Synthesis of Functionalized 2-(4-Hydroxyphenyl)-3-methylbenzofuran Allosteric Modulators of Hsp90 Activity

Article abstract of DOI:10.1002/ejoc.201600420

Hsp90 is a molecular chaperone that plays a pivotal role in the cell life cycle. ATP-regulated internal dynamics are critical to Hsp90 function and we recently demonstrated that these dynamics can be modulated in an allosteric fashion; the protein C-terminal domain (CTD) can be effectively targeted with a family of 2-phenyl-benzofuran derivatives. Here we describe the expansion of the initial library, reporting 28 new derivatives that explore the chemical space at opposite ends of the benzofuran scaffold. Interactions of the compounds with a full-length protein homolog were explored by Saturation Transfer Difference (STD) NMR spectroscopy. In this context we also report the interaction epitope of Novobiocin, a known CTD inhibitor.

Full text of DOI:10.1002/ejoc.201600420

DOI: 10.1002/ejoc.201600420
Full Paper
Allosteric Modulation
Synthesis of Functionalized 2-(4-Hydroxyphenyl)-3-
methylbenzofuran Allosteric Modulators of Hsp90 Activity
Sara Sattin*[a] Matteo Panza,[a] Francesca Vasile,[a] Francesca Berni,[a] Giulio Goti,[a]
Jiahui Tao,[b] Elisabetta Moroni,[c,d] David Agard,[b] Giorgio Colombo,[c] and Anna Bernardi[a]
Dedicated to Professor Carlo Scolastico on the occasion of his 80th birthday
Abstract: Hsp90 is a molecular chaperone that plays a pivotal rivatives that explore the chemical space at opposite ends of
role in the cell life cycle. ATP-regulated internal dynamics are the benzofuran scaffold. Interactions of the compounds with a
critical to Hsp90 function and we recently demonstrated that full-length protein homolog were explored by Saturation Trans-
these dynamics can be modulated in an allosteric fashion; the fer Difference (STD) NMR spectroscopy. In this context we also
protein C-terminal domain (CTD) can be effectively targeted report the interaction epitope of Novobiocin, a known CTD in-
with a family of 2-phenyl-benzofuran derivatives. Here we de- hibitor.
scribe the expansion of the initial library, reporting 28 new de-
Introduction
Hsp90 and to lead to proteosomal degradation of its clients by
inhibiting correct folding.
Heat shock proteins (HSPs) are a group of chaperone proteins
overexpressed by cells under stress conditions. Among these,
Hsp90 has been shown to play a central role in the maturation
of numerous client proteins involved in several cancer hall-
marks. As such, Hsp90 represents a hub in a signaling network
that controls cell cycle and apoptosis and is an established tar-
get in cancer therapy. This chaperone exists as a homodimer,
each protomer consisting of four regions: an N-terminal ATP
binding domain, a middle domain that completes the catalytic
site, a charged region, and a C-terminal homodimerization do-
main.[1] A complex internal dynamics of the domains within the
homodimer controls the chaperone activity of the protein and,
with it, Hsp90-driven downstream signalling pathways. This dy-
namic process is coupled to ATP hydrolysis and competitive in-
hibitors of ATP binding in the Hsp90 N-terminal domain, such
as geldanamycin or radicicol,[2] have shown potent antitumor
activity in a wide-range of malignancies.[3] The aminocoumarin
Recently, small molecule activators of Hsp90 have been re-
ported[5] and shown to act as allosteric modulators of ATPase
activity. In particular, we have described a group of functional-
ized 2-(4-hydroxyphenyl)-3-methylbenzofurans (1, Figure 1) that
target a computationally predicted allosteric site in Hsp90 and
able to modify its enzymatic, conformational, co-chaperone and
client-binding properties.[6] The best activators were shown to
stimulate Hsp90 ATPase activity while simultaneously accelerat-
ing the rate of interprotomer closure dynamics, as revealed by
FRET experiments. Some of these molecules were shown to af-
fect the viability of cancer cell lines, including cells resistant to
the geldanamycin derivative 17AAG, for which proper function-
ing of Hsp90 is required.[5b] In the absence of X-ray structures,
Molecular Dynamics (MD) simulations guided the ligand selec-
tion process and were also used to reveal the mechanisms of
Hsp90 modulation by the allosteric ligands.[7] An ensemble ap-
proach was adopted to characterize chaperone-ligand interac-
tions, with the aim of identifying the hot spots of the allosteric
site where key functional groups on the ligand best comple-
ment the receptor, while taking the dynamic exchange be-
tween the binding partners explicitly into account. This process
Novobiocin
and
its
analogues[4]
have been shown to bind the C-terminal domain (CTD) of
[a] Department of Chemistry, Università degli Studi di Milano,
Via Golgi 19, 20133 Milano, Italy
E-mail: sara.sattin@unimi.it
[b] Howard Hughes Medical Institute and Dept. of Biochemistry & Biophysics,
University of California,
San Francisco, California 94158 USA
[c] ICRM-CNR,
Via Mario Bianco 9, 20131 Milano, Italy
[d] IRCCS MultiMedica,
Via Fantoli 16/15, 20138 Milano, Italy
Supporting information and ORCID(s) from the author(s) for this article are
available on the WWW under http://dx.doi.org/10.1002/ejoc.201600420.
Figure 1. General structure of the 2-(4-hydroxyphenyl)-3-methylbenzofuran
modulators 1.
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led to the definition of two regions on the ligand where func-
tional group diversification could arguably translate into a mod-
ulated response of the chaperone: the 5-substituent of the
benzofuran ring (R1, Figure 1) and the phenol substituent (R2,
Figure 1).
Here we describe the synthesis of 28 new derivatives in the
active benzofuran series. These compounds were designed to
expand the structure–activity relationship (SAR) observed in the
initial generation for the R1 and R2 regions[7] and to explore
the role played by specific structural features in determining
compound potency. Hydrophilic and/or charged substituents
were privileged, in an effort to increase the water solubility of
the ligands. We also describe STD NMR experiments with the
yeast Hsp90 isoform, Hsc82, that confirm binding of the benzo-
furan ligands with the full protein. In this context, we present
the first STD interaction study of Novobiocin with the same
protein. STD data were combined with the MD-based character-
ization of the structures of Hsc82/allosteric ligand com-
plexes.[6b] The results indicate qualitative agreement between
the two approaches, allowing us to identify the ligand epitopes.
Scheme 1. Synthesis of 5-chloro-2-(4-hydroxyphenyl)-3-methylbenzofuran 1a
by enolate arylation. (i) Pd(OAc)2, rac-2-(di-tert-butylphosphanyl)-1,10-bi-
naphthyl (rac-DTBPB), NaOtBu, toluene. (ii) TFA/DCM (iii) EtSNa, DMF 43 %
over the three steps (from[8]).
al.,[9] in the one-pot version introduced by Contiero
(Scheme 2).[10] Condensation of aryloxyamine 2 with ketone 3
yields intermediate O-aryloxime 4, which is heated at 60 °C in
THF and with an excess of MeSO3H to induce sigmatropic rear-
rangement of the protonated enamine isomer. Re-aromatiza-
tion of the intermediate, followed by intramolecular cyclization
and ammonia elimination afford desired benzofuran 1 in 3–6 h
and with satisfactory yields (Scheme 2).
Conversion of 4-trifluoromethyl derivative 4d required
harsher conditions and was achieved in 45 % yield by replacing
THF with higher boiling dioxane and performing the reaction at
100 °C for 2 d. Starting aryloxyamines 2a–d were conveniently
synthesized from N-hydroxyphthalimide 5 and corresponding
boronic acids 6a–d (Scheme 3) using a Cu-mediated cross-cou-
pling procedure developed by Petrassi and co-workers.[11]
It has already been shown that 1a is a suitable substrate
for Stille coupling with tributyl(1-propenyl)tin.[8] 5-Bromo-2-(4-
hydroxyphenyl)-3-methylbenzofuran (1b) was instrumental to
Results and Discussion
Diversification at R1
The starting material for the group of benzofuran derivatives
described so far is the 5-chloro-2-(4-hydroxyphenyl)-3-methyl-
benzofuran (1a), synthesized from 2-bromo-4-chlorophenol by
Pd-catalyzed enolate arylation, as described previously
(Scheme 1).[8]
The introduction of substituents other than chlorine at posi- the exploration of additional Pd-catalyzed chemistry and to ex-
tion 5 required a different approach, based on a [3,3]-sigma- panding the library of 5-substituents. In particular, since calcula-
tropic rearrangement of O-aryloximes described by Miyata et tions had suggested that the Hsp90 allosteric pocket may in-
Scheme 2. Synthesis of 5-substituted 2-(4-hydroxyphenyl)-3-methylbenzofurans 1a–d by [3,3]-sigmatropic rearrangment of O-aryloximes 2a–d.
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ligands as stimulators of Hsp90. Thus, we focused our attention
on the introduction of amino groups in this region. This was
accomplished by phase-transfer-catalyzed (PTC) alkylation of
the phenol oxygen of 1a, 1c and 7 with 2-chloro-N,N-dimethyl-
ethylamine
9
or 3-chloro-N,N-dimethylpropylamine 10
(Scheme 5) to afford compounds 11–13 in good yields. Amino
tert-butyl esters 14 and 18 (Scheme 5), obtained by alkylation
of 7, were either directly deprotected (20 % CF3COOH in CH2Cl2)
to give the corresponding N,N-dimethylamino acids 15 and 19,
or hydrolyzed after reduction of the double bond (H2 and 10 %
Pd-C) to afford 17 and 21.
In an effort to improve the solubility of the ligands, a second
group of amine derivatives was prepared from epoxides 23 and
24, synthesized by alkylation of 1a and 1b with epichlorohydrin
22 (Scheme 6). Reaction of 23 with a set of four secondary
amines in a 9:1 CH2Cl2/EtOH mixture yielded compounds 25–
28. Similarly, 32 was obtained by reaction of 24 with N-methyl-
piperidine (Scheme 6). The primary amine 30 was prepared by
NaN3 opening of 23 [dioxane, 80 °C, cat. Yb(OTf)3, 81 % yield]
and reduction of the resulting azide 29 under carefully con-
trolled conditions (H2, PtO2, morpholine in MeOH, 71 %
yield),[13] to avoid hydrogenolysis of the arylchloride. Finally,
HCl-promoted hydrolysis of 23 in 3:1 dioxane/water mixture af-
forded diol 31 in 60 % yield (Scheme 6).
Scheme 3. Synthesis of the aryloxyamines 2a–d. (i) CuCl, 4 Å molecular sieves,
1,2-dichloroethane, air, room temperature, 48–72 h; (ii) H2NNH2·H2O, 10 %
MeOH in CH2Cl2; (iii) 4
M HCl in dioxane.
clude complementary basic sites, a Heck[12] reaction was se-
lected as the means by which to introduce an acidic function
to the benzofuran scaffold. Therefore, 1b and tBu acrylate were
reacted in toluene affording 5-substituted tBu ester 7 in 81 %
yield and with 100 % E selectivity (Scheme 4). Despite the acid
sensitivity of the substrate, ester deprotection could be per-
formed using 20 % CF3COOH in CH2Cl2 to afford acid 8 in quan-
titative yields. Double bond reduction was also successfully
achieved on a set of derivatives as described below.
Two additional derivatives, 34 and 35 were prepared by acti-
vation of known acid 33[5b] and reaction with morpholine or N-
methylpiperazine, respectively (Scheme 7).
We have previously reported that a set of monoglycosides
of 1a can modulate Hsp90 activity. In particular when R2 = α-
L-
Rha, α-D-Man and ꢀ-D-Gal the 1a congeners were found to
induce significant activation of Hsp90 ATPase.[5b] In order to
harness the effects of both a sugar and an amino group, the
synthesis of glycoconjugates of amino sugars was also at-
tempted (Scheme 8). Glycosylation of 1a with 6-azido-2,3,4-tri-
O-benzoylmannose 1-trichloroacetimidate 36[14] was executed
in good yields (62 %) under standard conditions (TMSOTf,
–30 °C) to afford 6-azidomannoside 37 (Scheme 8). It is worth
noting that, under the same conditions, mannosylation of 1a
with 2,3,4,6-tetra-O-benzoylmannose 1-trichloroacetimidate af-
Scheme 4. Heck reaction of 1b. (i) tBu acrylate, Pd(OAc)2, P(o-tol)3, TEA, tolu-
ene, reflux, 6 h, 81 %; (ii) 20 % CF3COOH in DCM, 2 h, quant.
Diversification at R2
Our initial set of data for R2 substituents had shown that basic forded only a modest 20 % yield.[8] Zemplén removal of the
groups at this position improve the activity of the benzofuran benzoyl esters occurred uneventfully to yield 38, which is barely
Scheme 5. PTC alkylation of the phenol with N,N-dimethylalkylamines. (i) Compounds 9·HCl, or 10·HCl, Bu4NHSO4, K2CO3, H2O (5 % w/w in K2CO3) in CHCl3.
(ii) 20 % CF3COOH in CH2Cl2; (iii) H2, Pd/C, MeOH, 2 h, quant.
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Scheme 6. Synthesis of epoxides 23 and 24 and nucleophilic opening reactions. (i) Compound 22, Bu4NHSO4, K2CO3, H2O (5 % w/w in K2CO3) in CHCl3.
(ii) Compounds 25–28 and 32: secondary amine, [9:1] CH2Cl2/EtOH; for 29: NaN3 in refluxing dioxane, Yb(OTf)3, 5 d, 81 %; for 31: [3:1] dioxane/H2O, HCl,
reflux, 5 h, 60 %; (iii) H2, PtO2-morpholine in MeOH, 71 %.
ever, but none could be properly isolated. Similar results were
obtained with the corresponding ꢀ-glucoside 41, obtained by
glycosylation of 1a with glucosylbromide 39[15] under PTC con-
ditions. Zemplén removal of the acetyl groups from 40 occurred
under unusually harsh conditions (MeOH, 40 °C, 2 d), due to
the low solubility of the starting material in MeOH, and yielded
6-azidoglucoside 41 after precipitation from CH2Cl2. The com-
pound was found to be soluble in acetone and could be fully
characterized. Again, though, we were unable to obtain clean
reduction of the azido group.
Glycosylations with other non-metabolic sugars were more
Scheme 7. Synthesis of amides 34 and 35. (i) EDC, morpholine, THF, 20 h,
successful. We focused on a limited group of commercially
35 %; (ii) a. Oxalyl chloride, b. N-methylpiperazine, CH2Cl2, 78 %.
available monosaccharides, all characterized by the presence of
an axial hydroxyl group either in position 2 or 4 of the pyranose
ring, as suggested by the set of active glycosides identified in
the previous campaign. The α-
tives 42 and 43 (Table 1) are enantiomers of previously de-
scribed derivatives[5b] and were synthesized accordingly. The
and α-arabinosides 44 and 45 were synthesized from the
corresponding bromides, as exemplified in Scheme 9 for the
enantiomer. PTC alkylation of 1a with ꢀ- -arabinopyranosyl
bromide 2,3,4-triacetate 50[16] followed by Zemplén hydrolysis
of the acetyl groups afforded -arabinoside 44 (60 % overall
L-Man and the ꢀ-D-Fuc deriva-
soluble in MeOH. Reduction of the azido group could not be
achieved under a variety of conditions mainly owing to the
poor solubility of 38 and of the resulting amine in most organic
solvents.
Hydrogenolysis with mild catalysts, such as Lindlar-Pd, left
the starting material unreacted even after 1 d. Under more se-
vere conditions [e.g. using Pd/C (AcOEt, 1 % AcOH) or PtO2 and
morpholine] several by-products (mostly from hydrogenolysis
L
D
L
L
L
of the aryl chloride bond) were observed by MS analysis, how- yield) exclusively in the α configuration. It is worth noting that
Scheme 8. Glycosylation of 1a with 6-azidosugar donors. (i) 1a, TMSOTf, –30 °C, 30 min, 62 %; (ii) MeONa, MeOH; (iii) 1a, Bu4NHSO4, K2CO3, H2O (5 % w/w in
K2CO3) in CHCl3, 40 %.
