Synthesis and photophysical characterisation of new fluorescent triazole adenine analogues

Article abstract of DOI:10.1039/c4ob00904e

Fluorescent nucleic acid base analogues are powerful probes of DNA structure. Here we describe the synthesis and photo-physical characterisation of a series of 2-(4-amino-5-(1H-1,2,3-triazol-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-7- yl) and 2-(4-amino-3-(1H-1,2,3-triazol-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl) analogues via Sonogashira cross-coupling and [3 + 2]-cycloaddition reactions as the key steps in the synthesis. Compounds with a nitrogen atom in position 8 showed an approximately ten-fold increase in quantum yield and decreased Stokes shift compared to analogues with a carbon atom in position 8. Furthermore, the analogues containing nitrogen in the 8-position showed a more red-shifted and structured absorption as opposed to those which have a carbon incorporated in the same position. Compared to the previously characterised C8-triazole modified adenine, the emissive potential was significantly lower (tenfold or more) for this new family of triazoles-adenine compounds. However, three of the compounds have photophysical properties which will make them interesting to monitor inside DNA.

Full text of DOI:10.1039/c4ob00904e

Organic &
Biomolecular Chemistry
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Synthesis and photophysical characterisation of
new fluorescent triazole adenine analogues†
Cite this: Org. Biomol. Chem., 2014,
12, 5158
Christopher P. Lawson,‡a Anke Dierckx,‡b Francois-Alexandre Miannay,b
Eric Wellner,c L. Marcus Wilhelmsson*b and Morten Grøtli*a
Fluorescent nucleic acid base analogues are powerful probes of DNA structure. Here we describe the syn-
thesis and photo-physical characterisation of a series of 2-(4-amino-5-(1H-1,2,3-triazol-4-yl)-7H-pyrrolo-
[2,3-d]pyrimidin-7-yl)
and
2-(4-amino-3-(1H-1,2,3-triazol-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)
analogues via Sonogashira cross-coupling and [3 + 2]-cycloaddition reactions as the key steps in the syn-
thesis. Compounds with a nitrogen atom in position 8 showed an approximately ten-fold increase in
quantum yield and decreased Stokes shift compared to analogues with a carbon atom in position 8. Fur-
thermore, the analogues containing nitrogen in the 8-position showed a more red-shifted and structured
absorption as opposed to those which have a carbon incorporated in the same position. Compared to the
previously characterised C8-triazole modified adenine, the emissive potential was significantly lower
(tenfold or more) for this new family of triazoles-adenine compounds. However, three of the compounds
have photophysical properties which will make them interesting to monitor inside DNA.
Received 2nd May 2014,
Accepted 3rd June 2014
DOI: 10.1039/c4ob00904e
www.rsc.org/obc
outwards from the nucleic acid chains and can therefore inter-
fere with the mobility and geometry of their targets, affecting
Introduction
Fluorescence has been shown to be an extremely valuable and their critical biological functions. For that reason, fluorescent
versatile tool for the investigation of biological systems.1,2 It base analogues (FBAs), which possess intrinsic fluorescence
complements other techniques such as NMR spectroscopy and and can be incorporated into DNA/RNA causing only minimal
X-crystallography which may sometimes be limited by the perturbations of the delicate balance of biochemical function
resolution which can be achieved.3 However most importantly, are potentially of great value.
fluorescence allows for real time observation/tracking of the
Over the years FBAs have been shown to be extremely
labelled molecules in living systems.4–6 Furthermore, it is powerful tools with a wide range of applications in biology and
straightforward and requires very little sample. The virtually biotechnology as molecular probes and reporters for nucleic
non fluorescent nature of naturally occurring nucleic acid acids.7,9,10 Extensively studied FBAs include the pteridines
bases highlights the need for synthetically labelled nucleo- developed by Hawkins et al.,11 the cytosine analogue pyrrolo
bases as tools for the investigation of nucleic acid systems. dC12 and derivatives thereof,13–16 the environmental less sensi-
These are typically synthesised by attaching a fluorophore to tive tricyclic cytosines tCO17 and tC18 as well as the emissive
naturally existing nucleic acid bases.7,8 However, the most RNA alphabet designed by Tor and colleagues,19 to name but a
commonly used fluorophores tend to suffer from certain limit- few (for recent reviews see for example Sinkeldam et al.,7
ations. Most dye molecules are large and hydrophobic which Wilhelmsson9 and Dodd et al.20). The adenine analogue
tends to impact negatively on the aqueous solubility of the 2-aminopurine, one of the most widely applied FBAs, has also
resultant nucleosides. The fluorescent tags also tend to project been thoroughly characterised.7,21
Purine analogues are commonly modified on the C8
position of natural adenine/guanine.22–31 Interest in these
compounds stems from their utility as fluorescent markers,
aDepartment of Chemistry, Medicinal Chemistry, University of Gothenburg, S-41296
Gothenburg, Sweden. E-mail: grotli@chem.gu.se
biomolecular probes, supramolecular building blocks, confor-
mational probes and the use they have in therapeutics. Even
though small modifications are reported to insignificantly
perturb the DNA duplex,25,31 bulky C8-substituents have
shown to be destabilizing22,23,29,30 and can shift the confor-
mation around the glycosidic bond from anti to syn.32,33 On
the other hand, modifications on the purine 7-position have
bDepartment of Chemical and Biological Engineering/Chemistry and Biochemistry,
Chalmers University of Technology, S-41296 Gothenburg, Sweden.
E-mail: marcus.wilhelmsson@chalmers.se
cAstraZeneca R&D Mölndal, S-431 83 Mölndal, Sweden
†Electronic supplementary information (ESI) available: NMR spectra of all new
compounds as well as additional photophysical characterisation. See DOI:
10.1039/c4ob00904e
‡These authors contributed equally to this work.
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been shown to be well tolerated by the DNA helix.34–37
Recently, we reported the synthesis and characterisation of the
photophysical and base-mimicking properties of a novel
C8-modified fluorescent adenine analogue, triazole adenine
(AT), in DNA.27 It showed promising photophysical properties,
both as a monomer as well as inside DNA. However, as for
other C8-modified adenines, AT destabilizes the DNA structure,
most likely because of the bulky C8-triazole group.30
Pursuant to our interest in this area, we herein present the
synthesis and photo-physical characterisation of a model
series of 2-(4-amino-5-(1H-1,2,3-triazol-4-yl)-7H-pyrrolo[2,3-d]-
pyrimidin-7-yl) and 2-(4-amino-3-(1H-1,2,3-triazol-4-yl)-1H-
pyrazolo[3,4-d]pyrimidin-1-yl) analogues which were designed
in an attempt to further minimize the structural perturbations
to B-DNA seen with the adenine analogue AT by shifting the
position of the triazole substituent to the adenine 7-position.30
We report dramatic changes of the emissive properties of this
new family of triazole adenine compounds compared to the
previously characterised C8-modified series that could be of
potential use in future applications in nucleic acid systems.
Our photophysical characterisation of this family of adenine
analogues also helps to create a better understanding of the
structure–fluorescence relationship, which is poorly under-
stood at the moment.
