Synthesis of triazole-linked SAM-adenosine conjugates: Functionalization of adenosine at N-1 or N-6 position without protecting groups

Article abstract of DOI:10.3390/molecules25143241

More than 150 RNA chemical modifications have been identified to date. Among them, methylation of adenosine at the N-6 position (m6A) is crucial for RNA metabolism, stability and other important biological events. In particular, this is the most abundant

Full text of DOI:10.3390/molecules25143241

molecules
Article
Synthesis of Triazole-Linked SAM-Adenosine
Conjugates: Functionalization of Adenosine at N-1 or
N-6 Position without Protecting Groups
Colette Atdjian, Dylan Coelho, Laura Iannazzo *, Mélanie Ethève-Quelquejeu *
and Emmanuelle Braud *
Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, Universit
F-75006 Paris, France; colette.atdjian@wanadoo.fr (C.A.); dylan.coelho@etu.parisdescartes.fr (D.C.)
Correspondence: laura.iannazzo@parisdescartes.fr (L.I.); melanie.etheve-quelquejeu@parisdescartes.fr
M.E.-Q.); emmanuelle.braud@parisdescartes.fr (E.B.)
é de Paris, CNRS UMR 8601,
*
(
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Katherine Seley-Radtke
Received: 21 June 2020; Accepted: 10 July 2020; Published: 16 July 2020
ꢀ
ꢁꢂꢃꢄꢅꢆ
Abstract: More than 150 RNA chemical modifications have been identified to date. Among them,
6
methylation of adenosine at the N-6 position (m A) is crucial for RNA metabolism, stability and
other important biological events. In particular, this is the most abundant mark found in mRNA
1
in mammalian cells. The presence of a methyl group at the N-1 position of adenosine (m A)
is mostly found in ncRNA and mRNA and is mainly responsible for stability and translation
6
1
fidelity. These modifications are installed by m A and m A RNA methyltransferases (RNA MTases),
6
respectively. In human, deregulation of m A RNA MTases activity is associated with many diseases
including cancer. To date, the molecular mechanism involved in the methyl transfer, in particular
substrate recognition, remains unclear. We report the synthesis of new SAM-adenosine conjugates
containing a triazole linker branched at the N-1 or N-6 position of adenosine. Our methodology
does not require protecting groups for the functionalization of adenosine at these two positions.
6
1
The molecules described here were designed as potential bisubstrate analogues for m A and m A RNA
MTases that could be further employed for structural studies. This is the first report of compounds
1
mimicking the transition state of the methylation reaction catalyzed by m A RNA MTases.
6
1
Keywords: RNA m A methyltransferase; RNA m A methyltransferase; bisubstrate analogues;
S-adenosyl-l-methionine; 1-N-alkylated adenosine; 1,2,3-triazole; click chemistry; CuAAC
1
. Introduction
Among the numerous post-transcriptional modifications of RNA identified to date, methylation
is currently one of the most studied [
1
]. This modification can occur at the terminal cap of RNAs or
0
at internal position, and the methyl group is found on nucleic acid bases or at the 2 position of the
ribose units. The adenine base can be methylated at the C-2 and C-8 atoms as well as at the nitrogen
6
6
atoms N-1 and N-6 [
2,3]. N -Methyladenosine (m A) is an abundant reversible modification found in
all types of RNA, involved in the regulation of RNA metabolism, protein expression or RNA-protein
recognition [
as cancers, obesity, infertility [
4
–
7
]. Abnormal methylation process is associated with the development of diseases such
1
8
–10]. Though less studied, the modification at the N-1 site (m A) is also
reversible and linked to the structural stability and the functions of RNAs [11,12].
1
6
Since m A can rearrange to m A under alkaline conditions by Dimroth rearrangement [13],
its presence in mRNA has been detected only recently in mammalian mRNAs [14 16]. The functional
–
consequence of this modification is poorly understood but could impact the regulation and the function
1
of m A for some positions in rRNA and tRNA in the human transcriptome. However, the unique
Molecules 2020, 25, 3241; doi:10.3390/molecules25143241
www.mdpi.com/journal/molecules

Molecules 2020, 25, 3241
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1
chemical properties of m A, with both a positive charge and a methyl group, potentially allow for a
strong effect in terms of RNA structure or protein-RNA interaction [14].
1
6
For these two modifications (m A and m A), the methyl group is introduced enzymatically by
RNA methyltransferases (RNA MTases) that catalyze the transfer of the methyl from the cofactor
6
S-adenosyl-l-methionine (SAM) to the nucleotides (Scheme 1). Most m A marks in mammals mRNA
are written by a dedicated methyltransferase complex involving the heterodimer METTL3/METTL14,
targeting the consensus sequence RRACH [17
–
19]. However, little is known about the recognition of the
6
RNA targets by this complex [20]. METTL16 has a distinct set of targets for m A modification, including
0
the 3 UTR of MAT2A mRNA and the U6 snRNA, a longer conserved sequence of UACAGAGAA [21
–
23].
6
Among human m A RNA MTases, only METTL16 has been crystallized with RNA substrate [24].
For m A modification of mRNA it is not clear whether or not specific methylation machinery could also
1
exist. It has been recently hypothesized that the tRNA methyltransferase complex TRMT6/61A could
catalyze the methyl transfer on the mRNA [16]. In conclusion, few RNA-bound MTases structures are
currently available. This lack of complex structures is due to the difficulties in crystallizing RNA/protein
complexes. As a consequence, RNA recognition patterns and methylation reaction mechanisms remain
poorly understood.
NH2
O2C
NH3
H3C
NH2
N
NH2
N
N
N
N
NH
NH2
N
O2C
S
NH3
O2C
S
NH3
N
N
N
N
N
N
N
N
N
H
H
S
+
O
N
+
N
N
N
N
NH
H
H3C
O
6
O
m A RNA MTase
N
N
OH OH
N
OH OH
SAM
OH OH
SAH
N
m6A RNA
RNA
Transition state for N6 methylation
NH2
O2C
NH3
O
N
N
NH2
N
NH2
N
N
NH2
NH2
N
O2C
S
NH3
N
O2C
S
NH3
N
N
N
N
N
N
CH3
+
NH2H
H
S
N
+
N
N
N
N
N
N
N
N
N
1
N
H
H3C
O
OH OH
O
1
m A RNA MTase
OH OH
SAM
OH OH
SAH
RNA
m A RNA
Transition state for 1-N methylation
Scheme 1. RNA MTases-catalyzed methylation of adenosine at the N-6 and N-1 positions.
In this context, we recently described the synthesis of SAM-adenosine conjugates as first transition
6
state analogues for m A RNA MTases and their use as tools for structural study [25
,26]. We showed
that a SAM-adenosine conjugate containing a three-carbon linker tethering the analogue of SAM to the
N-6 atom of the adenosine binds the bacterial RNA MTase RlmJ with a conformation close to the real
transition state. The structure of this bisubstrate analogue favors the correct positioning of the RNA
moiety mimicked by an adenosine and the methionine part of the cofactor into the catalytic site of the
MTase. However, a deviation was observed for the positioning of the adenosine in the cofactor part,
◦
which is rotated 120 out of the canonical binding pocket for SAH. This deviation indicates that our
bisubstrate analogues are not optimal. In this study, we pursue the development of SAM-adenosine
6
conjugates for m A RNA MTases and extend our work to the synthesis of the first potential bisubstrate
1
analogues for m A RNA MTases by covalently linking an analogue of SAM to the N-6 or N-1 atom of
the adenosine substrate, respectively.
6
N -alkylation of adenosine derivatives is mainly achieved through aromatic nucleophilic
substitution (S Ar) of diverse electrophilic adenosine derivatives [27
–
39] or by Dimroth rearrangement
N
of 1-N-alkylated adenosines [40
–52]. In the context of S Ar, synthetic strategies involving non classic
N

Molecules 2020, 25, 3241
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leaving groups were developed using peptide-coupling agents for activation of the amide group of
6
inosine derivatives [53
–
56]. Another study reports the reduction of N -acyl-adenosine derivatives with
6
LiAlH4 [57]. Selective N -alkylation of adenosine derivatives can also be achieved under phase transfer
catalysis conditions as described by Arimoto et al. [58]. Two groups used Mitsunobu reaction applied
6
0
0
0
6
to N -acetyl-2 ,3 ,5 -tri-O-acetyladenosine [59
,
60] or N -Boc protected adenosine [61] to regioselectively
6
synthesize N -alkylated products. Finally, functionalization of 6-chloroadenosine derivatives can be
achieved by palladium-catalyzed Buchwald–Hartwig coupling [62,63].
To date, the alkylation reaction at the N-1 position has been principally developed through the
N-1 nucleophilic attack of alkyl halides [40–52]. This approach allows for the introduction of methyl
and alkyl groups as well as benzyl, or allyl substituents. Propargyl group has been also installed on
6
0
0
0
N -acetyl-2 ,3 ,5 -tri-O-acetyladenosine but in a quite low yield of 14% [51]. In 2005, Terrazas et al.
synthesized 1-N-alkylated adenosines by reacting electrophilic inosine with primary amines [64].
In this context, we sought to introduce new chemical modifications at the N-6 and N-1 positions of
adenosine to synthesize new SAM-adenosine conjugates. Click chemistry, especially copper(I)-catalyzed
alkyne-azide cycloaddition (CuAAC), is an efficient strategy to rapidly synthesize complex structures.
On these bases, we designed new bisubstrate analogues bearing a 1,2,3-triazole ring instead of the alkyl
linker to increase rigidity between the mimic of the substrate and the SAM analogue while maintaining
an appropriate length between the two entities (Figure 1). The synthetic strategy used to obtain these
molecules relies on the efficient introduction of the propargyl group at the N-6 and N-1 positions of
adenosine derivatives.
A) Previous work
B) This work
NH2
N
NH2
N
HO2C
linker
NH2
N
N
N
N
N
N
N
HN
N
N
N
N
HN
N
n
N
N
O
N
O
N
linker
N
N
HO
OH
HO
HO
OH
HO
O
O
SAM analogue
SAM analogue
HO
OH
HO
OH
RNA mimic
RNA mimic
NH2
NH2
N
NH2
N
NH2
NR
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
HN
N
N
N
N
N
N
N
N
N
N
N
N
O
O
O
HO
HO
O
O
N
HO
HO
OH
HO
OH
HO
OH
O
HO
OH
HO
OH
HO
OH SAM-adenosine conjugate
functionalized at the N-6 position
SAM-adenosine conjugates functionalized at the N-1 position
Figure 1.
(A) Our previous work: structure of SAM-adenosine conjugates with an alkyl linker [25,26];
(
B) structure of SAM-adenosine conjugates with a 1,2,3-triazole linker synthesized in this study.
2
. Results and discussion
2
.1. Synthesis of SAM-Adenosine Conjugates Using Protecting Groups
First, we took advantage of the work of Sekine et al. who used tetrabutylammonium
6
bromide (TBABr) as the phase transfer catalyst to produce a mixture of N and 1-N-alkylated
6
adenosines from N -benzoylated adenosine [58]. A short study was carried out to investigate the
6
0
0
0
alkylation of N -benzoyl-2 ,3 ,5 -tris-O-(tert-butyldimethylsilyl)adenosine 1a [65] with propargyl
bromide (Scheme 2). Using tetrabutylammonium hydroxide (TBAOH) instead of TBABr led to the
formation of adenosines 2a and 3a in 57 and 28% yield with total conversion of the starting material
(
(
Scheme 2). Analysis of 1D and 2D NMR spectra confirmed the site of alkylation for each regioisomer
6
Figure 2) [51
,
58]. The signals for 2-H and 8-H appear at 8.49 and 8.14 ppm for N -alkylated compound
2a while they are upfield at 8.24 and 7.88 ppm in 3a as expected. Moreover, two correlations are

Molecules 2020, 25, 3241
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observed in the HMBC spectrum of 2a between protons of the methylene group of the propargyl
p
(
H ) and C6 and C=O of benzoyl group in the N-6 regioisomer (Figure 2A). For compound 3a
,
p
HMBC experiments show a correlation between 2-H and the carbon of the methylene group (C )
as well as two correlations between the H protons and C2 and C6 (Figure 2B). The same reaction
p
conditions applied to compound 1b [25] containing a tert-butoxycarbonyl protecting group at the
N-6 position of adenosine afforded 2b and 3b in 76 and 16% yield respectively, the presence of the
6
carbamate function favoring the N alkylation.
6
Scheme 2. Synthesis of N and 1-N-propargylated adenosines.
6
Figure 2. 2D HMBC spectra (500 MHz, CDCl ): (
A
) N -propargyladenosine derivative 2a. (
B) 1-
3
propargyladenosine derivative 3a.

