Table 1) to act as selective ligands
of the A3AR. Both small molecular model compounds (9−17,
22−40) and PAMAM dendrimeric precursors and nucleoside
conjugates (41−46) were included. There were two variations
of the (N)-methanocarba ring system: either bearing a 5′-N-
methyluronamide or unsubstituted, i.e., “truncated”, at the 4′
position. Prior to coupling the nucleosides to dendrimer
carriers, we explored the SAR of alkyne-functionalized chains
on the N6 substituent and the resultant click products, i.e.,
Schrodinger, LLC. The molecular docking of the compound
̈
was performed in a rigid binding site of the A3AR model with
Glide using the SP (standard precision) procedure. The best-
ranked complex receptor−ligand was subjected to conforma-
tional analysis by means of MCMM for further structural
optimization and energy minimization. During the conforma-
tional search, full flexibility was granted to the ligands and the
residues within a radius of 4 Å from the ligand.
Complex of A3AR with Dendrimer Conjugate 46.
Randomly, one of the N6-triazolylmethyl nucleoside moieties
of 46 was replaced with the docked conformation of the ligand
in the A3AR binding site. The dendrimer conjugate 46 with a
docked nucleoside terminal inside the A3AR binding site was
then subjected to minimization in MOE with MMFF94
forcefield to a root-mean-square gradient of 0.001.
240
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Scheme 4. Synthesis of 4′-Truncated (N)-Methanocarba Adenosine Derivatives Linked through a N6-Triazolylmethyl Group as
Model Compounds
1,2,3-triazoles. The modification of the N6-substituent was
accomplished either through the extension of a dialkynyl chain
at the 3-position of a N6-benzyl group with subsequent Cu(I)-
catalyzed click reaction of the terminal alkyne41 as shown in
Schemes 1 and 2 or by formation of an aromatic ring (i.e., 1,2,3-
triazole) at a N6-methylene carbon (Schemes 3 and 4). The
triazole was formed in the latter case from a N6-propargyl group
combined with an azido derivative using click chemistry. The
rationale for these two N6 variations were incorporation of
either a 3-substituted N6-benzyl group, which is known to
enhance A3AR selectivity, or a N6-triazolylmethyl group, with
the expectation that this newly formed 5-membered aromatic
ring would serve the same role in A3AR recognition as the six-
membered N6-benzyl ring. An arylmethyl group at the N6
position generally favors high affinity binding to the A3AR with
selectivity that is species-independent.
(Scheme 5A).32 Alternately, the dendrimer precursor 44 was
fully substituted with azido groups (Scheme 5B), formed using
a previously reported procedure.29,43 Then, some of the azido
groups on the dendrimer 41 were reacted with alkyne-
derivatized PEG chains for water solubility to form 42. These
azido-modified dendrimers 42 and 44 were then cross-linked
by click chemistry28 with terminal alkynyl groups at the N6
position of (N)-methanocarba nucleoside derivatives. Thus, a
dialkyne introduced at the 3-position of a N6-benzyl group in
5′-methyluronamide derivative 9 (i.e., based on full A3AR
agonists) or in the truncated nucleoside series 23 (i.e., based on
partial A3AR agonists) could be subjected for further reaction
with azido groups on the dendrimer. Alternately, a N6-
propargyl group on adenosine derivative 31 could react with
the azido-derivatized dendrimer 42 (Scheme 5C). The
substitution of the resulting conjugates 43, 45, and 46 was
determined by mass spectroscopy, indicating an average of 34,
64, and 30.7 nucleoside moieties, respectively, present out of
the 64 possible sites on each G4 dendrimer. Approximately 3.3
azido groups per molecule of 46 remained unreacted.
Quantification of Pharmacological Activity. We initially
tested the nucleoside derivatives in binding assays at three hAR
subtypes; we used standard nucleoside radioligands 47−49 and
membrane preparations from CHO cells (A1 and A3) or
HEK293 cells (A2A) stably expressing a hAR subtype (Table
1).34−36,44,45 We used the previously reported agonist
molecular probes of the A3AR (4 and 8) for comparison in
the biological assays.23 We included the parent dendrimers 41,
which contained both azido and amino groups, and the fully
azido-substituted 44 as controls; they were inactive or weakly
active in inhibiting AR binding.