Results and discussion
Synthetic procedures
Scheme 1 General synthetic pathway, conditions. Systematic number-
ing shown in red for X = N and in black for X = C. Red numbering was
used for the NMR assignment for consistency: (a) NIS (1.5 eq.), DMF
120 °C, 20 min, (b) Cs2CO3 (1.2 eq.), ethyl bromoacetate (1.1 eq.), DMF
0 °C to RT, o/n, (c) TMS-acetylene (1.5 eq.), Pd(PPh3)2Cl2 (10 mol%) and
CuI (25 mol%), ETN–DMF (1 : 4), RT, o/n, (d) polymer supported fluoride
(Amberlite® IRA 900 F− form) (2.0 eq.), CHCl3, (e) Alk-Br (16.1 eq.), NaN3
(32.6 eq.), H2O 140 °C, 30 min, then Et3N (1.0 eq.), RT o/n, EtOAc, (f)
LiOH–THF 50% (2.0 eq.), (g) Cs2CO3 (1.2 eq.), ethyl bromoacetate (1.1
eq.), DMF 0 °C to RT then DMF–DMA (5.0 eq.), DMF RT o/n, (h) Ar–I,
NaN3 (1.2 eq.), sodium ascorbate (40 mol%), sodium carbonate (20 mol%),
L-proline (20 mol%), Cu/C (40 mol%), then NH3–MeOH.
The key intermediates 9 and 10 were readily accessed in 3 to 4
steps utilizing a common protocol. Starting from commercially
available4-amino-7H-pyrrolo[2,3-d]pyrimidine(1),N-iodosuccin-
imide (NIS) mediated iodination in acetonitrile under micro-
wave irradiation afforded 3 in good yields (X = C 86%)
(Scheme 1).38
Similar conditions were applied to the synthesis of 4 with
comparable results from commercially available 4-aminopyra-
zolo[3, 4-d]pyrimidine (2) (X = N 71%). Treatment of DMF solu-
tions of both 3 and 4 respectively with ethyl bromoacetate, at
0 °C afforded the desired protected compounds 5 an d 6
respectively in reasonably good yields (X = C 81%, X = N
71%).39 Sonogashira couplings with TMS-acetylene afforded
the respective TMS acetylenes in excellent yields (X = C 91%,
X = N 90%).30,40 Subsequent deprotection with polymer sup-
ported fluoride afforded the key intermediates 9 and 10 in
good yields (X = C 70%, X = N 61%).30 A copper catalysed
[3 + 2] cycloaddition reaction afforded the desired compounds
in good yields.40 A more detailed inspection of the LCMS data
however revealed the presence of a small quantity of a con-
taminant, (<5%), which is believed to arise from the insertion
of iodine from the copper catalyst into the triazole ring
depicted as 21.41 Therefore in a slight modification to the
previously applied protocol for the synthesis of the alkyl ana-
logues, CuI was replaced with Cu/C to avoid this contami-
nant.42 Aryl analogues were synthesised utilising a protocol
previously reported by Klein et al. utilising azides generated
in situ from the corresponding aryl iodides (Table 1).40 In
initial attempts to synthesise the aryl analogues utilising this
protocol, the competing amination reaction afforded the
Ullman product as the major product. An alternate strategy to
avoid this competing reaction required the synthesis of the
N-protected acetylenes which were obtained by initial protec-
tion of the exocyclic amino group on 5 and 6 respectively
with N,N-dimethylformamide dimethyl acetal affording the
protected iodides 15 and 16 in good yields (X = C 75%, X = N
71%).
Sonogashira couplings, followed by subsequent deprotec-
tion with polymer supported fluoride afforded the key inter-
mediates 17 and 18 in good yields over the 2 steps (X = C 61%,
X = N 85%). The 1,2,3-triazole rings were then synthesized uti-
lising the appropriate copper catalysed [3 + 2] cycloaddition
reaction protocol between the respective alkynes 9, 10, 17 or 18
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Table 1 Synthesis of triazole analogues, conditions; Alk-Br (16.1 eq.),
NaN3 (32.6 eq.), H2O 140 °C, 30 min, then Et3N (1.0 eq.), RT o/n, EtOAc,
or Ar–I, NaN3 (1.2 eq.), sodium ascorbate (40 mol%), sodium carbonate
(20 mol%), L-proline (20 mol%), Cu/C (40 mol%). Then LiOH–THF 50%
(2.0 eq.), to obtain the corresponding acids where synthesised
Product
(yield %)
Acid
(yield %)
Compound #
Alkyne #
Azide
11a
12a
11b
12b
19
20a
20b
20c
9
10
9
10
17
18
18
18
Pent
Pent
Benz
Benz
Ph
Ph
3-NH2 Ph
Py
22
76
89
78
75
21
56
56
25 (13a)
54 (14a)
84 (13b)
91 (14b)
a
—
a
—
—
a
a
—
a Not synthesized.
Fig. 1 Absorption spectra of the triazole adenine analogues (Scheme 1)
normalized to the lowest energy absorption maximum. Compounds
were dissolved in methanol at room temperature.
and the corresponding azides.40,42,43 Deprotection with metha-
nolic ammonia afforded the desired aryl analogues. Treatment
with LiOH in THF converted 11 and 12 into 13 and 14
respectively.
Photophysical properties
The previously characterised sister compound of 11a and 12a,
AT, containing the same triazole-pentyl modification on C8
instead, exhibits a high quantum yield in both water (61%)
and methanol (49%). Also inside DNA, AT shows promising
fluorescence properties, but displays a destabilising effect on
the B-DNA helix as a consequence of the C8-triazole group,
which is suggested to shift the equilibrium of the glycosidic
bond from anti to syn.30 On the other hand, purine analogues
modified on the 7-position are known to be well accommo-
dated in the DNA duplex.34–37 In an attempt to obtain an ana-
logue that retains the advantageous photophysical properties
of AT while incorporating the non-perturbing nature of 7-modi-
fied purine analogues, the current series of 7-modified triazole
adenines has been synthesised and here we investigate its fluo-
rescence properties.
Fig. 2 Normalized emission of triazole adenine analogues (with Φf
0.2%) (Scheme 1) dissolved in methanol at 25 °C.
>
Table 2 Fluorescence quantum yield (Φf), absorption maximum of the
lowest energy absorption band (λA,max), corresponding extinction coeffi-
cients (εmax) (also at 260 nm, ε260 nm) and emission maximum (λEm,max) of
each compound dissolved in methanol
Absorption and emission envelopes of all triazole adenine
compounds (Scheme 1) in methanol are shown in Fig. 1 and 2,
respectively. Their characteristic photophysical properties are
summarised in Table 2.
Φfb
(%)
λA,max
(nm)
λEm,max
(nm)
εmax
ε260 nm
Compounda
(M−1 cm−1
)
(M−1 cm−1
)
As for many adenine analogues, all compounds presented
in Fig. 1 show a red-shifted absorption (lowest energy Amax
=
11a
11b
12a
12b
19
20a
20b
20c
0.75
282
281
290
290
280
288
293
293c
369
366
343
340
11 200
11 800
10 700
10 800
14 500
13 600
14 000
15 300e
9700
10 900
13 800
14 800
17 200
21 500
24 100
17 800e
280–293 nm) compared to natural adenine (Amax = 260 nm),
allowing for specific excitation in the red absorption tail
outside the absorbance of the natural nucleobases. Our
previous series of triazole adenines with similar substituents
but having the triazole group placed on C8 of adenine
showed absorption maxima in the same region in THF
(289–296 nm),27 methanol (286 nm, pentyl)30 and water
0.39
5.22
4.38
<0.1
<0.2
0.62
<0.1
d
—
345
468
359
a Compounds are shown in Scheme 1. b Quantum yields were
(282 nm, pentyl,30 isopentyl27). Also the C8-modified 8-vinyl- determined relative to 2-aminopyridine (Φf = 60%) in 0.05 M H2SO4 at
25 °C.44 c Absorption maximum estimated for lowest energy absorption
deoxyadenosine (8vdA) in methanol (294 nm)25 has its
shoulder. d Emission maximum omitted due to the weak fluorescence.
e Extinction coefficient only determined once due to a limited amount
maximum absorption in this wavelength range. As a compari-
son the absorbance of the most commonly applied FBA, 2-AP of substance.