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for pentapyranosides, the anomeric substituent is ꢀ(α) if in a 46 and 47. The
D
-enantiomers 48 and 49 were obtained using
trans (cis) orientation relative to the 4-hydroxy group[17]].
the same route, starting from the D-enantiomer of lyxose.
Table 1. Glycosylation of 1a with non-metabolic sugars.
Interaction of Compounds with Hsp90: STD-NMR
Experiments
Whereas full characterization of the compounds activity on
Hsp90 ATPase and functional dynamics is in progress, we set
out to characterize the interaction of selected library members
with Hsp90 using NMR spectroscopy. Saturation Transfer Differ-
ence NMR is one of the most widespread NMR methods to
study the interaction between small molecules and macro-
molecular receptors.[20] The technique exploits the nuclear
Overhauser effect and relies on the fast exchange equilibrium
for a ligand between the free and bound states, in the presence
of the receptor. Selective irradiation of the protein is followed
by transfer of magnetization to the ligand protons, which in
turn causes a partial saturation of the ligand signals, allowing
one to map the interaction epitope on the small molecule by
analyzing the difference spectrum obtained in the absence or
in the presence of the selective irradiation pulse. The ligand
protons that are in closer contact with the protein receive a
larger amount of magnetization transfer and give larger signals
in the difference spectrum.
42
43
44
45
46
47
48
49
α-
ꢀ-
α-
α-
α-
ꢀ-
α-
L
-Man
-Fuc
-Ara
-Ara
-Lyx
-Lyx
-Lyx
-Lyx
D
L
D
L
L
D
ꢀ-
D
To the best of our knowledge, no STD experiments have
been reported with full-length Hsp90. Thus, to test the feasibil-
ity of this experiment, we first analyzed Novobiocin 56 (Fig-
ure 2, A) in the presence of the yeast isoform of Hsp90, Hsc82.
Novobiocin is the first and most studied inhibitor of the Hsp90
carboxy terminus.[21] To date, no co-crystal structure of Hsp90
bound to C-terminal ligands has been solved. Thus, information
on the size and nature of the binding pocket and on ligand
positioning is based exclusively on computational SAR and mo-
lecular docking studies.[22] Novobiocin affinity for Hsp90 was
estimated to be in the millimolar range by SPR and other tech-
niques,[23] which makes it ideally suitable for STD analysis. How-
ever, NMR interaction experiments had not been reported thus
far.
Scheme 9. Glycosylation of 1a with non-metabolic sugars. (i) 1a, Bu4NHSO4,
K2CO3, H2O (5 % w/w in K2CO3), in CHCl3, 3 h, 83 %; (ii) MeONa, MeOH;
(iii) AgOBox, 24 %; (iv) 1a, TMSOTf, 1,2-dichloroethane, –78 °C, 69 % com-
bined yield (40 % 54 and 29 % 55).
The STD spectrum of Novobiocin 56 in the presence of
Hsc82 was obtained in deuterated phosphate buffer with a
200:1 ligand:protein ratio, using as the irradiating frequency the
upfield aliphatic region of the protein. The spectrum is reported
in Figure 2, C, together with the 1H spectrum of Novobiocin 56
under the same conditions (Figure 2, B). The molecule appears
to be deeply embedded in the protein binding site since differ-
ent degrees of magnetization transfer could be observed for
each proton (Figure 2, C). A quantitative analysis of these data
(Figure 2 and Supporting Information) suggests that the inter-
action epitope of Novobiocin strongly involves the prenylated
phenol ring, the coumarin moiety and, to a lesser extent, the
noviose ring. These observations are consistent with the results
The anomeric configuration of the compound could be es-
tablished based on the 1,2 coupling constant of 7 Hz, typical of
a 1,2-trans glycoside. Under the same conditions, glycosylation
of 1a with α-
-lyxopyranosyl bromide 2,3,4-triacetate 51[18]
L
(Scheme 9) afforded the desired product in less than 10 % yield.
Thus, O-benzoxazole (O-Box) donor 53 was prepared from
bromide 52 and used under Demchenko's conditions[19] to
glycosylate 1a in 69 % overall yield.
Both anomers α-
L
-Lyx 54 and ꢀ-
-Lyx 55 were formed in this of MD-based studies of Hsc82/Novobiocin complexes.[6b] In the
L
reaction in a 56:44 ratio and were separated by flash chroma- latter, extensive MD simulation of 56 docked to the putative
tography (9:1 toluene/hexane). Their anomeric configurations allosteric pocket were run and characterized by means of clus-
and main conformations (shown in Scheme 9) were established ter analysis. The representative structure (Figure 3, b, right
by NMR experiments described in the Supporting Information. panel) was largely consistent with STD data, showing the
Benzoate removal afforded final α-L-Lyx and ꢀ-L-Lyx glycosides prenyl, aromatic and sugar protons within contact distance with
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Figure 2. STD-NMR experiments with Novobiocin 56. (a) Structure of Novobiocin 56. (b) 1H-NMR spectrum of 56 in presence of Hsc82 in a 200:1 ligand/
protein ratio. (c) STD spectrum of 56 in presence of Hsc82 in a 200:1 L/P ratio. STD was obtained by irradiating the protein at –0.05 ppm with total saturation
times of 0.98 s. The water suppression was achieved by Watergate 3–9-19 pulse sequence. The relative STD values, grouped in four intensity ranges, are
marked with different colours and point up that the prenylated hydroxylphenyl ring is making the closest contacts with the protein in the bound state.
the protein. Additionally, the OCH3 substituent at noviose 4′ ing frequency the upfield methyl region of the protein. A se-
position was also calculated to point towards the interior of the lected spectrum is reported in Figure 4C and the corresponding
protein, consistent with the observed STD signal.
1D spectrum is shown in Figure 4 (B).
NMR analysis of the benzofuran derivatives in the presence
of Hsc82 was severely impaired by the low solubility of the
compounds in water. Finally, conditions were established
whereby compound 11 (Figure 4, A) could be dissolved in the
aqueous buffer at the appropriate concentration and main-
tained in solution after addition of the protein. Amine 11 has
already been characterised as a powerful Hsp90 allosteric acti-
vator.[5b] The predicted binding site (Figure 3, a) is close to the
region occupied by Novobiocin (Figure 3, b). However, MD sim-
ulations suggest that formation of the Hsp90:Novobiocin com-
plex blocks the protein in a partially inactive state (Figure 3, b,
left panel), whereas interaction with 11 results in higher fre-
quency turnover of the closed-active state (Figure 3, a, left
panel) and allosterically stimulates ATPase activity.
The results confirmed that binding occurs between com-
pound 11 and Hsc82 and that the recognition is strongly medi-
ated by aromatic protons (belonging both to the phenol and
benzofuran rings). An intense STD signal was also observed for
N-CH2 and N-methyl groups. In particular, the protons of the
aromatic rings show the largest saturation transfer and the N-
CH3 group the lowest effect, suggesting that the region of the
ligand comprising the aromatic rings makes the closest con-
tacts with the protein binding site. A competition experiment
between 11 and Novobiocin was attempted, but was frustrated
by precipitation of the ligand. In good agreement with STD, the
representative structures from MD simulations of the Hsc82/11
complex (Figure 3, a, right panel) show extensive packing of the
benzofuran and aromatic rings of the ligand with the protein,
STD NMR experiments were recorded with a 200:1 li- coupled with contacts involving both the N-CH2 and N-methyl
gand:protein ratio at different saturation times, using as irradiat- groups of the amine modification.[7] Within the limitations of
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Figure 3. Hsp90 complexes of benzofuran 11 (a) and Novobiocin 56 (b). Dynamic equilibrium between open and closed Hsp90 is differentially affected by 11
(a, left panel) and Novobiocin (b, left panel) resulting in activation and inhibition of the ATPase activity, respectively. The docked complexes of Hsc82 with
11 (a) and Novobiocin (b) are shown in the right panels.
MD sampling for such large and complex systems, and with a
caveat on the qualitative nature of the comparison, compara-
tive analysis of STD and MD results provides a consistent struc-
tural model for the complexes in solution. Moreover, it is worth
noting that such knowledge has been used to guide the devel-
opment of more potent derivatives, providing further support
to the validity of the model.
Conclusions
A library of Hsp90 stimulators characterized by a 2-(4-hydroxy-
phenyl)-3-methylbenzofuran structure has been significantly ex-
panded. Groups at position 5 of the benzofuran ring were diver-
sified using a [3,3]-sigmatropic rearrangement of O-aryloximes
for the synthesis of the benzofuran core and a Heck cross-cou-
pling reaction for further modifications. The synthesis of a sec-
ond set of compounds, diversified at position R2 was achieved
by alkylation of the phenol ring followed by further elabora-
tions. Glycosylation of 1a with amino sugars was attempted but
abandoned due to low solubility of the products, although
other non-metabolic sugars could be successfully introduced
to the scaffold. Overall, 28 new compounds were synthesized,
demonstrating the versatility of the benzofuran platform. STD-
NMR analysis of the interaction of the known allosteric modula-
tor 11 with the yeast isoform of Hsp90 (Hsc82) revealed that
the ligand actually binds to the protein and confirmed the role
of the benzofuran scaffold in establishing the interaction. The
spectra also showed that the substituents on the amino group
are in close contact with the protein, providing a viable struc-
tural model that can be used to guide the development of
more potent derivatives. Full characterization of Hsp90 ATPase
activity modulation by the 28 new compounds and functional
Figure 4. STD-NMR experiments with benzofuran derivative 11. (a) Formula
dynamics studies are currently underway.
of 11. (b) 1H-NMR spectrum of 11 in presence of Hsc82 in a 200:1 ligand/
protein ratio. (c) STD spectrum of 11 in the presence of Hsc82 in a 200:1 L/
P ratio. STD was obtained by irradiating the protein at –0.05 ppm with total
saturation times of 2.94 s. The water suppression was achieved by Watergate
Experimental Section
Protein Expression and Purification: The Hsc82 was cloned into
pET28b (Novagen, U.S.) and transformed into BL21 (DE3) cells (NEB,
3–9-19 pulse sequence. The relative STD values are grouped in four intensity
ranges and marked with different colors.
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U.S.). The protein expression was induced by 0.5 m
M
IPTG (Goldbio,
General Procedure I. Synthesis of the 5-Substituted 2-(4-
Hydroxyphenyl)-3-methylbenzofurans (1a–c):[10] The phenyloxy-
amine hydrochloride 2a–c (1 mmol) was suspended in THF (3 mL)
under nitrogen. Freshly activated, acid washed molecular sieves
(4 Å, 0.1 g) and 1-(4-hydroxyphenyl)propan-1-one 3 (1 mmol) were
added. The solution was stirred for 5 min at 60 °C, then methane-
sulfonic acid (2 mmol) was added. The mixture was stirred for 5 h
and reaction progress was followed by HP-TLC (8:2 toluene/DCM;
each sample was diluted with DCM and quenched with NaHCO3
before deposition on TLC). The reaction mixture was then diluted
with EtOAc, the organic phase was washed with NaHCO3 (2 × 5 mL),
and brine (2 × 5 mL), dried with anhydrous Na2SO4 and concen-
trated in vacuo. The crude product was purified by chromatography
as described below.
U.S.) at 20 °C for 16 h. The cells were lysed using EmulsiFlex-C3
(Avestin, Canada). The lysate was purified using Ni-NTA columns
(Qiagen, U.S.), monoQ ion exchange chromatography (GE, U.S.) and
Superdex200 gel filtration chromatography (GE·U.S.).
STD-NMR: All NMR spectra were recorded with a Bruker Avance
spectrometer operating at 600 MHz. All data were collected at
298 K. 1D, COSY, and NOESY (mixing time of 200 and 700 ms) were
recorded using standard sequences from the Bruker library on No-
vobiocin 56 (0.23 mg, MW = 634.6, 2 m
(0.3 mg, MW = 366.3, 4.5 m ) in order to assign all resonances. The
sample was prepared in deuterated 20 m PBS buffer. Data were
M) and Compound 11
M
M
analysed using Bruker Topspin software. After NMR characterization,
Hsc82 (10 mg/mL, MW = 80899.7) was added to each sample afford-
ing a 200:1 L/P ratio. For the acquisition of NMR Saturation Transfer
Difference (STD) experiments, a 1D-pulse sequence incorporating a
T1ρ filter to remove disturbing protein signals was used. The STD
spectra were performed with an on-resonance irradiation frequence
at –0.05 ppm, whereas 200 ppm was chosen as the off-resonance
frequency. Selective presaturation of the protein was achieved by a
train of Gauss shaped pulses, each of 49 ms in length. STD experi-
ments were acquired on each sample varying the total saturation
times from 0.49 to 3.92 s (with 0.49 s increments). In STD experi-
ments, water suppression was achieved by using the WATERGATE
5-Chloro-2-(4-hydroxyphenyl)-3-methylbenzofuran (1a): Gen-
eral procedure I performed starting from 2a. The crude material
was purified by flash chromatography (toluene/DCM gradient from
10 % to 25 % DCM) leading in 51 % yield to product 1a, whose
spectroscopic data corresponded with those reported in the litera-
ture.[8] 1H NMR (400 MHz, CDCl3): δ = 7.67 (d, Jo-m = 8.8 Hz, 2 H,
2 × H-m-Phe-O), 7.45 (d, J4-6 = 2 Hz, 1 H, H-4Bf), 7.35 (d, J7-6
=
8.6 Hz, 1 H, H-7Bf), 7.20 (dd, 1 H, H-6Bf), 6.96 (d, 2 H, 2 × H-o-Phe-
O), 2.39 (s, 3 H, CH3-Bf) ppm.
5-Bromo-2-(4-hydroxyphenyl)-3-methylbenzofuran (1b): Gen-
3–9-19 pulse sequence. For both products, blank experiments in eral procedure I performed starting from 2b. The crude product
the absence of protein were performed.
was purified using an automated chromatography system (SiO2,
Hex/EtOAc gradient from 30 % to 100 % EtOAc), to afford com-
pound 1b in 70 % yield as a white solid, whose spectroscopic data
corresponded with those reported in the literature.[9] 1H NMR
(400 MHz, CDCl3): δ = 7.68 (d, Jo-m = 8.3 Hz, 2 H, 2 × H-m-Phe-O),
7.62 (s, 1 H, H-4Bf), 7.38–7.29 (m, 2 H, H-6Bf, H-7Bf), 6.95 (d, 2 H,
2 × H-o-Phe-O), 2.40 (s, 3 H, CH3Bf) ppm.
