Triazolophostins: A library of novel and potent agonists of IP3 receptors

Article abstract of DOI:10.1039/c5ob00440c

IP₃ receptors are channels that mediate the release of Ca²⁺ from the intracellular stores of cells stimulated by hormones or neurotransmitters. Adenophostin A (AdA) is the most potent agonist of IP₃ receptors, with the β-anomeric adenine contributing to the increased potency. The potency of AdA and its stability towards the enzymes that degrade IP₃ have aroused interest in AdA analogs for biological studies. The complex structure of AdA poses problems that have necessitated optimization of synthetic conditions for each analog. Such lengthy one-at-a-time syntheses limit access to AdA analogs. We have addressed this problem by synthesizing a library of triazole-based AdA analogs, triazolophostins, by employing click chemistry. An advanced intermediate having all the necessary phosphates and a β-azide at the anomeric position was reacted with various alkynes under Cu(i) catalysis to yield triazoles, which upon deprotection gave triazolophostins. All eleven triazolophostins synthesized are more potent than IP₃ and some are equipotent with AdA in functional analyses of IP₃ receptors. We show that a triazole ring can replace adenine without compromising the potency of AdA and provide facile routes to novel AdA analogs. This journal is

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PAPER
Triazolophostins: A Library of Novel and Potent Agonists of IP3
Receptors†
Amol M. Vibhute,a Vera Konieczny,b Colin W. Taylor b and Kana M. Sureshan* a
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
5
DOI: 10.1039/b000000x
IP3 receptors are channels that mediate release of Ca2+ from the intracellular stores of cells stimulated by
hormones or neurotransmitters. Adenophostin A (AdA) is the most potent agonist of IP3 receptors, with
the β-anomeric adenine contributing to the increased potency. The potency of AdA and its stability
towards the enzymes that degrade IP3 have aroused interest in AdA analogs for biological studies. The
10 complex structure of AdA poses problems that have necessitated optimization of synthetic conditions for
each analog. Such lengthy one-at-a-time syntheses limit access to AdA analogs. We have addressed this
problem by synthesizing a library of triazole-based AdA analogs, triazolophostins, by employing click
chemistry. An advanced intermediate having all the necessary phosphates and a β-azide at the anomeric
position was reacted with various alkynes under Cu(I) catalysis to yield triazoles, which upon
15 deprotection gave triazolophostins. All eleven triazolophostins synthesized are more potent than IP3 and
some are equipotent with AdA in functional analyses of IP3 receptors. We show that a triazole ring can
replace adenine without compromising the potency of AdA and provide facile routes to novel AdA
analogs.
essential for AdA activity. The nucleobase/surrogate with β-
Introduction
45 stereochemistry is indispensible for the enhanced potency.9b,11
20 Many biological processes are regulated by changes in the
intracellular concentration of Ca2+.1 A major pathway for these
Ca2+ signals is the release of Ca2+ from intracellular stores
sequestered within the endoplasmic reticulum (ER) via IP3
receptors (IP3R).2 IP3R are a family of intracellular Ca2+ channels
25 that are expressed largely in ER membranes.3 IP3 (Fig. 1A, 1) is
produced when cell-surface receptors stimulate phospholipase C
activity. IP3 then binds to IP3R causing its channel to open and
release Ca2+ into the cytosol.4 The ability of IP3 to interact with
IP3R is terminated by its dephosphorylation or phosphorylation
30 by specific 5-phosphatase and 3-kinase enzymes.5 While
terminating the Ca2+-mobilizing ability of IP3, these steps also
initiate recycling of inositol to the lipid from which IP3 is
generated and the synthesis of more complex bioactive
phosphoinositols including the pyrophosphates,6 many of which
35 have been recent synthetic targets.7 Adenophostin A (AdA), a
fungal metabolite isolated from the culture broth of Penicillium
brevicompactum, is a more potent agonist than IP3 of IP3R.8
Moreover, AdA is resistant to the enzymes that metabolize IP3.
Many analogs of AdA have been synthesized to address
40 structure-activity relationships and the determinants of the
increased potency of AdA.9 These studies revealed that the three
phosphates and 2″-OH (color coded in Fig. 1A, 2), which
structurally mimic the essential pharmacophore of IP3,10 are
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Fig. 1 (A) Molecular structures of IP3 (1) and AdA (2); (B) Conventional
35
Imidophostin (Fig. 2A), though weaker than AdA, is more
potent than IP3, suggesting that even a 5-membered aromatic
heterocyclic ring can be a partial functional mimic of adenine. As
triazole is isosteric with imidazole, we envisioned that triazole-
based AdA analogs would also be potent agonists of IP3R. This
synthesis of AdA-analogs. The question marks represent the complexity of
conventional syntheses.
Recent studies suggested that a cation-π interaction between
5
adenine and a cationic residue (Arg504) of the IP3R might be
responsible for the increased potency of AdA.9b,12 Additional
base-modified analogs of AdA would be useful in both testing
this proposal and as potent and metabolically stable agonists of
IP3R to probe Ca2+ signaling mechanisms.
The major hurdle in developing such AdA analogs lies in
the synthesis of these structurally complex base-modified analogs
(disaccharide nucleotides).9e,13 The traditional synthesis involves
N-glycosylation of a nucleobase with an orthogonally protected
disaccharide derivative followed by chemoselective demasking
15 and phophorylation of specific hydroxyl groups in the nucleoside
(Fig. 1B). This early introduction of the nucleobase necessitates
an almost individualy tailored synthesis (branching at an early
stage of synthesis) for each base-modified analog. Further
complications arise from the lack of a generally applicable
20 method for glycosylation and phosphorylation of such complex
molecules. For instance glycosylation of nucleobases is
challenging with respect to yield14 and regioselectivity,13b,15 and
each glycosylation condition has to be optimized individually.
Chemoselective O-phosphorylation in the presence of
25 nucleobases having nucleophilic groups/sites is also
challenging.16 Thus syntheses of analogs by a one-at-a-time
method is cumbersome, time-consuming and impractical. We
herein report a general strategy for making a combinatorial
library of structurally diverse AdA analogs that are potent
30 agonists of IP3R.
40 and the earlier reports on use of click reaction in biologically
important molecules17 prompted us to adopt azide-alkyne click
chemistry to make a library of triazolophostins, AdA analogs
wherein the nucleobase is replaced by substituted triazoles. These
molecules can easily be prepared by reacting appropriately
45 protected tris-phosphate derivative II having an azide at anomeric
position with aryl or heteroaryl alkynes under Cu(I) catalysis
(Fig. 2B). Such an approach would solve three major issues
associated with conventional AdA analog synthesis: (i) Ensure
the necessary β-anomeric stereochemistry of the base-surrogate
50 (ii) As the triazole-based base-surrogate is introduced only at the
penultimate step (after phosphorylation), the interference of
nucleobases during phosphorylation can be avoided (iii)
Diversification at the penultimate step provides access to many
10
structurally diverse analogs from
55 intermediate.
a
common advanced
Scheme 1 Synthesis of triazolophostins. a) H2SO4-silica, Ac2O, DCM, rt, 12
h, 90%; b) TMSN3, AlCl3, DCM, 0 oC, 10 min, 99%; c) NaOMe, MeOH, rt, 30
min, 96%; d) i. (iPr)2NP(OBn)2, ImOTf, DCM, rt, 1 h, ii. m-CPBA, -78 oC, 45
60 min, 90%; e) i. Trimethylsilylacetylene, Cu, CuSO4, H2O:tBuOH, rt, 12 h; ii.
TBAF, THF, rt, 2 h, (95% for two steps); f) Alkyne, Cu, CuSO4, H2O:tBuOH,
rt, 12 h, 48-97%; g) Cyclohexene, Pd(OH)2, MeOH:H2O, 80 oC, 4 h, Bn =
benzyl, Ac = acetyl, Me = methyl.
We have synthesized orthogonally protected disaccharide 4 in
Fig. 2 (A) Imidophostin and Triazolophostins. (B) Retrosynthetic analysis of
65 thirteen steps from D-glucose and D-xylose in a convergent
manner as reported previously.18 Acetolysis19 of the ketal 4 gave
tetraacetate 5 as anomeric mixtures in very good yield. Lewis
acid catalysed azidation of the acetate 5 gave anomerically pure
triazolophostins.
Results and discussion
2
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β-azide 6.20 Methanolysis of the acetate groups in 6 provided the
express only type 1 IP3R.23 A low-affinity Ca2+ indicator trapped
triol 7 in excellent yield. Phosphitylation of the triol 7 using 25 within the ER allowed the effects of IP3 to be directly measured
phosphoramidite followed by in situ oxidation using m-CPBA
provided the fully protected trisphosphate 8. Compound 8 with
three phosphates in their appropriate relative position and β-
oriented azide is the common advanced intermediate for coupling
using a fluorescence plate-reader equipped to allow automated
additions. All the triazolophostins fully release the IP3-sensitive
Ca2+ stores and all are more potent than IP3 (Table 1, Fig. 3). It is
interesting to note that the parent analog triazolophostin 10a
5
with different alkynes. Copper (I) catalysed click reaction of 30 having only the five-membered ring is 13-fold more potent than
azide
8
with various alkynes provided fully protected
IP3. This remarkable AdA-like potency of 10a suggests that a
single triazole ring can effectively replace the adenine base in
AdA without compromising potency. In contrast, imidophostin
was previously reported to be only slightly (1.3-fold) more potent
triazolophostins 9a-k. As the reaction is catalyzed by copper(I),
10 1,4-substituted triazoles21 were obtained exclusively in all the
cases. Global removal of benzyl protecting groups by transfer
hydrogenolysis provided triazolophostins 10a-k in very good 35 than IP3, and it was therefore suggested that a fused bicyclic
yields. Though in a recent elegant communication, Potter et al
reported a functionally active triazole based cADPR analog,22
15 ours is the first report on the use of click chemistry to generate a
library of IP3R agonists.
nucleobase/surrogate is essential for AdA-like potency.18a Among
all triazolophostins, the 3-fluoro derivative 10f is the most potent,
and equipotent to AdA: it is the most potent AdA analog without
a purine nucleobase. Of the several AdA analogs reported in the
40 literature, very few match the potency of AdA.9c,12b,13b,14,18a,24,25 In
this context, our late-stage diversification method is important
because it allows many potent analogs to be made in an easy and
combinatorial way from the same advanced intermediate.
Table 1. Ca2+ release via IP3R evoked by triazolophostins
Ligand
R =
pEC50
EC50
(nM)
EC50
w.r.t.
1
max.
response
(%)
IP3 (1)
6.75 ± 0.08
178 ±
30
1
73 ± 3
AdA (2)
8.05 ± 0.04
7.87 ± 0.00
9 ± 1
20
13
73 ± 2
71 ± 1
13 ± 0
18 ± 2
10a
H
7.75 ± 0.03
10
72 ± 2
10b
45
7.81 ± 0.05
7.77 ± 0.03
7.70 ± 0.07
15 ± 1
17 ± 1
20 ± 3
11
10
9
65 ± 3
64 ± 1
75 ± 4
Fig. 3 Concentration-dependent release of Ca2+ (%) by IP3, AdA and
10c
10d
triazolophostins from the intracellular stores of cells expressing IP3R1.
Results show means ± SEM from at least 3 experiments.