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in water (303–304 nm)21 or methanol (309 nm),45 is situated in over, hydrogen bonding can also play an important role in
a similar region.
stabilizing the ICT-state.48 Therefore, the emission of 20b and
As shown in Fig. 1, 2 and Table 2, compounds 11a and 11b for comparison also of 20c was recorded in dichloromethane
exhibit very similar absorptive and emissive properties, which (DCM), an apolar and aprotic solvent. Whereas the emission
is also the case for compounds 12a and 12b. This finding is properties of 20c (Φf < 0.1%, λEm,max = 359 nm) remain un-
expected as the conjugated π-system does not extend further altered, the quantum yield of 20b increases dramatically in
than the triazole ring in these analogues, implying that the DCM (Φf ∼ 21%) and its emission maximum is shifted to
non-conjugated benzyl and pentyl-moieties should have a 393 nm. Also in acetonitrile (ACN), a polar aprotic solvent, the
minimal effect on the photophysical characteristics. Similar emission of 20b remains intense (Φf ∼ 24%) with the emission
observations were made for the previously characterised ana- maximum found at 428 nm. Hence, the emission energy of
logous set of triazole adenine compounds bearing the triazole 20b seems to decrease both with polarity and protic character
ring on C8 of adenine, for which those containing aliphatic of the solvent, whereas no significant changes can be detected
substituents showed very similar properties.27 The differences in the absorption spectra (see Fig. S5, ESI†). These findings
that are found between both pairs of analogues, 11 and 12, are in accordance to the suggested character of the excited
must be explained by the incorporation of the nitrogen on C8 state ICT.
of adenine. This yields an approximately ten-fold increase in
Apart from observed shifts in emission energy, 20b seems
quantum yield and decreased Stokes shift for compounds 12a to be efficiently quenched by protic solvents, as seen from the
and 12b (Φf ∼ 5%, Δ˜ν ∼ 5200 cm−1, Δλ ∼ 50 nm) compared to large decrease in fluorescence quantum yield in methanol
the two sister compounds, 11a and 11b (Φf ∼ 0.5%, Δ˜ν ∼ (Φf = 0.62%) compared to ACN (Φf ∼ 24%) and DCM (Φf ∼ 21%).
8300 cm−1, Δλ ∼ 85 nm) lacking this feature. However, these This may be due to a more efficient internal conversion in case
four compounds show similar extinction coefficients of hydrogen bonding with solvent molecules.47 Also for N6,N6-
(10 700–11 800 M−1 cm−1, Table 2), indicating no dramatic dimethyladenosine, significant quenching of the fluorescence
differences in their radiative rate constants according to the of the putative twisted intramolecular charge transfer (TICT)-
Strickler–Berg equation.46 This suggests that the significant state was observed by protic solvents.49 Similar as for 20b in
differences in emissive properties are mainly due to more DCM, the 3-aminophenyl modification also yielded a signifi-
efficient non-radiative processes depopulating the excited state cantly higher fluorescence quantum yield among the conju-
for 11a and 11b.
gated compounds (38% versus 3–5%) in an aprotic apolar
Generally, the analogues containing a nitrogen on the solvent (THF) in a previous study concerning C8-modified tri-
8-position of adenine (12a, 12b, 20a, 20b, 20c, λmax azole adenine analogues.27 However, we do not know whether
288–293 nm) show a more red-shifted and structured absorp- the behaviour of the latter compound would be similar as for
tion as opposed to those which have a carbon incorporated in 20b in a polar protic solvent.
this position (11a, 11b, 19, λmax 280–282; Table 2). Within each
Compared to the previously characterised C8-modified AT
group of compounds, the lowest energy absorption tail extends (Φf = 49% in methanol), the emissive potential is decreased
further into the lower energy wavelength region for com- tenfold for sister compound 12a (5%) and even more dramati-
pounds with aromatic extensions past the triazole ring (19, cally for 11a (0.75%) presented here. Within each group, dis-
20a–c; Fig. 1). They also exhibit the highest absorption coeffi- tinguished by a nitrogen or carbon in position 8, all other
cients which may be due to the larger conjugated system for compounds have even lower fluorescence quantum yields in
these compounds compared to those with aliphatic substitu- methanol (Table 2). It is unclear what causes these major
ents. Moreover, these aromatic derivatives show the lowest differences in emissive performance, since it remains challen-
quantum yields within their groups. This finding is in line ging to predict fluorescence behaviour based on chemical
with the related C8-modified family.27 As can be seen in Fig. 1, structure.
analogues 20b and 19 exhibit an extended absorption tail,
which may raise concerns about aggregation. However, no
changes in the shape of the absorption envelope could be
detected upon dilution of concentrated samples of these com-
Conclusions
pounds, nor by heating the solutions (ESI Fig. S1–S4†).
A series of adenine derivatives have been synthesised with
Interestingly, compound 20b shows an extensive Stokes Sonogashira cross-coupling and [3 + 2]-cycloaddition reactions
shift of ∼12 800 cm−1 (Δλ = 175 nm), due to an emission being the key steps in the synthesis. The cycloaddition reaction
maximum ∼100–125 nm red-shifted compared to the other provides a means for rapid tuning of the fluorescence pro-
compounds, including the related conjugated analogues 20a perties of the compounds. In general, derivatives with nitrogen
and 20c. This large shift in emission wavelength (λEm,max
=
in position 8 have a higher quantum yield and a more red-
468 nm) could be due to intramolecular charge transfer (ICT) shifted absorption compared to compounds with carbon in
in the excited state from the electron donating amino group. position 8. It is difficult to rationalise these differences.
Since ICT would result in a larger dipole moment in the However, the design and characterisation of new FBAs remains
excited state compared to the ground state, the emission important to the field due to the large potential for brighter
energy is expected to decrease with solvent polarity.47 More- and more photostable isomorphic probes. Although most
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FBAs show a decrease in fluorescence upon incorporation into 17.73 mmol, 1.2 eq.). The vial was then sealed and flushed
nucleic acid systems,7,11,12,20,22,26,31,34 increasing quantum with nitrogen. DMF (10 mL) was added and the resulting
efficiencies have been reported for some analogues.50 There- mixture heated at 100 °C by microwave irradiation for
fore it will be interesting to investigate the potential of 12a or 20 minutes using the fixed hold time setting. The solvent was
12b inside DNA as it is likely that the C7-triazole modification removed under reduced pressure and the resultant crude
will be well accommodated in the major groove, potentially product washed with hot ethanol (30 mL) to afford the desired
accompanied by interesting and applicable fluorescence pro- product (2.75 g, 10.53 mmol, 71%) as a pink solid. This was
perties. In addition the emission of 20b, which shows a high used in the following synthesis without further purification.
quantum yield in aprotic environments, will be interesting to LRMS (ES+): m/z (%) 262 (100) [M + H]+; 1H NMR (400 MHz,
monitor inside DNA where it is more shielded from protic d6-DMSO) δ 8.15 (1H, s, C6–H); 13C NMR (101 MHz, d6-DMSO)
solvent molecules.
δ 157.8 (C4), 156.2 (C6), 155.3 (C7a), 102.9 (C3a), 90.2 (C3).