The dialkynyl chains at the 3-position of N6-benzyl moieties
in 9−11 and 19−21 were introduced by a Sonogashira
reaction42 at the 3-iodo position of a N6-benzyl ring. In the
case of the 5′-N-methylcarboxamides, the 2′ and 3′ hydroxyl
groups were unprotected during this step, i.e., reaction of
derivative 4 to yield intermediates 9−11. However, for the
truncated series, Sonogashira reactions were performed on
isopropylidene-protected derivative 18 to provide intermediates
19−21. Compounds 19 and 21 were isolated as pure products,
but 20 was subjected to the next synthetic step without
isolation. The isopropylidene groups were then removed to
yield dialkyne intermediates 22−24.
Then, a dialkyne that was introduced at the 3-position of a
N6-benzyl group in 5′-methyluronamide derivatives 9−11 or in
the truncated nucleoside series, i.e., derivatives 22−24, could be
subjected for further reaction with azido groups, which
generally occurred exclusively at the distal alkyne. Alternately,
a N6-propargyl group in 31 and 38 could react with an azido
derivative, such as the commercially available 4-fluoro-3-nitro-
phenylazide. These arylfluoride derivatives provided an addi-
tional means of chain derivatization by nucleophilic sub-
stitution.
The small molecular ligands indicated a dependence of the
AR affinity on the chain length. For example, in diyne series 9−
11, the affinity tended to decrease upon homologation from 3
to 5 methylenes. Compound 9 displayed a Ki of 8.1 nM at the
A3AR, and its click product 12 with 4-fluoro-3-nitro-phenyl-
azide was also the most potent in that homologous series.
Reduction of the nitroaryl group to the corresponding amine as
in 16 and 17 slightly reduced A3AR affinity. However, the 4′-
truncated dialkyne 23 was weak in AR binding, and the click
products 25−27 were only slightly more potent. In that
homologous series of 1-(4-fluoro-3-nitro-phenyl)-triazole de-
Routes to the dendrimer conjugates involved similar click
chemistry. In the case of dendrimer precursor 41, most (56 out
of 64) of the peripheral amino groups of the precursor
PAMAM dendrimers were first substituted with azido groups
241
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a
Scheme 5. Synthesis of Dendrimeric Conjugates of Strategically Functionalized Nucleoside Derivatives
a
The derivatives contain (N)-methanocarba adenosine derivatives linked through a N6-benzyl (A,B) or N6-triazolylmethyl (C) group. A 5′-N-
methyluronamide (A,C) or 4′-truncated nucleoside (B) is present. Average structures are shown.
rivatives missing a 5′-uronamide, the binding affinity at the
A3AR was independent of chain length. Therefore, the 5′-
methyluronamide moiety appears to stabilize a binding mode of
the nucleoside such that it interacts with the extracellular
regions of the ARs in a more structure-sensitive manner.
The N6-propargyl intermediate 31 in the 5′-methyluronamide
series was potent in binding to the A3AR (Ki 1.5 nM) and 270-
fold selective in comparison to the A1AR. The click product 32,
a 1-(4-fluoro-3-nitrophenyl)-triazole, bound to the A3AR with a
Ki of 9.1 nM and over 100-fold selectivity, and the
corresponding 3-amino derivative 33 was slightly weaker. A
large 1-adamantyl group on the triazole ring of 35 was also
tolerated with respect to A3AR binding. The corresponding 4′-
truncated series 38−40 did not achieve high AR affinity; thus,
the 5′-methyluronamide was an important recognition element
for the small molecules.