Synthesis – General: Dry dichloromethane (DCM), methanol, 1,4-
dioxane and triethylamine (TEA) were distilled from calcium
hydride; tetrahydrofuran (THF) was distilled from sodium. Dichloro-
ethane (DCE) was dried with molecular sieves (4 Å) and hexane
(Hex) and toluene were used as purchased. Reactions requiring an-
hydrous conditions were performed under nitrogen, or argon where
indicated. AgOBox was synthesized as reported.[19] 1H and 13C spec-
tra were recorded at 400 MHz or 100 MHz, respectively, on a Bruker
5-Fluoro-2-(4-hydroxyphenyl)-3-methylbenzofuran (1c): General
procedure I performed starting from 2c. The crude product was
purified by flash chromatography (8:2 Hex/EtOAc, Rf = 0.3) leading
AVANCE-400 instrument. Chemical shifts (δ) for H and 13C spectra
1
1
to product 1c in 56 % yield. H NMR (400 MHz, CDCl3): δ = 7.68 (d,
are expressed in ppm relative to internal standard [CDCl3: 7.26 for
1H and 77.16 for 13C. CD3OD: 3.31 for 1H and 49.00 for 13C; (CD3)2CO:
2.05 for 1H and 29.84 for 13C]. Signals were abbreviated as s, singlet;
d, doublet; t, triplet; q, quartet; m, multiplet; b means broad as in
br. s, broad singlet. Quaternary carbon signals were abbreviated as
Cq. All assignments were confirmed with the aid of two-dimensional
1H-1H (COSY), 1H-13C (HSQC) and/or 1H-13C (HMBC) experiments us-
ing standard pulse programs. Processing of the spectra was per-
formed using TopSpin or MestReNova software. Sugar signals were
numbered as is customary; benzofuran signals were numbered as
is customary and indicated with the suffix Bf, and the substituents
of benzofuran do not have any numbering, but they are clearly
indicated in the characterizations. OBox signals were numbered as
is customary and indicated with the suffix Box. In the names of the
compounds the conventional numbering is used. Mass spectra were
obtained with a ThermoFisher LCQ apparatus (ESI ionization), or
iontrap ESI Esquire 6000 from Bruker, or Apex II ICR FTMS (ESI ioniza-
tion – HR-MS). Thin layer chromatography (TLC) was carried out
J
o-m = 8.9 Hz, 2 H, 2 × H-m-Phe-O), 7.37 (dd, J7-6 = 8.9, 4JHF = 4.2 Hz,
1 H, H-7Bf), 7.15 (dd, J4-6 = 2.6, 3JH-F = 8.6 Hz, 1 H, H-4Bf), 7.00–6.93
(m, 3 H, H-6Bf, 2 × H-o-Phe-O), 5.22 (br. s, 1 H, OH), 2.39 (s, 3 H,
CH3-Bf) ppm. 13C NMR (100 MHz, CDCl3): δ = 160.5 (Cq), 156.9 (d,
1JCF = 240 Hz, C-F), 155.7 (Cq), 152.7 (Cq), 149.9 (Cq), 132.3 (d, JCF
=
3
10 Hz, Cq), 128.6 (2 × C-m-Ph-O), 124.2 (Cq), 115.8 (2 × C-o-Ph-O),
2
3
111.6 (d, JCF = 12 Hz, C-6Bf), 111.4 (d, JCF = 4.4 Hz, C-7Bf), 110.1
2
(Cq), 104.8 (d, JCF = 30 Hz, C-4Bf), 9.5 (CH3-Bf) ppm. ESI-MS: calcd.
for C15H10O2F [M – H]– 241.2, found 241.0.
5-Trifluoromethyl-2-(4-hydroxyphenyl)-3-methylbenzofuran
(1d): Phenyloxyamine hydrochloride 2d (1 mmol) was suspended
in dioxane (3 mL) under nitrogen. Freshly activated, acid-washed
4 Å molecular sieves (0.1 g) and 1-(4-hydroxyphenyl)propan-1-one
3 (1 mmol) were added. The solution was stirred for 5 min at 40 °C,
then methanesulfonic acid (2 mmol) was added and the mixture
was stirred for 2 d at 100 °C. The reaction progress was followed by
RP-HPLC (C18, H2O/CH3CN gradient from 30 % to 100 % CH3CN;
with pre-coated Merck F254 silica gel plates. Flash chromatography retention time of 1d 1.36 min; retention time of the oxime interme-
(FC) was carried out with Macherey–Nagel silica gel 60 (230–
diate 4d 1.38 min; each sample was diluted with DCM and
quenched with NaHCO3 before injection). The reaction mixture was
400 mesh), according to the established Still procedure.[24] Auto-
mated chromatography was performed using a BiotageIsolera diluted with EtOAc, the organic phase was washed with NaHCO3
Prime. Compounds 1a, 11, 12, 33,[5b] 1b,[9] 39[15] and 50[16] have
been described previously. Glycosides 42 and 43 were character-
(2 × 5 mL), and brine (2 × 5 mL), dried with anhydrous Na2SO4 and
concentrated in vacuo. The crude material was purified by flash
ized based on the spectroscopic data reported for their enantio- chromatography (8:2 Hex/EtOAc, Rf = 0.4) leading to product 1c in
mers[8] and on the sign of their optical rotation. Boronic acids 57a–
d and ketone 3 are commercially available.
45 % yield. 1H NMR (400 MHz, CDCl3): δ = 7.79 (br. s, 1 H, H-4Bf),
7.71 (d, Jo-m = 8.8 Hz, 2 H, 2 × H-m-Phe-O), 7.55–7.48 (m, 2 H, H-7Bf,
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H-6Bf), 6.97 (d, 2 H, 2 × H-o-Phe-O), 5.07 (br. s, 1 H, OH), 2.46 (s, 3 CDCl3): δ = 7.75–7.69 (m, 3 H, =CH-Ar, 2 × H-m-Phe-O), 7.65 (br. s,
H, CH3-Bf) ppm. 13C NMR (100 MHz, CDCl3): δ = 155.9 (Cq), 154.0 1 H, H-4Bf), 7.44 (br. s, 2 H, H-6Bf, H-7Bf), 7.04 (d, Jo-m = 8.9 Hz, 2
1
(q, JCF = 241 Hz, CF3), 131.5 (Cq), 128.7 (2 × C-m-Ph-O), 125.1 (q,
H, 2 × H-o-Phe-O), 6.39 (d, Jtrans = 15.9 Hz, 1 H, =CH-C=O), 4.13 (t,
JCH2-CH2 = 5.7 Hz, 2 H, CH2-O), 2.77 (t, 2 H, CH2-N), 2.44 (s, 3 H,
CH3Bf), 2.36 (s, 6 H, NMe2), 1.55 (s, 9 H, 3 × CH3-tBu) ppm. 13C NMR
(100 MHz, CDCl3): δ = 166.8 (C=O), 158.8 (Cq-phenol), 154.8 (Cq-Bf),
152.1 (Cq-Bf), 144.4 (=CH-Ar), 132.0 (Cq-Bf), 129.4 (Cq-phenol), 128.3
(2 × C-m-Ph-O), 124.2 (C-6Bf), 124.0 (Cq-Bf), 119.1 (C-4Bf), 118.7 (=
C H - C = O ) , 1 1 5 . 0 ( 2 × C - o - P h - O ) , 1 1 1 . 3 ( C - 7 B f ) , 1 0 9 . 9
(Cq-Bf), 80.4 (Cq-tBu), 66.0 (CH2-O), 58.3 (CH2-N), 45.9 (NMe2), 28.4
(3 × CH3-tBu), 9.5 (CH3-Bf) ppm. ESI-MS: calcd. for C26H32NO4 [M +
H]+ 422.2331, found 422.2418.
2JCF = 32 Hz, CCF3), 123.8 (Cq), 121.1 (q, JCF = 3.7 Hz, C-6Bf), 116.8
3
3
(q, JCF = 4.1 Hz, C-4Bf), 115.9 (2 × C-o-Ph-O), 111.2 (C-7Bf), 110.0
(Cq), 9.4 (CH3-Bf ) ppm. ESI-MS: calcd. for C16H10F3O2 [M – H]–
291.064, found 291.092.
E-tert-butyl 3-[2-(4-Hydroxyphenyl)-3-methylbenzofuran-5-
yl]acrylate (7):[12] Compound 1b (131 mg, 0.43 mmol), Pd(OAc)2
(1 mg, 0.043 mmol) and (o-tol)3P (5 mg, 0.017 mmol) were dissolved
in toluene (800 μL) under nitrogen atmosphere. TEA (300 μL,
0.73 mmol) and tBu-acrylate (76 μL, 0.52 mmol) were added. The
mixture was warmed to reflux for 6 h (TLC 8:2 Hex/EtOAc). The
reaction was cooled to room temperature and diluted with EtOAc.
tert-Butyl (E)-3-(2-{4-[3-(dimethylamino)propoxy]phenyl}-3-
methylbenzofuran-5-yl)acrylate (18): Prepared from 7 and 10, fol-
lowing the general procedure II for phenol alkylation. The crude
product was purified using an automated chromatography system
[SiO2, CHCl3/MeOH/TEA (1 %) gradient from 0 to 20 % MeOH] to
afford the product in 62 % yield as a white foam. 1H NMR (400 MHz,
CDCl3): δ = 7.75–7.68 (m, 3 H, =CH-Ar, 2 × H-m-Phe-O), 7.64 (s, 1 H,
H-4Bf), 7.43 (br. s, 2 H, H-6Bf, H-7Bf), 7.00 (d, Jo-m = 8.8 Hz, 2 H,
2 × H-o-Phe-O), 6.39 (d, Jtrans = 15.9 Hz, 1 H, =CH-C=O), 4.09 (t,
JCH2-CH2 = 6.2 Hz, 2 H, CH2-O), 2.62 (t, 2 H, CH2-N), 2.44 (s, 3 H,
CH3Bf), 2.38 (s, 6 H, NMe2), 2.12–2.02 (m, 2 H, CH2-CH2-CH2), 1.55
(s, 9 H, 3 × CH3-tBu) ppm. 13C NMR (100 MHz, CDCl3): δ = 166.8 (C=
O), 158.9 (Cq-phenol), 154.9 (Cq-Bf), 152.1 (Cq-Bf), 144.4 (=CH-Ar),
132.0 (Cq-Bf), 129.4 (Cq-phenol), 128.3 (2 × C-m-Ph-O), 124.3 (C-6Bf),
124.0 (Cq-Bf), 119.2 (C-4Bf), 118.7 (=CH-C=O), 114.8 (2 × C-o-Ph-O),
111.3 (C-7Bf), 109.9 (Cq-Bf), 80.5 (Cq-tBu), 65.9 (CH2-O), 56.3 (CH2-
N), 44.8 (NMe2), 28.4 (3 × CH3-tBu), 26.7 (CH2-CH2-CH2), 9.5 (CH3-
Bf) ppm. ESI-MS: calcd. for C27H34NO4 [M + H]+ 436.2488, found
436.3021.
The mixture was washed with 1
M HCl (2 × 5 mL), water (2 × 5 mL)
and brine (1 × 5 mL), dried with anhydrous Na2SO4 and concen-
trated in vacuo. The crude product was purified using an automated
chromatography system (SiO2, Hex/EtOAc gradient from 30 % to
100 % EtOAc) to afford compound 7 (122 mg, 81 % yield, 100 % E
selectivity) as a white solid. H NMR (400 MHz, CDCl3): δ = 7.71 (m,
3 H, 2 × H-m-Phe-O, =CH-Ar), 7.64 (s, 1 H, H-4Bf), 7.44 (s, 2 H, H-6Bf,
1
H-7Bf), 6.96 (d, Jo-m = 8.7 Hz, 2 H, 2 × H-o-Phe-O), 6.39 (d, Jtrans
=
15.9 Hz, 1 H, =CH-C=O), 2.44 (s, 3 H, CH3Bf ), 1.56 (s, 9 H,
3 × CH3-tBu) ppm. 13C NMR (100 MHz, CDCl3): δ = 166.9 (C=O), 155.9
(Cq-phenol), 154.9 (Cq-Bf), 152.1 (Cq-Bf), 144.5 (=CH-Ar), 132.0 (Cq-
Bf), 129.5 (Cq-phenol), 128.6 (2 × C-m-Ph-O), 124.3 (C-6Bf), 124.0
(Cq-Bf), 119.2 (C-4Bf), 118.7 (=CH-C=O), 115.8 (2 × C-o-Ph-O), 111.3
(C-7Bf), 109.9 (Cq-Bf), 80.6 (Cq-tBu), 28.4 (3 × CH3-tBu), 9.5 (CH3-
Bf) ppm. ESI-MS: calcd. for C22H22NaO4 [M + Na]+ 373.1416, found
373.1462.
General Procedure II - Phenol Alkylation: Synthesis of 13, 14,
18, 23 and 24: The benzofuran (1a–c or 7; 0.3 mmol) and the
alkylating agent (9 or 10 or 22; 1 mmol) were dissolved in CHCl3
(3 mL). Water (19 μL), K2CO3 (2.7 mmol) and the phase-transfer
catalyst (Bu4NHSO4, 0.15 mmol) were added and the mixture was
kept overnight at 45 °C under vigorous stirring. When the reaction
was complete (TLC 95:5 CHCl3/MeOH) it was diluted with chloro-
form and brine, the aqueous phase was extracted with chloroform
(2 × 10 mL) and the organic phases were washed with water (2 ×
20 mL), dried with Na2SO4 and concentrated in vacuo.
5-Chloro-3-methyl-2-[4-(oxiran-2-ylmethoxy)phenyl]benzo-
furan (23): Obtained in quantitative yield by reaction of 1a and
epichlorohydrin 22, following the general procedure II for phenol
alkylation. TLC: 6:4 Hex/DCM, Rf = 0.3. No purification required. H
NMR (400 MHz, CDCl3): δ = 7.72 (d, Jo-m = 8.6 Hz, 2 H, 2 × H-m-Phe-
O), 7.46 (s, 1 H, H-4Bf), 7.36 (d, J7-6 = 8.6 Hz, 1 H, H-7Bf), 7.21 (d, 1
1
H, H-6Bf), 7.03 (d, 2 H, 2 × H-o-Phe-O), 4.30 (dd, Jgem = 11.0, Jvic
=
3.0 Hz, 1 H, CH2-OAr), 4.02 (dd, Jvic = 5.7 Hz, 1 H, CH2-OAr), 3.43–
3.37 (m, 1 H, CH-O), 2.94 (t, Jgem = 4.9, Jvic = 4.5 Hz, 1 H, CH2-O),
2.79 (dd, Jvic = 2.8 Hz, 1 H, CH2-O), 2.40 (s, 3 H, CH3Bf) ppm. 13C
NMR (100 MHz, CDCl3): δ = 158.6 (Cq-phenol), 152.3 (Cq-Bf), 151.1
(Cq-Bf), 132.9 (Cq), 128.4 (2 × C-m-Ph-O), 128.1 (Cq), 124.3 (Cq), 124.1
(C-6Bf), 118.9 (C-4Bf), 115.0 (2 × C-o-Ph-O), 111.9 (C-7Bf), 109.7 (Cq),
69.0 (CH2-OAr), 50.2 (CH-O), 44.8 (CH2-O), 9.5 (CH3Bf) ppm. ESI-MS:
calcd. for C18H15ClNaO3 [M + Na]+ 337.0, found 337.0.
5-Fluoro-2-{4-[2-(dimethylamino)ethoxy]phenyl}-3-methyl-
benzofuran (13): Prepared from 1c and 9, following the general
procedure II. The crude product was purified by flash chromatogra-
phy (CHCl3 + 1 %TEA, Rf = 0.26) leading to product 13 in 81 % yield.
1H NMR (400 MHz, 9:1 CD3OD/CDCl3): δ = 7.73 (d, Jo-m = 8.8 Hz, 2
4
H, 2 × H-m-Phe-O), 7.40 (dd, J6-7 = 8.9, JHF = 4.0 Hz, 1 H, H-7Bf),
5-Bromo-3-methyl-2-[4-(oxiran-2-ylmethoxy)phenyl]benzo-
furan (24): Prepared by reaction of 1b and epichlorohydrin 22,
following the general procedure II for phenol alkylation. TLC: 6:4
Hex/DCM, Rf = 0.4.The crude material was purified by flash chroma-
tography (85:15 toluene/DCM, Rf = 0.3) and the product was iso-
lated in 75 % yield. 1H NMR (400 MHz, CDCl3): δ = 7.71 (d, J = 9.0 Hz,
2 H, 2 × H-m-Phe-O), 7.61 (d, J4-6 = 1.8 Hz, 1 H, H-4Bf), 7.34 (dd,
J7-6 = 8.6, J4-6 = 1.8 Hz, 2 H, H-6Bf), 7.31 (d, 1 H, H7-Bf), 7.02 (d, 2
H, 2 × H-o-Phe-O), 4.29 (dd, Jgem = 11.0, Jvic = 3.0 Hz, 1 H, CH2-OAr),
4.00 (dd, Jvic = 5.9 Hz, 1 H, CH2-OAr), 3.42–3.37 (m, 1 H, CH-O), 2.94
(dd, Jgem = 4.7, Jvic = 4.3 Hz, 1 H, CH2-O), 2.79 (dd, Jvic = 3.2 Hz, 1
H, CH2-O), 2.41 (s, 3 H, CH3Bf) ppm. 13C NMR (100 MHz, CDCl3): δ =
158.5 (Cq), 152.4 (Cq), 152.0 (Cq), 133.4 (Cq), 128.3 (2 × C-m-Ph-O),
126.7 (C-6Bf), 124.1 (Cq), 121.8 (C-4Bf), 115.4 (Cq), 114.9 (2 × C-o-Ph-
O), 112.3 (C-7Bf), 109.4 (Cq), 68.8 (CH2-OAr), 50.1 (CH-O), 44.7 (CH2-
O), 9.4 (CH3Bf) ppm. ESI-MS: calcd. for C18H15BrO3 [M + H]+ 360.2,
found 360.4.