General procedure A for preparation of ethyl propionate
protected pyrimidines (5 & 6)
Experimental
To a mixture of the respective iodopyrimidine (3 or 4, 1.0 eq.)
and cesium carbonate (1.2 eq.) in a sealed flask flushed with
nitrogen, was added DMF (5 mL) and cooled to 0 °C in an ice
bath. Ethylbromoacetate (1.1 eq.) was then slowly added to the
slurry and the resulting mixture allowed to warm up to room
temperature and stirred at room temperature for 18 hours.
Water (50 mL) was then added and the resultant precipitate fil-
tered off washed and dried under reduced pressure. Purifi-
cation by flash column chromatography on silica gel eluting
with a gradient from 0% to 3% MeOH in CHCl3 afforded the
desired compound.
Chemistry
General methods. Reagents and solvents were either used
as received or purified according to standard techniques.
Microwave reactions were performed using Biotage Initiator
Microwave synthesiser, using single mode microwave
irradiation with temperature and pressure control with fixed
hold time on. Reactions were monitored by LC-MS (ESI/UV),
using a PerkinElmer PE Sciex API 150 EX mass spectrometer
equipped with a C8 column (Genesis Light C8 4 µm, length
50 mm, ID 4.6 mm) and H2O–MeCN (95 : 5) to H2O–MeCN
(5 : 95) eluent, H2O containing 1% formic acid. The chromato-
grams were analysed with Analyst 1.5.1 software. TLC was
carried out on silica gel plates (Merck 60 F254) and analyzed
under UV (254 nm). Column chromatography was performed
by automated column chromatography on a Biotage SP-4
instrument using pre-packed silica columns or NH silica
columns. 1H and 13C NMR spectra were obtained at 400 and
100 MHz, respectively, using a Varian 400/54 spectrometer.
Ethyl 2-(4-amino-5-iodo-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-
acetate (5)
Using general procedure A, 3 (1.02 g, 3.92 mmol, 1.0 eq.)
afforded the title compound (1.10 g, 3.18 mmol, 81%) as a
cream coloured solid. LRMS (ES+): m/z (%) 347 (100) [M + H]+,
1
693 (2) [2M + H]+; H NMR (400 MHz, d6-DMSO) δ 8.09 (1H, s,
C6–H), 7.44 (1H, s, C2–H), 6.66 (2H, br NH2), 4.98 (2H, s, CH2),
4.17–4.11 (2H, q, J = 7.1 Hz, CH2–OEt), 1.20 (3H, t, J = 7.1 Hz,
CH3–OEt); 13C NMR (101 MHz, d6-DMSO) δ 168.7 (CO2Et),
157.6 (C4), 152.5 (C6), 150.4 (C7a), 130.6 (C2), 103.1 (C3a), 61.6
CH2–OEt, 50.7 (C3), 45.5 N–CH2, 14.5 CH3–OEt.
5-Iodo-7H-pyrrolo[2,3-d]pyrimidin-4-amine (3)
To a microwave vial (2–5 mL) fitted with a magnetic stirrer was
added 4-amino-7H-pyrrolo[2,3-d]pyrimidine (1) (0.60 g,
4.47 mmol, 1.0 eq.) and N-iodosuccinimide (1.51 g,
6.71 mmol, 1.5 eq.). The vial was then sealed and flushed with
nitrogen. Acetonitrile (3.5 mL) was added and the resulting
mixture heated at 120 °C by microwave irradiation for
20 minutes using the fixed hold time setting. The resultant
precipitate was filtered off, washed with ice cold acetonitrile
(3 × 1 mL) and dried under reduced pressure to afford the
crude product (1.00 g, 3.85 mmol, 86%) as a dark brown solid.
This was used in the following synthesis without further purifi-
cation. LRMS (ES+): m/z (%) 261 (100) [M + H]+; 1H NMR
(400 MHz, d6-DMSO) δ 11.95 (NH), 8.05 (1H, s, C6–H), 7.35
(1H, s, C2–H), 6.52 (NH2); 13C NMR (101 MHz, d6-DMSO)
δ 157.5 (C4), 152.3 (C6), 151.0 (C7a), 127.1 (C2), 103.1 (C3a),
50.5 (C3).
Ethyl 2-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-
acetate (6)
Using general procedure A, 4 (2.00 g, 7.66 mmol, 1.0 eq.)
afforded the title compound (1.90 g, 5.47 mmol, 71%) as an
off white solid. LRMS (ES+): m/z (%) 348 (100) [M + H]+, 695 (5)
1
[2M + H]+; H NMR (400 MHz, d6-DMSO) δ 8.19 (1H, s, C6–H),
5.16 (2H, s, CH2), 4.16–4.10 (2H, q, J = 7.1 Hz, CH2–OEt), 1.18
(3H, t, J = 7.1 Hz, CH3–OEt); 13C NMR (101 MHz, d6-DMSO) δ
168.0 (CO2Et), 158.1 (C4), 156.7 (C6), 154.9 (C7a), 103.5 (C3a),
90.6 (C3), 61.8 CH2–OEt, 48.4 N–CH2, 14.4 CH3–OEt.
General procedure B for preparation of amidine protected
ethyl propionate pyrimidines (15 & 16)
To the respective iodopyrimidines (5 or 6, 1.0 eq.) in a sealed
flask flushed with nitrogen, was added DMF (5 mL). N,N-Di-
3-Iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (4)38
To a microwave vial (20 mL) fitted with a magnetic stirrer methylformamide dimethyl acetal (5.0 eq.) was then added
was added 1H-pyrazolo[3,4-d]pyrimidin-4-amine (2) (2.0 g, and the resulting mixture stirred at 60 °C for 18 hours. Sol-
14.81 mmol, 1.0 eq.) and N-iodosuccinimide (3.99 g, vents were removed under reduced pressure and purification
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by flash column chromatography on silica gel eluting with a Ethyl 2-(4-amino-3-((trimethylsilyl)ethynyl)-1H-pyrazolo-[3,4-d]-
gradient from 0% to 3% MeOH in CHCl3 afforded the desired pyrimidin-1-yl)acetate (8)
compound.
Using general procedure C, 6 (1.18 g, 3.40 mmol, 1.0 eq.)
afforded the title compound (0.97 g, 3.06 mmol, 90%) as a
brown solid. LRMS (ES+): m/z (%) 318 (100) [M + H]+, 635 (3)
Ethyl (E)-2-(4-(((dimethylamino)methylene)amino)-5-iodo-7H-
[2M + H]+; 1H NMR (400 MHz, CDCl3) δ 8.35 (1H, s, C6–H),
pyrrolo[2,3-d]pyrimidin-7-yl)acetate (15)
6.02 (2H, s, NH2), 5.13 (2H, s, CH2), 4.25–4.19 (2H, q, J = 7.1
Hz, CH2–OEt), 1.26 (3H, t, J = 7.1 Hz, CH3–OEt), 0.30 (9H, s, Si-
(CH3)3; 13C NMR (101 MHz, CDCl3) δ 167.0 (CO2Et), 157.7 (C4),
156.9 (C6), 154.0 (C7a), 127.4 (C3a), 101.8 (CuC–Si), 101.6
(CuC–Si), 95.9 (C3), 62.0 (CH2–OEt), 48.4 (N–CH2), 14.1(CH3–
OEt), 0.4 (Si(CH3)3.