The docking study suggests that triazolophostin 10a and AdA
50 exhibit similar interactions with the IP3-binding core of IP3R1
(IBC, residues 224-604).24 The triazolyl ring of 10a forms a
cation-π interaction with Arg504 similar to the proposed
interaction between the adenine of AdA and Arg504. This
supports previous reports that a cation-π interaction between the
55 base and IP3R contributes to the increased potency of AdA and
analogs.9c,24 All other triazolophostin analogs showed a similar
interaction. The small differences in activity among these analogs
seem to arise from steric, rather than electronic factors. For
instance, attaching aromatic rings with different electrostatic
60 surface potentials to the triazole ring did not cause any systematic
change in biological activity. The fact that all the analogs are both
more potent than IP3, and show a conserved cation-π interaction
between the triazole ring and Arg504 suggests that this critical
interaction is unaffected by aromatic substitution, irrespective of
65 the electronic nature of the aromatic substituent. The single bond
connection between the triazole ring and the substituent (aromatic
ring) allow torsional freedom (freely rotatable) and hence the
sterically favorable non-coplanar conformation of the two
aromatic rings. As a result of such non-coplanarity (lack of
70 conjugation), the electron density on the triazole ring cannot be
influenced by the aromatic unit. This could be the reason why
10e
10f
8.00 ± 0.03
7.86 ± 0.04
10 ± 1
14 ± 2
18
13
69 ± 1
71 ± 2
10g
7.64 ± 0.01
23 ± 1
8
67 ± 2
10h
10i
7.45 ± 0.03
7.78 ± 0.03
35 ± 2
17 ± 2
5
65 ± 1
68 ± 2
11
10j
10k
7.89 ± 0.02
13 ± 1
14
70 ± 1
Results show the maximal Ca2+ release (% of Ca2+ content of intracellular
20 stores), EC50 and pEC50 values (means ± SEM from at least 3 experiments).
Hill coefficients were not significantly different from 1.
The abilities of the newly synthesized ligands to evoke Ca2+
release via IP3R were assessed using permeabilized cells that
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there is no clear trend in activity when the aromatic ring is
45 molecules.
Experimental section
1. General Methods
Chromatograms were visualized under UV light and by dipping
plates into either phosphomolybdic acid in MeOH or
50 anisaldehyde in ethanol, followed by heating. H NMR, COSY,
modified with electron-deficient or electron-rich substituents.
One reason for the increased potency of AdA is believed to be its
conformational flexibility allowing it to adopt an optimal
conformation for binding to IP3R.26 The additional
conformational freedom due to the single bond linkage of this
hetero-biaryl unit (rather than fused rings in AdA) allows the
second ring to orient to an electronically favorable position, such
that it can have additional electrostatic interactions. Docking
5
1
NOESY and HMQC spectra were recorded on a 500 MHz NMR
spectrometer. Proton chemical shifts are reported in ppm (δ)
relative to the internal standard tetramethylsilane (TMS, δ 0.0
ppm) or with the solvent reference relative to TMS employed as
55 the internal standard (CDCl3, δ 7.26 ppm; D2O, δ 4.79 ppm). Data
are reported as follows: chemical shift (multiplicity [singlet (s),
doublet (d), doublet of doublet (dd), triplet (t), quartet (q), and
multiplet (m)], coupling constants [Hz], integration and peak
identification). All NMR signals were assigned on the basis of 1H
60 NMR, 13C NMR, COSY and HMQC experiments. 13C spectra
were recorded with complete proton decoupling. Carbon
chemical shifts are reported in ppm (δ) relative to TMS with the
respective solvent resonance as the internal standard. All NMR
data were collected at 25 °C. The concentration of the compounds
65 for 1H NMR was 5 mg per 0.5 mL and for 13C NMR it was 20 mg
per 0.5 mL for protected compounds and 5-7 mg per 0.5 mL for
final compounds in case of 1H and 13C NMR. Each
triazolophostin (10a-k) was quantified using Briggs’s/Ame’s
phosphate assay. Melting point was determined using melting
70 point apparatus and is uncorrected. Flash column chromatography
was performed using silica gel 230-400 mesh. Where ever
needed, the reactions were carried out under argon or nitrogen
atmosphere employing oven dried glassware.
X-ray intensity data measurements of freshly grown crystals
75 of 4 was carried out at 293-296 K on a Bruker-KAPPA APEX II
CCD diffractometer with graphite-monochromatized (MoK =
0.71073Å) radiation. The X-ray generator was operated at 50 kV
and 30 mA. Data were collected with scan width of 0.3° at
different settings of φ (0°, 90° and 180°) keeping the sample to
80 detector distance fixed at 40 mm and the detector position (2θ)
fixed at 24°. The X-ray data collection was monitored by
SMART program. All the data were corrected for Lorentzian,
polarization and absorption effects using SAINT and SADABS
programs. SHELX-97 was used for structure solution and full
85 matrix least-squares refinement on F2. Molecular and packing
diagrams were generated using Mercury-3.1. Geometrical
calculations were performed using SHELXTL and PLATON.
10 results suggest that the most potent analog, 10f, has additional
interactions between F and IP3R side-chains (D566, Q565 and
Q507) that may enhance its activity (Fig. 4).
15 Fig. 4 Docking of 3-fluorophenyl triazolophostin with IBC showing preferred
binding mode and the important cation-π and fluorine-oxygen interactions
with residues Arg504 and Asp566, respectively. The cation-π distance r =
2,97 Å. The fluorophenyl moiety orients perpendicular to the triazole ring.
Color code for ligand: carbon, gray; oxygen, red; nitrogen, blue;
20 phosphorous, yellow; fluorine, cyan.
Conclusions
In conclusion, addressing the problems associated with
conventional one-at-a-time synthesis of AdA analogs, we have
synthesized a library of new analogs using azide-alkyne click
25 chemistry. The simple reaction conditions, high yield and atom-
economy of the click reaction to install base-surrogates are
advantageous over the conventional Vorbruggen method of
installation of nucleobase. By this easy and rapid method, we
generated a variety of ligands with diverse structures from a
30 common advanced intermediate. All the analogs synthesized by
this method are more potent than IP3 and some (10a, 10f, 10g,
10k) are as potent as AdA. Though replacement of purine with
pyrimidines or other aromatic groups are reported to give
agonists that are more potent than IP3, none of the previously
35 reported non-purinated AdA analogs was as potent as AdA. We
have shown that AdA-like potency can be achieved without a
nucleobase. Our study reveals that a five-membered aromatic
ring, triazole, is sufficient as a base surrogate to exhibit AdA-like
potency. Easy access to potent, non-metabolizable ligands of
40 IP3R and the ease of conjugation with reporter groups such as
fluorescent probes, photoaffinity probes, etc offers avenues for
advanced biological exploration. This report that triazoles can be
functional mimics of nucleobases has broader implication and
might trigger such attempts in other nucleobase-derived signaling
2. Synthesis of 1,2,3′,4′-tetra-O-acetyl-2′,5,6′-tri-O-benzyl-3-
O-α-D-glucopyranosyl-D-ribofuranose (5)
90 To a solution of disaccharide 4 (1.0 g, 1.41 mmol) and Ac2O (0.4
mL, 4.24 mmol) in dry DCM (20 mL), 0.05 g of freshly prepared
H2SO4-silica19 was added. The reaction mixture was stirred at
room temperature for 12 h under nitrogen atmosphere and the
reaction was monitored using TLC. When the starting material
95 disappeared, the reaction mixture was quenched by the addition
of solid NaHCO3. Solid material was filtered off and the organic
layer was washed with a saturated aq. NaHCO3 solution. The
organic layer was dried over Na2SO4 and evaporated under
reduced pressure. The crude product thus obtained was purified
100 by flash column chromatography using 40% ethyl acetate in
petroleum ether as eluent to get the known18a tetraacetate 5 (0.96
g, 90%) as a sticky mass.
4
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3. Synthesis of 2,3′,4′-tri-O-acetyl-2′,5,6′-tri-O-benzyl-3-O-α-
D-glucopyranosyl-β-D-ribofuranosyl azide (6)
To a solution of tetraacetate 5 (0.51 g, 0.68 mmol) and TMSN3 60 ribofuranosyl azide (8)
5. Synthesis
of
2′,5,6′-tri-O-benzyl-2,3′,4′-
tris(dibenzyloxyphosphoryl)-3-O-α-D-glucopyranosyl-β-D-
(0.44 mL, 3.4 mmol) in anhydrous dichloromethane (10 mL) was
added AlCl3 (0.09 g, 0.68 mmol) at 0oC. The reaction mixture
was stirred at 0oC under nitrogen atmosphere for 10 min. When
the starting material disappeared (TLC), the reaction mixture was
A solution of the triol 7 (0.1 g, 0.16 mmol), bis(benzyloxy) N,N-
5
diisopropylaminophosphine (0.27 mL, 0.82 mmol), and
imidazolium triflate (0.20 g, 0.90 mmol) in anhydrous DCM (10
mL) was stirred at room temperature under N2 atmosphere for 30
quenched by the addition of aqueous NaHCO3. The reaction 65 min. The reaction mixture was then cooled to -78 °C, and then m-
mixture was partitioned between dichloromethane and aqueous
CPBA (0.093 g, 0.54 mmol) was added. The reaction mixture
was then stirred for 45 min at -78 °C and then at room
temperature for an additional 30 min. The reaction was quenched
by the addition of aqueous NaHCO3. The reaction mixture was
10 layers in a separating funnel. The dichloromethane layer was
washed with brine, dried over Na2SO4 and evaporated under
reduced pressure. The crude product thus obtained was purified
by flash column chromatography using 25% ethyl acetate in 70 partitioned between DCM and aqueous layer in a separating
petroleum ether as eluent to get triacetate azide 6 (0.495 g, 99%)
15 as a colourless gum. H NMR (500 MHz, CDCl3) δ: 1.79 (s, 6H,
2 x COCH3), 1.86 (s, 3H, COCH3), 3.22 (dd, J = 10.0, 4.0 Hz,
funnel. The DCM layer was washed with brine, dried over
Na2SO4 and evaporated under reduced pressure. The crude
product thus obtained was purified by flash column
1
1H, H-6A′), 3.26 (dd, J = 10.0, 2.0 Hz, 1H, H-6B′), 3.46 (dd, J =
10.0, 3.4 Hz, 1H, H-2′), 3.52 (dd, J = 11.0, 4.0 Hz, 1H, H-5A), 75 to get pure 8 (0.206 g, 90%) as a colourless gum: H NMR (500
chromatography using 25% acetone in petroleum ether as eluent
1
3.61 (dd, J = 11.0, 3.0 Hz, 1H, H-5B), 3.77 (d, J = 9.8, Hz, 1H, H-
20 5′), 4.22 (d, J = 12.0, Hz, 1H, 0.5 x PhCH2), 4.29-4.30 (m, 1H, H-
4), 4.38-4.43 (m, 3H, 1.5 x PhCH2), 4.46-4.54 (m, 3H, H-3 and 1
x PhCH2), 4.88 (d, J = 3.3 Hz, 1H, H-1′), 4.95 (t, J = 9.8 Hz, 1H,
MHz, CDCl3) δ: 3.43-3.58 (m, 5H, H-6A′, H-6B′, H-5B, H-5A, H-
2′), 3.70-3.72 (m, 1H, H-5′), 4.17-4.22 (m, 2H, H-4, and 0.5 x
PhCH2), 4.27-4.34 (m, 3H, H-3 and 1 x PhCH2), 4.37-4.42 (m,
2H, 1 x PhCH2), 4.47 (dd, 19.05, 9.75 Hz, 1H, H-4′), 4.55-4.59
H-4′), 5.02 (d, J = 2.6 Hz, 1H, H-2), 5.27-5.29 (m, 2H, H-1 and 80 (m, 2H, H-2, 0.5 x PhCH2), 4.65 (dd, 11.95, 8.5 Hz, 1H, 0.5 x
H-3′), 7.16-7.22 (m, 15H, 3 x Ph); 13C NMR (125 MHz, CDCl3)
25 δ: 20.5, 20.7, 20.9 (COCH3), 67.6 (C-6′), 69.0 (C-4′ and C-5′),
69.5 (C-5), 71.9 (C-3′), 73.3, 73.5, 73.5 (3 x CH2Ph), 73.9 (C-2),
74.5 (C-3), 76.6 (C-2′), 81.4 (C-4), 92.77 (C-1), 96.7 (C-1′),
PhCH2), 4.78-4.94 (m, 12H, H-3′ and 5.5 x PhCH2), 5.01 (d, 3.55
Hz, 1H, H-1′), 5.30 (d, 2.6 Hz, 1H, H-1), 6.96-7.25 (m, 45H, 9 x
Ph), 13C NMR (125 MHz, CDCl3) δ: 68.1 (C-6′), 69.1 (C-5), 69.3,
69.3, 69.4, 69.5, 69.8, 69.83, 69.9, (31P coupled, PhCH2OP), 70.0
127.5, 127.7, 127.7, 127.8, 128.0, 128.4, 128.4, 128.5 (Ar-C), 85 (C-5′), 72.0 (1 x PhCH2OC), 73.2 (C-3), 73.3, 73.5 (2 x
137.5, 137.7, 137.9 (ipso Ar-C), 169.8, 170.5, 170.5 (3 x
30 COCH3); Anal. calcd for C38H43N3O12: C, 62.20; H, 5.91; N,
5.73. found: C, 62.12; H, 6.09; N, 5.71.