Molecules 2020, 25, 3241
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Azide
position of 1a led to the alcohol
treatment of 5 with sodium azide provided azido adenosine 6 in 92% yield.
6 was synthesized in three steps [25] (Scheme 3). Briefly, removal of TBS group at the
0
5
4
that was mesylated to afford derivative
5
in 82% yield. Finally,
NHBz
NHBz
NHBz
NHBz
N
N
N
N
N
N
N
N
N
N
N
N
N
N
TFA/H
2
O (1/1)
MsCl
NaN
3
N
N
TBSO
HO
MsO
N
3
THF, 0 °C then
rt, 1h30
Pyr, rt, 16 h
DMF, 70 °C, 4 h
92 %
O
O
O
O
82 %
86 %
TBSO
OTBS
TBSO
OTBS
TBSO
OTBS
TBSO
OTBS
1a
4
5
6
Scheme 3. Synthesis of azide 6.
CuAAC reactions between adenosines 2a
-b and 3a-b and azides 6 or 7 [25] were conducted under
classic conditions in the presence of sodium ascorbate and copper sulfate in THF/H O to afford triazoles
2
8
–11 in 60–89% yield (Scheme 4).
Scheme 4. Synthesis of triazoles 8–11.
The fully deprotected SAM-adenosine conjugate 12 could be obtained from either 9a or 9b
(
(
Scheme 5). Compound 9a was successively treated with methylamine, ZnBr and cesium fluoride
CsF) to remove the benzoyl-, the Boc- and the TBS groups respectively to give 12 in 5% yield over
2
three steps after HPLC purification. In comparison, a two-step strategy from 9b, followed by HPLC
purification, led to the formation of 12 in 4% yield. These results seem to indicate that in the pathway
2
, the removal of the two Boc groups is less efficient than the two steps required for the deprotection of
the benzoyl and Boc groups in the pathway 1 (Scheme 5).
We applied the strategy used for the deprotection of compound 9a to compound 11a. Unfortunately,
efforts to remove the protecting group of the exocyclic amine were unsuccessful. Indeed, using the
same successive steps, we observed the formation of the N-methylated compound 13 in 8% yield as a
mixture of E and Z imines (Scheme 6). Other attempts were conducted with bases such as ammonia
or potassium carbonate which led to the recovery of the starting material in the first case and to
degradation in the second one. As an alternative, we chose to remove only the Boc and the TBS
groups in a two-step sequence allowing for the formation of SAM-adenosine conjugate 14 in 13% yield
(Scheme 6).

Molecules 2020, 25, 3241
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Scheme 5. Synthesis of SAM-adenosine conjugate 12.
Scheme 6. Access to SAM-adenosine conjugates 13 and 14.
By contrast and to our delight, treating derivative 11b with ZnBr and then CsF afforded the
2
fully deprotected SAM-adenosine conjugate 15 in 20% yield over two steps (Scheme 7). Of note,
deprotection of compounds 8a and 10a using methylamine and then CsF provided compounds 12 and
1
4, respectively that could not be properly purified in these particular conditions.
Scheme 7. Access to SAM-adenosine conjugate 15.

Molecules 2020, 25, 3241
7 of 19
2
.2. Synthesis of SAM-Adenosine Conjugates without Protecting Groups
Since our syntheses require numerous steps of protection and deprotection, we reinvestigated
the synthetic strategies to develop more efficient approaches to get the SAM-adenosine conjugates.
We also sought to introduce regioselectively the propargyl group at the N-6 and N-1 positions. We first
6
modified the synthesis of N -conjugate 12. Wan et al. previously reported the amination of unprotected
inosine using primary amines in the presence of BOP and DIPEA [55]. Following this methodology,
the propargyl group was introduced in one step at the N-6 position of adenosine leading to compound
1
6 in 71% yield (Scheme 8).
Scheme 8. Access to SAM-adenosine conjugate 12.
The unprotected azido partner 17 was prepared following a two-step procedure in 36% yield [66
,67].
Then, alkyne 16 and azide 17 were reacted in the presence of copper sulfate and sodium ascorbate in a
DMF/H O mixture to afford the expected SAM-adenosine conjugate 12 in 36% yield (Scheme 8).
2
We next investigated the synthesis of 1-propargyladenosine 18 (Scheme 9). Adenosine is known
to react with alkyl halides in polar solvents such as DMF or DMA at room temperature, to afford
1
-N-alkylated compounds [40
,
51]. Under these conditions, adenosine was treated with an excess of
◦
propargyl bromide in DMF at 50 C for 24 h and 1-N-propargylated adenosine 18 was obtained in
56% yield (Scheme 9). The synthesis of SAM-adenosine conjugate with a triazole linker connected
at the N-1 position was then achieved through the CuAAC between 17 and 18. The reaction was
carried out in the presence of sodium ascorbate and copper sulfate to afford the expected conjugate 19
in 53% yield (Scheme 9). Compound 19 corresponds to the protonated and positively charged form
1
of compound 15. This was confirmed by H NMR spectra analysis for both compounds 15 and 19
(
8
See the supporting file for NMR spectra of compounds 15 and 19). The signal for 2-H (H2a) appears at
.62 ppm for compound 19 while it is upfield at 8.22 ppm for compound 15 as expected [51].
Scheme 9. Access to SAM-adenosine conjugate 19.

Molecules 2020, 25, 3241
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3
. Materials and Methods
Reactions were carried out under argon atmosphere and solvents were dried using standard
methods and distilled before use. DCM, Pyridine and DMF were dried over calcium hydride and
THF over sodium and benzophenone. Unless otherwise specified, materials were purchased from
commercial suppliers and used without further purification. TLC was performed using Merck
commercial aluminum sheets coated with silica gel 60 F254 (Merck, Darmstadt, Germany). Compounds
were detected by charring with 10% H SO in ethanol followed by heating. Purification was performed
2
4
by flash chromatography on silica gel (60 Å, 180–240 mesh; Merck, Darmstadt, Germany). Preparative
HPLC was performed using a HPLC system with a reverse phase C-18 column (250 21.2 mm) using
a solvent system consisting of H O and CH CN (linear gradient from 0:100 to 100:0 in 30 min) at a
×
2
3
−
1
flow rate of 15 mL.min and UV detection at 254 nm. The purity of final compounds (
established by analytical HPLC, which was performed on Macherey Nagel C18 100-5 NUCLEOSIL
column (25 4.6, 5 m) with UV detection at 214 and 254 nm. NMR spectra were recorded on
Bruker spectrometers (Avance II 500 and Avance III HD 4000) (Bruker Biospin, Fällanden, Switzerland).
Chemical shifts ( ) are reported in parts per million (ppm) and referenced to the residual proton or
≥
95%) was
×
µ
δ
carbon resonance of the solvents: CDCl (
δ
7.26), MeOD (
δ
3.31), D O (
δ
4.79) or (CD ) SO (
δ
2.50)
3
2
3 2
1
13
for H and CDCl (
δ
77.16), MeOD (
δ
49.0) or (CD ) SO (
δ
39.52) for C. Signals were assigned using
3
3 2
1
13
1D ( H and C) and 2D (HSQC, COSY and HMBC) experiments. NMR coupling constants (J) are
reported in Hertz (Hz) and splitting patterns are indicated as follows: s (singlet), bs (broad singlet),
d (doublet), t (triplet), q (quartet), dd (doublet of doublet), m (multiplet). High-resolution mass
spectroscopy (HRMS) was recorded with an ion trap mass analyzer under electrospray ionization
(
(
ESI) in the negative or positive ionization detection mode, using Thermo Scientific LTQ Orbitrap XL
Thermo Scientific, Illkirch, France.
Compounds 2a and 3a: The trisilylated adenosine 1a (1.59 g, 2.22 mmol) and propargyl bromide
8
(
0% in toluene (674
1.78 g, 2.22 mmol) and 1M aqueous NaOH (22.2 mL) were added to the solution. After vigorous
stirring at room temperature for 1 h, the reaction mixture was diluted in DCM and washed with brine,
µL, 8.88 mmol) were dissolved in DCM (50 mL) and tetrabutylammonium hydroxyde
dried over MgSO , and the solvent was removed under reduced pressure. The residue was purified by
4
silica gel chromatography (eluent: Cyclohexane/EtOAc 9:1 then 7:3) to provide the desired compounds
1
as white foams (946 mg, 57% for 2a and 475 mg, 28% for 3a). 2a: H NMR (500 MHz, CDCl ):
(
δ
8.49
3
Bz
Bz
Bz
s, 1H, H2), 8.14 (s, 1H, H8), 7.39–7.37 (m, 2H, H ), 7.18–7.15 (m, 1H, H ), 7.06–7.03 (m, 2H, H ),
0 0
.95 (d, J = 5.9 Hz, 1H, H1 ), 5.7 (d, J = 4.9 Hz, 2H, CH N), 4.54–4.52 (m, 1H, H2 ), 4.17–4.15 (m, 1H,
2
5
0
0
0
0
H3 ), 4.03–4.01 (m, 1H, H4 ), 3.89 (dd, J = 4.4, 11.4 Hz, 1H, H5 ), 3.68 (dd, J = 2.8, 11.2 Hz, 1H, H5 ),
TBS
TBS
TBS
2
.01 (t, J = 4.7 Hz, 1H, C CH), 0.85 (s, 9H, tBu ), 0.83 (s, 9H, tBu ), 0.64 (s, 9H, tBu ), 0.02 (s, 3H,
≡
Me ), 0.01 (s, 3H, Me ), 0.00 (s, 6H, 2 MeTBS),
TBS
TBS
−
0.19 (s, 3H, MeTBS),
−
0.51 (s, 3H, MeTBS). 13C NMR
Bz
Bz
(
126 MHz, CDCl ):
δ
171.4 (C=O), 153.2 (C6), 152.8 (C4), 151.9 (C2), 142.8 (C8), 135.9 (Cq ), 130.9 (C ),
3
Bz
Bz
0
0
0
0
1
7
28.9 (2C, C ), 127.9 (2C, C ), 127.0 (C5), 88.2 (C1 ), 86.2 (C4 ), 79.0 (C≡CH), 75.8 (C2 ), 72.4 (C3 ),
0
TBS
TBS
TBS
TBS
1.7 (C≡CH), 62.8 (C5 ), 37.5 (CH N), 26.1 (3C, tBu ), 25.9 (3C, tBu ), 25.7 (3C, tBu ), 18.6 (Cq ),
2
TBS
TBS
TBS
4.4 (MeTBS),
TBS
TBS
TBS
18.1 (Cq ), 17.9 (Cq ),
−
4.3 (Me ),
−
−
4.5 (Me ),
−
5.0 (Me ),
−
5.2 (Me ),
−
5.3
TBS
+
1
(
Me ). HRMS (ESI) m/z: calcd for C H N NaO Si [M + Na] : 774.3878; found: 774.3904. 3a: H
3
8
61
5
5
3
Bz
NMR (500 MHz, CDCl ):
δ
Bz
8.24 (s, 1H, H2), 8.16–8.15 (m, 2H, H ), 7.88 (s, 1H, H8), 7.48–7.45 (m, 1H,
3
Bz
0
H ), 7.40–7.37 (m, 2H, H ), 5.90 (d, J = 5.0 Hz, 1H, H1 ), 4.97 (t, J = 2.8 Hz, 2H, CH N), 4.48–4.47 (m,
H, H2 ), 4.26–4.24 (m, 1H, H3 ), 4.08–4.06 (m, 1H, H4 ), 3.88 (dd, J = 4.0, 11.3 Hz, 1H, H5 ), 3.73 (dd,
J = 3.4, 11.4 Hz, 1H, H5 ), 2.59 (t, J = 2.5 Hz, 1H, C CH), 0.92 (s, 9H, tBu ), 0.89 (s, 9H, tBu ), 0.81
s, 9H, tBu ), 0.09 (s, 3H, Me ), 0.08 (s, 3H, Me ), 0.06 (s, 3H, Me ), 0.05 (s, 3H, Me ),
177.2 (C=O), 146.1 (C6), 145.4 (C2),
45.1 (C4), 138.8 (C8), 135.8 (Cq ), 132.0 (C ), 129.9 (2C, C ), 128.1 (2C, C ), 122.3 (C5), 88.2 (C1 ),
2
0
0
0
0
1
0
TBS
TBS
≡
TBS
TBS
TBS
TBS
TBS
(
3
1
8
2
−
0.03 (s,
H, MeTBS),
−
0.19 (s, 3H, MeTBS). C NMR (126 MHz, CDCl ):
13
δ
3
Bz
Bz
Bz
Bz
0
0
0
0
0
TBS
5.4 (C4 ), 76.7 (C≡CH), 76.1 (C2 ), 75.9 (C≡CH), 72.0 (C3 ), 62.7 (C5 ), 37.8 (CH N), 26.1 (3C, tBu ),
2
TBS
TBS
TBS
TBS
TBS
TBS
TBS
5.9 (3C, tBu ), 25.8 (3C, tBu ), 18.6 (Cq ), 18.2 (Cq ), 18.0 (Cq ),
−
4.2 (Me ), 4.4 (Me ),
−