Using general procedure B, 5 (1.10 g, 3.18 mmol, 1.0 eq.)
afforded the title compound (0.95 g, 2.37 mmol, 75%) as a
light brown solid. LRMS (ES+): m/z (%) 402 (100) [M + H]+;
1H NMR (400 MHz, CDCl3) δ 8.75 (1H, s, NvCH), 8.41 (1H, s,
C6–H), 7.12 (1H, s, C2–H), 4.90 (2H, s, CH2), 4.21–4.16 (2H, q,
J = 7.1 Hz, CH2–OEt), 3.27 (3H, s, NCH3), 3.14 (3H, s, NCH3),
1.23 (3H, t, J = 7.1 Hz, CH3–OEt); 13C NMR (101 MHz, d6-
DMSO) δ 167.9 (CO2Et), 160.6 (C4), 155.9 NvCH, 152.0 (C6),
151.4 (C7a), 130.9 (C2), 110.8 (C3a), 61.8 CH2–OEt, 52.6 (C3),
45.3 N–CH2, 40.8 (N–CH3), 35.3 (N–CH3), 14.4 CH3–OEt.
General procedure D for preparation of ethynyl pyrimidines
(9 & 10)
To a solution of either (7 or 8, 1.0 eq.) respectively, in chloro-
form was added Fluoride on polymer support (2.0 eq.) and the
resulting mixture flushed with nitrogen then stirred at room
temperature for 18 hours. The reaction mixture was then fil-
tered washed with THF and CHCl3 (3 × 20 mL), absorbed onto
celite under reduced pressure and purified by flash column
chromatography on silica gel eluting with a gradient from 0%
to 5% MeOH in CHCl3 to afford the desired compound.
Ethyl (E)-2-(4-(((dimethylamino)methylene)amino)-3-iodo-1H-
pyrazolo[3,4-d]pyrimidin-1-yl)acetate (16)
Using general procedure B, 6 (2.87 g, 8.27 mmol, 1.0 eq.)
afforded the title compound (2.41 g, 5.99 mmol, 71%) as a
light brown solid. LRMS (ES+): m/z (%) 403 (100) [M + H]+;
1H NMR (400 MHz, CDCl3) δ 8.92 (1H, s, NvCH), 8.49 (1H, s,
C6–H), 5.15 (2H, s, CH2), 4.24–4.19 (2H, q, J = 7.1 Hz, CH2–
OEt), 3.34 (3H, s, NCH3), 3.24 (3H, s, NCH3), 1.25 (3H, t, J =
7.1 Hz, CH3–OEt); 13C NMR (101 MHz, d6-DMSO) δ 167.3
(CO2Et), 157.4(C4), 159.9 NvCH, 155.8(C6), 154.9 (C7a), 109.9
(C3a), 90.7 (C3), 61.8 CH2–OEt, 48.4 N–CH2, 41.3 (N–CH3), 35.6
(N–CH3), 14.4 CH3–OEt.
Ethyl 2-(4-amino-5-ethynyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-
acetate (9)
Using general procedure D, 7 (0.46 g, 1.33 mmol, 1.0 eq.)
afforded the title compound (0.23 g, 0.94 mmol, 70%) as a
light brown solid. LRMS (ES+): m/z (%) 245 (100) [M + H]+, 489
(10) [2M + H]+; 1H NMR (400 MHz, CDCl3) δ 8.29 (1H, s, C6–H),
7.21 (1H, s, C2–H), 5.72 (2H, s, NH2), 4.92 (2H, s, CH2),
4.26–4.21 (2H, q, J = 7.1 Hz, CH2–OEt), 3.25 (1H, s, HCuC),
1.29 (3H, t, J = 7.1 Hz, CH3–OEt); 13C NMR (101 MHz, CDCl3) δ
167.7 (CO2Et), 157.4 (C4), 153.3 (C6), 150.0 (C7a), 129.6 (C2),
103.0 (C3a), 94.7 (C3) 79.9 (CuC–H), 77.4 (CuC–H), 62.0 CH2–
OEt, 45.4 N–CH2, 14.1 CH3–OEt.
General procedure C for preparation of silylethynyl
pyrimidines (7 & 8)
To a mixture of the respective iodopyrimidine (5 or 6, 1.0 eq.),
Pd(PPh3)2Cl2 (10 mol%) and CuI (25 mol%) in a sealed flask
flushed with nitrogen, was added a degassed mixture of DMF–
Et3N (4 : 1) followed by ethynyl-TMS (1.5 eq.) and the resulting
mixture stirred at room temperature for 18 hours. The reaction Ethyl 2-(4-amino-3-ethynyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-
mixture was then absorbed onto celite under reduced pressure acetate (10)
and purified by flash column chromatography on silica gel
eluting with a gradient from 0% to 2% MeOH in CHCl3 to
afford the desired compound.
Using general procedure D, 8 (0.97 g, 2.79 mmol, 1.0 eq.)
afforded the title compound (0.42 g, 1.71 mmol, 61%) as a
light brown solid. LRMS (ES+): m/z (%) 246 (100) [M + H]+, 491
(15) [2M + H]+; 1H NMR (400 MHz, CDCl3) δ 8.37 (1H, s, C6–H),
Ethyl 2-(4-amino-5-((trimethylsilyl)ethynyl)-7H-pyrrolo-[2,3-d]-
pyrimidin-7-yl)acetate (7)
6.00 (2H, s, NH2), 5.16 (2H, s, CH2), 4.27–4.21 (2H, q, J =
7.1 Hz, CH2–OEt), 3.50 (1H, s, HCuC), 1.27 (3H, t, J = 7.1 Hz,
CH3–OEt); 13C NMR (101 MHz, CDCl3) δ 167.0 (CO2Et), 157.7
(C4), 156.9 (C6), 154.1 (C7a), 126.4 (C3), 101.9 (C3a), 82.9
(CuC–H), 75.5 (CuC–H), 62.0 CH2–OEt, 48.4 N–CH2, 14.0
CH3–OEt.
Using general procedure C, 5 (0.50 g, 1.45 mmol, 1.0 eq.)
afforded the title compound (0.42 g, 1.33 mmol, 91%) as a
brown solid. LRMS (ES+): m/z (%) 317 (100) [M + H]+, 633 (5)
[2M + H]+; 1H NMR (400 MHz, CDCl3) δ 8.28 (1H, s, C6–H),
7.17 (1H, s, C2–H), 5.65 (2H, s, NH2), 4.91 (2H, s, CH2),
4.25–4.20 (2H, q, J = 7.1 Hz, CH2–OEt), 1.26 (3H, t, J = 7.1 Hz,
CH3–OEt), 0.26 (9H, s, Si(CH3)3; 13C NMR (101 MHz, CDCl3)
Ethyl (E)-2-(4-(((dimethylamino)methylene)amino)-3-ethynyl-
1H-pyrazolo[3,4-d]pyrimidin-1-yl)acetate (17)
δ 167.7 (CO2Et), 157.5 (C4), 153.3 (C6), 150.0 (C7a), 129.0 (C2), Using general procedure C, 15 (0.40 g, 1.00 mmol, 1.0 eq.)