Two of the multivalent dendrimer derivatives, 43 and 46,
obtained through click conjugation of the corresponding 5′-
methyluronamide alkynyl nucleoside precursors, retained
potent binding activity at the A3AR and displayed enhanced
242
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Bioconjugate Chemistry
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selectivity over the monomer precursors. Water-solubilizing
PEG chains, also incorporated by click linkage, did not prevent
receptor binding. The binding assays were performed on
conjugate preparations subjected to 8 sequential cycles of
ultrafiltration to ensure that no small molecular weight
contaminants were present. The N6-benzyl derivative 43 had
a Ki app value of 24 nM in binding to the A3AR, which is 3-fold
less potent than the alkynyl precursor 9, and the affinity at A1
and A2AARs was greatly decreased. The multivalent N6-
triazolylmethyl derivative 46 was also potent in binding to
the A3AR (Ki app 55 nM) and selective in comparison to the
A1AR, but it was 37-fold less potent at the A3AR than the
propargyl precursor 31. However, the multivalent 4′-truncated
N6-benzyl dendrimer 45 was nearly inactive in binding at the
A3AR, consistent with the weak affinity of the alkyne precursor
23.
degrees of maximal inhibition of cyclic AMP production at 1 or
10 μM The concentration selected was generally in >100-fold
excess of the Ki value for each 5′-methyluronamide nucleoside,
to approximate the maximal effect. The relative efficacy was
compared to that of NECA (10 μM), taken as a reference
standard. The small molecular 5′-methyluronamide derivatives
9, 13, 15, 17, and 31−35 displayed ≥80% of the maximal effect
of NECA, indicated nearly full agonism. Other 5′-uronamide
derivatives reached only intermediate efficacy despite a high
affinity in A3AR binding, e.g., terminal alkyne intermediates 10
and 11 and aryl fluorides 14 and 16. 4′-Truncated small
molecules 23, 25−27, and 38−40 displayed low efficacy
(≤27% of the maximal effect) at the A3AR, consistent with their
roughly micromolar Ki values and with the tendency of
truncation to reduce A3AR efficacy. Consistent with the binding
results, only dendrimer conjugates of 5′-methyluronamide
derivatives, 43 and 46, displayed considerable activity in the
functional assay. Both were full A3AR agonists at 1 μM.
Molecular Modeling. With X-ray crystallographic struc-
tures of agonist-bound forms of the hA2AAR now known,39,46 it
is possible to extend the modeling analysis of agonist binding
by homology to other AR subtypes. In the X-ray crystal
structure (PDB ID: 3QAK)39 of the A2AAR bound to agonist
UK-432097 (complex designated here: UK-A2AAR), large N6
and C2 substituents project toward the opening of the
nucleoside binding cleft, consistent with the biological activity
of dendrimer-linked conjugates.
A homology model of the human A3AR was built using UK-
A2AAR as template, which was chosen among the available
agonist-bound A2AAR crystal structures39,46 because it was
found to be suitable for the modeling of the binding pocket
embedding other N6-substituted agonists. In fact, in the UK-
A2AAR crystal structure the conformation of the third
extracellular loop (EL3), connecting TM6 and TM7, is open
allowing the accommodation of the bulky N6 substituent of the
agonist UK-432097. In the adenosine-bound or NECA-bound
structures,46 instead, EL3 appears closer to EL2 with an H-
bond interaction between His264(EL3) and Glu169(EL2). The
agonist-bound model of A3AR was built with the Homology
Modeling module of MOE and utilized for the initial ligand
docking.
We chose for receptor docking the conjugate 46, containing
a triazole ring in place of the usual phenyl ring of the typical N6-
benzyl derivatives, rather than the more potent conjugate 43.
Because the receptor binding of N6-benzyl derivatives has been
modeled in previous studies,19,21,23,30 we wanted to probe the
similarity between the two aromatic rings in their docked
conformations. Initially, a fragment of 46, i.e., a chain-extended
version of a N6-triazolylmethyl derivative (related to the 5′-
methyluronamide (N)-methanocarba nucleoside series 32−35)
was drawn (designated compound 47, Figure S1). After this
theoretical agonist-containing terminal portion of the PAMAM
conjugate was docked to the A3AR model binding site with
Glide, and the best scored docking pose was further optimized
by means of a Monte Carlo Multiple Minima (MCMM)
conformational search of the ligand and the residues in the
binding site, in order to take into account the flexibility of the
receptor residues.