3
7.21 (dd, J4-6 = 2.6, JHF = 8.8 Hz, 1 H, H-4Bf), 7.08 (d, 2 H, 2 × H-o-
Phe-O), 7.04–6.94 (m, 1 H, H-6Bf), 4.19 (t, Jgem = Jvic = 5.3 Hz, 2 H,
CH2-O), 2.96–2.86 (m, 2 H, CH2-N), 2.45 (br. s, 6 H, 2 × CH3-N), 2.40
(s, 3 H, CH3Bf) ppm. 13C NMR (100 MHz, 9:1 CD3OD/CDCl3): δ =
1
161.8 (Cq), 160.6 (d, JCF = 238 Hz, C-F), 160.0 (Cq), 153.9 (Cq), 151.1
(Cq), 129.2 (2 × C-m-Ph-O), 125.2 (Cq), 115.8 (2 × C-o-Ph-O), 112.3 (d,
2
3JCF = 5.3 Hz, C-7Bf), 112.2 (d, JCF = 11.4 Hz, C-6Bf), 105.5 (2JCF
=
25 Hz, C-4Bf), 66.2 (CH2-O), 58.8 (CH2-N), 45.6 (NMe2), 9.4 (CH3-
Bf) ppm. ESI-MS: calcd. for C19H20FNNaO2 [M + Na]+ 336.3, found
336.0.
tert-Butyl (E)-3-(2-{4-[2-(dimethylamino)ethoxy]phenyl}-3-
methylbenzofuran-5-yl)acrylate (14): Prepared from 7 and 9, fol-
lowing the general procedure II for phenol alkylation. The crude
product was purified using an automated chromatography system
[SiO2, CHCl3/MeOH/TEA (1 %) gradient from 0 to 20 % MeOH] to
1
afford the product as a white foam (50 % yield). H NMR (400 MHz,
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Double Bond Reduction. Synthesis of 16 and 20: To a solution
of tert-butyl (E)-acrylate (14 or 18, 0.1 mmol) in MeOH (1 mL), Pd/
170.7 (C=O), 159.1 (Cq-phenol), 156.2 (Cq-Bf), 152.9 (Cq-Bf), 147.0 (=
CH-Ar), 133.1 (Cq-Bf), 130.7 (Cq-phenol), 129.4 (2 × C-m-Ph-O), 126.1
C was added. The mixture was stirred for 2 h at room temperature (Cq-Bf), 125.6 (C-6Bf), 120.7 (C-4Bf), 117.8 (=CH-C=O), 116.0 (2 × C-
under H2 atmosphere. The crude reaction was filtered through celite
to remove the catalyst and the filtrate concentrated in vacuo to
afford the product (16 or 20, respectively) as a white foam.
o-Ph-O), 112.2 (C-7Bf), 111.5 (Cq-Bf), 63.2 (CH2-O), 57.7 (CH2-N), 43.9
+
(NMe2), 9.3 (CH3-Bf) ppm. HRMS (ESI): calcd. for C22H24NO4 [M +
H]+ 366.16998, found 366.17028.
3-(2-{4-[2-(Dimethylamino)ethoxy]phenyl}-3-methylbenzo-
furan-5-yl)propanoic Acid (17): Isolated as the trifluoroacetate salt
after tBu ester removal from 16. 1H NMR (400 MHz, CD3OD): δ =
7.74 (d, Jo-m = 8.9 Hz, 2 H, 2 × H-m-Phe-O), 7.37 (d, J4-6 = 1.1 Hz, 1
H, H-4Bf), 7.33 (d, J7-6 = 8.4 Hz, 1 H, H-7Bf), 7.15–7.10 (m, 3 H, 2 × H-
o-Phe-O, H-6Bf), 4.40–4.36 (m, 2 H, CH2-O), 3.64–3.58 (m, 2 H, CH2-
N), 3.03–2.97 (m, 8 H, CH2-Ar, NMe2), 2.64 (t, JCH2-CH2 = 7.7 Hz, 2 H,
CH2-C=O), 2.39 (s, 3 H, CH3Bf) ppm. 13C NMR (100 MHz, CD3OD):
δ = 176.8 (C=O), 158.8 (Cq-phenol), 153.8 (Cq-Bf), 151.9 (Cq-Bf), 136.4
(Cq-Bf), 132.6 (Cq-phenol), 129.2 (2 × C-m-Ph-O), 126.6 (Cq-Bf), 125.8
(C-6Bf), 119.5 (C-4Bf), 115.9 (2 × C-o-Ph-O), 111.4 (C-7Bf), 111.2 (Cq-
Bf), 63.2 (CH2-O), 57.7 (CH2-N), 43.9 (NMe2), 37.4 (CH2-C=O), 32.1
tert-Butyl 3-(2-{4-[2-(Dimethylamino)ethoxy]phenyl}-3-methyl-
benzofuran-5-yl)propanoate (16): Obtained from 14 in 94 % yield.
1H NMR (400 MHz, CDCl3): δ = 7.72 (d, Jo-m = 8.9 Hz, 2 H, 2 × H-m-
Phe-O), 7.35 (d, J7-6 = 8.3 Hz, 1 H, H-7Bf), 7.32 (d, J4-6 = 1.6 Hz, 1 H,
H-4Bf), 7.10 (dd, 1 H, H-6Bf), 7.02 (d, J = 8.9 Hz, 2 H, 2 × H-o-Phe-
O), 4.13 (t, JCH2-CH2 = 5.7 Hz, 2 H, CH2-O), 3.02 (t, JCH2-CH2 = 7.8 Hz,
2 H, CH2-Ar), 2.77 (t, 2 H, CH2-N), 2.59 (t, 2 H, CH2-C=O), 2.42 (s, 3
H, CH3Bf), 2.37 (s, 6 H, NMe2), 1.43 (s, 9 H, tBu) ppm. 13C NMR
(100 MHz, CDCl3): δ = 172.4 (C=O), 156.6 (Cq-phenol), 152.4 (Cq-Bf),
150.5 (Cq-Bf), 135.0 (Cq-Bf), 131.3 (Cq-phenol), 128.3 (2 × C-m-Ph-O),
124.7 (C-6Bf), 118.5 (C-4Bf), 114.8 (2 × C-o-Ph-O), 110.6 (C-7Bf),
110.3 (Cq-Bf), 80.3 (Cq-tBu), 63.0 (CH2-O), 56.7 (CH2-N), 46.0 (NMe2),
43.93, 37.9 (CH2-C=O), 31.2 (CH2-Ar), 28.1 (CH3-tBu), 9.4 (CH3-
Bf) ppm. ESI-MS: calcd. for C26H34NO4 [M + H]+ 424.2488, found
424.2982.
+
(CH2-Ar), 9.40 (CH3-Bf) ppm. HRMS (ESI): calcd. for C22H26NO4
[M + H]+ 368.18563, found 368.18594.
(E)-3-(2-{4-[3-(Dimethylamino)propoxy]phenyl}-3-methylbenzo-
furan-5-yl)acrylic Acid (19): Isolated as the trifluoroacetate salt
after tBu ester removal from 18. 1H NMR (400 MHz, CD3OD): δ =
tert-Butyl 3-(2-{4-[3-(Dimethylamino)propoxy]phenyl}-3-meth-
ylbenzofuran-5-yl)propanoate (20): Obtained from 18 in 82 %
yield. 1H NMR (400 MHz, CDCl3): δ = 7.71 (d, Jo-m = 8.8 Hz, 2 H,
7.83–7.71 (m, 4 H, =CH-Ar, 2 × H-m-Phe-O, H-4Bf), 7.55 (dd, J6-7
=
8.6, J6-4 = 1.5 Hz, 1 H, H-6Bf), 7.46 (d, 1 H, H-7Bf), 7.08 (d, Jo-m
=
2 × H-m-Phe-O), 7.38–7.30 (m, 2 H, H-4Bf, H-7Bf), 7.10 (dd, J6-7
=
8.9 Hz, 2 H, 2 × H-o-Phe-O), 6.49 (d, J = 15.9 Hz, 1 H, =CH-C=O),
4.18 (t, JCH2-CH2 = 5.7 Hz, 2 H, CH2-O), 3.39 (t, 2 H, CH2-N), 2.96 (s, 6
H, NMe2), 2.44 (s, 3 H, CH3Bf), 2.31–2.19 (m, 2 H, CH2-CH2-CH2) ppm.
13C NMR (100 MHz, CD3OD): δ = 170.7 (C=O), 159.9 (Cq-phenol),
156.2 (Cq-Bf), 153.1 (Cq-Bf), 147.1 (=CH-Ar), 133.2 (Cq-Bf), 130.6 (Cq-
phenol), 129.2 (2 × C-m-Ph-O), 125.4 (C-6Bf), 125.4 (Cq-Bf), 120.6 (C-
4Bf), 117.7 (=CH-C=O), 115.8 (2 × C-o-Ph-O), 112.1 (C-7Bf), 111.1
(Cq-Bf), 66.0 (CH2-O), 56.8 (CH2-N), 43.6 (NMe2), 25.7 (CH2-CH2-CH2),
8.3, J6-4 = 1.5 Hz, 1 H, H-6Bf), 6.98 (d, 2 H, 2 × H-o-Phe-O), 4.09 (t,
JCH2-CH2 = 6.1 Hz, 2 H, CH2-O), 3.02 (t, JCH2-CH2 = 7.8 Hz, 2 H, CH2-
Ar), 2.83–2.77 (t, 2 H, CH2-N), 2.59 (t, 2 H, CH2-C=O), 2.51 (s, 6 H,
NMe2), 2.41 (s, 3 H, CH3Bf), 2.18–2.08 (m, 2 H, CH2-CH2-CH2), 1.43
(s, 9 H, tBu) ppm. 13C NMR (100 MHz, CDCl3): δ = 172.6 (C=O), 158.4
(Cq-phenol), 152.6 (Cq-Bf), 151.1 (Cq-Bf), 135.0 (Cq-Bf), 131.5 (Cq-
phenol), 128.3 (2 × C-m-Ph-O), 124.6 (C-6Bf), 118.6 (C-4Bf), 114.7
(2 × C-o-Ph-O), 110.6 (C-7Bf), 109.8 (Cq-Bf), 102 (Cq-Bf), 80.5 (Cq-
tBu), 65.7 (CH2-O), 56.2 (CH2-N), 44.5 (NMe2), 38.0 (CH2-C=O), 31.4
(CH2-Ar), 29.84, 28.2 (CH3-tBu), 26.2 (CH2-CH2-CH2), 9.5 (CH3-
Bf) ppm. ESI-MS: calcd. for C27H36NO4 [M + H]+ 438.3, found 438.4.
9.3 (CH3-Bf) ppm. HRMS (ESI): calcd. for C23H28NO4 [M + H]+
+
382.20128, found 382.20177.
3-(2-{4-[3-(Dimethylamino)propoxy]phenyl}-3-methylbenzo-
furan-5-yl)propanoic Acid (21): Isolated as the trifluoroacetate salt
after tBu ester removal from 20. 1H NMR (400 MHz, CD3OD): δ =
7.75 (d, Jo-m = 8.9 Hz, 2 H, 2 × H-m-Phe-O), 7.40 (d, 1 H, H-4Bf), 7.35
(d, 1 H, H-7Bf), 7.15 (dd, J6-7 = 8.4, J6-4 = 1.7 Hz, 1 H, H-6Bf), 7.09
(d, 2 H, 2 × H-o-Phe-O), 4.19 (t, JCH2-CH2 = 5.7 Hz, 2 H, CH2-O), 3.39
(t, JCH2-CH2 = 8.0 Hz, 2 H, CH2-N), 3.03 (t, JCH2-CH2 = 7.6 Hz, 2 H, CH2-
Ar), 2.96 (s, 6 H, NMe2), 2.66 (t, 2 H, CH2-C=O), 2.42 (s, 3 H, CH3Bf),
2.31–2.21 (m, 2 H, CH2-CH2-CH2) ppm. 13C NMR (100 MHz, CD3OD):
δ = 177.0 (C=O), 159.6 (Cq-phenol), 153.8 (Cq-Bf), 152.1 (Cq-Bf), 136.4
(Cq-Bf), 132.6 (Cq-phenol), 129.1 (2 × C-m-Ph-O), 125.9 (Cq-Bf), 125.7
(C-6Bf), 119.5 (C-4Bf), 115.8 (2 × C-o-Ph-O), 111.3 (C-7Bf), 110.9 (Cq-
Bf), 66.0 (CH2-O), 56.8 (CH2-N), 43.7 (NMe2), 37.5 (CH2-C=O), 32.1
(CH2-Ar), 25.7 (CH2-CH2-CH2), 9.4 (CH3-Bf) ppm. HRMS (ESI): calcd.
for C22H26NO4+ [M + H]+ 382.20128, found 382.20177.
Hydrolysis of tert-Butyl Esters: Synthesis of 8, 15, 17, 19, 21: To
a DCM solution (1.8 mL) of tert-butyl ester (7, 14, 16, 18 or 20;
0.1 mmol) under a flux of nitrogen, TFA (400 μL) was added drop-
wise. The reaction was stirred for 2 h then concentrated in vacuo.
TFA was co-evaporated with toluene to afford pure product (quanti-
tative yield) without any further purification.
(E)-3-[2-(4-Hydroxyphenyl)-3-methylbenzofuran-5-yl]acrylic
1
Acid (8): H NMR (400 MHz, CD3OD): δ = 7.82 (d, Jtrans = 16.0 Hz, 1
H, =CH-Ar), 7.78 (d, 1 H, H-4Bf), 7.66 (d, Jo-m = 8.8 Hz, 2 H, 2 × H-m-
Phe-O), 7.55 (dd, J6-7 = 8.5, J6-4 = 1.6 Hz, 1 H, H-6Bf), 7.47 (d, 1 H,
H-7Bf), 6.92 (d, 2 H, 2 × H-o-Phe-O), 6.52 (d, 1 H, =CH-C=O), 2.45 (s,
3 H, CH3Bf) ppm. 13C NMR (100 MHz, CD3OD): δ = 176.1 (C=O),
159.0 (Cq-phenol), 156.2 (Cq-Bf), 153.8 (Cq-Bf), 146.9 (=CH-Ar), 133.3
(Cq-Bf), 131.0 (=CH-COOH), 130.6 (Cq-phenol), 129.4 (2 × C-m-Ph-O),
125.2 (C-6Bf), 123.6 (Cq-Bf), 120.4 (C-4Bf), 116.6 (2 × C-o-Ph-O),
112.0 (C-7Bf), 110.3 (Cq-Bf), 9.3 (CH3-Bf) ppm. HRMS (ESI): calcd. for
C18H13O4 [M – H]– 293.08193, found 293.08172.