Molecules 2020, 25, 3241
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TBS
4.8 (MeTBS),
5.2 (MeTBS),
5.3 (MeTBS). HRMS (ESI) m/z: calcd for C H N NaO Si
3
−
4.5 (Me ),
−
−
−
38
61
5
5
+
[
M + Na] : 774.3878; found: 774.3911.
Compounds 2b and 3b: To a stirred solution of compound 1b (1.5 g, 2.11 mmol) in DCM (47.5 mL)
was added propargyl bromide (80% in toluene) (0.795 mL, 8.44 mmol), tetrabutylammonium hydroxide
1.68 g, 1.68 mmol) and 1M aqueous NaOH (21.1 mL). The reaction mixture was stirred at room
temperature for 1 h. The reaction mixture was dissolved in DCM and washed with brine. The organic
(
layer was dried over MgSO , concentrated in vacuo and purified on silica gel chromatography (eluent:
4
cyclohexane/EtOAc 9:1 then 7:3) to afford the desired compounds as white foams (1.2 g, 76% for 2b
1
and 0.251 g, 16% for 3b). 2b: H NMR (500 MHz, CDCl ):
δ
8.77 (s, 1H, H2), 8.41 (s, 1H, H8), 6.10 (d,
3
0
0
J = 4.9 Hz, 1H, H1 ), 4.80 (d, J = 2.4 Hz, 2H, CH N), 4.64 (t, J = 4.6 Hz, 1H, H2 ), 4.33 (t, J = 4.0 Hz, 1H,
H3 ), 4.14 (q, J = 3.8 Hz, 1H, H4 ), 4.04 (dd, J = 11.4, 4.1 Hz, 1H, H5 ), 3.80 (dd, J = 11.4, 2.8 Hz, 1H,
2
0
0
0
0
Boc
TBS
TBS
H5 ), 2.16 (t, J = 2.4 Hz, 1H, C CH), 1.48 (s, 9H, tBu ), 0.96 (s, 9H, tBu ), 0.93 (s, 9H, tBu ), 0.80 (s,
≡
H, tBu ), 0.15 (s, 3H, Me ), 0.14 (s, 3H, Me ), 0.10 (s, 3H, Me ), 0.09 (s, 3H, MeTBS),
TBS
TBS
TBS
TBS
−
0.04 (s,
9
3
1
H, MeTBS),
−
0.22 (s, 3H, MeTBS). C NMR (126 MHz, CDCl ):
13
δ
Boc
152.7 (C6), 152.5 (C4), 152.4 (C=O),
3
0
0
0
0
51.8 (C2), 142.2 (C8), 127.60 (C5), 88.7 (C1 ), 85.5 (C4 ), 82.9 (Cq ), 79.9 (C≡CH), 76.1 (C2 ), 72.0 (C3 ),
1.2 (C≡CH), 62.6 (C5 ), 37.3 (CH N), 28.1 (3C, tBuBoc), 26.3 (3C, tBuTBS), 26.0 (3C, tBu ), 25.8 (3C,
0
TBS
7
2
TBS
TBS
TBS
TBS
4.2 (MeTBS),
4.5 (MeTBS),
TBS
TBS
tBu ), 18.7 (Cq ), 18.2 (Cq ), 18.0 (Cq ),
−
−
−
4.6 (Me ),
−
4.8 (Me ),
5.2 (MeTBS),
−
5.2 (MeTBS). HRMS (ESI) m/z: calcd for C H N O Si [M + H] : 748,4315; found
+
−
36
66
5
6
3
1
7
48.4304. 3b: H NMR (500 MHz, CDCl ):
H1 ), 4.83 (d, J = 2.7 Hz, 2H, CH N), 4.36–4.30 (m, 2H, H2 , H3 ), 4.11–4.08 (m, 1H, H4 ), 3.99 (dd,
δ
8.22 (s, 1H, H2), 8.12 (s, 1H, H8), 5.91 (d, J = 3.3 Hz, 1H,
3
0
0
0
0
0
2
0
J = 11.6, 3.4 Hz, 1H, H5 ), 3.78 (dd, J = 11.6, 2.5 Hz, 1H, H5 ), 2.56 (t, J = 2.6 Hz, 1H, C
≡
CH), 1.60 (s,
Boc
TBS
TBS
TBS
TBS
9
3
H, tBu ), 0.94 (s, 9H, tBu ), 0.92 (s, 9H, tBu ), 0.86 (s, 9H, tBu ), 0.12 (s, 3H, Me ), 0.11 (s,
H, Me ), 0.10 (s, 3H, Me ), 0.08 (s, 3H, Me ), 0.02 (s, 3H, MeTBS),
TBS
TBS
TBS
−
0.04 (s, 3H, Me ).
TBS
13
C
NMR (126 MHz, CDCl ):
(
δ
Boc
161.0 (C=O), 147.1 (C6), 145.1 (C2), 144.2 (C4), 138.2 (C8), 122.0 (C5), 88.7
3
0
0
0
0
0
C1 ), 84.5 (C4 ), 80.9 (Cq ), 76.8 (C≡CH), 76.7 (C2 ), 76.4 (C≡CH), 70.9 (C3 ), 61.9 (C5 ), 37.0 (CH N),
2
8.3 (3C, tBuBoc), 26.3 (3C, tBuTBS), 26.0 (3C, tBu ), 25.9 (3C, tBu ), 18.7 (Cq ), 18.2 (Cq ), 18.1
TBS
TBS
TBS
TBS
2
TBS
4.1 (MeTBS),
TBS
4.7 (MeTBS),
5.2 (MeTBS),
TBS
(Cq ),
−
−
4.6 (2C, Me ),
calcd for C H N O Si [M + H] : 748.4315; found 748.4309.
−
−
−
5.3 (Me ). HRMS (ESI) m/z:
+
36
66
5
6
3
◦
Compound
4
: To a stirred solution of compound 1a (1.42 g, 1.97 mmol) in THF (20 mL) at 0 C,
was added dropwise an aqueous solution of TFA (1:1, 7.55 mL, 98.72 mmol). The solution was stirred
at room temperature for 1.5 h. The reaction mixture was neutralized with saturated aqueous NaHCO3
solution and dissolved in EtOAc. The organic layer was washed with brine, dried over MgSO4,
filtered and concentrated in vacuo. The residue was purified on silica gel chromatography (eluent:
1
Cyclohexane/EtOAc 6:4) to afford compound
4
as a white foam (1.02 g, 86%). H NMR (500 MHz,
Bz
Bz
CDCl3):
δ 8.83 (s, 1H, H2), 8.13 (s, 1H, H8), 8.07–8.03 (m, 2H, H ), 7.65–7.60 (m, 1H, H ), 7.56–7.51 (m,
H, H ), 5.88 (d, J = 7.6 Hz, 1H, H1 ), 5.03 (dd, J = 7.6, 4.5 Hz, 1H, H2 ), 4.35 (d, J = 4.5 Hz, 1H, H3 ),
.20 (d, J = 1.7 Hz, 1H, H4 ), 3.98 (dd, J = 13.1, 1.8 Hz, 1H, H5 ), 3.74 (dd, J = 13.0, 1.6 Hz, 1H, H5 ),
Bz
0
0
0
2
4
0
−
0
0
0
TBS
TBS
TBS
TBS
TBS
.96 (s, 9H, tBu ), 0.75 (s, 9H, tBu ), 0.14 (s, 3H, Me ), 0.13 (s, 3H, Me ), 0.12 (s, 3H, Me ),
−
TBS 13
0.60 (s, 3H, Me ). C NMR (126 MHz, CDCl ): δ 164.4 (C=O), 152.4 (C6), 150.6 (C2), 150.5 (C4),
3
Bz
Bz
Bz
Bz
0 0
1
7
43.4 (C8), 133.7 (Cq ), 133.1 (C ), 129.1 (2C, C ), 127.9 (2C, C ), 124.5 (C5), 91.4 (C1 ), 89.7 (C4 ),
0
0
0
TBS
TBS
TBS
TBS
4.2 (C2 ), 74.0 (C3 ), 63.1 (C5 ), 25.9 (3C, tBu ), 25.8 (3C, tBu ), 18.2 (Cq ), 17.9 (Cq ),
−
4.4
TBS
TBS
TBS
TBS
(Me ),
−
4.4 (Me ),
−
4.5 (Me ),
−
5.7 (Me ). HRMS (ESI) m/z: calcd for C H N O Si [M +
2
9
46
5
5
2
+
H] : 600.3032; found: 600.3022.
Compound
dropwise to a solution of compound
5
: Methanesulfonyl chloride (0.26 mL, 3.33 mmol) in pyridine (10 mL) was added
◦
4
(1 g, 1.66 mmol) in pyridine (7 mL) at 0 C. The reaction mixture
was stirred at room temperature for 16 h. The reaction was quenched by addition of water and then
diluted in DCM. The organic layer was washed with a saturated solution of NaHCO and brine.
3
The combined organic layer was dried over MgSO and concentrated to dryness. The residue was
4
purified on silica gel chromatography (eluent: Cyclohexane/EtOAc 6:4) and compound
5 was isolated
1
as a white foam (0.92 g, 82%). H NMR (500 MHz, CDCl ):
δ 8.82 (s, 1H, H2), 8.20 (s, 1H, H8), 8.04 (d,
3