PhCH2OC), 74.4 (31P coupled, C-4′), 76.9 (C-2′), 77.6 (C-3′, 31P
coupled), 78.1 (C-2, 31P coupled), 81.3 (C-4), 92.4 (C-1), 95.2
(C-1′), 127.6, 127.65, 127.70, 127.8, 128.06, 128.08, 128.26,
128.28, 128.31, 128.34, 128.39, 128.44, 128.49, 128.52, 128.62,
90 128.64, 128.68, 128.69 (aromatic carbons), 135.40, 135.45,
135.70, 135.77, 135.85, 135.90, 136.14, 136.16, 136.20, 136.23,
4. Synthesis of 2′,5,6′-tri-O-benzyl-3-O-α-D-glucopyranosyl-β-
D-ribofuranosyl azide (7)
137.6, 137.9, 138.1, (31P coupled, ipso carbons of POCH2Ph); 31
P
To the suspension of triacetate 6 (0.42 g, 0.57 mmol) in MeOH
35 (10 mL) was added sodium methoxide (0.015 g, 0.28 mmol) and
stirred at room temperature. Reaction mixture became clear after
30 min. When TLC showed complete disappearance of starting
material, the reaction was quenched by adding Dowex 50-ion
exchange resin. The reaction mixture was then filtered through a
40 filter paper and washed successively with methanol (2 times).
The filtrate was concentrated under reduced pressure to get a
thick mass. The thick mass was then purified by flash column
chromatography using 50% ethyl acetate in petroleum ether as 100
eluent to get triol azide 7 (0.34 g, 96%) as a white solid. mp: 69-
NMR (202.4 MHz, CDCl3) δ: -1.20, -1.90, -2.15; Anal. calcd for
C74H76N3O18P3: C, 64.02; H, 5.52; N, 3.03. Found: C, 64.33; H,
95 5.45; N, 3.19.
6. Synthesis of fully protected triazolophostins:
a) Synthesis of 1-{5-O-benzyl-2-O-bisbenzyloxyphosphoryl-3-
O-[2,6-di-O-benzyl-3,4-di-O-(bisbenzyloxyphosphoryl)-α-D-
glucopyranosyl]-β-D-ribofuranosyl}-1,2,3-triazole (9a)
A
mixture of tris-phosphate
8
(0.1 g, 0.07 mmol),
trimethylsilylacetylene (0.011 mL, 0.071 mmol), Cu (0.018 g,
0.28 mmol) and CuSO4 (0.004 g, 0.014 mmol) in H2O/tBuOH
(1/1, v/v, 1 mL) was stirred at room temperature for 12 h. After
complete consumption of the starting material (TLC, 12 h),
o
1
45 71 C; H NMR (500 MHz, CDCl3) δ: 3.33 (dd, J = 9.5, 3.5 Hz,
1H, H-2′), 3.43–3.54 (m, 5H, H-4′, H-5A, H-5B, H-6A′, H-6B′),
3.66 (ddd, J = 9.8, 8.7, 3.9, 1H, H-5′), 3.82-3.85 (m, 2H, H-3′ and
H-2), 4.16-4.18 (m, 2H, 3-H and H-4), 4.38-4.48 (m, 4H, 2 x 105 TBAF (1.0 M in THF, 0.5 mL) was added to the reaction and the
CH2Ph), 4.61-4.70 (m, 3H, H-1′ and 1 x CH2Ph), 5.20 (d, J = 1.4
50 Hz, 1H, H-1), 7.18–7.29 (m, 15H, 3 x Ar-H); 13C NMR (125
MHz, CDCl3) δ: 69.2 (C-5 or C-6′), 70.2 (C-5 or C-6′), 70.6 (C-
5′), 71.1 (C-4′), 73.3 (C-3′), 73.5 and 73.6 (2 x CH2Ph), 74.0 (C-
mixture was stirred for additional 2 h at room temperature. The
reaction mixture was then filtered and washed with ethyl acetate.
The filtrate was extracted with ethyl acetate (2 x 10 mL) and
washed with water and brine. The organic layer was dried over
2), 74.3 (1 x CH2Ph), 78.3 (C-3 or C-4), 78.4 (C-2′), 81.4 (C-3 or 110 MgSO4 and concentrated under reduced pressure. The crude
C-4), 95.1 (C-1), 98.0 (C-1′), 127.6, 127.7, 127.8, 127.9, 128.4,
55 128.5, 128.6, 128.8 (Ar-C), 137.1, 137.6, 137.9 (Ipso Ar-C);
Anal. calcd for C32H37N3O9: C, 63.25; H, 6.14; N, 6.92; found: C,
63.23; H, 6.16; N, 6.90.
product thus obtained was purified by flash column
chromatography using a mixture of acetone and petroleum ether
(3:7 v/v) as eluent to get pure 9a (0.098 g, 97%) as a colourless
1
gum. H NMR (500 MHz, CDCl3) δ: 3.43-3.45 (m, 1H, H-5A′),
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DOI: 10.1039/C5OB00440C
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Page 6 of 12
3.47-3.57 (m, 3H, H-2′′, H-5B′, H-6A′′), 3.59-3.61 (m, 1H, H-6B′′)
3.75 (ddd, J = 9.2, 4.65, 4.2 Hz, 1H, H-5′′), 4.21-4.23 (m, 1H, 0.5
x PhCH2), 4.29-4.32 (m, 4H, H-4′, 1.5 x PhCH2), 4.39-4.46 (m,
glucopyranosyl]-β-D-ribofuranosyl}-4-phenyl-1,2,3-triazole
(9c)
A
mixture of tris-phosphate
8
(0.1 g, 0.07 mmol),
3H, H-3′, H-4′′ and 0.5 x PhCH2), 4.58-4.66 (m, 3H, 1.5 x 60 phenylacetylene (0.016 mL, 0.158 mmol), Cu (0.018 g, 0.28
5
PhCH2), 4.68-4.72 (m, 3H, 1.5 x PhCH2), 4.80-4.96 (m, 8H, H-
3′′, 3.5 x PhCH2), 5.13 (d, J = 3.0 Hz, 1H, H-1′′), 5.27 (ddd, J =
8.75, 8.2, 4.6 Hz, 1H, H-2′), 6.31 (d, 4.9 Hz, 1H, H-1′), 6.99-7.27
(m, 45H, 9 x C6H5), 7.48 (s, 1H, H-5), 7.61 (s, 1H, H-4); 13C
mmol) and CuSO4 (0.004 g, 0.014 mmol) in H2O/tBuOH (1/1,
v/v, 1 mL) was stirred at rt for 12 h. After completion of the
reaction (TLC) the mixture was filtered. The filtrate was
extracted with ethyl acetate and washed successively with water
NMR (125 MHz, CDCl3) δ: 68.5 (C-6′′), 69.1 (C-5′), 69.2, 69.36, 65 and brine. The organic layer was dried over MgSO4 and
10 69.40, 69.5, 69.6, 69.8, 69.83, 69.9, 70.0, 70.02, (31P coupled,
PhCH2OP), 70.3 (C-5′′), 71.9, 73.4, 73.6 (3 x PhCH2OC), 73.9
(C-3′), 74.5 (31P coupled, C-4′′), 76.9 (C-2′), 78.0 (C-3′′, 31P
coupled), 78.6 (C-2′, 31P coupled), 82.81 (C-4′), 90.0 (C-1′), 95.6
concentrated under reduced pressure. The crude product thus
obtained was purified by flash column chromatography using a
mixture of acetone and petroleum ether (3:7 v/v) as eluent to get
pure 9c (0.101 g, 94%) as a colourless gum. 1H NMR (500 MHz,
(C-1′′), 122.4 (C-5), 127.6, 127.7, 127.74, 127.77, 127.8, 127.9, 70 CDCl3) δ: 3.46-3.52 (m, 2H, H-2′′ and H-5A′), 3.55-3.58 (m, 2H,
15 128.0, 128.07, 128.1, 128.2, 128.3, 128.34, 128.4, 128.5, 128.52,
128.55, 128.59, 128.7, (aromatic carbons), 135.2, 135.28, 135.7,
135.74, 135.8, 135.87, 136.1, 136.2, 137.4, 137.6, 138.0, (31P
coupled, ipso carbons of POCH2Ph); 31P NMR (202.4 MHz,
H-6B′′ and H-5B′), 3.64-3.66 (m, 1H, H-6A′′), 3.77-3.80 (m, 1H,
H-5′′), 4.21-4.35 (m, 5H, H-4′ and 2 x PhCH2), 4.41-4.46 (m, 3H,
H-3′, H-4′′ and 0.5 x PhCH2), 4.58-4.64 (m, 3H, 1.5 x PhCH2),
4.68-4.72 (m, 3H, 1.5 x PhCH2), 4.81-4.94 (m, 8H, H-3′′, and 3.5
CDCl3) δ: -1.427, -1.908, -2.128; Anal. calcd for C76H78N3O18P3: 75 x PhCH2), 5.16 (d, J = 3.5 Hz, 1H, H-1′′), 5.33 (ddd, J = 9.4,
20 C, 64.54; H, 5.56; N, 2.97. Found: C, 64.31; H, 5.42; N, 3.01.
8.55, 5.0, Hz, 1H, H-2′), 6.33 (d, 5.4 Hz, 1H, H-1′), 6.99-7.47 (m,
50H, 10 x C6H5), 7.83 (s, 1H, H-5); 13C NMR (125 MHz, CDCl3)
δ: 68.7 (C-6′′), 69.2 (C-5′), 69.2, 69.4, 69.45, 69.5, 69.55, 69.6,
69.85, 69.88, 70.0, 70.02, 70.1 (31P coupled, PhCH2OP), 70.4 (C-
80 5′′), 72.0, 73.5, 73.7 (3 x PhCH2OC), 74.5 (C-3′), 74.6 (31P
coupled, C-4′′), 77.0 (31P coupled, C-2′′), 78.1 (C-3′′, 31P
coupled), 78.8 (C-2′), 83.0 (C-4′), 90.1 (C-1′), 95.9 (C-1′′), 118.2
(C-5), 125.8 (ArC), 127.6, 127.7, 127.8, 127.86, 128.0, 128.1,
128.2, 128.3, 128.4, 128.45, 128.50, 128.53, 128.6, 128.7
85 (aromatic carbons), 130.3, 135.2, 135.23, 135.3, 135.7, 135.8,
135.87, 135.93, 136.2, 136.25, 137.4, 137.6, 138.1, 148.1; (31P
coupled, ipso carbons of POCH2Ph); 31P NMR (202.4 MHz,
CDCl3) δ: -1.551, -1.932, -2.105; Anal. calcd for C82H82N3O18P3:
C, 66.08; H, 5.55; N, 2.82. Found: C, 65.95; H, 5.72; N, 2.97.