103.1 (C3a), 98.4 (C3), 97.4 (CuC–Si), 96.7(CuC–Si), 61.6 afforded the title compound (0.15 g, 0.50 mmol, 50%) as a
1
(CH2–OEt), 45.4 (N–CH2), 14.1 CH3–OEt), 0.1 (Si(CH3)3.
cream solid. LRMS (ES+): m/z (%) 300 (100) [M + H]+; H NMR
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(500 MHz, CDCl3) δ 8.77 (1H, s, NvCH), 8.32 (1H, s, C6–H), (C2), 109.5 (C3), 99.7 (C3a), 62.4 (CH2–OEt), 50.9 (C1″), 45.8
7.63 (1H, s, C2–H), 5.03 (2H, s, CH2), 4.15–4.14 (2H, q, J = (N–CH2), 29.8 (C2″), 28.5 (C3″), 22.0 (C4″), 14.0 (CH3–OEt),
7.5 Hz, CH2–OEt), 3.94 (1H, s, HCuC), 3.17 (3H, s, NCH3), 3.14 13.8 (5″).
(3H, s, NCH3), 1.2 (3H, t, J = 7.5 Hz, CH3–OEt); 13C NMR
Ethyl 2-(4-amino-3-(1-pentyl-1H-1,2,3-triazol-4-yl)-1H-pyrazolo-
(125 MHz, CDCl3) δ 168.2 (CO2Et), 160.8 NvCH, 156.4 (C4),
[3,4-d]pyrimidin-1-yl)acetate (12a)
152.0 (C6), 150.9 (C7a), 132.4 (C2), 110.9 (C3), 109.6 (C3a), 95.1
(CuC–H), 80.5 (CuC–H), 61.1 CH2–OEt, 45.1 N–CH2, 34.4 Using general procedure D, 10 (90.00 mg, 0.37 mmol, 1.0 eq.)
(N–CH3), 13.9 CH3–OEt.
and 1-bromopentane (0.79 g, 0.55 mL, 5.91 mmol, 16.1 eq.)
afforded the title compound (118.0 mg, 0.33 mmol, 89%) as a
Ethyl (E)-2-(4-(((dimethylamino)methylene)amino)-3-ethynyl-
1H-pyrazolo[3,4-d]pyrimidin-1-yl)acetate (18)
1
white solid. LRMS (ES+): m/z (%) 359(100) [M + H]+; H NMR
(400 MHz, CDCl3) δ 9.56 (1H, br s, NH), 8.42 (1H, br s, C6–H),
Using general procedure C, 16 (2.35 g, 5.845 mmol, 1.0 eq.) 8.10 (1H, s, C5′–H), 6.11 (1H, br s, NH), 5.15 (2H, br, s, CH2),
afforded the title compound (1.81 g, 4.86 mmol, 85%) as a 4.42 (2H, t, J = 8.0 Hz, C1″–CH2); 4.26–4.21 (2H, q, J = 8.0 Hz,
light brown solid. 1.25 g of this was treated with polymer sup- CH2–OEt), 1.99–1.95 (2H, m, C2″–CH2), 1.39–1.25 (7H, m, C3″–
ported Fluoride according to general procedure D affording CH2, C4″–CH2, CH3–OEt), 0.93–0.89 (3H, m, C5″–CH3);
the target compound as a cream solid (0.85 g, 2.83 mmol, 13C NMR (101 MHz, CDCl3) δ 167.5 (CO2Et), 158.7 (C4), 158.8
85%). LRMS (ES+): m/z (%) 301 (100) [M + H]+, 601 (10) [2M + (C7a), 155.3 (C6), 141.9 (C3) 137.1 (C1′), 123.4 (C5′), 109.5
H]+; 1H NMR (400 MHz, CDCl3) δ 8.87–8.82 (1H, s, NvCH), (C3a), 62.9 (CH2–OEt), 50.8 (C1″), 48.1 (N–CH2), 29.8 (C2″),
8.47(1H, s, C6–H), 5.13 (2H, s, CH2), 4.19–4.14 (2H, q, J = 28.5 (C3″), 22.0 (C4″), 14.0 (CH3–OEt), 13.8 (5″).
7.1 Hz, CH2–OEt), 3.33 (1H, s, HCuC), 3.22 (3H, s, NCH3), 3.17
Ethyl 2-(4-amino-5-(1-benzyl-1H-1,2,3-triazol-4-yl)-7H-pyrrolo-
(3H, s, NCH3), 1.19 (3H, t, J = 7.1 Hz, CH3–OEt); 13C NMR
[2,3-d]pyrimidin-7-yl)acetate (11b)
(101 MHz, CDCl3) δ 167.1 (CO2Et), 162.6 NvCH, 157.3 (C4),
156.1 (C6), 154.9 (C7a), 128.3(C3), 108.6 (C3a), 80.8 (CuC–H), Using general procedure D, 9 (70.00 mg, 0.29 mmol, 1.0 eq.)
76.0 (CuC–H), 61.8 CH2–OEt, 48.4 N–CH2, 41.3 (N–CH3), 35.6 and benzyl bromide (0.89 g, 0.73 mL, 4.62 mmol, 16.1 eq.)
(N–CH3), 14.0 CH3–OEt.
afforded the title compound (81.00 mg, 0.22 mmol, 76%) as a
white solid.
General procedure D for preparation of triazoles from alkyl
bromides (11 & 12)
LRMS (ES+): m/z (%) 378 (100) [M + H]+; 1H NMR (400 MHz,
CDCl3) δ 8.25 (1H, br s, C6–H), 7.57 (1H, s, C5′–H), 7.39–7.37
To a mixture of sodium azide (32.6 eq.) and the corresponding (3H, m, C4″, C5″, C6″), 7.30–7.26 (2H, m, C3″, C7″), 7.12 (1H,
bromide respectively (16.1 eq.) in a microwave vial was added s, C2–H), 5.52 (2H, s, C1″–CH2), 4.90 (2H, s, CH2) 4.23–4.17
water (3 mL) and the resulting solution heated at 140 °C by (2H, q, J = 7.1 Hz, CH2–OEt), 1.25 (3H, t, J = 7.1 Hz, CH3–OEt);
microwave irradiation for 1 hour using the fixed hold time 13C NMR (101 MHz, CDCl3) δ 168.2 (CO2Et), 157.9 (C4), 152.6
setting. The reaction was cooled, extracted with ethyl acetate (C7a), 151.1 (C6), 142.9 (C1′), 134.2 (C2″), 129.2 (C3″, 7″), 128.9
(2 × 3 mL) and dried (via fritted syringe containing MgSO4). (C4″, C6″), 128.1 (C5″), 121.5 (C5′), 118.9 (C2), 106.7 (C3a), 61.9
The combined organic fractions were then added to a mixture (CH2–OEt), 54.4 (C1″), 45.2 (N–CH2), 14.1 (CH3–OEt).
of either (9 or 10, 1.0 eq.) respectively, Cu/C (40 mol%) and
Ethyl 2-(4-amino-3-(1-benzyl-1H-1,2,3-triazol-4-yl)-1H-pyrazolo-
[3,4-d]pyrimidin-1-yl)acetate (12b)
Et3N (1.0 eq.) in a sealed flask flushed with nitrogen and the
resulting mixture stirred at room temperature for 18 hours.