4-[5-Chloro-3-methylbenzofuran-2-yl]-phenoxy-2-hydroxy-3-
dimethylaminopropane (25): Dimethylamine (33 % in EtOH,
0.1 mL, 0.56 mmol) was added to a solution of epoxide 23 (39.2 mg,
0.125 mmol) in anhydrous ethanol (2 mL) and the reaction was
stirred at 80 °C for 1 h (TLC 6:4 Hex/EtOAc and 9:1 CHCl3/MeOH+
1 % TEA). The solvent and the excess of amine were evaporated at
reduced pressure obtaining the crude product (44 mg) that was
purified by flash chromatography (9:1 CHCl3/MeOH + 1 % TEA). De-
sired product 25 was obtained as a white solid in 82 % yield
(36.7 mg). 1H NMR (400 MHz, CDCl3): δ = 7.71 (d, Jo-m = 8.9 Hz, 2
(E)-3-(2-{4-[2-(Dimethylamino)ethoxy]phenyl}-3-methylbenzo-
furan-5-yl)acrylic Acid (15): Isolated as the trifluoroacetate salt
after tBu ester removal from 14. 1H NMR (400 MHz, CD3OD): δ =
7.84–7.76 (m, 4 H, =CH-Ar, 2 × H-m-Phe-O, H-4Bf), 7.56 (dd, J6-7
=
8.5, J6-4 = 1.5 Hz, 1 H, H-6Bf), 7.47 (d, 1 H, H-7Bf), 7.17 (d, Jo-m
=
8.9 Hz, 2 H, 2 × H-o-Phe-O), 6.49 (d, Jtrans = 16.0 Hz, 1 H, =CH-C=O),
4.46–4.41 (m, 2 H, CH2-O), 3.67–3.61 (m, 2 H, CH2-N), 3.02 (s, 6 H, H, 2 × H-m-Phe-O), 7.45 (d, J4-6 = 2.1 Hz, 1 H, H4-Bf), 7.36 (d, J7-6
=
NMe2), 2.46 (s, 3 H, CH3Bf) ppm. 13C NMR (100 MHz, CD3OD): δ =
8.6 Hz, 1 H, H7-Bf), 7.20 (dd, 1 H, H6-Bf), 7.03 (d, 2 H, 2 × H-o-Phe-
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O), 4.11 (m, 1 H, CHOH), 4.04 (d, Jvic = 4.9 Hz, 2 H, CH2O), 2.59 (dd,
Jgem = 12.2, Jvic = 9.9 Hz, 1 H, CH2N), 2.43 (dd, Jvic = 3.6 Hz, 1 H,
CH2N), 2.40 (s, 3 H, CH3-Bf), 2.36 (s, 6 H, NMe2) ppm. 13C NMR
(100 MHz, CDCl3): δ = 158.9 (Cq), 152.4 (Cq), 152.1 (Cq), 132.9 (Cq),
128.3 (C-m-Ph-O), 128.0 (Cq), 124.1 (C6-Bf), 124.0 (Cq), 118.8 (C4-Bf),
114.9 (C-o-Ph-O), 111.8 (C7-Bf), 109.5 (Cq), 70.6 (CH2O), 66.2 (CHOH),
61.9 (CH2N), 45.7 (NMe2), 9.5 (CH3-Bf) ppm. MS (ESI+): calcd. for
119.9 (C-4Bf), 116.1 (2 × C-o-Ph-O), 112.9 (C-7Bf), 110.8 (Cq), 71.2
(CH2-O), 67.0 (CH-O), 61.0 (CH-N), 50.6 (CH2-N), 30.8 (CH2-cycle), 30.7
(CH2-cycle), 25.2 (CH2-cycle), 9.5 (CH3-Bf) ppm. ESI-MS: calcd. for
C23H27ClNO3 [M + H]+ 400.9, found 400.2.
1-Azido-3-[4-(5-chloro-3-methylbenzofuran-2-yl)phenoxy]-
propan-2-ol (29): Epoxide 23 (39 mg, 0.12 mmol) was dissolved in
1,4-dioxane (0.7 mL). A solution of NaN3 (24 mg, 0.37 mmol) and
Yb(OTf)3 (15.5 mg, 36 μmol) in H2O (0.18 mL) was added and the
solution was stirred for 5 d at 80 °C. The reaction was followed by
TLC (7:3 Hex/EtOAc, Rf = 0.39). The reaction mixture was diluted
with EtOAc, washed once with H2O and once with brine. The or-
ganic layer was dried with Na2SO4 and concentrated in vacuum.
The crude mixture was purified by flash chromatography (55:45 tol-
uene/DCM, Rf = 0.3) leading to product 29 (34 mg, 97 μmol) in
C
20H22ClNO3 [M + H]+ 360.1, found 360.3.
1-[4-(5-Chloro-3-methylbenzofuran-2-yl)phenoxy]-3-(N-methyl-
piperazyl)propan-2-ol (26): To a solution of epoxide 23 (0.1 mmol)
in dry EtOH (1 mL) 1-methylpiperazine (0.5 mmol) was added. The
reaction was stirred for 2 h at 80 °C, monitoring by TLC (7:3 toluene/
EtOAc and 9:1 CHCl3/MeOH). The solvent was evaporated under
vacuum and the crude purified by flash chromatography (8:2 CHCl3/
MeOH) to afford 26 in 58 % yield. 1H NMR (400 MHz, CDCl3): δ =
7.72 (d, Jo-m = 8.9 Hz, 2 H, 2 × H-m-Phe-O), 7.46 (d, J4-6 = 2.0 Hz, 1
H, H4-Bf), 7.36 (d, J7-6 = 8.6 Hz, 1 H, H7-Bf), 7.21 (dd, 1 H, H6-Bf),
1
81 % yield. H NMR (400 MHz, CDCl3): δ = 7.73 (d, Jo-m = 8.8 Hz, 2
H, 2 × H-m-Phe-O), 7.47 (d, J4-6 = 2 Hz, 1 H, H-4Bf), 7.37 (d, J7-6
=
8.8 Hz, 2 H, H-7Bf), 7.21 (dd, 1 H, H-6Bf), 7.03 (d, 2 H, H-o-Phe-O),
4.26–4.18 (m, 1 H, CH-O), 4.08 (d, Jvic = 5 Hz, 2 H, CH2-O), 3.60 (dd,
7.03 (d, 2 H, 2 × H-o-Phe-O), 4.15 (m, 1 H, CHOH), 4.05 (d, Jvic
=
4.9 Hz, 2 H, CH2O), 2.81–2.56 (m, 10 H, CH2N), 2.40 (s, 3 H, CH3-Bf),
2.37 (s, 3 H, NMe) ppm. 13C NMR (100 MHz, CDCl3): δ = 158.9 (Cq),
152.1 (Cq), 132.9 (Cq), 128.4 (C-m-Ph-O), 128.1 (Cq), 124.1 (C6-Bf),
118.9 (C4-Bf ), 114.9 (C-o-Ph-O), 111.9 (C7-Bf), 109.6 (Cq), 70.4
( C H 2 O ) , 6 5 . 6 ( C H O H ) , 6 0 . 5 ( C H 2 N C H 2 C H 2 N M e ) , 5 5 . 0
(CH2NCH2CH2NMe), 45.8 (NCH3), 9.5 (CH3-Bf) ppm. MS (ESI+): calcd.
for C23H28ClN2O3 [M + H]+ 415.2, found 415.5.
Jgem = 12.7, Jvic = 4.7 Hz, 1 H, CH2-N3), 3.54 (dd, Jgem = 12.7, Jvic =
6.0 Hz, 1 H, CH2-N3), 2.41 (s, 3 H, CH3-Bf) ppm. 13C NMR (100 MHz,
CDCl3): δ = 158.3 (Cq), 132.8 (Cq), 128.4 (2 × C-m-Ph-O), 128.1 (Cq),
124.6 (Cq), 124.2 (C-6Bf), 118.9 (C-4Bf), 114.9 (2 × C-o-Ph-O), 111.9
(C-7Bf), 109.8 (Cq), 69.4 (CH-O), 69.3 (CH2-O), 53.5 (CH2-N3), 9.5 (CH3-
Bf) ppm. ESI-MS: calcd. for C18H17ClN3O3 [M + H]+ 358.8, found
358.4.
1-[4-(5-Chloro-3-methylbenzofuran-2-yl)phenoxy]-3-(cyclo-
propylamino)propan-2-ol (27): Epoxide 23 (20 mg, 63 μmol) was
suspended in a solution of DCM/EtOH (0.6 mL, 9:1 ratio). Cyclo-
1-Amino-3-[4-(5-chloro-3-methylbenzofuran-2-yl)phenoxy]-
propan-2-ol (30): Azide 29 (20 mg, 56 μmol) was dissolved in
MeOH (0.46 mL). Morpholine (1.3 μL, 15 μmol) and PtO2 (1.3 mg,
propylamine (13 μL, 0.19 mmol) was added to the reaction mixture. 2 μmol) were added to the solution. The reaction mixture was
The reaction was stirred for 24 h at 40 °C. The reaction was followed
by TLC chromatography (9:1:0.1 DCM/MeOH/TEA, Rf = 0.47). The
solution was concentrated in vacuo. The crude material was purified
by flash chromatography (96:4 CHCl3/MeOH) leading to product 27
(13 mg, 23 μmol) in 60 % yield. 1H NMR (400 MHz, CD3OD): δ =
stirred for 1 h under H2 atmosphere; the reaction progress was fol-
lowed by TLC (8:2:0.2 CHCl3/MeOH/AcOH, Rf = 0.49). The reaction
mixture was filtered through a celite pad and concentrated in vac-
uum. The crude product was purified by flash chromatography
(75:25:0.2 CHCl3/MeOH/AcOH, Rf = 0.35) leading to product 30
7.76 (d, Jo-m = 8.5 Hz, 2 H, 2 × H-m-Phe-O), 7.53 (d, J4-6 = 2 Hz, 1 H, (13 mg, 40 μmol) in 71 % yield. 1H NMR (400 MHz, CD3OD): δ =
H-4Bf), 7.41 (d, J7-6 = 8.5 Hz, 2 H, H-7Bf), 7.23 (dd, 1 H, H-6Bf), 7.12
(d, 2 H, 2 × H-o-Phe-O), 4.36–4.29 (m, 1 H, CH-O), 4.15–4.06 (m, 2 H,
7.71 (d, Jo-m = 8.8 Hz, 2 H, 2 × H-m-Phe-O), 7.50 (d, J4-6 = 2 Hz, 1 H,
H-4Bf), 7.39 (d, J7-6 = 8.8 Hz, 2 H, H-7Bf), 7.20 (dd, 1 H, H-6Bf), 7.08
CH2-O), 3.88 (br. s, 1 H, NH), 3.43 (dd, Jgem = 12, Jvic = 2 Hz, 1 H, (d, 2 H, H-o-Phe-O), 4.06–4.00 (m, 2 H, CH2-O), 3.99–3.92 (m, 1 H,
CH2-N), 3.30–3.25 (m, 1 H, CH2-N), 2.85–2.78 (m, 1 H, CH-N), 2.41 (s,
3 H, CH3-Bf), 1.00–0.91 (m, 4 H, CH2-cycle) ppm. 13C NMR (100 MHz,
CD3OD): δ = 160.0 (Cq), 153.5 (Cq), 153.3 (Cq), 134.0 (Cq), 129.3
CH-O), 2.96–2.84 (m, 1 H, CH2-NH2), 2.84–2.72 (m, 1 H, CH2-NH2),
2.38 (s, 3 H, CH3-Bf) ppm. 13C NMR (100 MHz, CD3OD): δ = 160.5
(Cq), 153.7 (Cq), 153.4 (Cq), 134.1 (Cq), 129.3 (2 × C-m-Ph-O), 129.1
(2 × C-m-Ph-O), 129.1 (Cq), 125.3 (Cq), 125.1 (C-6Bf), 119.7 (C-4Bf), (Cq), 125.0 (C-6Bf), 125.0 (Cq), 119.7 (C-4Bf), 115.9 (2 × C-o-Ph-O),
115.9 (2 × C-o-Ph-O), 112.7 (C-7Bf), 110.7 (Cq), 71.0 (CH2-O), 66.3
112.7 (C-7Bf), 110.5 (Cq), 71.6 (CH-O), 71.4 (CH2-O), 45.1 (CH2-NH2),
(CH-O), 51.9 (CH2-N), 31.5 (CH-N), 9.3 (CH3-Bf), 4.3 (CH2-cycle), 4.0
9.3 (Cp, CH3-Bf) ppm. ESI-MS: calcd. for C18H19ClNO3 [M + H]+ 332.1,
(CH2-cycle) ppm. ESI-MS: calcd. for C23H27ClNO3 [M + H]+ 372.9, found 332.0.
found 372.8.
3-[4-(5-Chloro-3-methylbenzofuran-2-yl)phenoxy]propan-1,2-
diol (31): Epoxide 23 (20 mg, 0.064 mmol) was dissolved in 1,4-
dioxane (0.3 mL). H2O (75 μL) and 4 solution of HCl in dioxane
1-[4-(5-Chloro-3-methylbenzofuran-2-yl)phenoxy]-3-(cyclo-
pentylamino)propan-2-ol (28): Obtained from 23 and cyclo-
pentylamine with the same procedure described above for 27. The
reaction was followed by TLC chromatography (6:4 Hex/EtOAc, Rf =
0.1). The solution was concentrated in vacuo. The crude reaction
was purified by flash chromatography (97:3 CHCl3/MeOH, Rf = 0.3)
leading to product 28 (24 mg, 59 μmol) in 93 % yield. 1H NMR
(400 MHz, 9:1 CD3OD:1,4-[D8]dioxane): δ = 7.73 (d, Jo-m = 8.8 Hz, 2
M
(32 μL) were added to the reaction mixture. The reaction was stirred
for 5 h at 80 °C and its progress was followed by TLC chromatogra-
phy (7:3 Hex/EtOAc, Rf = 0.4). NaOH was added until pH = 7 was
reached. The reaction mixture was diluted with EtOAc and washed
with H2O. The water layer was extracted with EtOAc three times.
The organic layer was dried with Na2SO4 and concentrated in vac-
uum. The crude product was purified by flash chromatography (8:2
H, 2 × H-m-Phe-O), 7.50 (d, J4-6 = 2 Hz, 1 H, H-4Bf), 7.40 (d, J7-6
=
8.8 Hz, 2 H, H-7Bf), 7.21 (dd, 1 H, H-6Bf), 7.10 (d, 2 H, 2 × H-o-Phe-
O), 4.34–4.26 (m, 1 H, CH-O), 4.14–4.05 (m, 2 H, CH2-O), 3.69–3.55 60 % yield. H NMR (400 MHz, CD3OD): δ = 7.73 (d, Jo-m = 8.9 Hz, 2
Hex/EtOAc, Rf = 0.28) leading to product 31 (6 mg, 38 μmol) in
1
(m, 1 H, CH-N), 3.36–3.28 (m, 1 H, CH2-N), 3.21–3.12 (m, 1 H, CH2- H, 2 × H-m-Phe-O), 7.51 (d, J4-6 = 2 Hz, 1 H, H-4Bf), 7.40 (d, J7-6
=
N), 2.37 (s, 3 H, CH3-Bf), 2.22–2.10 (m, 2 H, CH2-cycle), 1.89–1.78 (m,
8.8 Hz, 2 H, H-7Bf), 7.22 (dd, 1 H, H-6Bf), 7.09 (d, 2 H, 2 × H-o-Phe-
O), 4.13 (dd, Jgem = 9.3, Jvic = 3.9 Hz, 1 H, CH2-O), 4.06–3.97 (m, 3 H,
2 H, CH2-cycle), 1.62 (m, 4 H, CH2-cycle) ppm. 13C NMR (100 MHz,
9:1 CD3OD:1,4-[D8]dioxane): δ = 160.2 (Cq), 153.6 (Cq), 153.5 (Cq), CH2-O, CH-O), 3.71 (dd, Jgem = 11.2, Jvic = 5.3 Hz, 1 H, CH2-OH), 3.67
134.2 (Cq), 129.4 (2 × C-m-Ph-O), 129.3 (Cq), 125.4 (Cq), 125.2 (C-6Bf),
(dd, Jvic = 5.3 Hz, 1 H, CH2-OH), (s, 3 H, CH3-Bf) ppm. 13C NMR
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(100 MHz, CD3OD): δ = 160.6 (Cq), 153.8 (Cq), 134.1 (Cq), 130.2 (Cq), (C=O), 157.9 (Cq), 152.2 (Cq), 132.8 (Cq), 128.5 (2 × C-m-Ph-O), 128.1
129.3 (2 × CH-m-Phe-O), 129.1 (Cq), 125.0 (C-6Bf), 124.9 (Cq), 119.7
(C-4Bf), 115.9 (2 × C-o-Phe-O), 112.7 (C-7Bf), 110.5 (Cq), 71.8 (CH-
O), 70.4 (CH2-O), 64.1 (CH2-OH), 9.3 (CH3-Bf) ppm. ESI-MS: calcd. for
C18H18ClO4 [M + H]+ 333.1, found 333.2.