Molecules 2020, 25, 3241
10 of 19
Bz
Bz
Bz
J = 7.3 Hz, 2H, H ), 7.62 (t, J = 7.4 Hz, 1H, H ), 7.54 (t, J = 7.6 Hz, 2H, H ), 6.00 (d, J = 4.8 Hz, 1H,
0
0
0
0
H1 ), 4.99 (t, J = 4.4 Hz, 1H, H2 ), 4.62 (dd, J = 11.2, 4.1 Hz, 1H, H5 ), 4.50 (dd, J = 11.2, 4.6 Hz, 1H, H5 ),
0 0
.40 – 4.35 (m, 2H, H3 , H4 ), 3.03 (s, 3H, CH ), 0.95 (s, 9H, tBu ), 0.82 (s, 9H, tBu ), 0.15 (s, 3H,
3
TBS
TBS
4
TBS
TBS
TBS
TBS 13
Me ), 0.13 (s, 3H, Me ), 0.00 (s, 3H, Me ),
−
0.20 (s, 3H, Me ). C NMR (126 MHz, CDCl ):
3
Bz
Bz
Bz
δ
168.3 (C=O), 152.8 (C6), 151.5 (C2), 149.9 (C4), 142.5 (C8), 134.8 (Cq ), 132.9 (C ), 129.0 (2C, C ),
Bz
0 0 0 0 0
27.9 (2C, C ),123.9 (C5), 89.9 (C1 ), 82.4 (C4 ), 74.3 (C2 ), 72.1 (C3 ), 67.9 (C5 ), 37.8 (CH ), 25.9 (3C,
3
1
TBS
TBS
TBS
TBS
TBS
TBS
TBS
tBu ), 25.8 (3C, tBu ), 18.2 (Cq ), 18.00 (Cq ),
−
4.3 (Me ),
−
4.5 (Me ),
−
4.7 (Me ), 4.8
−
+
(
MeTBS). HRMS (ESI) m/z: calcd for C H N O SSi [M + H] : 678.2807; found: 678.2788.
3
0
48
5
7
2
Compound
6
: To a stirred solution of compound
5
(920 mg, 1.35 mmol) in DMF (7 mL) was added
◦
sodium azide (264 mg, 4.06 mmol) and the mixture was heated at 70 C for 4 h. The reaction mixture
was cooled to room temperature and was dissolved in EtOAc and washed with brine. The organic
layer was dried over MgSO , concentrated in vacuo and purified on silica gel chromatography (eluent:
4
1
Cyclohexane/EtOAc 8:2) to afford compound
CDCl3):
J = 7.8, 2H, H ), 5.98 (d, J = 4.0 Hz, 1H, H1 ), 4.88 (t, J = 4.1 Hz, 1H, H2 ), 4.31 (t, J = 4.6 Hz, 1H, H3 ),
6
as a white foam (770 mg, 92%). H NMR (500 MHz,
Bz
Bz
δ
8.80 (s, 1H, H2), 8.28 (s, 1H, H8), 8.03 (d, J = 7.3, 2H, H ), 7.60 (t, J = 7.3, 1H, H ), 7.52 (t,
Bz
0
0
0
0
0
TBS
TBS
4
.23 (q, J = 4.6 Hz, 1H, H4 ), 3.80–3.67 (m, 2H, H5 ), 0.94 (s, 9H, tBu ), 0.84 (s, 9H, tBu ), 0.12 (s, 3H,
TBS
TBS
TBS
0.12 (s, 3H, MeTBS). C NMR (126 MHz, CDCl ):
13
Me ), 0.11 (s, 3H, Me ),
164.6 (C=O), 152.8 (C6), 151.5 (C2), 149.8 (C4), 142.4 (C8), 133.8 (Cq ), 132.9 (C ), 129.0 (2C, C ),
−
0.01 (s, 3H, Me ),
−
3
Bz
Bz
Bz
δ
Bz
0
0
0
0
0
TBS
1
2
27.9 (2C, C ), 123.90 (C5), 90.1 (C1 ), 82.8 (C4 ), 74.8 (C2 ), 72.3 (C3 ), 51.6 (C5 ), 25.9 (3C, tBu ),
TBS
TBS
TBS
TBS
TBS
TBS
TBS
5.8 (3C, tBu ), 18.2 (Cq ), 18.0 (Cq ),
−
4.2 (Me ),
−
4.5 (Me ),
−
4.6 (Me ), 4.7 (Me ).
−
+
HRMS (ESI) m/z: calcd for C H N O Si [M + H] : 625.3096; found 625.3088.
29
45
8
4
2
General procedure A for CuAAC reaction: To a solution of alkyne (1 eq) in THF (13 mL/mmol),
were successively added azido compound or (1.2 eq), CuSO (0.3 eq, in water 3 mL/mmol) and
6
7
4
sodium ascorbate (0.6 eq, in water 3 mL/mmol). The heterogeneous mixture was stirred at room
temperature for 16 h. EtOAc was added and the organic layer was washed with brine, dried over
MgSO , and concentrated in vacuo. The crude was purified by flash chromatography to afford the
4
desired compounds.
Compound 8a: Following the general procedure A for CuAAC, starting from alkyne 2a (200 mg,
0.26 mmol) and azido compound 6 (194 mg, 0.31 mmol) and using Cyclohexane/EtOAc 6:4 as eluent
for flash chromatography purification, compound 8a was obtained as a white foam (245 mg, 68%).
1
H NMR (500 MHz, CDCl3):
δ 8.84 (s, 1H, H2a or H2b), 8.52 (s, 1H, H2a or H2b), 8.21 (s, 1H, H8a or
Bz
Triazole
Bz
Bz
H8b), 8.02 (bs, 3H, H8a or H8b and H ), 7.81 (s, 1H, H
7
), 7.60 (bs, 1H, H ), 7.53 (bs, 2H, H ),
Bz
Bz
Bz
.42 (d, J = 7.1 Hz, 2H, H ), 7.22 (t, J = 7.4 Hz, 1H, H ), 7.10 (t, J = 7.6 Hz, 2H, H ), 5.99 (d, J = 5.6 Hz
,
0 0 0 0
H, H1 a), 5.90 (bs, 1H, H1 b), 5.64 (s, 2H, CH N), 5.07 (bs, 1H, H2 b), 4.89–4.81 (m, 1H, H5 b), 4.65–4.58
2
1
(
0 0 0 0 0
m, 2H, H5 b and H2 a), 4.43 (bs, 1H, H3 b), 4.36 (bs, 1H, H4 b), 4.26–4.24 (m, 1H, H3 a), 4.11–4.08 (m,
0
0
0
TBS
1
0
0
−
H, H4 a), 3.97 (dd, J = 11.3, 4.3 Hz, 1H, H5 a), 3.76 (dd, J = 11.3, 2.9 Hz, 1H, H5 a), 0.94 (s, 9H, tBu ),
TBS
TBS
TBS
TBS
TBS
.92 (s, 9H, tBu ), 0.89 (s, 9H, tBu ), 0.76 (s, 9H, tBu ), 0.74 (s, 9H, tBu ), 0.11 (s, 3H, Me ),
.11 (s, 3H, Me ), 0.09 (s, 3H, Me ), 0.09 (s, 3H, Me ), 0.04 (s, 3H, MeTBS),
0.05 (s, 3H, Me ),
TBS
TBS
TBS
−
TBS
0.08 (s, 3H, MeTBS),
−
0.11 (s, 3H, MeTBS),
−
0.37 (s, 3H, MeTBS),
0.42 (s, 3H, Me ). C NMR (126
−
TBS 13
MHz, CDCl ):
δ
172.2 (2C, C=O), 164.5 (Cq), 153.7 (Cq), 152.8 (Cq), 152.0 (C2a or C2b), 151.5 (Cq),
3
Triazole
Bz
Bz
1
1
1
50.1 (C2a or C2b), 144.7 (Cq
), 143.2 (C8a or C8b), 143.0 (C8a or C8b), 136.0 (Cq ) 133.9 (Cq )
Bz
Bz
Bz
Bz
Bz
Triazole
32.9 (C ), 130.9 (C ), 129.1 (2C, C ), 129.0 (2C, C ), 127.9 (4C, C ), 127.30 (Cq), 124.9 (CH
),
0
0
0
0
0
0
0
24.2 (Cq), 90.1 (C1 b), 88.4 (C1 a), 86.0 (C4 a), 84.1 (C4 b), 75.8 (C2 a), 73.4 (C3 b), 73.3 (C2 a), 72.3
0 0 0
C3 a), 62.8 (C5 a), 51.7 (C5 b), 44.0 (CH N), 26.2 (3C, tBu ), 26.0 (3C, tBu ), 25.9 (3C, tBu ), 25.8
2
TBS
TBS
TBS
(
(
6C, tBu ), 18.6 (Cq ), 18.2 (Cq ), 18.1 (Cq ), 17.9 (CqTBS), 17.8 (Cq ),
TBS
TBS
TBS
TBS
TBS
−
4.3 (Me ),
−
TBS
−
4.5 (3C,
TBS
TBS
4.7 (MeTBS),
4.9 (MeTBS),
5.0 (MeTBS),
5.2 (MeTBS),
5.3 (MeTBS). HRMS
Me ),
−
4.6 (Me ),
−
−
−
−
+
(ESI) m/z: calcd for C H N O Si [M + H] : 1376.7077; found 1376.7056.
67
106 13
9
5
Compound 9a: Following the general procedure A for CuAAC, starting from alkyne 2a (578 mg,
0
.77 mmol) and azido compound
7
(277 mg, 0.38 mmol) and using Cyclohexane/EtOAc 7:3 as eluent
1
for flash chromatography purification, compound 9a was obtained as a white foam (384 mg, 60%). H