The reaction mixture was then filtered and absorbed onto Using general procedure D, 10 (90.00 mg, 0.37 mmol, 1.0 eq.)
celite under reduced pressure and purified by flash column and 1-bromopentane (0.79 g, 0.55 mL, 5.91 mmol, 16.1 eq.)
chromatography on silica gel eluting with a gradient from 0% afforded the title compound (110.90 mg, 0.29 mmol, 78%) as a
1
to 5% MeOH in CHCl3 to afford the desired compound.
white solid. LRMS (ES+): m/z (%) 379 (100) [M + H]+; H NMR
(400 MHz, CDCl3) δ 9.53 (1H, br s, NH), 8.33 (1H, s, C6–H),
8.02 (1H, s, C5′–H), 7.42–7.36 (3H, m, C4″, C5″, C6″), 7.34–7.31
(2H, m, C3″, C7″), δ 6.14 (1H, br s, NH), 5.58 (2H, s, CH2), 5.11
Ethyl 2-(4-amino-5-(1-pentyl-1H-1,2,3-triazol-4-yl)-7H-pyrrolo-
[2,3-d]pyrimidin-7-yl)acetate (11a)
Using general procedure D, 9 (70.00 mg, 0.28 mmol, 1.0 eq.) (2H, s, CH2), 4.23–4.18 (2H, q, J = 7.1 Hz, CH2–OEt), 1.24 (3H,
and 1-bromopentane (0.69 g, 0.57 mL, 4.62 mmol, 16.1 eq.) t, J = 7.0 Hz, 3H, CH3–OEt); 13C NMR (101 MHz, CDCl3) δ 167.5
afforded the title compound (22.51 mg, 6.30 Exp−5 mol, 22%) (CO2Et), 158.5 (C4), 156.9 (C6), 155.3 (C7a), 142.3 (C3), 136.9
as a white solid. LRMS (ES+): m/z (%) 358(100) [M + H]+; 1H (C1″), 133.7 (C1′), 129.3 (C3″, C7″), 129.3 (C5″), 128.3 (C4″,
NMR (400 MHz, CDCl3) δ 10.77 (1H, s, NH), 10.15 (1H, s, NH), C6″), 121.2 (C5′), 98.5 (C3a), 61.8 (CH2–OEt), 54.7 (C1″), 48.0
7.99 (1H, s, C6–H), 7.77 (1H, s, C5′–H), 7.39 (1H, s, C2–H), 4.99 (N–CH2), 14.1 (CH3–OEt).
(2H, s, CH2), 4.39 (2H, t, J = 7.2 Hz, C1″–CH2); 4.29–4.24 (2H,
q, J = 7.1 Hz, CH2–OEt), 1.99–1.93 (2H, m, C2″–CH2), 1.37–1.29
(7H, m, C3″–CH2, C4″–CH2, CH3–OEt), 0.91 (t, J = 7.0 Hz, 3H,
General procedure E for preparation of triazoles from aryl
iodides (20)
C5″–CH3); 13C NMR (101 MHz, CDCl3) δ 167.2 (CO2Et), 152.9 To a mixture of sodium azide (1.2 eq.), the corresponding
(C4), 147.9 (C6), 142.8 (C7a), 140.4 (C1′), 123.4 (C5′), 119.4 iodide (1.2 eq.), sodium ascorbate (40 mol%), sodium carbon-
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ate (20 mol%), L-proline (20 mol%), Cu/C (40 mol%) 17, (1H, d, J = 8 Hz, C4″), 5.54 (2H, br s, NH2), 5.24 (2H, s, N–CH2)
(1.0 eq.) in a microwave vial was added DMSO–H2O (3 mL) and 4.19–4.13 (2H, q, J = 8.0 Hz, CH2–OEt), 1.20 (3H, t, J = 8.0 Hz,
the resulting solution heated at 130 °C by microwave CH3–OEt); 13C NMR (101 MHz, d6-DMSO) δ 168.1 (CO2Et),
irradiation for 30 minutes using the fixed hold time setting. 158.7 (C4), 157.2 (C6), 155.3 (C7a), 150.6 (C3″), 142.0 (C3),
The reaction mixture was then cooled ammonia in ethanol 137.5 (C1″), 136.1 (C1′), 130.7 (C5″), 121.6 (C5′), 115.0 (C6″),
(7 M 2 mL) added and stirred for 2 hours and poured into 109.9 (C3a), 107.8 (C4″), 105.7 (C2″), 61.8 (CH2–OEt), 48.4
aqueous ammonia solution (10 mL, 20% v/v) and extracted (N–CH2), 14.4 (CH3–OEt).
with ethyl acetate (3 × 10 mL) and. The combined organic
layers were washed with brine then dried (MgSO4) and
absorbed onto celite under reduced pressure. Purification
by flash column chromatography on silica gel eluting with a
Ethyl 2-(4-amino-3-(1-(pyridin-2-yl)-1H-1,2,3-triazol-4-yl)-1H-
pyrazolo[3,4-d]pyrimidin-1-yl)acetate (20c)
gradient from 0% to 5% MeOH in CHCl3 afforded the desired Using general procedure E, 18 (100.0 mg, 0.34 mmol, 1.0 eq.)
compound.
and 2-iodopyridine (82.2 mg, 42.6 µL, 0.40 mmol, 1.2 eq.)
afforded the title compound (69.3 mg, 0.19 mmol, 56%) as a
Ethyl 2-(4-amino-3-(1-phenyl-1H-1,2,3-triazol-4-yl)-1H-pyrazolo-
[3,4-d]pyrimidin-1-yl)acetate (19)
1
cream solid. LRMS (ES+): m/z (%) 366 (100) [M + H]+; H NMR
(400 MHz, d6-DMSO) δ 9.24 (1H, d, J = 8.0 Hz, C5′–H), 8.89
Using general procedure E, 17 (100.0 mg, 0.34 mmol, 1.0 eq.) (1H, br s, NH), 8.65–8.63 (1H, m, C3″–H), 8.24 (1H, s, C6–H),
and Iodobenzene (81.8 mg, 44.9 µL, 0.40 mmol, 1.2 eq.) 8.20–8.14 (2H, m, C5″, C6″), 7.26–7.59 (1H, m, C4″), 5.23 (2H,
afforded the title compound (92.6 mg, 0.25 mmol, 75%) as a s, N–CH2) 4.17–4.12 (2H, q, J = 7.1, CH2–OEt), 1.20 (3H, t, J =
1
brown solid. LRMS (ES+): m/z (%) 364 (100) [M + H]+; H NMR 8.0 Hz, CH3–OEt); 13C NMR (101 MHz, d6-DMSO) δ 168.1
(400 MHz, d6-DMSO) δ 8.27 (1H, s, C 6–H), 8.07 (1H, s, C5′), (CO2Et), 158.6 (C4), 157.2 (C6), 155.3 (C7a), 149.6 (C3″), 142.1
7.73–7.72 (2H, d, J = 7.4 Hz, C2″, C6″), 7.54 (2H, t, J = 7.4 Hz, (C3), 140.7 (C5″), 135.6 (C1′), 125.58 (C4″), 119.6 (C5′), 114.9
C3″, C5″), 7.46 (1H, s, C4″), 7.27 (H, s, C2), 4.97 (2H, s, CH2), (C6″), 98.0 (C3a), 61.8 (CH2–OEt), 48.5 (N–CH2), 14.4
4.26–4.22 (2H, q, J = 7.2 Hz, CH2–OEt), 1.28 (3H, t, J = 7.2 Hz, (CH3–OEt).