b) Synthesis of 1-{5-O-benzyl-2-O-bisbenzyloxyphosphoryl-3-
O-[2,6-di-O-benzyl-3,4-di-O-(bisbenzyloxyphosphoryl)-α-D-
glucopyranosyl]-β-D-ribofuranosyl}-4-(1-methylimidazol-5-
yl)-1,2,3-triazole (9b)
25 A mixture of tris-phosphate 8 (0.2 g, 0.14 mmol), 5-ethynyl-1-
methyl-1H-imidazole (0.016 mL, 0.15 mmol), Cu (0.036 g, 0.57
mmol) and CuSO4 (0.008 g, 0.028 mmol) in H2O/tBuOH (1/1,
v/v, 2 mL) was stirred at room temperature for 12 h. After
completion of the reaction, the mixture was filtered and washed
30 successively with ethyl acetate. The filtrate was extracted with
ethyl acetate and washed with water and brine. The organic layer
was dried over MgSO4 and concentrated under reduced pressure.
The resulting crude product was purified by flash column
chromatography using a mixture of acetone, diethyl ether and 90 d) Synthesis of 1-{5-O-benzyl-2-O-bisbenzyloxyphosphoryl-3-
35 petroleum ether (4:1:15 v/v/v) as eluent to get pure 9b (0.205 g,
95%) as a colourless gum. H NMR (500 MHz, CDCl3) δ: 3.55-
3.76 (m, 8H, 1 x N-CH3, 6A′′, H-6B′′, H-5B′, H-2′′ and H-5A′), 3.88
(bs, 1H, H-5′′), 4.31-4.45 (m, 5H, H-4′ and 2 x PhCH2), 4.51-4.54
O-[2,6-di-O-benzyl-3,4-di-O-(bisbenzyloxyphosphoryl)-α-D-
glucopyranosyl]-β-D-ribofuranosyl}-4-(2-pyridyl)-1,2,3-
triazole (9d)
1
A
mixture of tris-phosphate 8 (0.2 g, 0.14 mmol), 2-
(m, 3H, H-4′′ and 1 x PhCH2), 4.70-4.82 (m, 6H, H-3′, and 2.5 x 95 ethynylpyridine (0.016 mL, 0.15 mmol), Cu (0.036 g, 0.57 mmol)
40 PhCH2), 4.94-5.03 (m, 8H, H-3′′ and 3.5 x PhCH2), 5.26 (bs, 1H,
H-1′′), 5.41 (bs, 1H, H-2′), 6.42 (s, 1H, H-1′), 6.97-7.34 (m, 47H,
Ar-H), 7.75 (s, 1H, H-5); 13C NMR (125 MHz, CDCl3) δ: 33.4
(N-CH3), 68.6 (C-6′′), 69.2 (C-5′), 69.4, 69.42, 69.5, 69.59, 69.8,
and CuSO4 (0.008 g, 0.028 mmol) in H2O/tBuOH (1/1, v/v, 2
mL) was stirred at rt for 12 h. After completion of the reaction
(TLC) the mixture was filtered. The filtrate was extracted with
ethyl acetate and washed successively with water and brine. The
69.9, 70.03, (31P coupled PhCH2OP), 70.3 (C-5′′), 71.92, 73.4, 100 organic layer was dried over MgSO4 and concentrated under
45 73.7 (3 x PhCH2OC), 74.3 (C-3′), 74.5 (C-4′′, 31P coupled), 77.0
(C-2′, 31P coupled), 78.0 (C-2′′), 78.8 (C-3′′, 31P coupled), 83.1
(C-4′), 90.4 (C-1′, 31P coupled), 95.77 (C-1′′), 119.41 (C-5),
127.64, 127.67, 127.71, 127.80, 127.8, 127.96, 128.0, 128.1,
reduced pressure. The crude product thus obtained was purified
by flash column chromatography using a mixture of acetone and
petroleum ether (3:7 v/v) as eluent to get pure 9d (0.104 g, 48%)
as a colourless gum. 1H NMR (500 MHz, CDCl3) δ: 3.43-3.45 (m,
128.14, 128.3, 128.5, 128.52, 128.6, 128.67, (aromatic carbons), 105 1H, H-5A′), 3.48-3.59 (m, 4H, H-2′′, H-5B′, H-6B′′and H-6A′′),
50 135.1, 135.2, 135.6, 135.76, 135.80, 135.9, 136.1, 136.18, 137.2,
137.6, (31P coupled, ipso carbons of POCH2Ph), 138.0, 138.8
(ipso carbons of COCH2Ph); 31P NMR (202.4 MHz, CDCl3) δ: -
1.453, -1.894, -2.080; Anal. calcd for C80H82N5O18P3: C, 64.29;
H, 5.53; N, 4.69. Found: C, 63.99; H, 5.23; N, 4.81.
3.64-3.71-3.76 (m, 1H, H-5′′), 4.20-4.23 (m, 1H, 0.5 x PhCH2),
4.27-4.37 (m, 4H, H-4′ and 1.5 x PhCH2), 4.41-4.45 (m, 3H, H-3′,
H-4′′ and 0.5 x PhCH2), 4.60-4.62 (m, 1H, 0.5 x PhCH2), 4.66-
4.72 (m, 5H, 2.5 x PhCH2), 4.80-4.93 (m, 8H, H-3′′, and 3.5 x
110 PhCH2), 5.15 (d, J = 2.9 Hz, 1H, H-1′′), 5.34-5.35 (m, 1H, H-2′),
6.30 (d, J = 5.0 Hz, 1H, H-1′), 6.99-7.19 (m, 45H, 9 x C6H5),
7.28 (d, J = 7.0 Hz, 1H, pyr-H), 7.72 (t, J = 7.0 Hz, 1H, pyr-H),
8.03 (d, J = 7.0 Hz, 1H, pyr-H), 8.44 (s, 1H, H-5), 8.52 (d, J = 4.0
55 c) Synthesis of 1-{5-O-benzyl-2-O-bisbenzyloxyphosphoryl-3-
O-[2,6-di-O-benzyl-3,4-di-O-(bisbenzyloxyphosphoryl)-α-D-
6
| Org. Biomol. Chem., 2015, 13, 00–00
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DOI: 10.1039/C5OB00440C
Page 7 of 12
Organic & Biomolecular Chemistry
Hz, 1H, pyr-H); 13C NMR (125 MHz, CDCl3) δ: 68.4 (C-6′′),
acetate and washed successively with water and brine. The
organic layer was dried over MgSO4 and concentrated under
reduced pressure. The crude product thus obtained was purified
69.1 (C-5′), 69.13, 69.18, 69.4, 69.42, 69.5, 69.55, 69.8, 69.85,
69.87, 69.9, 70.0, 70.04 (31P coupled, PhCH2OP), 70.3 (C-5′′),
72.0, 73.38, 73.6 (3 x PhCH2OC), 74.2 (C-3′), 74.5 (31P coupled, 60 by flash column chromatography using a mixture of acetone and
5
C-4′′), 77.1 (31P coupled, C-2′′), 78.1 (C-3′′, 31P coupled), 78.7
(C-2′), 83.1 (C-4′), 90.4 (C-1′), 95.7 (C-1′′), 120.6 (C-5), 123.0
(ArC), 127.6, 127.7, 127.8, 127.9, 128.0, 128.1, 128.2, 128.3,
128.4, 128.43, 128.49, 128.5, 128.55 (aromatic carbons), 135.2,
petroleum ether (1:3 v/v) as eluent to get 9f (0.188 g, 86%) as a
colourless gum; H NMR (500 MHz, CDCl3) δ: 3.58 (dd, J =
10.85, 2.45 Hz, 1H, H-5A′), 3.62 (dd, J = 9.65, 3.65 Hz, 1H, H-
2′′), 3.66-3.69 (m, 2H, H-6A′′, H-5B′), 3.78 (dd, J = 10.8, 1.5 Hz,
1
135.25, 135.28, 135.30, 136.19, 136.25, 137.37, 137.60, 138.11; 65 1H, H-6B′′) 3.90 (ddd, J = 10.8, 9.2, 4.2 Hz, 1H, H-5′′), 4.33-4.38
10 (31P coupled, ipso carbons of POCH2Ph); 31P NMR (202.4 MHz,
CDCl3) δ: -1.60, -1.95, -2.13; Anal. calcd for C81H81N4O18P3: C,
65.23; H, 5.47; N, 3.76. Found: C, 65.01; H, 5.21; N, 3.92.
(m, 2H, 1 x PhCH2), 4.41-4.44 (m, 2H, 1 x PhCH2), 4.46-4.47 (m,
1H, H-4′), 4.52-4.56 (m, 3H, H-3′, H-4′′ and 0.5 x PhCH2), 4.70-
4.75 (m, 3H, 1.5 x PhCH2), 4.80-4.84 (m, 3H, 1.5 x PhCH2),
4.92-5.06 (m, 8H, H-3′′, 3.5 x PhCH2), 5.28 (d, J = 3.6 Hz, 1H,
70 H-1′′), 5.43 (ddd, J = 8.85, 5.2, 4.65 Hz, 1H, H-2′), 6.44 (d, 5.6
Hz, 1H, H-1′), 7.09-7.39 (m, 49H, Ar-H), 7.90 (s, 1H, H-5); 13C
NMR (125 MHz, CDCl3) δ: 68.7 (C-6′′), 69.1 (C-5′), 69.4, 69.5,
69.8, 69.6, 69.7, 69.87, 69.9, 69.94, 70.07, 70.1 (31P coupled,
PhCH2OP), 72.0 (C-5′′), 72.1, 73.5, 73.7 (3 x PhCH2OC), 74.5
75 (C-3′), 74.53 (31P coupled, C-4′′), 77.1 (31P coupled, C-2′′), 78.1
(C-3′′, 31P coupled), 78.9 (C-2′), 83.1 (C-4′), 90.3 (C-1′), 95.9 (C-
1′′), 112.7, 114.8 (Ar-C), 118.5 (C-5), 121.4 (Ar-C), 127.6, 127.7,
127.8, 127.83, 127.9, 128.0, 128.07, 128.14, 128.31, 128.34,
128.43, 128.48, 128.5, 128.51, 128.69, (aromatic carbons), 130.2,
80 130.27, 136.2, 136.23, 137.31, 137.61, 138.1, 148.1, 162.3, 164.2
(31P coupled, ipso carbons of POCH2Ph); 31P NMR (202.4 MHz,
CDCl3) δ: -1.515, -1.920, -2.064; 19F NMR (470.68 MHz, CDCl3)
δ: -112.766; Anal. calcd for C82H81FN3O18P3: C, 65.29; H, 5.41;
N, 2.79. Found: C, 65.50; H, 5.21; N, 2.66.