(Cq), 124.8 (Cq), 124.2 (C-6Bf), 118.9 (C-4Bf), 115.0 (2 × C-o-Ph-O),
111.9 (C-7Bf), 109.9 (Cq), 67.8 (CH2OAr), 55.1 (CH2N), 54.6 (CH2N),
46.0 (NCH3), 45.1 (CH2NMe), 41.9 (CH2NMe), 9.5 (CH3-Bf) ppm. ESI-
MS: calcd. for C22H24ClN2O3 [M + H]+ 399.1475, found 399.1793.
1-[4-(5-Bromo-3-methylbenzofuran-2-yl)phenoxy]-3-(N-methyl-
piperazyl)propan-2-ol (32): To a solution of the epoxide 24
(0.1 mmol) in dry EtOH (1 mL) 1-methylpiperazine was added
(0.5 mmol). The reaction was stirred for 2 h at 80 °C, monitoring
by TLC (7:3 Toluene/EtOAc and 9:1 CHCl3/MeOH). The solvent was
evaporated under vacuum and the crude purified by flash chroma-
tography (95:5 CHCl3/MeOH) to afford 32 in 45 % yield. 1H NMR
(400 MHz, CDCl3): δ = 7.72 (d, Jo-m = 9.2 Hz, 2 H, 2 × H-m-Phe-O),
7.62 (d, J4-6 = 1.5 Hz, 1 H, H4-Bf), 7.35 (dd, J6-7 = 8.4 Hz, 2 H, H6Bf),
7.32 (d, 1 H, H7-Bf), 7.03 (d, 2 H, 2 × H-m-Phe-O), 4.18–4.12 (m, 1
H, CH-O), 4.05 (d, Jvic = 4.9 Hz, 2 H, CH2-O), 2.18–2.16 (br. s, 2 H,
4-[5-Chloro-3-methylbenzofuran-2-yl]phenyl 2,3,4-Tri-O-ben-
zoyl-6-azido-α-D
-mannopyranoside (37): Azide 36[14] (0.17 g,
0.25 mmol) and phenol 1a (58 mg, 0.23 mmol) were co-evaporated
three times with toluene and dissolved in dry DCM (4.6 mL) under
nitrogen atmosphere. To the solution freshly activated molecular
sieves (4 Å) were added. The reaction mixture was cooled to –30 °C
and TMSOTf (8.3 μL, 46 μmol) was slowly added. The reaction was
stirred for 30 min and followed by TLC (9:1 toluene/EtOAc, Rf =
0.91). To the solution TEA (0.13 mL) was added. The reaction was
warmed to room temperature and concentrated in vacuo. The crude
material was purified by flash chromatography (8:1:1 Hex/EtOAc/
NCH2CH2NMe), 2.60–2.58 (m, 8 H, CH2NCH2CH2NMe), 2.40 (s, 3 H, toluene) leading to product 37 (87 mg, 0.13 mmol) in 50 % yield.
CH3-N), 2.39 (s, 3 H, CH3Bf) ppm. 13C NMR (100 MHz, CDCl3): δ =
158.9 (Cq), 152.5 (Cq), 152.2 (Cq), 133.5 (Cq), 128.4 (2 × C-m-Phe-O),
126.8 (C-6Bf), 124.1 (Cq), 122.0 (C-4Bf), 115.5 (Cq), 115.0 (2 × C-m-
Phe-O), 112.4 (C-7Bf ), 70.4 (CH2 -O), 65.7 (CH-OH), 60.5
(CH2NCH2CH2NMe), 55.0 (CH2NCH2CH2NMe), 45.7 (CH3-N), 9.3
(CH3Bf) ppm. ESI-MS: calcd. for C23H28BrN2O3 [M + H]+ 460.4, found
459.9.
1H NMR (400 MHz, CDCl3): δ = 8.16 (d, Jo-m = 7.6 Hz, 2 H, 2 × o-CH-
Bz), 7.98 (d, Jo-m = 7.6 Hz, 2 H, 2 × o-CH-Bz), 7.87 (d, Jo-m = 7.8 Hz,
2 H, 2 × o-CH-Bz), 7.81 (d, Jp-m = 9.0 Hz, 2 H, 2 × m-CH-Bz), 7.70–
7.63 (m, 1 H, p-CH-Bz), 7.57–7.50 (m, 3 H, p-CH-Bz, 2 × m-CH-Bz),
7.49 (d, J4-6 = 2.2 Hz, 1 H, H-4Bf), 7.47–7.44 (m, 1 H, p-CH-Bz), 7.42–
7.37 (m, 2 H, 2 × m-CH-Bz), 7.39 (d, J7-6 = 8.5 Hz, 1 H, H-7Bf), 7.34
(d, 2 H, H-o-Phe-O), 7.32–7.27 (m, 2 H, 2 × m-CH-Bz), 7.23 (dd, 1 H,
H-6Bf), 6.14 (dd, J3′-4′ = 10.0, J3′-2′ = 3.2 Hz, 1 H, H-3), 6.00 (dd,
4-[5-Chloro-3-methylbenzofuran-2-yl]phenoxyacetylmorph-
oline (34): To a solution of 33 (19.7 mg, 0.062 mmol) in dry THF
(1.2 mL) were added EDC·HCl (15.5 mg, 0.081 mmol) and TEA (22 μL,
0.155 mmol) and the reaction was stirred at 30 °C under nitrogen
atmosphere for 20 h. Morpholine (11 μL, 0.124 mmol) was added
and the reaction stirred for 2 h at 45 °C. After the reaction was
complete (TLC 95:5:0.1 CHCl3/MeOH/HCOOH) the reaction mixture
J4′-5′ = 10.0 Hz, 1 H, H-4′), 5.93–5.90 (m, 1 H, H-2′), 5.9 (d, J1′-2′
=
1.9 Hz, 1 H, H-1′), 4.43–4.37 (m, 1 H, H-5′), 3.54 (dd, J5′-6′a = 6, J6′a-6′
= 13.3 Hz, 1 H, H-6′a), 3.45 (dd, J5′-6′b = 2.5 Hz, 1 H, H-6′b), 2.44
b
(s, 3 H, CH3-Bf) ppm. 13C NMR (100 MHz, CDCl3): δ = 165.6 (C=O
Bz), 165.5 (C=O), 155.6 (Cq Bf), 152.1 (Cq Bf), 151.8 (Cq Bf), 133.8
(p-CH-Bz), 133.7 (p-CH-Bz), 133.4 (p-CH-Bz), 132.7 (Cq), 130.0 (o-CH-
Bz), 129.9 (o-CH-Bz), 129.8 (o-CH-Bz), 129.0 (CqBz), 128.9 (CqBz),
128.8 (m-CH-Bz), 128.7 (CqBz), 128.6 (m-CH-Bz), 128.4 (C-m-Phe-O),
128.0 (Cq Bf), 126.0 (Cq Bf), 124.3 (C-6Bf), 118.9 (C-4Bf), 116.7 (C-o-
Phe-O), 111.9 (C-7Bf), 110.2 (Cq Bf), 95.7 (C-1′), 71.2 (C-5′), 70.1
(C-2′), 69.5 (C-3′), 67.4 (C-4′), 51.2 (C-6), 9.4 (CH3-Bf) ppm. ESI-MS:
calcd. for C42H32ClN3NaO9 [M + Na]+ 781.2, found 781.1.
was diluted with Et2O and washed with 0.1
M HCl. The organic
phase was dried with anhydrous Na2SO4 and the solvent was evap-
orated in vacuo. The crude product was purified by flash chroma-
tography (4:6 Hex/EtOAc) to render desired product 34 (8.4 mg) as
1
a white solid in 35 % yield. H NMR (400 MHz, CDCl3): δ = 7.73 (d,
Jo-m = 8.0 Hz, 2 H, 2 × CH-m-Ph-O), 7.47 (s, 1 H, H-4Bf), 7.37 (d,
J7-6 = 8.4 Hz, 1 H, H-7Bf), 7.21 (d, 1 H, H-6Bf), 7.06 (d, 2 H, 2 × C-o-
Ph-O), 4.76 (s, 2 H, CH2OAr), 3.66 (m, 8 H, CH2-Morf), 2.41 (s, 3 H,
CH3-Bf) ppm. 13C NMR (100 MHz, CDCl3): δ = 166.5 (C=O), 157.8
4-[5-Chloro-3-methylbenzofuran-2-yl]phenyl 6-Azido-α-D-
mannopyranoside (38): Compound 37 (20 mg, 26 μmol) was dis-
solved in MeOH (0.26 mL) under nitrogen atmosphere. To the reac-
(Cq), 152.2 (Cq), 132.8 (Cq), 128.5 (2 × C-m-Ph-O), 128.1 (Cq), 124.9 tion mixture MeONa (3 mg, 53 μmol) was added. The reaction was
(Cq), 124.2 (C-6Bf), 118.9 (C-4Bf), 115.0 (2 × C-o-Ph-O), 111.9 (C-7Bf), stirred for 2.5 h and was followed by TLC (7:3 Hex/EtOAc). The solu-
67.8 (CH2OAr), 67.0 (2 × CH2O), 46.1 (CH2N), 42.6 (CH2N), 9.5 (CH3- tion was diluted with MeOH and treated with Amberlite IR120 to
Bf) ppm. HRMS (ESI): calcd. for C21H20ClNNaO4 [M + Na]+ 408.0979,
found 408.1089.
reach pH = 7. The crude product was purified by flash chromatogra-
phy (96:4 DCM/MeOH, Rf = 0.28) leading to product 38 (7 mg,
17 μmol) in 79 % yield. [α]2D5 = +81 (c = 0.1, MeOH). 1H NMR
(400 MHz, CD3OD): δ = 7.74 (d, Jo-m = 8.8 Hz, 2 H, H-m-Phe-O), 7.50
(d, J4-6 = 2.0 Hz, 1 H, H-4Bf), 7.40 (d, J7-6 = 8.7 Hz, 1 H, H-7Bf), 7.26
(d, 2 H, H-o-Phe-O), 7.21 (d, 1 H, H-6Bf), 5.59 (d, J1-2 = 1.6 Hz, 1 H,
H-1′), 4.07 (dd, J2′-3′ = 3.4 Hz, 1 H, H-2′), 3.92 (dd, J3′-4′ = 8.8 Hz, 1
H, H-3′), 3.78–3.67 (m, 2 H, H-4′,H-5′), 3.50–3.40 (m, 2 H, H-6′), 2.44
(s, 3 H, CH3-Bf) ppm. 13C NMR (100 MHz, CD3OD): δ = 157.4 (Cq),
153.2 (Cq), 133.8 (Cq), 129.1 (C-m-Phe-O), 129.0 (Cq), 126.1 (Cq), 125.0
(C-6Bf), 119.6 (C-4Bf), 117.5 (C-m-Phe-O), 112.6 (C-7Bf), 110.8 (Cq),
99.4 (C-1′), 74.5 (C-5′), 72.0 (C-3′), 71.5 (C-2′), 69.0 (C-4′), 52.6 (C-6′),
9.4 (CH3-Bf) ppm. ESI-MS: calcd. for C21H21ClN3NaO9 [M + H]+ 446.1,
found 446.5.
4-[5-Chloro-3-methylbenzofuran-2-yl]-phenoxyacetyl-1-methyl-
piperazine (35): Oxalyl chloride (0.27 mmol, 2.0
M solution in dry
DCM) was added to a solution of 33 (28.8 mg, 0.091 mmol) in dry
DCM (0.9 mL) and the reaction was stirred at reflux under nitrogen
atmosphere for 2 h. The solvent was co-evaporated with toluene at
reduced pressure, the crude reaction was re-dissolved in dry DCM
(0.1 mL) and 1-methylpiperazine (300 μL, 2.7 mmol) was added. The
reaction was stirred at 40 °C overnight (TLC 9:1:0.1 CHCl3/MeOH/
TEA). The solvent was evaporated in vacuo and the crude product
was purified using an automatic system (SiO2, DCM/MeOH gradient
from 0 to 14 % MeOH) to afford desired product 35 (28 mg) as a
white solid in 78 % yield. 1H NMR (400 MHz, CDCl3): δ = 7.73 (d,
Jo-m = 8.9 Hz, 2 H, 2 × CH-m-Ph-O), 7.46 (d, J4-6 = 2.1 Hz, 1 H, 4-[5-Chloro-3-methylbenzofuran-2-yl]phenyl 2,3,4-Tri-O-acetyl-
H-4Bf), 7.36 (d, J7-6 = 8.6 Hz, 1 H, H-7Bf), 7.21 (dd, 1 H, H-6Bf), 7.06
(d, 2 H, 2 × CH-o-Ph-O), 4.75 (s, 2 H, CH2OAr), 3.70 (m, 2 H, CH2N),
6-azido-ꢀ-
D
-glucopyranoside (40): 2,3,4-O-Acetyl-6-azido-α-
D-
glucopyranosyl bromide 39[15] (189 mg, 0.48 mmol) and benzofuran
3.66 (m, 2 H, CH2N), 2.46 (m, 4 H, 2 × CH2NMe), 2.40 (s, 3 H, CH3- 1a (40 mg, 0.16 mmol) were diluted in CHCl3 (1.6 mL). To the reac-
Bf), 2.34 (s, 3 H, NMe) ppm. 13C NMR (100 MHz, CDCl3): δ = 166.3
tion mixture, Bu4NHSO4 (27 mg, 0.08 mmol) was added, followed
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Full Paper
by K2CO3 (132.7 mg, 138.1 mmol) and water (6.63 μL, 18 mmol).