We tested the small molecular derivatives and nucleoside
conjugates in a functional assay at the A3AR consisting of
inhibition of forskolin-stimulated cyclic AMP production in
A3AR-expressing CHO cells (Table 2). These GLiDe
conjugates displayed A3AR agonist properties with varying
Table 2. Maximal Efficacy of (N)-Methanocarba Adenosine
Derivatives in a Functional Assay at the hA3AR Consisting of
Inhibition of Cyclic AMP Accumulation
a,b
compound
% inhibition
b
Cl-IB-MECA
97
b
NECA
100
c
4
100
c
8
44
6
9
80.0 6.4
10
11
12
13
14
15
16
17
23
25
26
27
31
32
33
34
35
38
39
40
57.3 10.6
57.8 21.2
92.0 7.6
101
8
64.1 11.5
79.8 4.4
66.7 5.6
96.6 14.3
7.8 1.4
6.6 2.6
9.9 2.1
20.1 5.2
103
107
8
5
88.3 4.3
81.5 4.8
106
5
14.3 0.7
6.4 2.8
27.1 4.3
86.8 3.2
4.0 2.4
91.3 3.4
b
43
b
45
b
46
a
The efficacy at the hA3AR was determined by inhibition of forskolin-
stimulated cyclic AMP production in AR-transfected CHO cells, as
described in the text. Percent inhibition is shown, in comparison to the
maximal effect of a full agonist NECA at 10 μM. Data are expressed as
Separately, the model of the entire PEGylated dendrimer
conjugate 46 was built and optimized by means of MD. To take
into account the remainder of the PAMAM moiety of the
dendrimer conjugate 46 in the receptor docking model, the
extended N6-triazolylmethyl chain of the docked nucleoside
b
mean standard error (n = 3−5). Concentrations used were Cl-IB-
MECA and NECA at 10 μM and dendrimer derivatives 43, 45, and 46
at 1.0 μM, and all other compounds were determined at 10 μM. ND,
c
not determined. Values from Tosh et al.23 4, MRS1898; 8, MRS5127.
243
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ligand 47 was fused to a portion of the preoptimized conjugate
46. In other words, after the MD simulation of 46, one of the
nucleoside terminals of this dendrimer conjugate was randomly
chosen and replaced by the ligand previously docked in the
binding site of the A3AR model. The model of the docked
dendrimer conjugate 46 in complex with A3AR was then
subjected to further minimization in order to release possible
overlays and steric clashes between the dendrimer and the
receptor.
The docking pose of the functionalized nucleoside in the
binding site of A3AR model was stabilized by several H-bond
and van der Waals (vdW) interactions with the residues in the
binding cavity, as shown in Figure 1A. In particular, the adenine
ring of the molecule was anchored to Asn250(6.55) (the
close proximity to Gln167(EL2), Leu264(7.35), Ile249(6.54),
Met174(5.35), Ile253(6.58), and Val169(EL2), similar to the
docked position of the N6-benzyl ring of known A3AR agonists
(not shown).
The molecular model of the A3AR-dendrimer conjugate
complex showed how the hydrophilic PEG chains surrounded
the dimethylamide moiety of the hydrophobic dendrimer
(Figure 1B), possibly favoring the solubility of the conjugate.
Nevertheless, the ligand moieties attached to the dendrimer
were still exposed to the solvent.
DISCUSSION
■
We previously demonstrated that coupling of strategically
functionalized A3AR agonists to PAMAM dendrimers by click
chemistry through functionalized chains attached at the adenine
C2 position achieved higher affinities than coupling by amide
formation.29 The present study focused exclusively on coupling
through the N6 position of methanocarba nucleoside ligands
using several different approaches to linkage by click chemistry.
The small molecules were selected for probing the effects of
sterically bulky groups at the end of a functionalized chain.
Within the four separate structural series examined here, a
terminal adamantyl group generally served as a suitable high
affinity replacement for a substituted phenyl ring in the small
model compounds, i.e., in compounds 15 and 35. However, the
same substitution in truncated nucleoside 40, containing a
shorter tether from the adenine exocyclic amine, did not
preserve binding affinity. The results indicated that the N6
position of functionalized nucleosides, like the C2 position, is
also viable for the click conjugation to macromolecular carriers.