e) Synthesis of 1-{5-O-benzyl-2-O-bisbenzyloxyphosphoryl-3-
O-[2,6-di-O-benzyl-3,4-di-O-(bisbenzyloxyphosphoryl)-α-D-
15 glucopyranosyl]-β-D-ribofuranosyl}-4-(2-fluorophenyl)-1,2,3-
triazole (9e)
A mixture of tris-phosphate 8 (0.2 g, 0.14 mmol), 2-fluorophenyl
acetylene (0.021 mL, 0.15 mmol), Cu (0.036 g, 0.57 mmol) and
CuSO4 (0.008 g, 0.028 mmol) in H2O/tBuOH (1/1, v/v, 2 mL)
20 was stirred at rt for 12 h. After completion of the reaction (TLC)
the mixture was filtered. The filtrate was extracted with ethyl
acetate and washed successively with water and brine. The
organic layer was dried over MgSO4 and concentrated under
reduced pressure. The crude product thus obtained was purified
25 by flash column chromatography using a mixture of acetone and
petroleum ether (1:3 v/v) as eluent to get 9e (0.203 g, 93%) as a
colourless gum. 1H NMR (500 MHz, CDCl3) δ: 3.53-3.55 (m, 1H,
H-5A′), 3.61-3.73 (m, 4H, H-2′′, H-5B′, H-6A′′ and H-6B′′), 3.88
(bs, 1H, H-5′′), 4.33-4.35 (m, 1H, 0.5 x PhCH2), 4.42-4.48 (m, 85 g) Synthesis of 1-{5-O-benzyl-2-O-bisbenzyloxyphosphoryl-3-
30 4H, H-4′ and 1.5 x PhCH2), 4.53-4.58 (m, 3H, H-3′, H-4′′ and 0.5
x PhCH2), 4.72-4.81 (m, 6H, 3 x PhCH2), 4.96-5.06 (m, 8H, H-
3′′, 3.5 x PhCH2), 5.27 (bs, 1H, H-1′′), 5.46 (bs, 1H, H-2′), 6.42
(bs, 1H, H-1′), 7.13-7.40 (m, 48H, Ar-H), 8.21 (s, 1H, H-5), 8.30
O-[2,6-di-O-benzyl-3,4-di-O-(bisbenzyloxyphosphoryl)-α-D-
glucopyranosyl]-β-D-ribofuranosyl}-4-(4-fluorophenyl)-1,2,3-
triazole (9g)
A mixture of azide 8 (0.2 g, 0.14 mmol), 4-fluorophenyl
(bs, 1H, Ar-H); 13C NMR (125 MHz, CDCl3) δ: 68.4 (C-6′′), 69.0 90 acetylene (0.018 mL, 0.15 mmol), Cu (0.036 g, 0.57 mmol) and
35 (C-5′), 69.1, 69.2, 69.4, 69.42, 69.5, 69.6, 69.8, 69.85, 69.89,
70.0, 70.05 (31P coupled, PhCH2OP), 70.2 (C-5′′), 71.9, 73.4,
73.6 (3 x PhCH2OC), 74.2 (C-3′), 74.4 (31P coupled, C-4′′), 77.1
(31P coupled, C-2′′), 78.0 (C-3′′, 31P coupled), 78.6 (C-2′), 83.0
CuSO4 (0.008 g, 0.028 mmol) in H2O/tBuOH (1/1, v/v, 2 mL)
was stirred at rt for 12 h. After completion of the reaction (TLC),
the mixture was filtered, the filtrate was extracted with ethyl
acetate and washed successively with water and brine. The
(C-4′), 90.2 (C-1′), 95.7 (C-1′′), 115.7 (C-5), 121.6, 121.7, 124.5, 95 organic layer was dried over MgSO4 and concentrated under
40 124.6, 127.6, 127.9, 128.0, 128.1, 128.5, 128.6 (aromatic
carbons), 129.4, 129.5, 135.2, 135.22, 135.3, 135.7, 135.8,
135.84, 135.89, 136.2, 136.22, 137.3, 137.6, 138.1, 141.48,
141.5, 158.3, 160.3 (31P coupled, ipso carbons of POCH2Ph); 31P
reduced pressure. The resulting crude product thus obtained was
purified by flash column chromatography using a mixture of 25%
acetone in petroleum ether as eluent to get pure 9g (0.207 g, 95%)
1
as a colourless gum; H NMR (500 MHz, CDCl3) δ: 3.47-3.52
NMR (202.4 MHz, CDCl3) δ: -1.632, -1.930, -2.134; 19F NMR 100 (m, 2H, H-2′′ and H-5A′), 3.55-3.59 (m, 2H, H-6A′′, H-5B′), 3.65-
45 (470.68 MHz, CDCl3) δ: -114.22; Anal. calcd for
C82H81FN3O18P3: C, 65.29; H, 5.41; N, 2.79. Found: C, 65.43; H,
5.59; N, 2.67.
3.67 (m, 1H, H-6B′′), 3.77-3.80 (m, 1H, H-5′′), 4.21-4.28 (m, 2H,
1 x PhCH2), 4.30-4.35 (m, 3H, H-4′), 4.39-4.45 (m, 3H, H-4′′ and
1 x PhCH2), 4.61-4.65 (m, 3H, H-3′, and 1 x PhCH2), 4.67-4.73
(m, 3H, 1.5 x PhCH2), 4.81-4.97 (m, 8H, H-3′′, 3.5 x PhCH2),
105 5.16 (d, J = 3.6 Hz, 1H, H-1′′), 5.32 (ddd, J = 9.2, 8.5, 5.0 Hz,
1H, H-2′), 6.34 (d, 5.6 Hz, 1H, H-1′), 6.94-7.37 (m, 49H, Ar-H),
7.79 (s, 1H, H-5); 13C NMR (125 MHz, CDCl3) δ: 68.6 (C-6′′),
69.2 (C-5′), 69.22, 69.4, 69.45, 69.5, 69.56, 69.6, 69.8, 69.9,
69.91, 70.03, 70.07 (31P coupled, PhCH2OP), 70.3 (C-5′′), 72.0,
110 73.5, 73.7 (3 x PhCH2OC), 74.4 (C-3′), 74.5 (31P coupled, C-4′′),
77.3 (31P coupled, C-2′′), 78.0 (C-3′′, 31P coupled), 78.9 (C-2′),
83.0 (C-4′), 90.2 (C-1′), 95.9 (C-1′′), 115.6 (C-5), 117.9, 127.5,
127.51, 127.62, 127.67, 127.7, 127.8, 127.87, 128.0, 128.01,
f) Synthesis of 1-{5-O-benzyl-2-O-bisbenzyloxyphosphoryl-3-
O-[2,6-di-O-benzyl-3,4-di-O-(bisbenzyloxyphosphoryl)-α-D-
50 glucopyranosyl]-β-D-ribofuranosyl}-4-(3-fluorophenyl)-1,2,3-
triazole (9f)
A mixture of azide 8 (0.2 g, 0.14 mmol), 3-fluorophenyl
acetylene (0.021 mL, 0.15 mmol), Cu (0.036 g, 0.57 mmol) and
CuSO4 (0.008 g, 0.028 mmol) in H2O/tBuOH (1/1, v/v, 2 mL)
55 was stirred at rt for 12 h. After completion of the reaction (TLC),
the mixture was filtered, the filtrate was extracted with ethyl
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Organic & Biomolecular Chemistry
Page 8 of 12
128.07, 128.1, 128.16, 128.20, 128.35, 128.39, 128.47, 128.5,
128.55, 128.65, 128.67 (aromatic carbons), 136.14, 136.20,
137.4, 137.6, 138.0, 147.5, 162.1, 164.4 (31P coupled, ipso
organic layer was dried over MgSO4 and concentrated under
reduced pressure. The crude product thus obtained was purified
by flash column chromatography using a mixture of acetone and
carbons of POCH2Ph); 31P NMR (202.4 MHz, CDCl3) δ: -1.49, - 60 petroleum ether (1:3 v/v) as eluent to get 9i (0.193 g, 88%) as a
1
5
1.920, -2.092; 19F NMR (470.68 MHz, CDCl3) δ: -113.585; Anal.
calcd for C82H81FN3O18P3: C, 65.29; H, 5.41; N, 2.79. Found: C,
64.98; H, 5.11; N, 2.93.
colourless gum; H NMR (500 MHz, CDCl3) δ: 3.49-3.56 (m,
5H, 6A′′, H-6B′′, H-5B′, H-2′′ and H-5A′), 3.77 (bs, 4H, 1 x OCH3
and H-5′′), 4.21-4.35 (m, 5H, H-4′ and 2 x PhCH2), 4.40-4.45 (m,
3H, H-3′, H-4′′ and 0.5 x PhCH2), 4.58-4.72 (m, 6H, and 3 x
65 PhCH2), 4.84-4.93 (m, 8H, H-3′′ and 3.5 x PhCH2), 5.16 (bs, 1H,
H-1′′), 5.32 (bs, 1H, H-2′), 6.33 (d, J = 3.2 Hz, 1H, H-1′), 6.80 (d,
J = 7.5 Hz, 2H, Ar-H), 7.00-7.28 (m, 45H, 9 x C6H5), 6.38 (d, J =
7.5 Hz, 2H, Ar-H), 7.75 (s, 1H, H-5); 13C NMR (125 MHz,
CDCl3) δ: 55.4 (OCH3), 68.6 (C-6′′), 69.16 (C-5′), 69.2, 69.4,
70 69.44, 69.5, 69.58, 69.85, 69.9, 70.07, (31P coupled PhCH2OP),
70.3 (C-5′′), 72.0, 73.4, 73.6 (3 x PhCH2OC), 74.4 (C-3′ and C-
4′′, 31P coupled), 77.0 (C-2′, 31P coupled), 78.0 (C-2′′), 78.6 (C-
3′′, 31P coupled), 83.0 (C-4′), 90.3 (C-1′, 31P coupled), 95.9 (C-
1′′), 114.2, 117.5 (C-5), 127.1, 127.2, 127.6, 127.7, 127.8, 127.82,
75 127.85, 128.0, 128.03, 128.07, 128.1, 128.2, 128.3, 128.4,
128.45, 128.5, 128.52, 128.6, 128.64, (aromatic carbons), 135.1,
135.15, 135.2, 135.65, 135.70, 135.8, 135.84, 136.1, 136.16,
137.4, 137.6, (31P coupled, ipso carbons of POCH2Ph), 138.0
(ipso carbon of COCH2Ph); 31P NMR (202.4 MHz, CDCl3) δ: -
80 1.576, -1.941, -2.118; Anal. calcd for C83H84N3O19P3: C, 65.56;
H, 5.57; N, 2.76. Found: C, 65.66; H, 5.77; N, 2.95.