The conversion was detected through TLC (7:3 Hex/EtOAc, Rf =
0.35). After 15 h of stirring, a change in color from yellow to brown
(OCOCH3), 169.1 (OCOCH3), 92.1 (C-1α), 90.2 (C-1ꢀ), 69.9 (C-3α), 68.5,
68.1, 67.1, 67.01, 66.7, (C-2ꢀ, C-3ꢀ, C-4ꢀ, C-2α, C-4α), 63.9, 62.8 (C-
5ꢀ, C-5α), 20.9–20.6 (OCOCH3) ppm. HBr (33 % in AcOH, 21.9 mL,
was observed and the reaction was diluted with CHCl3. 1
M
HCl was
12.3 mmol) was added dropwise to L-Ara(OAc)4 (1.3 g, 4.1 mmol) at
added until acidic pH. The organic layer was extracted with NaHCO3
(2 × 20 mL), dried with Na2SO4 and concentrated in vacuo. Desired
0 °C under nitrogen atmosphere. The reaction mixture was warmed
to room temperature and after 15 min DCM was added (2.4 mL).
product 40 (35 mg, 0.06 mmol) was purified by flash chromatogra- The reaction was then stirred at room temperature for 25 min (TLC
phy (8:2 Hex/EtOAc) and obtained in 40 % yield. 1H NMR (400 MHz,
CDCl3): δ = 7.74 (d, Jo-m = 8.8 Hz, 2 H, H-m-Phe-O), 7.48 (d, J4-6
2.0 Hz, 1 H, H-4Bf), 7.37 (d, J7-6 = 8.8 Hz, 1 H, H-7Bf), 7.22 (d, 1 H,
65:35 Hex/EtOAc). The reaction was quenched adding ice and DCM
at 0 °C. The organic phase was washed with cold water and cold
saturated NaHCO3 solution (3 × 10 mL) and finally dried with anhy-
=
H-6Bf), 7.18 (d, 2 H, H-o-Phe-O), 5.34–5.29 (m, 2 H, H-2′, H-3′), 5.18– drous Na2SO4. The solvent was evaporated in vacuo and the crude
5.14 (m, 1 H, H-1′), 5.10–5.03 (m, 1 H, H-4′), 3.95–3.88 (m, 1 H, H-5′ product 50 (1.1 g) was obtained in 79 % yield as the single ꢀ isomer
), 3.51 (dd, J5′-6′a = 2.4, J6′a-6′b = 11 Hz, 1 H, H-6′a), 3.43 (dd, J5′-6′b
8 Hz, 1 H, H-6′b), 2.41 (s, 3 H, CH3-Bf), 2.09 (s, 3 H, OCOCH3), 2.08 silica). H NMR (400 MHz, CDCl3): δ = 6.69 (d, J1-2 = 3.7 Hz, 1 H, H-
(s, 3 H, OCOCH3), 2.05 (s, 3 H, OCOCH3) ppm. 13C NMR (100 MHz,
1), 5.39 (m, 2 H, H-3, H-4), 5.08 (m, 1 H, H-2), 4.20 (d, J5eq-5ax
=
and used without further purification (the product is not stable on
1
=
CDCl3): δ = 169.6 (Cq=O), 169.4 (Cq=O), 169.3 (Cq=O), 156.7 (Cq), 13.3 Hz, 1 H, H-5eq), 3.93 (dd, J5ax-4 = 1.9 Hz, 1 H, H-5ax), 2.15 (s, 3
152.1 (Cq), 151.8 (Cq), 132.7 (Cq), 128.3 (C-m-Ph-O), 128.1 (Cq), 126.3 H, OCOCH3), 2.11 (s, 3 H, OCOCH3), 2.02 (s, 3 H, OCOCH3) ppm. 13C
(Cq), 124.3 (C-6Bf), 119.0 (C-4Bf), 117.2 (C-o-Ph-O), 111.9 (C-7Bf), NMR (100 MHz, CDCl3): δ = 170.2 (OCOCH3), 170.2 (OCOCH3), 169.9
110.3 (Cq), 98.9 (C-1′), 74.3 (C-5′), 72.5 (C-3′), 71.3 (C-2′), 71.0 (C-4′),
(OCOCH3), 89.8 (C-1), 68.0, 67.9, 67.7 (C-3, C-2, C-4), 64.8 (C-5), 20.9
30.5 (C-6′), 20.7 (3 × CH3-Ac), 9.5 (CH3-Bf) ppm. ESI-MS: calcd. for (OCOCH3), 20.8 (OCOCH3), 20.7 (OCOCH3) ppm.
C27H26ClN3NaO9 [M + Na]+ 595.1, found 594.9.
2,3,4-Tri-O-acetyl-ꢀ-
described above for the
D
-arabinopyranosyl Bromide: Synthesized as
-enantiomer with similar yields and identi-
4-[5-Chloro-3-methylbenzofuran-2-yl]phenyl 6-Azido-ꢀ-
D
-
L
glucopyranoside (41): Compound 40 (37 mg, 0.06 mmol) was sus-
cal spectroscopic data.
pended in MeOH (900 μL) at 40 °C under nitrogen atmosphere. A
4-[5-Chloro-3-methylbenzofuran-2-yl]phenoxy α-L-Arabino-
1
M
solution of MeONa in MeOH (180 μL, 0.18 mmol) was added.
The reaction was followed by TLC (9:1 CHCl3/MeOH, Rf = 0.37) After
stirring at 40 °C for 3 d, the reaction was quenched with 1 acetic
pyranoside (44): TBAHSO4 (20 mg, 0.056 mmol), potassium carb-
onate (98.7 mg, 0.71 mmol) and water [5 μL, 5 % w/w in K2CO3]
were added to a mixture of 1a (30.7 mg, 0.12 mmol) and 2,3,4-tri-
M
acid in MeOH (189 μL, 0.18 mmol) and concentrated in vacuo. DCM
was added to the crude and after centrifugation, the liquid phase
containing all the impurities was pipetted out. The resulting pellet
was re-suspended in MeOH and stirred at 50 °C for 5 min before
centrifuging. Unreacted starting material (40, 4 mg) was recovered
in the supernatant. The remaining pellet contained final product 41,
obtained in 64 % yield (17 mg, 0.038 mmol) and sufficient purity,
O-acetyl-ꢀ- -arabinopiranosyl bromide 50 (80.5 mg, 0.24 mmol) in
chloroform (0.6 mL). The reaction was vigorously stirred at room
temperature for 3 h (TLC 6:4 Hex/EtOAc). The reaction mixture was
L
diluted with chloroform, the organic phase was washed with 0.1
M
HCl aq., saturated NaHCO3 solution and brine. The organic phase
was dried with anhydrous Na2SO4. The solvent was evaporated in
vacuo and the crude product (97.1 mg) was purified using an auto-
matic system (SiO2, Hex/EtOAc gradient) to render the desired prod-
uct (50.8 mg) as a white solid in 83 % yield. [α]2D5 = +24 (c = 0.5,
DCM). 1H NMR (400 MHz, CDCl3): δ = 7.73 (d, Jo-m = 8.8 Hz, 2 H,
1
as judged by H NMR ([D6]acetone). [α]2D5 = –58 (c = 0.1, acetone).
1H NMR [400 MHz, (CD3)2CO]: δ = 7.79 (d, Jo-m = 8.8 Hz, 2 H, H-m-
Phe-O), 7.62 (d, J4-6 = 2.5 Hz, 1 H, H-4Bf), 7.51 (d, J7-6 = 8.8 Hz, 1 H,
H-7Bf), 7.35–7.24 (m, 3 H, H-o-Phe-O, H-6Bf), 5.13 (d, J1′-2′ = 4.6 Hz,
1 H, H-1′), 3.91 (d, J5′-6′a = 2.0, J6′a-6′b = 11.0 Hz, 1 H, H-6′a), 3.85–
3.74 (m, 1 H, H-5′), 3.66–3.50 (m, 3 H, H-2′, H-3′, H-6′), 3.49–3.38 (m,
1 H, H-4′), 2.46 (s, 3 H, CH3-Bf) ppm. 13C NMR [100 MHz, (CD3)2CO]:
δ = 158.9 (Cq), 133.7 (Cq), 128.9 (C-m-Ph-O), 128.6 (Cq), 125.6
(2 × Cq), 125.0 (C-6Bf), 119.8 (C-4Bf), 117.8 (C-o-Ph-O), 112.8 (C-7Bf),
110.8 (Cq), 101.6 (C-1′), 77.5 (C-3′), 76.6 (C-5′), 74.6 (C-2′), 73.2 (C-4′),
45.5 (C-6′), 9.3 (CH3-Bf) ppm. ESI-MS: calcd. for C21H20ClNaN3O6 [M
+ Na]+ 468.9, found 468.7.
2 × H-m-Phe-O), 7.47 (d, J4-6 = 2.1 Hz, 1 H, H-4Bf), 7.37 (d, J7-6
=
8.6 Hz, 1 H, H-7Bf), 7.22 (dd, 1 H, H-6Bf), 7.12 (d, 2 H, 2 × H-o-Phe-
O), 5.46 (dd, J1′-2′ = 8.7, J3′-2′ = 6.3 Hz, 1 H, H-2′), 5.39–5.31 (m, 1 H,
H-4′), 5.23–5.12 (m, 2 H, H-1′, H-3′), 4.15 (dd, J5′ax-5′eq = 12.8, J4′-5′
eq = 4.1 Hz, 1 H, H-5′eq), 3.79 (dd, J4′-5′ax = 2.1 Hz, 1 H, H-5′ax), 2.41
(s, 3 H, CH3 Bf), 2.16 (s, 3 H, OCOCH3), 2.11 (s, 3 H, OCOCH3), 2.10
(s, 3 H, OCOCH3) ppm. 13C NMR (100 MHz, CDCl3): δ = 170.4
(OCOCH3), 170.3 (OCOCH3), 169.6 (OCOCH3), 156.7 (Cq), 152.2 (Cq),
151.9 (Cq), 132.7 (Cq), 128.4 (C-m-Ph-O), 128.1 (Cq), 126.1 (Cq), 124.4
(C-6Bf), 119.0 (C-4Bf), 117.1 (C-o-Ph-O), 112.0 (C-7Bf), 110.2 (Cq),
98.7 (C-1′), 69.8 (C-3′), 69.0 (C-2′), 67.2 (C-4′), 62.8 (C-5′), 21.0
(OCOCH3), 20.9 (OCOCH3), 20.9 (OCOCH3), 9.5 (CH3-Bf) ppm. ESI-MS:
2,3,4-Tri-O-acetyl-ꢀ-
(12 mg, 0.02 mmol) was added to a suspension of
6.7 mmol) in acetic anhydride (2.6 mL) and the reaction was stirred
L
-arabinopyranosyl Bromide 50:[16] La(OTf)3
-arabinose (1.0 g,
L
at 30 °C under nitrogen atmosphere for 1 h (TLC 6:4 Hex/EtOAc). calcd. for C26H25ClKO9 [M + K]+ 555.2, found 554.9. An aliquot of
The reaction mixture was diluted with DCM, the organic phase
washed with NaHCO3 and brine and dried with anhydrous Na2SO4.
The solvent was evaporated in vacuo and the crude product
(547 mg) was purified using an automatic system (SiO2, Hex/EtOAc
1
M
MeONa in MeOH (200 μL) was added to a solution of 4-[5-
chloro-3-methylbenzofuran-2-yl]phenoxy 2,3,4-triacetyl-α- -arabino-
L
pyranoside (50.7 mg, 0.098 mmol) in MeOH (0.8 mL). The reaction
was stirred under nitrogen atmosphere at room temperature for 1 h
(TLC 6:4 Hex/EtOAc) and then quenched with Amberlite IR-120 H+.
gradient) obtaining the 1,2,3,4-tetra-O-acetyl-α/ꢀ-
side [
yield). H NMR (400 MHz, CDCl3): δ = 6.36 (d, J1-2 = 3.1 Hz, 1 H, H-
1ꢀ), 5.67 (d, J1-2 = 7.0 Hz, 1 H, H-1α), 5.41–5.35 (m, 3 H, H-2ꢀ, H-3ꢀ,
L-arabinopyrano-
L
-Ara(OAc)4] as a white solid in α:ꢀ ratio of 1:1 (1.3 g, 60 % The resin was filtered off, the solvent was evaporated in vacuo and
1
the crude product (43.5 mg) was purified using an automatic sys-
tem (SiO2, DCM/MeOH gradient from 0 to 25 % MeOH) to render
desired product 44 (28.1 mg) as a white solid in 73 % yield. 1H NMR
(400MHz,CD3OD):δ=7.74(d,Jo-m=8.9Hz,2H,2 × H-m-Phe-O),7.53(d,
H-4ꢀ), 5.33–5.27 (m, 2 H, H-2α, H-4α), 5.12 (dd, J2-3 = 9.1, J3-4
=
3.5 Hz, 1 H, H-3α), 4.11–4.02 (m, 2 H, H-5bα, H-5bꢀ), 3.87–3.75 (m,
2 H, H-5aα, H-5aꢀ), 2.22–2.01 (m, 24 H, OCOCH3) ppm. 13C NMR J4-6 = 2.0 Hz, 1 H, H4-Bf), 7.42 (d, J7-6 = 8.6 Hz, 1 H, H7-Bf), 7.25–
(100 MHz, CDCl3): δ = 170.5 (OCOCH3), 170.3 (OCOCH3), 170.2
(OCOCH3), 169.9 (OCOCH3), 169.9 (OCOCH3), 169.4 (OCOCH3), 169.1
7.18 (m, 3 H, H6-Bf, 2 × H-o-Phe-O), 4.94 (d, J1′-2′ = 7.2 Hz, 1 H, H-1′
), 3.96 (dd, J5′eq-5′ax = 12.4, J5′eq-4′ = 2.8 Hz, 1 H, H-5′eq), 3.90 (m, 1
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Full Paper
H, H-4′), 3.86 (dd, J3′-2′ = 9.0 Hz, 1 H, H-2′), 3.75 (d, 1 H, H-5′ax), 3.67 2,3,4-Tri-O-benzoyl-α/ꢀ-
L
-lyxopyranosyl-O-Box (53): AgOBox
(dd, J3′-4′ = 3.5 Hz, 1 H, H-3′), 2.40 (s, 3 H, CH3 Bf) ppm. 13C NMR
(100 MHz, CD3OD): δ = 159.1 (Cq), 153.5 (Cq), 153.4 (Cq), 134.0 (Cq),
129.2 (Cq), 129.1 (C-m-Ph-O), 126.2 (Cq), 125.2 (C6-Bf), 119.8 (C4-Bf),
118.0 (C-o-Ph-O), 112.8 (C7-Bf), 111.0 (Cq), 102.5 (C-1′), 74.1 (C-3′),
72.2 (C-2′), 69.6 (C-4′), 67.2 (C-5′), 9.3 (CH3 Bf) ppm. HRMS: calcd.
(534 mg, 2.2 mmol), 2,6-lutidine (225 μL, 1.9 mmol) and TBAI
(28 mg, 0.08 mmol) were added to a solution of 2,3,4-tri-O-benzoyl-
α/ꢀ-L-lyxopyranosyl bromide (580 mg, 1.1 mmol) in dry DCM
(8.6 mL) under argon atmosphere. The reaction mixture was stirred
at 50 °C for 2 h (TLC 8:2 Hex/EtOAc) and then diluted with DCM.
The organic phase was washed with 0.1 M NaOH (2 × 10 mL), water
for C20H19ClNaO6 [M + Na]+ 413.07624, found 413.07695. [α]2D5
–9.2 (c = 0.1, MeOH).
=
(2 × 10 mL) and brine (1 × 10 mL). The organic phase was dried
with anhydrous Na2SO4, the solvent was evaporated in vacuo and
the crude product (150 mg) was purified using an automatic system
(SiO2, Hex/EtOAc gradient from 0 % to 40 % EtOAc) to afford the
desired product (135.7 mg) in an 84:16 α:ꢀ ratio (from H1 1H-NMR
signal ratio) as a white solid in 24 % yield. 1H NMR (400 MHz, CDCl3):
δ = 8.33–7.16 (m, 19 H, CH-Bz, CH OBox), 6.75 (d, J1ꢀ-2ꢀ = 4 Hz, 1 H,
H-1ꢀ), 6.55 (d, J1α-2α = 2.6 Hz, 1 H, H-1α), 6.09 (dd, J3α-4α = 9.6,
J2α-3α = 3.4 Hz, 1 H, H-3α), 5.98 (dd, 1 H, H-2α), 5.96 (m, 1 H, H-3ꢀ),
5.89 (m, 1 H, H-2ꢀ), 5.86 (td, J4α-5αeq = 5.2 Hz, 1 H, H-4α), 5.47 (m,
1 H, H-4ꢀ), 4.62 (dd, J5ꢀeq-5ꢀax = 13.4, J5ꢀeq-4ꢀ = 1.6 Hz, 1 H, H-5ꢀeq),
4-[5-Chloro-3-methylbenzofuran-2-yl]phenoxy α-
pyranoside (45): Synthesized as described above for the
D
-Arabino-
-enantio-
L
mer with similar yields and identical spectroscopic data.