Molecules 2020, 25, 3241
11 of 19
NMR (500 MHz, CDCl3):
δ
8.75 (s, 1H, H8 or H2), 8.51 (s, 1H, H8 or H2), 8.19 (s, 1H, H8 or H2), 8.04
Triazole
Bz
(
bs, 1H, NH), 7.92 (bs, 1H, H8 or H2), 7.80 (s, 1H, H
), 7.42–7.40 (m, 2H, H ), 7.23–7.20 (m, 1H,
Bz
Bz
0
0
H ), 7.11–7.08 (m, 2H, H ), 5.98 (d, J = 5.6 Hz, 1H, H1 a), 5.86 (d, J = 6.2 Hz, 1H, H1 b), 5.63 (s, 2H,
0
0
0
0
CH N), 5.04–5.02 (m, 1H, H2 b), 4.84 (dd, J = 6.3, 14.2 Hz, 1H, H5 b), 4.61–4.57 (m, 2H, H5 b and H2 a),
2
0 0 0 0
4
.39–4.38 (m, 1H, H3 b), 4.35–4.32 (m, 1H, H4 b), 4.25–4.23 (m, 1H, H3 a), 4.10–4.08 (m, 1H, H4 a), 3.96
0
0
Boc
(
dd, J = 4.4, 11.3 Hz, 1H, H5 a), 3.75 (dd, J = 3.0, 11.4 Hz, 1H, H5 a), 1.56 (s, 9H, tBu ), 0.93 (s, 9H,
TBS
TBS
TBS
TBS
TBS
tBu ), 0.91 (s, 9H, tBu ), 0.87 (s, 9H, tBu ), 0.73 (s, 18H, tBu ), 0.11 (s, 3H, Me ), 0.10 (s, 3H,
Me ), 0.08 (s, 6H, Me ), 0.02 (s, 3H, MeTBS),
MeTBS). 13C NMR (126 MHz, CDCl3):
172.1 (C=O), 153.7 (C2 or C8), 153.1 (Cq), 152.8 (Cq), 152.0
C2 or C8), 150.6 (Cq), 150.4 (Cq), 149.6 (Cq), 144.7 (Cq), 142.9 (C2 or C8), 142.6 (C2 or C8), 135.9 (Cq),
TBS
TBS
−
0.08 (s, 3H, MeTBS),
−
0.11 (s, 6H, MeTBS),
0.43 (s, 6H,
−
δ
(
1
Bz
Bz
Bz
Bz
Triazole
0
), 122.9 (Cq), 89.8 (C1 b), 88.3
30.9 (C ), 129.0 (2C, C ), 127.9 (2C, C ), 127.3 (Cq ), 124.9 (CH
0
0
0
Boc
0
0
0
0
0
(C1 a), 86.0 (C4 a), 84.2 (C4 b), 82.4 (Cq ), 75.8 (C2 a), 73.4 (C3 b), 73.1 (C2 b), 72.2 (C3 a), 62.7 (C5 a),
0
1.7 (C5 b), 44.0 (CH N), 28.3 (3C, tBu ), 26.2 (3C, tBu ), 25.9 (3C, tBu ), 25.8 (3C, tBu ), 25.7
2
Boc
TBS
TBS
TBS
5
(
6C, tBu ), 18.6 (Cq ), 18.2 (Cq ), 18.1 (CqTBS), 17.9 (2C, CqTBS),
TBS
TBS
TBS
−
4.3 (MeTBS), 4.5 (3C, 3 MeTBS),
−
−
4.6 (MeTBS),
−
4.8 (MeTBS),
−
5.0 (Me ),
TBS
−
5.1 (MeTBS),
−
5.2 (MeTBS),
5.3 (Me ). HRMS (ESI) m/z:
TBS
−
−
calcd for C H N O Si [M − H] : 1370.7188; found: 1370.7132.
65
108 13 10
5
Compound 9b: Following the general procedure A for CuAAC, starting from alkyne 2b (50 mg,
0
.067 mmol) and azido compound
7
(49 mg, 0.08 mmol) and using Cyclohexane/EtOAc 7:3 as eluent
1
for flash chromatography purification, compound 9b was obtained as a white foam (68 mg, 74%). H
NMR (500 MHz, CDCl3):
δ 8.74 (s, 1H, H2b), 8.70 (s, 1H, H2a), 8.38 (s, 1H, H8a), 8.00 (s, 1H, NH), 7.90
Triazole
0 0
), 6.07 (d, J = 4.7 Hz, 1H, H1 a), 5.86 (d, J = 6.2 Hz, 1H, H1 b), 5.30 (s,
(
2
s, 1H, H8b), 7.70 (s, 1H, H
0 0
H, CH N), 5.10 (dd, J = 6.2, 4.3 Hz, 1H, H2 b), 4.89 (dd, J = 14.3, 6.3 Hz, 1H, H5 b), 4.65–4.58 (m, 2H,
2
0
0
0
0
0
0
H5 b, H2 a), 4.41 (dd, J = 4.3, 2.4 Hz, 1H, H3 b), 4.36–4.31 (m, 2H, H4 b, H3 a), 4.15–4.12 (m, 1H, H4 a),
0
0
Boc
4
.03 (dd, J = 11.4, 4.1 Hz, 1H, H5 a), 3.79 (dd, J = 11.4, 2.9 Hz, 1H, H5 a), 1.57 (s, 9H, tBu ), 1.38 (s, 9H,
Boc
TBS
TBS
TBS
TBS
tBu ), 0.95 (s, 9H, tBu ), 0.93 (s, 9H, tBu ), 0.88 (s, 9H, tBu ), 0.80 (s, 9H, tBu ), 0.74 (s, 9H,
tBu ), 0.13 (s, 3H, Me ), 0.12 (s, 3H, Me ), 0.10 (s, 3H, Me ), 0.09 (s, 3H, Me ), 0.04 (s, 3H,
MeTBS),
TBS
TBS
TBS
TBS
TBS
0.04 (s, 3H, MeTBS),
−
0.06 (s, 3H, Me ),
TBS
−
0.10 (s, 3H, MeTBS),
−
0.21 (s, 3H, MeTBS),
0.42
−
−
TBS 13
(
(
s, 3H, Me ). C NMR (126 MHz, CDCl ):
C2a), 150.6 (Cq), 150.4 (C=O), 149.6 (C=O), 145.8 (Cq
δ
153.4 (C2b), 153.1 (Cq), 152.9 (Cq), 152.5 (Cq), 151.9
3
Triazole
), 142.6 (C8b), 142.2 (C8a), 127.7 (Cq), 124.1
Triazole
0 0 0 0 0
Boc
(
CH
), 122.9 (Cq), 90.1 (C1 b), 88.8 (C1 a), 85.3 (C4 a), 84.4 (C4 b), 82.5 (2C, Cq ), 75.9 (C2 a), 73.5
0
0
0
0
0
Boc
Boc
(
C3 b), 73.1 (C2 b), 71.8 (C3 a), 62.5 (C5 a), 51.8 (C5 b), 43.4 (CH N), 28.3 (3C, tBu ), 28.0 (3C, tBu ),
2
6.2 (3C, tBu ), 26.0 (3C, tBu ), 25.9 (3C, tBu ), 25.8 (3C, tBu ), 25.7 (3C, tBu ), 18.7 (CqTBS),
TBS
TBS
TBS
TBS
TBS
2
1
8.2 (Cq ), 18.1 (Cq ), 18.0 (Cq ), 17.9 (CqTBS),
TBS
TBS
TBS
−
4.2 (Me ),
TBS
−
4.4 (Me ),
TBS
−
4.5 (Me ), 4.5
TBS
−
TBS
TBS
4.7 (MeTBS),
TBS
5.1 (MeTBS), 5.2 (MeTBS),
TBS
(
(
Me ),
−
4.6 (Me ),
−
−
4.8 (Me ),
−
−
−
5.3 (Me ). HRMS
+
ESI) m/z: calcd for C H N O Si [M + H] : 1368.7601; found 1368.7601.
6
3
114 13 11
5
Compound 10a: Following the general procedure A for CuAAC, starting from alkyne 3a (200 mg,
.26 mmol) and azido compound (194 mg, 0.31 mmol) and using Cyclohexane/EtOAc 5:5 as eluent
0
6
for flash chromatography purification, compound 10a was obtained as a white foam (258 mg, 72%).
1
Bz
H NMR (500 MHz, CDCl ):
δ 8.70 (s, 1H, H2b), 8.26 (s, 1H, H2a), 8.05 (d, J = 7.4 Hz, 2H, H ), 7.95 (d,
3
Bz
Triazole
Bz
J = 7.2 Hz, 2H, H ), 7.79 (s, 2H, H8a and H8b), 7.74 (s, 1H, H
), 7.62 (t, J = 7.3 Hz, 1H, H ), 7.54
Bz
Bz
Bz
0
(
t, J = 7.5 Hz, 2H, H ), 7.40 (t, J = 7.3 Hz, 1H, H ), 7.31 (t, J = 7.6 Hz, 2H, H ), 5.85–5.80 (m, 2H, H1 a
0
0
0
and H1 b), 5.43–5.31 (m, 2H, CH N), 5.13–5.07 (m, 1H, H2 b), 4.85 (dd, J = 14.2, 4.8 Hz, 1H, H5 b), 4.73
2
0 0 0
(
dd, J = 14.2, 7.2 Hz, 1H, H5 b), 4.50 (t, J = 3.8 Hz, 1H, H3 b), 4.44 (t, J = 4.4 Hz, 1H, H2 a), 4.40–4.36 (m,
0 0 0
H, H4 b), 4.23 (t, J = 4.3 Hz, 1H, H3 a), 4.05 (q, J = 3.9 Hz, 1H, H4 a), 3.86 (dd, J = 11.4, 4.1 Hz, 1H,
1
0
0
TBS
TBS
TBS
H5 a), 3.69 (dd, J = 11.4, 3.4 Hz, 1H, H5 a), 0.91 (s, 9H, tBu ), 0.90 (s, 9H, tBu ), 0.86 (s, 9H, tBu ),
0
0
TBS
TBS
TBS
TBS
TBS
.80 (s, 9H, tBu ), 0.79 (s, 9H, tBu ), 0.10 (s, 3H, Me ), 0.07 (s, 3H, Me ), 0.06 (s, 3H, Me ),
.05 (s, 3H, Me ), 0.04 (s, 3H, Me ), 0.03 (s, 3H, MeTBS),
TBS
TBS
−
0.04 (s, 3H, MeTBS),
0.06 (s, 3H, Me ),
−
TBS
TBS
0.34 (s, 3H, MeTBS). C NMR (126 MHz, CDCl ):
13
−
0.19 (s, 3H, Me ),
−
δ
177.1 (C=O), 164.7 (C=O),
3
Triazole
1
1
52.7 (C2b), 151.2 (Cq), 150.1 (Cq), 146.7 (C2a), 146.6 (Cq), 145.2 (Cq), 143.1 (C8b), 141.9 (Cq
),
Bz
Bz
Bz
Bz
Bz
Bz
38.8 (C8a), 135.8 (Cq ), 133.8 (Cq ), 133.0 (C ), 131.9 (C ), 129.8 (2C, C ), 129.0 (2C, C ), 128.1