The two chemical linking strategies used here were (1)
reaction of a dialkyne moiety introduced at the N6 position
(similar to the linker chemistry used at the C2 position) and
(2) formation of a triazolylmethylene moiety, intended to
promote A3AR selectivity, by the click reaction of a N6-
propargyl group. One objective of this study was to determine if
a N6-triazolylmethyl group formed by Cu(I)-catalyzed click
chemistry could substitute for a N6-benzyl group in the receptor
binding site. In the triazolylmethyl products, high A3AR affinity
and selectivity was maintained in the 5′-methyluronamide
series, but not in the 4′-truncated series. In the case of linking
through a N6-benzyl group, only the 5′-methyluronamide
derivatives (Schemes 1 and 5A) maintained high AR affinity
consistently, while the 4′-truncated derivatives were only weakly
active both in the form of chain-extended small molecules and
as a multivalent conjugate (Scheme 5B). Thus, truncated
monomer 23 was not a suitable intermediate for dendrimeric
conjugation to achieve high A3AR affinity.
Figure 1. (A) Docking pose of (N)-methanocarba adenosine
derivative 47 (structure in Figure S1), which is a fragment of
conjugate 46, in the binding site of the A3AR model based on
homology to the agonist-bound A2AAR crystal structure.39 The
nucleoside moiety of the ligand is anchored in the binding site by
key H-bond interactions (highlighted with dotted lines) with the
residues Thr94 (TM3), Asn250 (TM6), and Ser271 and His272
(TM7). The ligand, in ball-and-stick representation, is depicted with
carbon atoms colored in orange. The receptor residues are shown in
stick model with carbons in gray. (B) A molecular model of the
complex A3AR-dendrimer conjugate 46. The receptor is represented as
a cartoon colored in light blue. The dendrimer conjugate 46 is
represented by a molecular surface, with the PEG chains colored in
red, the ligand moieties in green, and the PAMAM moiety in gray. The
likely glycosylation of the receptor in EL2 is not included in the model.
Graphic images were made with PYMOL v 0.99, Delano Scientific
LLC, CA, USA.
number in parentheses refer to the Ballesteros-Weinstein
numbering system47) by H-bond interactions with the N6
amino group and the N7 of the nucleoside. Moreover, the
aromatic adenine ring of the ligand was stabilized by a π−π
stacking interaction with the aromatic ring of Phe168(EL2) and
favorable vdW interactions with the hydrophobic residues
Leu246(6.51) and Ile268(7.39). The ribose moiety of the N6-
triazolylmethyl nucleoside derivative was found deeper in the
binding pocket, locked by H-bond interactions of the 2′- and 3′-
OH groups with His272(7.43) and Ser271(7.42), respectively.
The carbonyl group of the 5′-N-ethylcarboxamido moiety was
H-bonded to Thr93(3.36). Other favorable vdW interactions
were found between the ribose moiety and hydrophobic
residues, i.e., Trp243(6.48), Leu90(3.32), Leu91(3.33),
Ile98(3.40), and Ile186(5.47). The long N6 chain of the
agonist was oriented toward the extracellular solvent, exiting
the binding pocket among TM5, TM6, TM7, EL3, and EL2. In
particular, the triazole moiety of the N6 chain was located in
The results were found to be consistent with the recently
elucidated X-ray structure of the A2A receptor with a N6
substituted adenosine derivative bound. In a docking model,
the nucleoside moiety of the dendrimer conjugate 46 was found
to be located deep in the binding pocket anchored by strong H-
bond and hydrophobic interactions with the receptor residues,
while the long chain at the N6 position of the nucleoside is
oriented toward the extracellular part of the receptor exiting to
the solvent among the extracellular terminals of TM5, TM6,
and TM7, between EL3 and EL2. The long PEG chains are
embedding the ligand-functionalized dendrimer moiety expos-
ing the hydrophilic monomers to the solvent and coating the
PAMAM dendrimer chains. Nevertheless, the nucleoside
terminals of the dendrimer conjugate are still exposed to the
solvent and clearly accessible for the binding to the receptor.
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Article
A3AR homodimers have been proposed to exist at the cell
surface.48 Whether this GLiDe conjugate participates in
multivalent interaction with higher order receptor aggregates
is unknown.
ACKNOWLEDGMENTS
■
We thank Dr. John Lloyd and Dr. Noel Whittaker (NIDDK)
for the mass spectral determinations and Angela Kecskes for
technical assistance. This research was supported by the
Intramural Research Program of the NIH, NIDDK.