h) Synthesis of 1-{5-O-benzyl-2-O-bisbenzyloxyphosphoryl-3-
O-[2,6-di-O-benzyl-3,4-di-O-(bisbenzyloxyphosphoryl)-α-D-
10 glucopyranosyl]-β-D-ribofuranosyl}-4-(3,5-difluorophenyl)-
1,2,3-triazole (9h)
A mixture of azide 8 (0.2 g, 0.14 mmol), 3,5-difluorophenyl
acetylene (0.018 mL, 0.15 mmol), Cu (0.036 g, 0.57 mmol) and
CuSO4 (0.008 g, 0.028 mmol) in H2O/tBuOH (1/1, v/v, 2 mL)
15 was stirred at rt for 12 h. After completion of the reaction (TLC),
the mixture was filtered. The filtrate was extracted with ethyl
acetate and washed successively with water and brine. The
organic layer was dried over MgSO4 and concentrated under
reduced pressure. The crude product thus obtained was purified
20 by flash column chromatography using a mixture of 25% acetone
in petroleum ether as eluent to get pure 9h (0.2 g, 91%) as a
1
colourless gum;. H NMR (500 MHz, CDCl3) δ: 3.46 (dd, J =
10.8, 2.1 Hz, 1H, H-5A′), 3.51 (dd, J = 9.65, 3.6 Hz, 1H, H-2′′),
3.54-3.58 (m, 2H, H-6A′′ and H-5B′), 3.68 (dd, J = 10.7, 1.3 Hz,
25 1H, H-6B′′), 3.79 (ddd, J = 5.95, 4.85, 3.95 Hz, 1H, H-5′′), 4.24
(dd, J = 11.5, 7.5 Hz, 2H, 1 x PhCH2), 4.29-4.34 (m, 2H, 1 x
PhCH2), 4.36 (d, J = 2.3 Hz, 1H, H-4′), 4.38-4.45 (m, 3H, H-3′,
H-4′′ and 0.5 x PhCH2), 4.59-4.62 (m, 3H, 1.5 x PhCH2), 4.68-
j) Synthesis of 1-{5-O-benzyl-2-O-bisbenzyloxyphosphoryl-3-
O-[2,6-di-O-benzyl-3,4-di-O-(bisbenzyloxyphosphoryl)-α-D-
glucopyranosyl]-β-D-ribofuranosyl}-4-(2-formylphenyl)-1,2,3-
4.72 (m, 3H, 1.5 x PhCH2), 4.81-4.94 (m, 8H, H-3′′, 3.5 x 85 triazole (9j)
30 PhCH2), 5.18 (d, J = 3.6 Hz, 1H, H-1′′), 5.31 (ddd, J = 8.85, 5.2,
4.65 Hz, 1H, H-2′), 6.33 (d, J = 5.75 Hz, 1H, H-1′), 6.97-7.29 (m,
48H, Ar-H), 7.77 (s, 1H, H-5); 13C NMR (125 MHz, CDCl3) δ:
68.7 (C-6′′), 69.2 (C-5′), 69.23, 69.4, 69.45, 69.5, 69.57, 69.6,
A mixture of azide 8 (0.2 g, 0.14 mmol), 2-ethynylbenzaldehyde
(0.02 g, 0.15 mmol), Cu (0.036 g, 0.57 mmol) and CuSO4 (0.008
g, 0.028 mmol) in H2O/tBuOH (1/1, v/v, 2 mL) was stirred at rt
for 12 h. After completion of the reaction (TLC) the mixture was
69.8, 69.85, 69.9, 70.02, 70.07 (31P coupled, PhCH2OP), 70.4 (C- 90 filtered. The filtrate was extracted with ethyl acetate and washed
35 5′′), 72.0, 73.48, 73.7 (3 x PhCH2OC), 74.5 (C-3′), 74.54 (31P
coupled, C-4′′), 77.1 (31P coupled, C-2′′), 78.0 (C-3′′, 31P
coupled), 78.9 (C-2′), 83.3 (C-4′), 90.4 (C-1′), 95.8 (C-1′′), 108.7
(C-5), 118.9 127.6, 127.7, 127.73, 127.85, 127.92, 128.0, 128.1,
successively with water and brine. The organic layer was dried
over MgSO4 and concentrated under reduced pressure. The crude
product thus obtained was purified by flash column
chromatography using a mixture of acetone and petroleum ether
128.14, 128.3, 128.35, 128.39, 128.5, 128.51, 128.54, 128.58, 95 (1:3 v/v) as eluent to obtain pure 9j (0.19 g, 87%) as a colourless
40 128.66, 128.79 (aromatic carbons), 135.2, 135.23, 135.25, 135.7,
135.71, 135.8, 135.89, 136.1, 136.2, 137.2, 137.23, 137.6,
146.45, 162.5, 163.3 (31P coupled, ipso carbons of POCH2Ph);
31P NMR (202.4 MHz, CDCl3) δ: -1.502, -1.902, -2.068; 19F
gum;. 1H NMR (500 MHz, CDCl3) δ: 3.57-3.64 (m, 2H, H-2′′ and
H-5A′), 3.68-3.70 (m, 2H, H-5B′ and H-6A′′), 3.76-3.78 (m, 1H, H-
6B′′), 3.90 (bs, 1H, H-5′′), 4.33-4.44 (m, 4H, 2 x PhCH2), 4.49-
4.58 (m, 4H, H-4′, H-4′′ and 1 x PhCH2), 4.72-4.86 (m, 6H, H-3′,
NMR (470.68 MHz, CDCl3) δ: -109.331; Anal. calcd for 100 and 2.5 x PhCH2), 4.93-5.07 (m, 8H, H-3′′, 3.5 x PhCH2), 5.29
45 C82H80F2N3O18P3: C, 64.52; H, 5.28; N, 2.75. Found: C, 64.37; H,
5.58; N, 2.91.
(bs, 1H, H-1′′), 5.47 (bs, 1H, H-2′), 6.48 (d, 5.0 Hz, 1H, H-1′),
7.13-7.41 (m, 46H, Ar-H), 7.51-7.59 (m, 2H, Ar-H), 8.00 (s, 1H,
H-5), 8.05 (d, J = 7.4 Hz, 1H, Ar-H), 10.35 (s, 1H, CHO); 13C
NMR (125 MHz, CDCl3) δ: 68.6 (C-6′′), 68.66 (C-5′), 69.2,
105 69.22, 69.3, 69.4, 69.45, 69.6, 69.61, 69.87, 69.9, 69.94, 70.07,
70.1 (31P coupled, PhCH2OP), 70.4 (C-5′′), 72.0, 73.5, 73.7 (3 x
PhCH2OC), 74.3 (C-3′), 74.5 (31P coupled, C-4′′), 77.3 (31P
coupled, C-2′′), 78.0 (C-3′′, 31P coupled), 78.9 (C-2′), 83.2 (C-4′),
90.5 (C-1′), 95.8 (C-1′′), 121.7 (C-5), 127.7, 127.71, 127.73,
110 127.8, 127.83, 127.86, 128.0, 128.1, 128.14, 128.19, 128.35,
128.38, 128.5, 128.54, 128.59, 128.69 (aromatic carbons), 129.9,
132.8, 134.1, 136.2, 137.2, 137.6, 138.1, 145.6 (31P coupled, ipso
carbons of POCH2Ph), 192.21 (CHO); 31P NMR (202.4 MHz,
i) Synthesis of 1-{5-O-benzyl-2-O-bisbenzyloxyphosphoryl-3-
O-[2,6-di-O-benzyl-3,4-di-O-(bisbenzyloxyphosphoryl)-α-D-
glucopyranosyl]-β-D-ribofuranosyl}-4-(4-methoxyphenyl)-
50 1,2,3-triazole (9i)
A mixture of azide 8 (0.2 g, 0.14 mmol), 4-methoxyphenyl
acetylene (0.02 mL, 0.15 mmol), Cu (0.036 g, 0.57 mmol) and
CuSO4 (0.008 g, 0.028 mmol) in H2O/tBuOH (1/1, v/v, 2 mL)
was stirred at rt for 12 h. After completion of the reaction (TLC)
55 the mixture was filtered. The filtrate was extracted with ethyl
acetate and washed successively with water and brine. The
8
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Organic & Biomolecular Chemistry
CDCl3) δ: -1.32, -1.89, -2.07; Anal. calcd for C83H82N3O19P3: C,
65.65; H, 5.44; N, 2.77. Found: C, 65.41; H, 5.73; N, 2.90.
triazolophostin 10a was obtained as a white hygroscopic solid: 1H
NMR (500 MHz, D2O) δ: 3.74-3.80 (m, 7H, H-4′′, H-5′′, H-5A′,
H-5B′, H-6A′′, H-6B′′ and H-2′′), 4.03 (bs, 1H, H-4′), 4.41 (bs, 2H,
H-3′ and H-3′′), 5.14 (bs, 1H, H-2′), 5.23 (bs, 1H, H-1′′), 6.38 (bs,
60 1H, H-1′), 7.80 (s, 1H, H-5), 8.18 (s, 1H, H-4); 13C NMR (125
MHz, D2O) δ: 60.3 (C-5′), 60.8 (C-6′′), 70.9 (C-2′′, 31P coupled),
k) Synthesis of 1-{5-O-benzyl-2-O-bisbenzyloxyphosphoryl-3-
O-[2,6-di-O-benzyl-3,4-di-O-(bisbenzyloxyphosphoryl)-α-D-
glucopyranosyl]-β-D-ribofuranosyl}-4-(6-methoxynaphth-2-
yl)-1,2,3-triazole (9k)
5
71.8 (C-5′′, 31P coupled), 72.6 (C-4′′, 31P coupled), 73.6 (C-3′, 31
coupled), 76.3 (C-2′, 31P coupled), 77.3 (C-3′′, 31P coupled), 83.8
(C-4′), 90.8 (C-1′, 31P coupled), 98.0 (C-1′′), 125.1 (C-4, C-5); 31
P
The mixture of azide
8 (0.1 g, 0.07 mmol),2-ethynyl-6-
P
methoxynaphthalene (0.014 g, 0.077 mmol), Cu (0.018 g, 0.28
mmol) and CuSO4 (0.004 g, 0.014 mmol) in H2O/tBuOH (1/1,
65 NMR (202.4 MHz, CDCl3) δ: 3.496, 3.570, 4.382; HRMS (ESI)
mass calcd for C13H24N3O18P3 [M]+, 603.0268, found: 603.0271.