Molecules 2020, 25, 3241
12 of 19
Bz
Triazole
0 0 0 0
), 124.2 (Cq), 122.5 (Cq), 90.5 (C1 b), 88.5 (C1 a), 85.1 (C4 a), 83.5 (C4 b), 76.0
(
(
(
(
4C, C ), 125.6 (CH
0 0 0 0 0 0
C2 a), 73.5 (C3 b), 73.4 (C2 b), 71.7 (C3 a), 62.6 (C5 a), 51.8 (C5 b), 43.7 (CH N), 26.2 (3C, tBu ), 26.0
2
TBS
TBS
TBS
TBS
TBS
TBS
TBS
3C, tBu ), 25.9 (3C, tBu ), 25.8 (3C, tBu ), 25.80 (3C, tBu ), 18.6 (Cq ), 18.2 (Cq ), 18.1
TBS
TBS
TBS
4.3 (MeTBS),
−
4.4 (MeTBS),
4.5 (MeTBS), 4.6 (2C MeTBS),
Cq ), 18.0 (2C, Cq ),
−
4.2 (Me ),
5.2 (MeTBS),
5.3 (Me ). HRMS (ESI) m/z: calcd for C H N O Si
67 106 13 9 5
−
−
−
−
TBS
TBS
TBS
−
4.7 (Me ),
M + H] : 1376.7077; found 1376.7060.
−
4.9 (Me ),
−
+
[
Compound 11a: Following the general procedure A for CuAAC, starting from alkyne 3a (45 mg,
.059 mmol) and azido compound (19 mg, 0.03 mmol) and using Cyclohexane/EtOAc 7:3 as eluent
0
7
for flash chromatography purification, compound 11a was obtained as a white foam (37 mg, 89%).
1
Bz
H NMR (500 MHz, CDCl3):
δ 8.67 (s, 1H, H2b), 8.30 (s, 1H, H2a), 7.97 (m, 2H, H ), 7.85 (s, 1H, H8a),
Triazole
Bz
Bz
7
.78 (s, 1H, H8b), 7.77 (s, 1H, H
), 7.46–7.43 (m, 1H, H ), 7.36–7.32 (m, 2H, H ), 5.85 (d, J = 4.5
0
0
Hz, 1H, H1 a), 5.78 (d, J = 5.4 Hz, 1H, H1 b), 5.44 (d, J = 14.6 Hz, 1H, CH N), 5.36 (d, J = 14.6 Hz, 1H,
CH N), 5.12 (m, 1H, H2 b), 4.87 (dd, J = 5.2, 14.3 Hz, 1H, H5 b), 4.70 (dd, J = 7.3, 14.2 Hz, 1H, H5 b),
4
4
2
0
0
0
2
0 0 0 0
.48–4.47 (m, 1H, H3 b), 4.46–4.44 (m, 1H, H2 a), 4.37–4.34 (m, 1H, H4 b), 4.24–4.23 (m, 1H, H3 a),
0
0
.07–4.05 (m, 1H, H4 a), 3.87 (dd, J = 4.0, 11.3 Hz, 1H, H5 a), 3.71 (dd, J = 3.3, 11.3 Hz, 1H, H5a’), 1.57
Boc
TBS
TBS
TBS
TBS
(s, 9H, tBu ), 0.91 (s, 9H, tBu ), 0.89 (s, 9H, tBu ), 0.87 (s, 9H, tBu ), 0.80 (s, 9H, tBu ), 0.77 (s,
TBS
TBS
TBS
TBS
TBS
9
3
H, tBu ), 0.08 (s, 3H, Me ), 0.07 (s, 6H, Me ), 0.05 (s, 3H, Me ), 0.04 (s, 3H, Me ), 0.02 (s,
H, MeTBS),
−
0.04 (s, 3H, MeTBS),
−
0.08 (s, 3H, MeTBS),
−
0.18 (s, 3H, Me ),
TBS
−
0.39 (s, 3H, MeTBS).
13
C
NMR (126 MHz, CDCl ):
Cq), 145.4 (Cq), 142.6 (C8b), 141.9 (Cq
δ
176.8 (C=O), 152.9 (C2b), 150.3 (2C, Cq), 149.6 (C=O), 146.7 (2C, C2a and
3
Triazole
Bz
Bz
), 139.0 (C8a), 135.6 (Cq), 131.9 (C ), 129.7 (2C, C ), 128.1
Bz
Triazole
0 0 0 0
), 122.8 (Cq), 122.6 (Cq), 90.4 (C1 b), 88.5 (C1 a), 85.1 (C4 a), 83.6 (C4 b), 82.5
(
2C, C ), 125.6 (CH
Boc
0
0
0
0
0
0
(
Cq ), 76.0 (C2 a), 73.5 (C3 b), 73.3 (C2 b), 71.7 (C3 a), 62.6 (C5 a), 51.9 (C5 b), 43.8 (CH N), 28.2 (3C,
2
Boc
TBS
TBS
TBS
TBS
TBS
tBu ), 26.1 (3C, tBu ), 26.0 (3C, tBu ), 25.9 (3C, tBu ), 25.8 (3C, tBu ), 25.7 (3C, tBu ), 18.6
CqTBS), 18.2 (Cq ), 18.1 (Cq ), 18.0 (CqTBS), 17.9 (CqTBS),
TBS
TBS
−
4.2 (Me ),
TBS
−
4.3 (Me ),
5.3 (MeTBS). HRMS (ESI)
TBS
−
4.4 (MeTBS),
(
4.5 (MeTBS),
−
4.6 (MeTBS),
−
4.7 (2C, MeTBS),
−
5.0 (Me ),
TBS
−
5.2 (Me ),
TBS
−
−
−
m/z: calcd for C H N O Si [M − H] : 1370.7188; found: 1370.7138.
65
108 13 10
5
Compound 11b: Following the general procedure A for CuAAC, starting from alkyne 3b (50 mg,
.067 mmol) and azido compound (49 mg, 0.08 mmol) and using Cyclohexane/EtOAc 6:4 as eluent
0
7
for flash chromatography purification, compound 11b was obtained as a white foam (54 mg, 60%).
1
H NMR (500 MHz, CDCl3):
δ 8.72 (s, 1H, H2a or H2b), 8.10 (s, 1H, H2a or H2 b), 8.10 (s, 1H, H8a
Triazole
0
or H8b), 8.01 (s, 1H, NH), 7.93 (s, 1H, H8a or H8b), 7.90 (s, 1H, H
H1 b), 5.30–5.21 (m, 2H, CH N), 5.17 (dd, J = 5.8, 4.3 Hz, 1H, H2 b), 4.92 (dd, J = 14.3, 6.5 Hz, 1H,
H5 b), 4.62 (dd, J = 14.3, 6.5 Hz, 1H, H5 b), 4.48–4.45 (m, 1H, H3 b), 4.39–4.35 (m, 1H, H4 b), 4.32–4.27
), 5.85–5.83 (m, 2H, H1 a and
0
0
0
2
0
0
0
0 0 0 0
(m, 2H, H2 a, H3 a), 4.09–4.06 (m, 1H, H4 a), 3.98 (dd, J = 11.6, 3.4 Hz, 1H, H5 a), 3.75 (dd, J = 11.6,
0
Boc
TBS
TBS
TBS
2
0
0
.5 Hz, 1H, H5 a), 1.56 (s, 18H, tBu ), 0.92 (s, 9H, tBu ), 0.90 (s, 9H, tBu ), 0.86 (s, 9H, tBu ),
TBS
TBS
TBS
TBS
TBS
.85 (s, 9H, tBu ), 0.76 (s, 9H, tBu ), 0.10 (s, 3H, Me ), 0.10 (s, 3H, Me ), 0.07 (s, 3H, Me ),
.05 (s, 3H, Me ), 0.03 (s, 3H, Me ), 0.01 (s, 3H, MeTBS),
TBS
TBS
−
0.03 (s, 3H, MeTBS),
−
0.08 (s, 6H, Me ),
TBS
−
0.40 (s, 3H, MeTBS). 13C NMR (126 MHz, CDCl3):
δ 153.0 (C2a or C2b), 150.5 (Cq), 150.4 (Cq), 149.6
Triazole
(
2C, C=O), 146.9 (Cq
), 146.5 (C2a or C2b), 144.2 (Cq), 142.7 (Cq), 142.3 (C8a or C8b), 138.1 (C8a
Triazole
0 0 0 0 0
), 123.0 (Cq), 122.3 (Cq), 90.4 (C1 a or C1 b), 88.9 (C1 a or C1 b), 84.1 (C4 a),
or C8b), 125.7 (CH
0
Boc
0
0
0
0
0
0
8
4
2
3.9 (C4 b), 82.4 (2C, Cq ), 76.7 (C2 a), 73.5 (C3 b), 73.2 (C2 b), 70.5 (C3 a), 61.7 (C5 a), 51.9 (C5 b),
Boc
Boc
TBS
TBS
TBS
3.1 (CH N), 28.3 (3C, tBu ), 28.3 (3C, tBu ), 26.3 (3C, tBu ), 26.0 (3C, tBu ), 25.9 (6C, tBu ),
2
5.8 (3C, tBu ), 18.7 (Cq ), 18.2 (CqTBS), 18.1 (CqTBS), 18.0 (CqTBS), 17.9 (Cq ),
TBS
TBS
TBS
−
4.1 (Me ),
−
TBS
−
4.4
5.3
TBS
4.5 (MeTBS),
4.6 (MeTBS),
4.7 (MeTBS),
4.9 (MeTBS),
TBS
TBS
(
2C, 2 Me ),
−
−
−
−
−
5.1 (Me ), 5.2 (Me ),
−
TBS
+
(Me ). HRMS (ESI) m/z: calcd for C H N O Si [M + H] : 1368.7601; found 1368.7611.
63
114 13 11
5
Synthesis of compound 12 following pathway 1: Protected compound 9a (768 mg, 0.56 mmol) was
◦
dissolved in DCM/MeOH 4:1 (10 mL) and MeNH 33% in EtOH (3.6 mL, 28.0 mmol) was added at 0 C
2
to the solution. The reaction was stirred at room temperature for 16 h, then diluted in DCM and washed
with brine. The combined organic layers were dried over MgSO and concentrated. The residue was
4
purified by silica gel chromatography (eluent: Cyclohexane/EtOAc 5:5) to provide the debenzoylated