́
In addition to therapeutic applications,49 dendrimer-based
drug conjugates have been applied to biological imaging
modalities, such as luminescent, fluorescent, magnetic reso-
nance, and radioactive.50,51 Given the high A3AR affinity
observed in the small molecular series, there is now a possibility
of introducing radioisotopes to produce new radioligands for
receptor characterization in vitro and in vivo. Prosthetic groups
for targeting, therapeutic applications or diagnostic purposes,
such as fluorescent reporter groups, might be introduced.
Several fluorinated analogues and other small molecular
analogues bound to the A3AR with high affinity and raised
the possibility of their use as radioligands. This suggested the
application of these structures for incorporation of the positron
emitter 18F for in vivo imaging of the A3AR.25,52 The fluorine in
aryl amino analogues 16 and 17 would be stable toward
nucleophilic displacement, while the 4-fluoro group of the
corresponding nitro analogues could be readily substituted.
Therefore, a radioisotope (18F) could potentially be introduced
by nucleophilic fluoride attack at the nitro stage, e.g., with 12 as
substrate, followed by reduction to the more stable amine. The
most potent of the compounds in this series was 3-amino-4-
fluoro derivative 16 with a Ki value of 14.3 nM at the A3AR,
while 3-amino-4-fluoro derivative 33 was less potent in binding.
In conclusion, we have extended the SAR of small
monomeric adenosine agonists derivatized at the N6 position
to the multivalent state. We have explored a new linking
strategy to achieve the high A3AR agonist affinity and selectivity
in two PEGylated GLiDe conjugates of A3AR-binding nucleo-
sides from chemical, pharmacological, and structural perspec-
tives. The structural features of the pharmacophore, the
chemical nature of the linking and terminal groups of the
dendrimer all modulate the biological properties of a
multivalent conjugate. The success of this approach was
demonstrated for G4 PAMAM dendrimers as carriers, and
the application of the same triazole linkage to other dendrimer
carriers, with different degrees of loading or in combination
with other terminal groups, remains to be investigated.
ABBREVIATIONS:
■
AR, adenosine receptor; DMSO, dimethylsulfoxide; EDC, N-
ethyl-N′-dimethylaminopropylcarbodiimide; cyclic AMP, ad-
enosine 3′,5′-cyclic phosphate; CGS21680, 2-[p-(2-
carboxyethyl)phenyl-ethylamino]-5′-N-ethylcarboxamidoade-
nosine; CHO, Chinese hamster ovary; Cl-IB-MECA, 2-chloro-
N6-(3-iodobenzyl)-5′-N-methylcarboxamidoadenosine;
DMEM, Dulbecco’s modified Eagle’s medium; GPCR, G
protein-coupled receptor; HEK, human embryonic kidney;
HRMS, high resolution mass spectroscopy; NECA, 5′-N-
ethylcarboxamidoadenosine; NMR, nuclear magnetic reso-
nance; DIPEA, diisopropylethylamine; DMF, N,N-dimethylfor-
mamide; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesul-
fonic acid; MCMM, Monte Carlo Multiple Minima;
PAMAM, polyamidoamine; PEG, polyethylene glycol; PET,
positron emission tomography; R-PIA, (R)-N6 -
(phenylisopropyl)adenosine; TBTA, tris[(1-benzyl-1H-1,2,3-
triazol-4-yl)methyl] amine; TEA, triethylamine; TLC, thin
layer chromatography; UK-432097, 6-(2,2-diphenylethylami-
no)-9-((2R,3R,4S,5S)-5-(ethylcarbamoyl)-3,4-dihydroxytetra-
hydrofuran-2-yl)-N-(2-(3-(1-(pyridin-2-yl)piperidin-4-yl)-
ureido)ethyl)-9H-purine-2-carboxamide
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ASSOCIATED CONTENT
■
S
* Supporting Information
1H NMR and mass spectra of representative compounds and
modeling procedures. This material is available free of charge
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AUTHOR INFORMATION
■
Corresponding Author
*E-mail: kajacobs@helix.nih.gov. Molecular Recognition Sec-
tion, Bldg. 8A, Rm. B1A-19, NIH, NIDDK, LBC, Bethesda,
MD 20892-0810. Tel.: 301-496-9024, Fax: 301-480-8422.
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