10 v/v, 1 mL) was stirred at rt for 12 h. After completion of the
reaction (TLC) the mixture was filtered. The filtrate was
extracted with ethyl acetate and washed successively with water
and brine. The organic layer was dried over MgSO4 and
concentrated under reduced pressure. The crude product thus
b) 4-(1-methyl-imidazol-5-yl)-triazolophostin (10b)
By using 105 mg (0.07 mmol) of 9b, 45 mg (94%) of
triazolophostin 10b was obtained as a white hygroscopic solid:
15 obtained was purified by flash column chromatography using 70 1H NMR (500 MHz, D2O) δ: 3.74-3.81 (m, 5H, H-2′′, H-5A′, H-
25% acetone in petroleum ether as eluent to get pure, protected
trizolophostin 9k (93 mg, 82%) as a colourless gum; H NMR
(500 MHz, CDCl3) δ: 3.47-3.52 (m, 2H, H-2′′ and H-5A′), 3.55-
3.59 (m, 2H, H-5B′ and H-6A′′), 3.64-3.66 (m, 1H, H-6B′′), 3.77-
5B′, H-6A′′ and H-6B′′), 3.87 (dd, 1H, J = 12.8, 3.05 Hz, H-4′′),
3.94 (s, 3H, N-CH3), 4.08-4.10 (m, 1H, H-5′′), 4.47-4.50 (m, 2H,
H-4′ and H-3′′), 4.66-4.70 (m, 1H, obscured by HDO peak, H-3′),
5.24 (dd, 1H, J = 8.8, 4.45 Hz,, H-2′), 5.27 (d, J = 3. 3 Hz, 1H, H-
1
20 3.80 (m, 1H, H-5′′), 3.85 (s, 3H, OCH3), 4.21-4.24 (m, 1H, 0.5 x 75 1′′), 6.46 (d, J = 3.9 Hz, 1H, H-1′), 7.76 (s, 1H, ImH-4), 8.63 (s,
PhCH2), 4.28-4.36 (m, 4H, H-4′ and 1.5 x PhCH2), 4.41-4.48 (m,
3H, H-3′, H-4′′ and 1 x PhCH2), 4.59-4.65 (m, 3H, 1.5 x PhCH2),
4.69-4.72 (m, 3H, 1.5 x PhCH2), 4.80-4.97 (m, 8H, H-3′′, 3.5 x
PhCH2), 5.18 (d, J = 3.3 Hz, 1H, H-1′′), 5.37 (ddd, J = 9.1, 8.5,
1H, H-5), 8.80 (s, 1H, ImH-2); 13C NMR (125 MHz, D2O) δ: 34.8
(N-CH3), 60.3 (C-5′), 60.7 (C-6′′), 70.6 (C-2′′, 31P coupled), 71.7
(C-5′′, 31P coupled), 72.8 (C-4′′, 31P coupled), 73.6 (C-3′, 31P
coupled), 76.5 (C-2′, 31P coupled), 77.7 (C-3′′, 31P coupled), 84.2
25 4.9 Hz, 1H, H-2′), 6.35 (d, 5.3 Hz, 1H, H-1′), 7.00-7.19 (m, 45H, 80 (C-4′), 91.2 (C-1′, 31P coupled), 98.0 (C-1′′), 118.8 (C-5), 124.8
9 x C6H5), 7.29 (d, J = 7.5 Hz, 2H, Ar-H), 7.48 (d, J = 7.5 Hz,
1H, Ar-H), 7.63 (t, J = 7.5 Hz, 2H, Ar-H), 7.89 (s, 1H, Ar-H),
8.05 (s, 1H, H-5); 13C NMR (125 MHz, CDCl3) δ: 55.4 (OCH3),
68.7 (C-6′′), 69.2 (C-5′), 69.4, 69.6, 69.8, 69.9, 70.1 (31P coupled,
30 PhCH2OP), 70.3 (C-5′′), 72.0, 73.5, 73.6 (3 x PhCH2OC), 74.5
(C-3′), 74.54 (31P coupled, C-4′′), 77.0 (31P coupled, C-2′′), 78.1
(C-3′′, 31P coupled), 78.8 (C-2′), 83.1 (C-4′), 90.1 (C-1′), 95.9 (C-
1′′), 105.9, 119.20 (C-5), 124.5, 127.2, 127.6, 127.63, 127.7,
127.8, 127.86, 127.89, 127.97, 128.0, 128.1, 128.13, 128.2,
35 128.3, 128.36, 128.38, 128.45, 128.5, 128.52, 128.57, 128.6,
128.7, 129.0, (aromatic carbons), 129.8, 134.4, 136.2, 137.2,
137.5, 137.6, 138.1 (31P coupled, ipso carbons of POCH2Ph); 31P
NMR (202.4 MHz, CDCl3) δ: -1.524, -1.920, -2.082; Anal. calcd
for C87H86N3O19P3: C, 66.53; H, 5.52; N, 2.68. Found: C, 66.74;
40 H, 5.39; N, 2.88.
(ImC-4), 125.0 (C-4), 134.1 (ImC-5), 136.4 (ImC-2); 31P NMR
(202.4 MHz, CDCl3) δ: 3.62, 3.69, 4.41; HRMS (ESI) mass calcd
for C17H28N5O18P3 [M+H]+, 684.072, found 684.0699.
c) 4-phenyl triazolophostin (10c)
85 By using 100 mg (0.067 mmol) of 9c, 41 mg (91%) of
triazolophostin 10c was obtained as a white hygroscopic solid: 1H
NMR (500 MHz, D2O) δ: 3.73-3.85 (m, 6H, H-2′′, H-5A′, H-4′′,
H-5B′, H-6A′′, H-6B′′), 4.07-4.09 (m, 1H, H-5′′), 4.43 (bs, 1H, H-
4′), 4.47-4.49 (m, 1H, H-3′′), 4.64-4.75 (m, 1H, H-3′), 5.19-5.20
90 (m, 1H, H-2′), 5.24 (s, 1H, H-1′′), 6.39 (d, 1H, 3.65 Hz, H-1′),
7.38-7.41 (m, 1H, Ar-H), 7.46 (t, J = 7.5 Hz, 2H, Ar-H), 7.78 (d,
J = 7.5 Hz, 2H, Ar-H), 8.48 (s, 1H, H-5); 13C NMR (125 MHz,
D2O) δ: 60.2 (C-5′), 60.8 (C-6′′), 70.4 (C-2′′, 31P coupled), 71.6
(C-5′′, 31P coupled), 73.0 (C-4′′, 31P coupled), 73.8 (C-3′, 31P
95 coupled), 76.6 (C-2′, 31P coupled), 78.1 (C-3′′, 31P coupled), 83.9
(C-4′), 91.0 (C-1′, 31P coupled), 98.1 (C-1′′), 121.3 (C-5), 125.9,
126.0, 129.1, 129.2, 129.3 (Ar-H); 31P NMR (202.4 MHz, D2O)
δ: 3.551, 3.597, 4.343; HRMS (ESI) mass calcd for
C19H28N3O18P3 [M-H]+, 678.0503, found 678.0482.
7. General Procedure for the Synthesis of Triazolophostins:
To a solution of protected triazolophostins 9a-k (80-200 mg,
0.06-0.13 mmol) and cyclohexene (2 mL) in a mixture of
methanol and water (9:1 v/v, 5 mL), Pd(OH)2 (20% on carbon, 50
45 mg) was added and the mixture was stirred at 80 °C for 4 h. The
reaction mixture was then cooled, filtered through a membrane
filter washing successively with methanol and water. The
combined filtrate was evaporated under reduced pressure. The
crude product thus obtained was purified by ion-exchange
50 column chromatography on Q-Sepharose matrix using 0 → 1.0 M
triethylammonium bicarbonate (TEAB) as eluent to get pure
triazolophostins 10a-k.
100 d) 4-(2-pyridyl)-triazolophostin (10d)
By using 100 mg (0.067 mmol) of 9d, 21.2 mg (47%) of
triazolophostin 10d was obtained as a sticky mass: 1H NMR (500
MHz, D2O) δ: 3.79-3.90 (m, 5H, H-6A′′, H-6B′′, H-5A′, H-5B′, H-
2′′), 4.11-4.12 (m, 1H, H-5′′), 4.49-4.51 (m, 2H, H-4′ and H-4′′),
105 4.72 (m, 2H, obscured by HDO peak, H-3′ and H-3′′), 5.26-5.29
(m, 2H, H-2′ and H-1′′), 6.51 (s, 1H, H-1′), 7.98 (s, 1H, Pyr-H),
8.40 (d, J = 6.5 Hz, 1H, Pyr-H), 8.62 (s, 1H, Pyr-H), 8.74 (s, 1H,
Pyr-H), 9.04 (s, 1H, H-5); 13C NMR (125 MHz, D2O) δ: 60.3 (C-
5′), 60.7 (C-6′′), 70.6 (C-2′′, 31P coupled), 71.8 (C-5′′, 31P
a) 3′-O-α-D-glucopyranosyl-1-β-D-ribofuranosyl-1,2,3-
triazole 2′,3′′,4′′-trisphosphate [triazolophostin] (10a)
55 By using 80 mg (0.056 mmol) of 9a, 33 mg (97%) of 110 coupled), 73.0 (C-4′′, 31P coupled), 73.8 (C-3′, 31P coupled), 76.7
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Page 10 of 12
(C-2′, 31P coupled), 78.0 (C-3′′, 31P coupled), 84.5 (C-4′), 91.4
(C-1′, 31P coupled), 98.1 (C-1′′), 124.9 (C-5), 126.2, 139.4, 141.3,
142.4, 147.6 (Ar-C); 31P NMR (202.4 MHz, D2O) δ: 3.637,
3.720, 4.448; HRMS (ESI) mass calcd for C18H27N4O18P3 [M-
H]+, 679.0455, found 679.0431.
mass calcd for C19H27FN3O18P3 [M]+, 697.0486, found 697.0481.
h) 4-(3,5-diflurorophenyl)-triazolophostin (10h)
By using 200 mg (0.13 mmol) of 9h, 79 mg (84%) of
triazolophostin 10h was obtained as a white hygroscopic solid:
60 1H NMR (500 MHz, MeOD) 3.59-3.79 (m, 6H, H-2′′, H-5′′, H-
6A′′, H-6B′′, H-5A′, H-5B′), 4.10 (bs, 1H, H-4′′), 4.30 (bs, 1H, H-
4′), 4.52 (bs, 1H, H-3′′), 4.66 (bs, 1H, H-3′), 5.15 (bs, 1H, H-1′′),
5.22 (bs, 1H, H-2′), 6.28 (s, 1H, H-1′), 6.83 (t, J = 8.9 Hz, 1H,
Ar-H), 7.36 (d, J = 6.2 Hz, 2H, Ar-H), 8.60 (s, 1H, H-5); 13C
5
e) 4-(2-Fluorophenyl)-triazolophostin (10e)
By using 190 mg (0.12 mmol) of 9e, 79 mg (91%) of
triazolophostin 10e was obtained as a white hygroscopic solid: 1H
NMR (500 MHz, MeOD) 3.58-3.81 (m, 6H, H-5′′, H-6A′′, H-6B′′,
10 H-5A′, H-5B′, H-2′′), 4.09-4.11 (m, 1H, H-4′′), 4.30 (bs, 1H, H-4′), 65 NMR (125 MHz, MeOD) δ: 61.9 (C-5′), 62.1 (C-6′′), 72.7 (C-2′′,
4.51-4.53 (m, 1H, H-3′′), 4.68 (t, J = 5.0 Hz, 1H, H-3′), 5.15 (bs,
1H, H-1′′), 5.22 (bs, 1H, H-2′), 6.31 (bs, 1H, H-1′), 7.10-7.14 (m,
1H, Ar-H), 7.18 (t, J = 7.5 Hz, 1H, Ar-H), 7.28 (dd, J = 13.0, 6.5
Hz, 1H, Ar-H), 8.02 (t, J = 7.5 Hz, 1H, Ar-H), 8.51 (s, 1H, H-5);
31P coupled), 73.6 (C-5′′, 31P coupled), 74.7 (C-4′′, 31P coupled),
76.7 (C-3′, 31P coupled), 79.0 (C-2′, 31P coupled), 79.5 (C-3′′, 31
P
coupled), 85.4 (C-4′), 92.9 (C-1′, 31P coupled), 100.6 (C-1′′),
104.2 (C-5), 109.3, 122.4, 135.2, 146.9, 163.9, 165.9 (Ar-C); 31P
15 13C NMR (125 MHz, MeOD) δ: 61.9 (C-5′), 62.1 (C-6′′), 72.7 (C- 70 NMR (202.4 MHz, MeOD) δ: -0.650, 0.47, 0.81; 19F NMR
2′′, 31P coupled), 73.6 (C-5′′, 31P coupled), 74.6 (C-4′′, 31P
coupled), 76.8 (C-3′, 31P coupled), 79.1 (C-2′, 31P coupled), 79.5
(C-3′′, 31P coupled), 85.3 (C-4′), 93.0 (C-1′, 31P coupled), 100.6
(C-1′′), 116.9 (C-5), 124.0, 125.8, 128.8, 131.1, 142.4, 159.8,
20 161.7 (Ar-C); 31P NMR (202.4 MHz, MeOD) δ: 0.601 (2P), -
0.550; 19F NMR (470.68 MHz, MeOD) δ: -116.02; HRMS (ESI)
mass calcd for C19H27FN3O18P3 [M]+, 697.0486, found 697.0490.