Molecules 2020, 25, 3241
13 of 19
compound as a white foam. The residue was then dissolved in DCM (1.4 mL) and ZnBr (686 mg,
2
2.80 mmol) was added. The reaction mixture was vigorously stirred at room temperature for 24 h.
Then, water (5.8 mL) was added and the reaction mixture was stirred for 2 additional hours. A work
up was performed with DCM and brine. The combined organic layers were dried over MgSO and
4
concentrated. The residue was purified by silica gel chromatography (eluent: Cyclohexane/EtOAc
3:7 then DCM/MeOH 9:1) to afford a yellow powder. The resulting compound was engaged in the
last deprotection step and was dissolved in MeOH (10 mL) and CsF (8.5 g, 112 mmol) was added.
◦
The reaction mixture was stirred at 60 C for 24 h, concentrated and diluted in water. The residue was
purified by HPLC to afford compound 12 as a white foam (17 mg, 5% over 3 steps).
Synthesis of compound 12 following pathway 2: Protected compound 9b (60 mg, 0.046 mmol)
was dissolved in DCM (1 mL) and ZnBr (58 mg, 0.21 mmol) was added. The reaction mixture was
2
vigorously stirred at room temperature for 24 h. Then, water (2 mL) was added and the reaction mixture
was stirred for 2 additional hours. A work up was performed with DCM and brine. The combined
organic layers were dried over MgSO and concentrated. The residue was purified by silica gel
4
chromatography (eluent Cyclohexane/EtOAc 2:8) to afford a yellow pale powder. The resulting
compound was engaged in the last deprotection step and was dissolved in MeOH (10 mL) before
◦
adding CsF (480 mg, 3.2 mmol). The reaction mixture was stirred at 60 C for 24 h, concentrated and
purified by HPLC to afford compound 12 as a white foam (1.2 mg, 4% over 2 steps).
1
Compound 12: H NMR (500 MHz, (CD ) SO):
δ
8.35 (s, 1H, H2 or H8), 8.26 (bs, 1 H, NH), 8.23
3
2
Triazole
(
s, 1H, H2 or H8), 8.19 (s, 1H, H2 or H8), 8.14 (s, 1H, H2 or H8), 7.81 (s, 1H, H
NH ), 5.90–5.89 (m, 2H, H1 a and H1 b), 5.55 (d, J = 5.9 Hz, 1H, OH2 b), 5.43–5.41 (m, 2H, OH2 a and
), 7.28 (bs, 2H,
0
0
0
0
2
0
0
0
0
OH3 b), 5.36–5.34 (m, 1H, OH5 a), 5.16 (d, J = 4.6 Hz, 1H, OH3 a), 4.74–4.67 (m, 4H, H5 b and CH N),
2
0 0 0 0
4
.67–4.63 (m, 1H, H2 b), 4.63–4.60 (m, 1H, H2 a), 4.25–4.20 (m, 2H, H4 b and H3 b), 4.16–4.14 (m, 1H,
0
0
0
0
13
H3 a), 3.97–3.95 (m, 1H, H4 a), 3.69–3.65 (m, 1H, H5 a), 3.58–3.53 (m, 1H, H5 a). C NMR (126 MHz,
(
1
(
(
CD ) SO): δ 156.1 (Cq), 154.2 (Cq), 152.6 (C2 or C8), 152.2 (C2 or C8), 149.3 (Cq), 148.5 (Cq), 145.3 (Cq),
3 2
Triazole
0 0
), 119.8 (Cq), 119.2 (Cq), 87.9 (C1 a or C1 b), 87.7
39.9 (C2 or C8), 139.8 (C2 or C8), 123.6 (CH
0
0
0
0
0
0
0
0
0
C1 a or C1 b), 85.8 (C4 a), 82.5 (C4 b), 73.4 (C2 a), 72.5 (C2 b), 71.0 (C3 b), 70.6 (C3 a), 61.6 (C5 a), 51.3
0
−
C5 b), 35.3 (CH N). HRMS (ESI) m/z: calcd for C H N O [M
−
H] : 596.2078; found: 596.2065.
2
23 26 13
7
HPLC purity: 96.3%; t = 17.8 min (MeCN/H O 0:100 to 100:0 over 30 min).
R
2
Compound 13: Protected compound 11a (400 mg, 0.3 mmol) was dissolved in DCM/MeOH 4:1
◦
(
5 mL) and MeNH 33% in EtOH (1.8 mL, 15.0 mmol) was added at 0 C to the solution. The reaction
2
was stirred at room temperature for 16 h, then diluted in DCM and washed with brine. The combined
organic layers were dried over MgSO and concentrated. The residue was purified by silica gel
4
chromatography (eluent: EtOAc/MeOH 9:1) to provide the debenzoylated compound as a white
foam. The residue was then dissolved in DCM (1 mL) and ZnBr (117 mg, 0.47 mmol) was added.
2
The reaction mixture was vigorously stirred at room temperature for 24 h. Then, water (2 mL) was
added and the reaction mixture was stirred for 2 additional hours. A work up was performed with
DCM and brine. The combined organic layers were dried over MgSO and concentrated. The resulting
4
compound was engaged in the last deprotection step and was dissolved in MeOH (10 mL) and CsF
◦
(
2.9 g, 19 mmol) was added. The reaction mixture was stirred at 60 C for 24 h, concentrated and
diluted in water. The residue was purified by HPLC to afford compound 13 as a white foam (15 mg, 8%
1
over 3 steps). H NMR (500 MHz, CD OD):
δ 8.35 (2s, 1H, H2a), 8.23 (s, 1H, H8a or H8b), 8.13 (2s, 1H,
3
Triazole
Triazole
0
H8a or H8b), 8.03 (2s, 1H, H2b), 7.84 (s, 0.5H, H
.95–5.93 (m, 1H, H1 b), 5.32 (2s, 2H, CH N), H5 b masked in the residual pick of water, 4.79–4.75 (m,
), 7.69 (s, 0.5H, H
), 6.01–5.98 (m, 1H, H1 a),
0
0
5
0
2
0
0
0
0
.5H, H2 b), 4.71–4.68 (m, 0.5H, H2 b), 4.61 (t, J = 5 Hz, 1H, H2 a), 4.47–4.41 (m, 0.5H, H3 b), 4.37–4.31
0 0 0 0 0
(
m, 1.5H, H3 b and H3 a), 4.14–4.13 (m, 2H, H4 a and H4 b), 3.88–3.85 (m, 1H, H5 a), 3.78–3.74 (m, 1H,
0
13
H5 a), 3.56 (2s, 3H, CH ). C NMR (126 MHz, CD OD):
δ 178.3 (Cq), 163.0 (Cq), 157.3 (Cq), 153.7 (C8a
3
3
or C8b), 150.4 (Cq), 150.0 (Cq), 149.8 (Cq), 149.4 (Cq), 147.8 (Cq), 147.5 (Cq), 142.0 (C2a or C2b), 126.6
Triazole
Triazole
0 0 0 0
), 126.2 (Cq), 120.8 (Cq), 90.9 (C1 b), 90.4 (C1 a), 87.6 (2C, C4 a and C4 b),
(
8
CH
), 125.7 (CH
0
0
0
0
0
0
3.9 (C3 a), 76.1 (C2 a), 74.2 (C2 b), 72.2 (C3 b), 62.9 (C5 a), 52.5 (C5 b), 45.1 (CH N), 43.9 (CH N), 37.9
2
2

Molecules 2020, 25, 3241
14 of 19
+
(
CH ), 35.0 (CH ). LRMS (ESI) m/z: calcd for C H N O [M + H] : 612.23; found: 612.33. HPLC
3 3 24 30 13 7
purity: 95.3%; rt = 14.6 min (MeCN/H O 0:100 to 100:0 over 30 min).
2
Compound 14: Compound 11a (107 mg, 0.08 mmol) was dissolved in DCM (1 mL) and ZnBr2
(95 mg, 0.40 mmol) was added. The reaction mixture was vigorously stirred at room temperature for
24 h. Water (2 mL) was added and the reaction mixture was stirred for 2 additional hours. A work
up was performed with DCM and brine. The combined organic layers were dried over MgSO and
4
concentrated. The residue was purified by silica gel chromatography (eluent: Cyclohexane/EtOAc 2:8)
to afford a white foam (52 mg). The purified intermediate was then dissolved in MeOH (5 mL) and
◦
CsF (1.2 g, 8 mmol) was added. The reaction mixture was stirred at 60 C for 24 h, concentrated and
diluted in water. The residue was purified by HPLC to afford compound 14 as a white foam (7 mg,
1
1
(
7
3% over 2 steps). H NMR (500 MHz, CD OD):
δ
8.53 (s, 1H, H2 or H8), 8.12 (s, 1H, H2 or H8), 8.06
3
Triazole
Bz
Bz
s, 1H, H2 or H8), 7.90 (s, 2H, H2 or H8 and H
), 7.83–7.81 (m, 2H, H ), 7.45–7.42 (m, 1H, H ),
Bz
0
0
.33–7.30 (m, 2H, H ), 5.96 (d, J = 5.0 Hz, 1H, H1 a), 5.90 (d, J = 5.0 Hz, 1H, H1 b), 5.43 (s, 2H, CH N),
2
0
0
0
H5 b masked in the residual pick of water, 4.64–4.60 (m, 2H, H2 b and H2 a), 4.47 (t, J = 5.0 Hz, 1H,
H3 b), 4.37–4.34 (m, 1H, H4 b), 4.32–4.30 (m, 1H, H3 a), 4.11 (q, J = 3.3 Hz, 1H, H4 a), 3.83 (dd, J = 5.0,
5.0 Hz, 1H, H5 a), 3.74 (dd, J = 5.0, 15.0 Hz, 1H, H5 a). C NMR (126 MHz, CD OD):
0
0
0
0
0
0
13
1
δ
178.8 (C=O),
3
1
57.2 (C2 or C8), 153.7 (C2 or C8), 149.2 (Cq), 148.5 (Cq), 146.9 (Cq), 143.3 (Cq), 141.5 (C2 or C8), 141.2
Bz
Bz
Bz
Triazole
(
Cq), 136.7 (C2 or C8), 133.2 (Cq), 130.6 (C ), 129.1 (2C, C ), 129.0 (2C, C ), 127.0 (CH
), 123.2
0
0
0
0
0
0
0
(Cq), 120.6 (Cq), 91.0 (C1 a), 90.4 (C1 b), 87.4 (C4 a), 83.4 (C4 b), 76.0 (C2 a), 74.5 (C2 b), 72.2 (C3 b),
0
0
0
+
7
7
2.0 (C3 a), 62.8 (C5 a), 52.4 (C5 b), 45.0 (CH N). HRMS (ESI) m/z: calcd for C H N O [M + H] :
2
30 32 13
8
02.2497; found: 702.2470. HPLC purity: 97.1%; t = 19.0 min (MeCN/H O 0:100 to 100:0 over 30 min).
R
2
Compound 15: Protected compound 11b (50 mg, 0.036 mmol) was dissolved in DCM (1 mL) and
ZnBr (44 mg, 0.18 mmol) was added. The reaction mixture was vigorously stirred at room temperature
2
for 24 h then water (2 mL) was added and the reaction mixture was stirred for 2 additional hours.
A work up was performed with DCM and brine. The combined organic layers were dried over MgSO4
and concentrated. The residue was purified by silica gel chromatography (eluent Cyclohexane/EtOAc
1
:9) to afford a yellow pale powder. The resulting compound was engaged in the last deprotection step
and was dissolved in MeOH (10 mL) and CsF (636 mg, 4.2 mmol) was added. The reaction mixture
◦
was stirred at 60 C for 24 h, concentrated and purified by HPLC to afford compound 15 as a white
1
foam (4.3 mg, 20% over 2 steps). H NMR (500 MHz, (CD ) SO):
.15 (s, 1H, H2b), 8.14 (s, 1H, H8a), 7.96 (s, 1H, H
H1 b), 5.75 (d, J = 5.9 Hz, 1H, H1 a), 5.28–5.17 (m, 2H, CH N), 4.74–4.70 (m, 2H, H5 b), 4.64–4.61 (m,
H, H2 b), 4.48–4.44 (m, 1H, H2 a), 4.27–4.20 (m, 2H, H3 b and H4 b), 4.10 (d, J = 4.9 Hz, 1H, H3 a),
.92 (q, J = 3.8 Hz, 1H, H4 a), 3.64 (dd, J = 12.0, 4.0 Hz, 1H, H5 a), 3.53 (dd, J = 12.0, 3.9 Hz, 1H, H5 a).
C NMR (126 MHz, (CD ) SO):
), 141.2 (Cq), 139.8 (C8b), 137.9 (C8a), 124.5 (CH
C1 b), 87.6 (C1 a or C1 b), 85.6 (C4 a), 82.3 (C4 b), 73.9 (C2 a), 72.5 (C2 b), 70.9 (C3 b), 70.4 (C3 a), 61.4
δ 8.27 (s, 1H, H8b), 8.22 (s, 1H, H2a),
3
2
Triazole
8
), 7.28 (bs, 2H, NH ), 5.89 (d, J = 5.5 Hz, 1H,
2
0
0
0
2
0
0
0
0
0
0
1
0
0
3
13
δ 156.1 (Cq), 153.2 (Cq), 152.6 (C2b), 149.3 (Cq), 148.2 (C2a), 142.5
3
2
Triazole
Triazole
0
), 122.8 (Cq), 119.2 (Cq), 87.6 (C1 a or
(
Cq
0
0
0
0
0
0
0
0
0
0
0
+
(
C5 a), 51.4 (C5 a), 41.2 (CH N). HRMS (ESI) m/z: calcd for C H N O [M + H] : 598.2229; found:
2
23 28 13
7
5
98.2229. HPLC purity: 96.2%; t = 17.5 min (MeCN/H O 0:100 to 100:0 over 30 min).
R
2
Compound 16: Propargyl amine (1.4
µ
L, 0.031 mmol), BOP (13 mg, 0.031 mmol) and DIPEA
◦
(6.8
µL, 0.039 mmol) were added at 0 C to a solution of inosine (7 mg, 0.026 mmol) in DMF (1 mL).
The reaction mixture was stirred at room temperature for 24 h and then concentrated under reduced
pressure. The crude was diluted in water and purified by HPLC to afford compound 16 as a white
1
foam (5.7 mg, 71%). H NMR (500 MHz, D O):
δ
8.31 (s, 1H, H8), 8.27 (s, 1H, H2), 6.07 (d, J = 5 Hz,
2
0
0
0
1
H, H1 ), H2 masked in the residual pick of water, 4.47–4.45 (m, 1H, H3 ), 4.35 (s, 2H, CH N), 4.33 (q,
2
0
0
0
J = 3.3 Hz, 1H, H4 ), 3.97 (dd, J = 5, 11 Hz, 1H, H5 ), 3.88 (dd, J = 5, 10 Hz, 1H, H5 ), 2.69 (t, J = 2.5 Hz
,
1
(
(
H, C
≡
CH). 13C NMR (126 MHz, D O):
δ
= 153.9 (C5), 152.3 (C2), 147.8 (C4), 140.4 (C8), 119.6 (C6), 88.4
2
0
0
0
0
0
C1 ), 85.8 (C4 ), 80.3 (C
≡
C-CH ), 73.7 (C2 ), 71.9 (C
≡
CH), 70.6 (C3 ), 61.5 (C5 ), 30.2 (CH N). HRMS
2
2
−
ESI) m/z: calcd for C H N O [M − H] : 304.1045; found: 304.1048.
13
14
5
4