CH3–OEt); 13C NMR (101 MHz, d6-DMSO) δ 168.3 (CO2Et),
158.1 (C4), 153.7 (C6), 151.3 (C7a), 142.3 (C3), 136.7 (C1″),
129.8 (C1′), 129.7 (C4″), 121.7 (C3″, C5″), 120.5 (C5′), 116.9
General procedure F for preparation of acids (13 & 14)
(C2″, C6″), 106.2 (C2), 100.3 (C3a), 61.89 (CH2–OEt), 45.2 To a solution of the respective triazole in THF (2 mL) was
(N–CH2), 14.1 (CH3–OEt).
added LiOH (2.0 eq. as a solution in H2O 2 mL) and stirred at
room temperature for 18 hours. The pH of the reaction
mixture was then adjusted to approximately pH 3 with HCl
(1 M). The resultant precipitate was filtered off and washed
Ethyl 2-(4-amino-3-(1-phenyl-1H-1,2,3-triazol-4-yl)-1H-pyrazolo-
[3,4-d]pyrimidin-1-yl)acetate (20a)
Using general procedure E, 18 (100.0 mg, 0.34 mmol, 1.0 eq.) with H2O to afford the desired compound.
and Iodobenzene (81.8 mg, 44.9 µL, 0.40 mmol, 1.2 eq.)
afforded the title compound (92.6 mg, 0.25 mmol, 75%) as a
1
2-(4-Amino-5-(1-pentyl-1H-1,2,3-triazol-4-yl)-7H-pyrrolo-[2,3-d]-
pyrimidin-7-yl)acetic acid (13a)
brown solid. LRMS (ES+): m/z (%) 365 (100) [M + H]+; H NMR
(400 MHz, d6-DMSO) δ 9.54 (1H, NH2), 9.44 (1H, s, C5′), 9.04
(1H, br s, NH), 8.26 (1H, s, C6–H), 8.20 (1H, br s, NH), Using general procedure F, 11a (22.00 mg, 6.16 Exp−5 mol,
8.07–8.04 (2H, qd, J = 3.0 Hz, J = 1.7 Hz, C2″, C6″), 7.65–7.60 1.0 eq.) afforded the title compound (5.00 mg, 1.51 Exp−5 mol,
(2H, dd, J = 7.2 Hz, J = 2.4 Hz, C3″, C5″), 7.56–7.55 (1H, qd, J = 25%) as a white solid. LRMS (ES+): m/z (%) 330 (100) [M + H]+;
3.0 Hz, J = 1.7 Hz, C4″), 5.24 (2H, s, CH2), 4.19–4.13 (2H, q, J = 1H NMR (400 MHz, d6-DMSO) δ 8.54 (1H, s, C6–H), 8.20 (1H, s,
7.1 Hz, CH2–OEt), 1.20 (3H, t, J = 7.1 Hz, CH3–OEt); 13C NMR C5′–H), 7.74 (1H, C2–H), 4.98 (2H, s, CH2), 4.42 (2H, t, 3J =
(101 MHz, d6-DMSO) δ 168.1 (CO2Et), 158.7 (C4), 157.2 (C6), 7.1 Hz, C1″–CH2); 1.89–1.85 (2H, m, C2″–CH2), 1.34–1.22 (4H,
155.3 (C7a), 142.3 (C3), 136.7 (C1″), 136.0 (C1′), 130.4 (C2″, m, C3″–CH2, C4″–CH2), 0.86 (t, 3J = 7.1 Hz, 3H, C5″–CH3).
C6″), 129.7 (C4″), 121.3 (C5′), 120.95 (C3″, C5″), 97.9 (C3a),
61.8 (CH2–OEt), 48.4 (N–CH2), 14.1 (CH3–OEt).
2-(4-Amino-3-(1-pentyl-1H-1,2,3-triazol-4-yl)-1H-pyrazolo-
Ethyl-2-(4-amino-3-(1-(3-aminophenyl)-1H-1,2,3-triazol-4-yl)-
1H-pyrazolo[3,4-d]pyrimidin-1-yl)acetate (20b)
[3,4-d]pyrimidin-1-yl)acetic acid (14a)
Using general procedure F, 12a (58.80 mg, 1.56 mmol, 1.0 eq.)
Using general procedure E, 18 (100.0 mg, 0.34 mmol, 1.0 eq.) afforded the title compound (46 mg, 1.31 mmol, 84%) as a
1
and 3-iodoaniline (87.8 mg, 0.40 mmol, 1.2 eq.) afforded the white solid. LRMS (ES+): m/z (%) 331 (100) [M + H]+; H NMR
title compound (27.0 mg, 0.07 mmol, 21%) as a white solid.
(400 MHz, d6-DMSO) δ 13.22 (1H, S COOH), 9.15 (1H, NH2),
LRMS (ES+): m/z (%) 380 (100) [M + H]+; 1H NMR (400 MHz, 8.76 (1H, s, C5′–H), 8.23 (1H, s, C6–H), 8.08 (1H, NH2), 5.10
3
d6-DMSO) δ 9.24 (1H, s, C5′–H), 9.07 (1H, br s, NH), 8.26 (1H, (2H, s, CH2), 4.47 (2H, t, J = 7.1 Hz, C1″–CH2); 1.93–1.89 (2H,
s, C6–H), 8.19 (1H, br s, NH), 7.22–7.20 (2H, dt, J = 8.0 Hz, J = m, C2″–CH2), 1.34–1.18 (4H, m, C3″–CH2, C4″–CH2), 0.86 (t,
4.0 Hz, C5″, C6″), 7.11–7.08 (1H, d, J = 12 Hz, C2″), 6.71–6.69 3J = 7.1 Hz, 3H, C5″–CH3.
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Organic & Biomolecular Chemistry
2-(4-Amino-5-(1-benzyl-1H-1,2,3-triazol-4-yl)-7H-pyrrolo-[2,3-d]-
pyrimidin-7-yl)acetic acid (13b)
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Using general procedure F, 11b (80.00 mg, 0.22 mmol, 1.0 eq.)
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1
white solid. LRMS (ES+): m/z (%) 350 (100) [M + H]+; H NMR
(400 MHz, d6-DMSO) δ 8.53 (1H, s, C6–H), 8.10 (1H, s, C5′–H),
7.67 (1H, s, C6–H), 7.42–7.36 (5H, m, Ar–H), 5.68 (2H, s, C1″–
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2-(4-Amino-3-(1-benzyl-1H-1,2,3-triazol-4-yl)-1H-pyrazolo-
[3,4-d]pyrimidin-1-yl)acetic acid (14b)
Using general procedure F, 12b (81.5 mg, 0.22 mmol, 1.0 eq.)
afforded the title compound (69.00 mg, 0.20 mmol, 91%) as a
1
white solid. LRMS (ES+): m/z (%) 351 (100) [M + H]+; H NMR
(400 MHz, d6-DMSO) δ 9.09 (1H, NH2), 8.81 (1H, s C5′–H), 8.22
(1H, s, C6–H), 8.11 (1H, NH2), 7.41–7.36 (5H, m, Ar–H), 5.71
(2H, s, C1″–H), 5.09 (2H, s, CH2).
Photophysical measurements
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The triazole adenine analogues were dissolved in methanol,
acetonitrile (20b) or dichloromethane (20b, 20c) for all photo-
physical measurements. Absorption spectra were recorded on
a Varian Cary 4000 or 5000 spectrophotometer. Extinction
coefficients were determined by dissolving small amounts of
the compounds (typically 1.5–3 mg) in a known volume of
methanol. Extinction coefficient values reported are averages
of at least two measurements (except for 20c). Emission
spectra were recorded on a Horiba Spex fluorolog 3 and cor-
rected for Raman scattering. Quantum yields were determined
relative to 2-aminopyridine (Φf = 60%) in 0.05 M H2SO4 (aq.)
at 25 °C and an excitation wavelength of 290 nm.44 Values
reported were determined by at least two independent
measurements.
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Acknowledgements
The authors thank Carl Tryggers Stiftelse, Stiftelsen Olle
Engkvist Byggmästare and the Swedish Research Council for
financial support and Professor Bo Albinsson and Munefumi
Tokugawa for helpful discussions.
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