(470.68 MHz, MeOD) δ: -111.10; HRMS (ESI) mass calcd for
C19H26F2N3O18P3 [M]+, 715.0392, found 715.0402.
i) 4-(4-methoxyphenyl)-triazolophostin (10i)
By using 195 mg (0.12 mmol) of 9i, 87 mg (96%) of
1
75 triazolophostin 10i was obtained as a white hygroscopic solid: H
NMR (500 MHz, D2O) δ: 3.65-3.76 (m, 9H, H-2′′, H-5′′, H-6A′′,
H-6B′′, H-5A′, H-5B′ and 1 x OCH3), 4.00-4.02 (m, 1H, H-4′′),
4.33 (bs, 1H, H-4′), 4.40-4.41 (m, 1H, H-3′′), 4.54 (t, J = 5.1 Hz,
1H, H-3′), 5.08-5.15 (m, 1H, H-2′), 5.16 (bs, 1H, H-1′′), 6.27 (d, J
f) 4-(3-Fluorophenyl)-triazolophostin (10f)
By using 180 mg (0.11 mmol) of 9f, 78 mg (93%) of
25 triazolophostin 10f was obtained as a white hygroscopic solid: 1H 80 = 3.2 Hz, 1H, H-1′), 6.89 (d, J = 8.6 Hz, 1H, Ar-H), 7.54 (d, J =
NMR (500 MHz, MeOD) 3.61-3.62 (m, 1H, H-2′′), 3.69-3.84 (m,
5H, H-5′′, H-6A′′, H-6B′′, H-5A′, H-5B′), 4.13-4.18 (m, 1H, H-4′′),
4.32-4.33 (m, 1H, H-4′), 4.53-4.55 (m, 1H, H-3′′), 4.69 (t, J = 5.0
Hz, 1H, H-3′), 5.18 (d, J = 3.0 Hz, 1H, H-1′′), 5.27 (t, J = 3.8 Hz,
8.6 Hz, 1H, Ar-H), 8.25 (s, 1H, H-5); 13C NMR (125 MHz, D2O)
δ: 55.6 (OCH3), 60.3 (C-5′), 61.0 (C-6′′), 70.7 (C-2′′, 31P
coupled), 71.7 (C-5′′, 31P coupled), 73.0 (C-4′′, 31P coupled), 73.9
(C-3′, 31P coupled), 76.5 (C-2′, 31P coupled), 78.0 (C-3′′, 31P
30 1H, H-2′), 6.32 (d, J = 3.0 Hz, 1H, H-1′), 7.01 (dt, J = 8.5, 2.0 Hz, 85 coupled), 83.8 (C-4′), 91.2 (C-1′, 31P coupled), 98.2 (C-1′′), 114.8
1H, Ar-H), 7.38 (dd, J = 8.0, 14.0 Hz, 1H, Ar-H), 7.51 (d, J = 9.7
Hz, 1H, Ar-H), 7.57 (d, J = 7.5 Hz, 1H, Ar-H), 8.58 (s, 1H, H-5);
13C NMR (125 MHz, MeOD) δ: 61.9 (C-5′), 62.0 (C-6′′), 72.5 (C-
2′′, 31P coupled), 73.4 (C-5′′, 31P coupled), 75.2 (C-4′′, 31P
35 coupled), 76.6 (C-3′, 31P coupled), 79.3 (C-2′, 31P coupled), 80.0
(C-3′′, 31P coupled), 85.4 (C-4′), 92.7 (C-1′, 31P coupled), 100.5
(C-1′′), 113.4 (C-5), 116.2, 121.8, 122.6, 132.0, 133.9, 148.0 (Ar-
C); 31P NMR (202.4 MHz, MeOD) δ: -0.755, -0.131, 0.279; 19F
NMR (470.68 MHz, MeOD) δ: -114.722; HRMS (ESI) mass
40 calcd for C19H27FN3O18P3 [M]+, 697.0486, found 697.0472.
(C-5), 120.6, 122.1, 127.5, 147.4, 159.5 (Ar-C); 31P NMR (202.4
MHz, MeOD) δ: -0.55, 0.66 (2P); HRMS (ESI) mass calcd for
C20H30N3O19P3 [M]+, 709.0686, found 709.0691.
j) 4-(2-formylphenyl)-triazolophostin (10j)
90 By using 190 mg (0.12 mmol) of 9j, 78 mg (88%) of
triazolophostin 10j was obtained as a white hygroscopic solid: 1H
NMR (500 MHz, D2O) δ: 3.75-3.87 (m, 6H, H-2′′, H-5′′, H-6A′′,
H-6B′′, H-5A′ and H-5B′), 4.09-4.11 (m, 1H, H-4′′), 4.44 (bs, 1H,
H-4′), 4.50 (bs, 1H, H-3′′), 4.65-4.68 (m, 1H, H-3′), 5.21 (bs, 1H,
95 H-2′), 5.25 (bs, 1H, H-1′′), 6.40-6.42 (m, 1H, H-1′), 7.34 (bs, 1H,
Ar-H), 7.46-7.47 (m, 1H, Ar-H), 7.60-7.61 (m, 1H, Ar-H), 7.77
(s, 1H, H-5 (triazole)), 8.36-8.48 (m, 1H, CH(OH)2); 13C NMR
(125 MHz, D2O) δ: 60.3 (C-5′), 60.9 (C-6′′), 71.5 (C-2′′, 31P
coupled), 72.0 (C-5′′, 31P coupled), 72.2 (C-4′′, 31P coupled), 73.4
g) 4-(4-Fluorophenyl)-triazolophostin (10g)
By using 200 mg (0.13 mmol) of 9g, 88 mg (95%) of
triazolophostin 10g was obtained as a white hygroscopic solid: 1H
NMR (500 MHz, MeOD) 3.60-3.83 (m, 6H, H-2′′, H-5′′, H-6A′′,
45 H-6B′′, H-5A′, H-5B′), 4.13 (bs, 1H, H-4′′), 4.31 (bs, 1H, H-4′), 100 (C-3′, 31P coupled), 76.1 (C-2′, 31P coupled), 77.1 (C-3′′, 31P
4.54 (bs, 1H, H-3′′), 4.68 (bs, 1H, H-3′), 5.17 (bs, 1H, H-1′′), 5.25
(bs, 1H, H-2′), 6.30 (bs, 1H, H-1′), 7.09 (t, J = 8.5 Hz, 2H, Ar-H),
7.76 (d, J = 5.5 Hz, 2H, Ar-H), 8.50 (s, 1H, H-5); 13C NMR (125
MHz, MeOD) δ: 61.9 (C-5′), 62.1 (C-6′′), 72.6 (C-2′′, 31P
50 coupled), 73.5 (C-5′′, 31P coupled), 74.9 (C-4′′, 31P coupled), 76.7
(C-3′, 31P coupled), 79.1 (C-2′, 31P coupled), 79.7 (C-3′′, 31P
coupled), 85.4 (C-4′), 92.8 (C-1′, 31P coupled), 100.5 (C-1′′),
116.8 (C-5), 116.9, 121.2, 128.0, 128.8, 128.9, 148.2, 163.3,
165.2 (Ar-C); 31P NMR (202.4 MHz, MeOD) δ: -0.602, -0.269,
55 0.473; 19F NMR (470.68 MHz, MeOD) δ: -115.406; HRMS (ESI)
coupled), 83.8 (C-4′), 91.2 (C-1′, 31P coupled), 97.8 (C-1′′), 121.2
(C-5), 125.8, 126.3, 129.0, 129.3, 130.9, 136.5, 146.7, 147.4, (Ar-
C); 31P NMR (202.4 MHz, D2O) δ: 3.51, 3.63, 4.32; HRMS (ESI)
mass calcd for C20H28N3O19P3 [M]+, 707.0530, found 707.0549.
105 k) 4-(6-methoxynaphth-2-yl)-triazolophostin (10k)
By using 90 mg (0.057 mmol) of 9k, 42 mg (97%) of
triazolophostin 10k was obtained as a white hygroscopic solid:
1H NMR (500 MHz, MeOD) δ: 3.06 (d, J = 7.7 Hz, 1H, H-2′′),
3.69-3.85 (m, 8H, H-5′′, H-5A′, H-5B′, H-6A′′, H-6B′′ and OCH3),
10 | Org. Biomol. Chem., 2015, 13, 00–00
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DOI: 10.1039/C5OB00440C
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Organic & Biomolecular Chemistry
4.13-4.15 (m, 1H, H-4′′), 4.32-4.33 (m, 1H, H-4′), 4.53-4.55 (m,
1H, H-3′′), 4.70 (t, J = 5.0 Hz, 1H, H-3′), 5.17 (bs, 1H, H-1′′),
5.28 (bs, 1H, H-2′), 6.32 (d, J = 2.7 Hz, 1H, H-1′), 7.05 (dd, J =
9.0, 2.4 Hz, 1H, Ar-H), 7.15 (d, J = 2.2 Hz, 1H, Ar-H), 7.69 (d, J
= 9.0 Hz, 1H, Ar-H), 7.73 (d, J = 9.0 Hz, 1H, Ar-H), 7.79 (s, 1H,
H-5), 8.13 (s, 1H, Ar-H), 8.57 (s, 1H, Ar-H); 13C NMR (125
MHz, MeOD) δ: 55.9 (OCH3), 61.9 (C-5′), 62.1 (C-6′′), 72.6 (C-
2′′, 31P coupled), 73.5 (C-5′′, 31P coupled), 75.0 (C-4′′, 31P
coupled), 77.0 (C-3′, 31P coupled), 79.3 (C-2′, 31P coupled), 79.5
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15 8. Biological Assay:
Ca2+ release from the intracellular stores of saponin-
permeabilized DT40 cells expressing only type 1 IP3R was
measured using a low-affinity Ca2+ indicator (Mag-fluo-4) as
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containing p-trifluoromethoxyphenylhydrazone (FCCP) to inhibit
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Acknowledgements
A. M. V. thanks the University Grants Commission (UGC) India
for a Senior Research Fellowship (SRF) during this work. K. M.
S. thanks Department of Science and Technology (DST) India for
30 Swarnajayanti Fellowship, Ramanujan Fellowship and for
financial support. C. W. T. and V. K. were supported by the
Wellcome Trust, Biotechnology and Biological Sciences
Research Council, and the German Academic Exchange Service
(V. K.).
8
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35 Notes and rererences
a
School of Chemistry, Indian Institute of Science Education and
Research,
Fax:
Thiruvananthapuram,
914712597427,
KERALA-695016,
E-mail: kms@iisertvm.ac.in
India.
(+)
Homepage:
www.iisertvm.ac.in/scientists/kana-m-sureshan/personal-
40 information.html
b
Department of Pharmacology, Tennis Court Road, University of
Cambridge, Cambridge CB2 1PD, U.K.
† Electronic Supplementary Information (ESI) available: Synthetic
procedures and spectral data for all new compounds, crystal data (CCDC
45 1022279) for disaccharide 4 and details of the docking study. See
DOI: 10.1039/b000000x/
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