Molecules 2020, 25, 3241
15 of 19
Compound 17: Pyridine (610
µL, 7.48 mmol) and thionyl chloride (1.4 mL, 18.7 mmol) were added
◦
at 0 C, over 5 min to a solution of adenosine (1 g, 3.74 mmol) in MeCN (10 mL). The reaction mixture
◦
was stirred at 0 C for 3 h before being warmed to room temperature and stirred for 16 h. The resulting
precipitate was filtered and dissolved in water/MeOH (5:1) and aqueous ammonia (25%, 2 mL) was
added. The reaction mixture was stirred at room temperature for 30 min and the solvent was removed
0
0
under reduced pressure to provide 5 -chloroadenosine [66]. The resulting 5 -chloroadenosine was then
solubilized in DMF (5 mL) and sodium azide (1.2 g, 18.7 mmol) was added. The reaction mixture was
◦
heated at 80 C for 5 h, and cooled to room temperature. The excess of sodium azide was removed by
filtration and the filtrate purified by flash chromatography (DCM/MeOH 9:1) to give 17 as a white
1
foam (393 mg, 36% over 2 steps). H NMR (500 MHz, CD OD):
δ
8.29 (s, 1H, H8), 8.21 (s, 1H, H2),
3
0
0
0
0
6
.03 (s, 1H, H1 ), 4.80–4.78 (m, 1H, H2 ), 4.40–4.36 (m, 1H, H4 ), 4.27 (q, J = 5 Hz, 0.5H, H3 ), 4.18 (q,
0
0
0
J = 5 Hz, 0.5H, H3 ), 3.94 (dd, J = 10, 5 Hz, 0.5H, H5 ), 3.84 (dd, J = 10, 5 Hz, 0.5H, H5 ), 3.69–3.62 (m,
0
H, H5 ). HRMS (ESI) m/z: calcd for C H N O [M + H] : 293.1110; found: 293.1098. Analytical
10 13 8 3
+
1
data were in accordance with the literature [67].
General procedure B for CuAAC reaction: To a solution of alkyne 16 or 18 (1 eq) in DMF (1 mL),
were successively added azido compound 17 (1.5 eq), CuSO (0.3 eq, in water 500 L) and sodium
L). The mixture was stirred at room temperature for 16 h and then
µ
4
ascorbate (0.6 eq, in water 500
µ
concentrated in vacuo. The crude product was purified by HPLC to afford the desired compounds.
Synthesis of compound 12 following general procedure B for CuAAC: Following the general
procedure B, starting from alkyne 16 (5.7 mg, 0.019 mmol) and azido compound 17 (8.2 mg, 0.027 mmol),
compound 12 was obtained as a white foam (4 mg, 36%).
Compound 18: Propargyl bromide (80% in toluene) (143
adenosine (100 mg, 0.37 mmol) in DMF (1 mL) and the reaction mixture was stirred at 50 C for 24 h.
µ
L, 1.8 mmol) was added to a solution of
◦
After removal of the solvent under reduced pressure, the crude was diluted in water and purified by
1
HPLC to afford compound 18 as a white foam (64 mg, 56%). H NMR (500 MHz, D O):
δ
8.73 (s, 1H,
2
0
0
H2), 8.61 (s, 1H, H8), 6.20 (d, J = 5 Hz, 1H, H1 ), 5.27 (s, 2H, CH N), 4.84–4.82 (t, J = 5 Hz, 1H, H2 ),
.50 (t, J = 5 Hz, 1H, H3 ), 4.33–4.31 (m, 1H, H4 ), 3.97 (dd, J = 5, 10 Hz, 1H, H5 ), 3.90 (dd, J = 5, 10 Hz,
H, H5 ), 3.19 (bs, 1H, C CH). C NMR (126 MHz, D O): δ 150,2 (C6), 146.9 (C2), 146.8 (C4), 143.4
2
0
0
0
4
1
0
13
≡
2
0
0
0
0
0
(
C8), 119.6 (C5), 88.6 (C1 ), 85.5 (C4 ), 78.7 (C≡CH), 74.2 (C2 ), 73.1 (C≡CH), 70.1 (C3 ), 61.1 (C5 ), 40.8
+
(
CH N). HRMS (ESI) m/z: calcd for C H N O [M] : 306.1196; found: 306.1193.
2
13 16
5
4
Compound 19: Following the general procedure B for CuAAC, starting from alkyne 18 (6.3 mg,
0
.020 mmol) and azido compound 17 (9.1 mg, 0.03 mmol), compound 19 was obtained as a white foam
1
(
6.5 mg, 53%). H NMR (500 MHz, CD OD):
δ
8.62 (s, 2H, H2a and H8b), 8.14 (s, 1H, H2b), 8.05 (s, 1H,
3
Triazole
0 0
), 6.09 (d, J = 5 Hz, 1H, H1 b), 5.95 (d, J = 5 Hz, 1H, H1 a), 5.52 (s, 2H, CH N),
2
H8a), 7.96 (s, 1H, H
4
0
0
0
.92–4.96 (m, 1H, H5 b), H5 b masked in the residual pick of water, 4.76–4.74 (m, 1H, H2 a), 4.62 (t,
0
0
0
0
J = 5 Hz, 1H, H2 b), 4.48 (t, J = 5 Hz, 1H, H3 a), 4.38–4.33 (m, 2H, H3 b and H4 a), 4.15 (q, J = 3.1 Hz,
0
0
0
13
1
1
1
H, H4 b), 3.89–3.85 (m, 1H, H5 a), 3.76–3.70 (m, 1H, H5 a). C NMR (126 MHz, CD OD):
54.0 (C2b), 152.2 (Cq), 150.3 (Cq), 148.4 (C2a), 148.0 (Cq), 144.1 (C8b), 141.7 (C8a), 140.8 (Cq
26.7 (CH
δ
157.2 (Cq),
3
Triazole
),
Triazole
0 0 0 0 0
), 121.3 (Cq), 120.7 (Cq), 91.2 (C1 a), 90.4 (C1 b), 87.6 (C4 b), 83.6 (C4 a), 76.5 (C2 b),
0
0
0
0
0
7
4.5 (C2 a), 72.4 (C3 a), 71.8 (C3 b), 62.5 (C5 a), 52.7 (C5 b), 45.9 (CH N). HRMS (ESI) m/z: calcd for
2
+
C H N O [M + H] : 598.2234; found: 598.2222. HPLC purity: 97.6%; t = 14.6 min (MeCN/H O
23
28 13
7
R
2
0
:100 to 100:0 over 30 min).
4
. Conclusions
We reported in this study the synthesis of new SAM-adenosine conjugates with a 1,2,3-triazole
linker that covalently links the SAM analogue to the N-6 or N-1 position of the adenosine substrate.
6
The use of protecting groups allowed the formation of the N and 1-N-conjugates but required
numerous steps of protection and deprotection. Revisiting the synthetic strategy, we were able to avoid
the steps of protection and deprotection of the hydroxyl and exocyclic amine functions and to propose
6
more straightforward and efficient syntheses. The N and 1-N conjugates were obtained in 2 steps

Molecules 2020, 25, 3241
16 of 19
with overall yield of 26% and 30%, respectively. In addition, we developed an efficient methodology
based on CuAAC to get access to conjugates by connecting through a triazole linker an analogue of
RNA MTases substrate (an adenosine modified at the N-6 or N-1 position) to a SAM analog cofactor.
We think that this approach could be applied for the preparation of modified oligonucleotides to get
6
1
more complex bisubstrate analogues for the study of m A and m A RNA MTases.
Supplementary Materials: The following are available online: H and 13C NMR spectra of compounds 12
1
–15
and 19, and HPLC chromatograms of compounds 12–15 and 19.
Author Contributions: Conceptualization, L.I., M.E.-Q., and E.B.; methodology, C.A., D.C. and L.I.;
writing—original draft preparation, E.B.; writing—review and editing, L.I., M.E.-Q., and E.B.; project
administration, M.E.-Q.; funding acquisition, M.E.-Q. All authors have read and agreed to the published
version of the manuscript.
◦
Funding: This research was funded by ANR, project N ANR-19-CE07-0028-01, ANRTools.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are not available from the authors.
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