1,2,3,4-Tetra-O-benzoyl-α/ꢀ-
(3 mL, 26.7 mmol) was added dropwise to a solution of
L
-lyxopyranose:[25] Benzoyl chloride
-Lyxose
L
(501.7 mg, 3.3 mmol) in pyridine (8 mL) at 0 °C under nitrogen
atmosphere. DMAP (40 mg, 0.33 mmol) was added and the reaction
was warmed to room temperature and stirred for 2 h (TLC 7:3 Hex/
EtOAc). The reaction mixture was quenched with ice and diluted
4.40 (dd, J5αeq-5αax = 11.5 Hz, 1 H, H-5αeq), 4.12 (dd, J5αax-4α
=
with EtOAc. The organic phase was washed with 2
M HCl (2 ×
10.5 Hz, 1 H, H-5αax), 4.11 (m, 1 H, H-5ꢀax) ppm. 13C NMR (100 MHz,
CDCl3): δ = 165.8, 165.5, 165.3 (C=O Bz), 161.3 (Cq OBox), 148.7 (Cq
OBox), 140.6 (Cq OBox), 134.0, 133.9, 133.8, 133.7, 133.6, 133.6,
133.5, 130.7, 130.4, 130.2, 130.1, 130.0, 129.9 (CH-Bz), 129.0 (Cq Bz),
128.9 (Cq Bz), 128.8 (Cq Bz), 128.8, 128.7, 128.7, 128.6, 128.6, 128.5,
125.7, 124.8, 124.1, 123.7, 123.4, 118.9, 110.2 (CH Ar), 98.0 (C-1α),
96.8 (C-1ꢀ), 69.0 (C-2α, C-4ꢀ), 68.7 (C-3α), 67.1 (C-4α), 66.6 (C-3ꢀ),
66.0 (C-2ꢀ), 62.3 (C-5α) ppm. ESI-MS: calcd. for C33H25NNaO9 [M +
Na]+ 602.1, found 602.2; calcd. for [2M + Na]+ 1181.3, found 1180.9.
30 mL), water (1 × 30 mL), saturated NaHCO3 solution (1 × 40 mL)
and brine (1 × 30 mL). The organic phase was dried with anhydrous
Na2SO4, the solvent was evaporated in vacuo and the crude product
(2.5 g) was purified using an automatic system (SiO2, Hex/EtOAc
gradient from 0 to 60 % EtOAc) to render the desired product (1.8 g)
as a white solid in 73:27 α:ꢀ ratio (from H1 1H-NMR signal ratio) in
1
quantitative yield. H NMR (400 MHz, CDCl3): δ = 8.20–7.27 (m, 20
H, CH-Bz), 6.66 (d, J1-2 = 3.5 Hz, 1 H, H-1ꢀ), 6.53 (d, J1-2 = 3.1 Hz, 1
H, H-1α), 6.07 (dd, J3α-4α = 9.3, J3α-2α = 3.5 Hz, 1 H, H-3α), 5.96–5.90
(m, 2 H, H-2ꢀ, H-3ꢀ), 5.89 (t, 1 H, H-2α), 5.81 (dt, J4α-5αax = 9.2, J4α-
5αeq = 5.0 Hz, 1 H, H-4α), 5.55 (m, 1 H, H-4ꢀ), 4.61 (dd, J5ꢀeq-5ꢀax = 13.0,
J5ꢀeq-4ꢀ = 2.4 Hz, 1 H, H-5ꢀeq), 4.37 (dd, J5αeq-5αax = 11.5 Hz, 1 H,
H-5αeq), 4.11–4.01 (m, 1 H, H-5αax, H-5ꢀax) ppm. 13C NMR
(100 MHz, CDCl3): δ = 165.8, 165.7, 165.5, 165.5, 165.4, 165.2, 164.4
(C=O Bz), 134.1, 133.9, 133.8, 133.7, 133.7, 133.6, 130.3, 130.3, 130.2,
130.1, 130.0, 129.9, (CH-Bz), 129.5, 129.4, 129.2, 129.1, 128.9 (Cq Bz),
128.9, 128.8, 128.7, 128.7, 128.6, 128.6 (CH-Bz), 91.7 (C-1α), 90.2 (C-
1ꢀ), 69.4, 69.4 (C-2α, C-3α), 69.0 (C-4ꢀ), 67. 7 (C-2ꢀ), 67.6 (C-4α),
66.6 (C-3ꢀ), 62.5 (C-5α), 60.8 (C-5ꢀ) ppm.
4-[5-Chloro-3-methylbenzofuran-2-yl]phenoxy 2,3,4-Tribenzoyl-
α-
yl]phenoxy 2,3,4-Tribenzoyl-ꢀ-
benzoyl-α/ꢀ- -lyxopyranosyl-O-Box (96.5 mg, 0.17 mmol) and 1a
L-lyxopyranoside (54) and 4-[5-Chloro-3-methylbenzofuran-2-
L-lyxopyranoside (55): 2,3,4-Tri-O-
L
(35.9 mg, 0.14 mmol) were dissolved in dry 1,2-dichloroethane
(0.7 mL) under argon atmosphere. The solution was cooled to
–78 °C and 0.1
M TMSOTf in dry 1,2-dichloroethane (170 μL,
0.017 mmol) was added dropwise to the frozen mixture. The reac-
tion mixture was warmed to –30 °C (just above the m.p. of the
solvent) and stirred for 1–2 min. After completion (TLC 9:1 toluene/
EtOAc) the reaction mixture was diluted with DCM and the organic
phase was washed with 0.1 NaOHaq. (2 × 5 mL) and water (1 ×
5 mL). The organic phase was dried with anhydrous Na2SO4, the
solvent was evaporated in vacuo and the crude product (107.6 mg)
was purified using an automatic system (SiO2, Hex/EtOAc gradient
from 0 % to 20 % EtOAc) obtaining a 56:44 α:ꢀ anomeric mixture
(from H5eq1H-NMR signal ratio). The anomers were separated by
flash chromatography (9:1 toluene/Hex) obtaining 38.6 mg of the
α product 54 (Rf = 0.3, 40 % yield) and 28.1 mg of ꢀ product 55
(Rf = 0.1, 29 % yield).
2,3,4-Tri-O-benzoyl-α/ꢀ-
in AcOH, 2 mL, 35.3 mmol) was added dropwise to α/ꢀ-
L
-lyxopyranosyl Bromide:[25] HBr (33 %
-Lyx(OBz)4
L
(1.0 g, 1.4 mmol) at 0 °C under nitrogen atmosphere. The reaction
mixture was warmed to room temperature and after 15 min DCM
was added (0.8 mL). The reaction was then stirred at room tempera-
ture for 20 min (TLC 7:3 Hex/EtOAc). The reaction was quenched
adding ice and DCM at 0 °C. The organic phase was washed with
cold water and cold saturated NaHCO3 solution (3 × 10 mL), dried
with anhydrous Na2SO4. The solvent was evaporated in vacuo and
the crude product (680 mg) was obtained in 9:1 α:ꢀ ratio (from
H1 1H-NMR signal ratio) in 73 % yield and used without further α Anomer 54: [α]D25 = –29 (c = 0.43, DCM). 1H NMR (400 MHz,
1
purification (the product is not stable on silica). H NMR (400 MHz,
CDCl3): δ = 8.32–7.27 (m, 15 H, CH-Bz), 6.82 (d, J1ꢀ-2ꢀ = 4.1 Hz, 1 H,
H-1ꢀ), 6.54 (d, J1α-2α = 1.5 Hz, 1 H, H-1α), 6.29 (dd, J3α-2α = 3.5,
J3α-4α = 10.3 Hz, 1 H, H-3α), 5.89 (dd, 1 H, H-2α), 5.86 (m, 1 H, H-
3ꢀ), 5.84 (td, J4α-5αax = 10.6, J4α-5αeq = 5.7 Hz, 1 H, H-4α), 5.69 (t, J =
CDCl3): δ = 8.15 (d, Jo-m = 7.3 Hz, 2 H, 2 × o-CH-Bz), 8.01 (d, Jo-m =
7.3 Hz, 2 H, 2 × o-CH-Bz), 7.92 (d, Jo-m = 7.3 Hz, 2 H, 2 × o-CH-Bz),
7.77 (d, Jp-m = 8.8 Hz, 2 H, 2 × m-CH-Bz), 7.69–7.19 (m, 14 H, CH-Bz,
H-4Bf, H-6Bf, H7-Bf, H-m-Phe-O, H-o-Phe-O), 6.16 (dd, J3′-4′ = 9.9, J3′-
= 3.4 Hz, 1 H, H-3′), 5.90 (dd, 1 H, H-2′), 5.86 (dd, 1 H, H-4′), 5.82
2′
4.2 Hz, 1 H, H-2ꢀ), 5.39 (m, 1 H, H-4ꢀ), 4.60 (dd, J5ꢀeq-5ꢀax = 13, (br. d, J1′-2′ = 2.1 Hz, 1 H, H-1′), 4.28 (dd, J5′eq-5′ax = 11.2, J5′eq-4′
J5ꢀeq-4ꢀ = 1 Hz, 1 H, H-5ꢀeq), 4.39 (dd, J5αeq-5αax = 11.3, J5αeq-4α 5.5 Hz, 1 H, H-5′eq), 4.02 (br. t, J5′ax-4′ = 10.7 Hz, 1 H, H-5′ax), 2.43
5.7 Hz, 1 H, H-5αeq), 4.14 (m, 1 H, H-5ꢀax), 4.09 (t, J5αax-4α = 11.3 Hz, (s, 3 H, CH3 Bf) ppm. 13C NMR (100 MHz, CDCl3): δ = 165.9 (C=O
1 H, H-5αax) ppm. 13C NMR (100 MHz, CDCl3): δ = 165.9, 165.5,
Bz), 165.7 (2 × C=O Bz), 155.9 (Cq Bf), 152.2 (Cq Bf), 152.0 (Cq Bf),
165.3, 165.2 (C=O Bz), 134.0, 134.0, 133.9, 133.8, 133.8, 133.5 (p-CH- 133.9 (p-CH-Bz), 133.7 (p-CH-Bz), 133.5 (p-CH-Bz), 132.8 (Cq), 130.1
Bz), 130.5, 130.2, 130.1, 130.0, 129.9 (o-CH-Bz), 129.1, 129.1, 129.1, (2 × o-CH-Bz), 130.0 (2 × o-CH-Bz), 129.9 (2 × o-CH-Bz), 129.3 (Cq Bz),
129.0, 128.9 (Cq Bz), 128.9, 128.8, 128.8, 128.7, 128.6, 128.5 (m-CH- 129.2 (Cq Bz), 129.2 (Cq Bz), 128.8 (C-m-Ph-O), 128.6 (2 × m-CH-Bz),
=
=
Bz), 85.4 (C-1ꢀ), 84.5 (C-1α), 73.3 (C-2α), 68.9 (C-4ꢀ), 68.3 (C-3α), 67.1
(C-4α), 66.5 (C-3ꢀ), 66.3 (C-2ꢀ), 63.5 (C-5α), 61.4 (C-5ꢀ) ppm.
128.6 (2 × m-CH-Bz), 128.4 (2 × m-CH-Bz), 128.1 (Cq Bf), 125.9 (Cq
Bf), 124.3 (C6-Bf), 119.0 (C4-Bf), 116.9 (C-o-Ph-O), 112.0 (C7-Bf),
Eur. J. Org. Chem. 0000, 0–0
www.eurjoc.org
14
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
110.2 (Cq Bf), 96.1 (C-1′), 70.4 (C-2′), 69.2 (C-3′), 67.7 (C-4′), 60.8
D
-Lyxopyranoside (49): Synthesized as described above for the L-
(C-5′), 9.5 (CH3 Bf) ppm. ESI-MS: calcd. for C41H31ClNaO9 [M + Na]+ enantiomer with similar yields and identical spectroscopic data.
725.2, found 725.1; calcd. for [2M + Na]+ 1427.3, found 1426.7.
ꢀ Anomer (55): [α]2D5 = +66 (c = 0.3, DCM) 1H NMR (400 MHz,
CDCl3): δ = 8.27 (br. d, Jo-m = 7.7 Hz, 2 H, 2 × o-CH-Bz), 8.15 (br. d,
Supporting Information (see footnote on the first page of this
article): Procedures for the synthesis of 2a–d and 58a–d; NMR spec-
tra of all new compounds; STD-NMR quantitative analysis; configu-
rational assignment of 42–49.
Jo-m = 7.4 Hz, 2 H, 2 × o-CH-Bz), 7.95 (br. d, Jo-m = 7.2 Hz, 2 H, 2 × o-
CH-Bz), 7.74 (br. d, 2 H, 2 × m-CH-Bz), 7.69–7.19 (m, 14 H, CH-Bz, H-
4Bf, H-6Bf, H7-Bf, H-m-Phe-O, H-o-Phe-O), 5.95 (m, 2 H, H-1′, H-3′),
5.82 (br. t, J = 3.7 Hz, 1 H, H-2′), 5.46 (m, 1 H, H-4′), 4.58 (br. dd, J5′
Acknowledgments
= 13.2, J5′eq-4′ = 1.7 Hz, 1 H, H-5′eq), 4.01 (br. dd, J5′ax-4′
=
This work was supported by Premio Fondazione Cariplo-Ricerca
di frontiera 2011 (project: Chemical control of signalling path-
ways by modulation of hub proteins, CheCOSP). The authors
thank Loredana Stefania Sedda for help with the synthesis of
non-metabolic sugar derivatives. S. S. was supported by a post-
doctoral fellowship from Univeristà degli Studi di Milano, Italy
(Assegno di tipo A). G. C. was supported by the Associazione
Italiana Ricerca sul Cancro (AIRC) through grant IG 15420. The
HRMS spectra were obtained from the Centro Interdipartimen-
tale Grandi Apparecchiature (CIGA), Università degli Studi di
Milano.
eq-5′ax
2.0 Hz, 1 H, H-5′ax), 2.41 (s, 3 H, CH3 Bf) ppm. 13C NMR (100 MHz,
CDCl3): δ = 165.6 (C=O Bz), 165.3 (2 × C=O Bz), 157.2 (Cq Bf), 152.1
(Cq Bf), 151.9 (Cq Bf), 133.7 (p-CH-Bz), 133.6 (p-CH-Bz), 133.5 (p-CH-
Bz), 132.6 (Cq), 130.1 (2 × o-CH-Bz), 130.0 (2 × o-CH-Bz), 129.9 (2 × o-
CH-Bz), 129.6 (Cq Bz), 129.2 (Cq Bz), 129.1 (Cq Bz), 128.6 (2 × m-
CH-Bz), 128.6 (2 × m-CH-Bz), 128.5 (2 × m-CH-Bz), 128.4 (C-m-Ph-O),
128.0 (Cq Bf), 125.6 (Cq Bf), 124.2 (C6-Bf), 118.9 (C4-Bf), 116.9 (C-o-
Ph-O), 111.8 (C7-Bf), 110.0 (Cq Bf), 95.0 (C-1′), 69.4 (C-4′), 67.3 (C-3′
), 67.2 (C-2′), 59.1 (C-5′), 9.4 (CH3 Bf) ppm.
4-[5-Chloro-3-methylbenzofuran-2-yl]phenoxy α-
oside (46): An aliquot of 1 MeONa in MeOH (85 μL) was added
to a solution of 4-[5-chloro-3-methylbenzofuran-2-yl]phenoxy 2,3,4-
tribenzoyl-α- -lyxopyranoside (30 mg, 0.043 mmol) in MeOH Keywords: Oxygen heterocycles · Allosteric modulators · Heat
L-Lyxopyran-
M
L
(350 μL). The reaction was stirred under nitrogen atmosphere at
room temperature for 1 h (TLC 7:3 Hex/EtOAc) and then quenched
with Amberlite IR-120 H+. The resin was filtered off, the solvent was
evaporated in vacuo and the crude product (16.8 mg, quantitative
yield) was used without further purifications. 1H NMR (400 MHz,
CD3OD): δ = 7.71 (d, Jo-m = 8.9 Hz, 2 H, 2 × H-m-Phe-O), 7.46 (br. d,
J4-6 = 2 Hz, 1 H, H-4Bf), 7.36 (d, J7-6 = 8.8 Hz, 1 H, H-7Bf), 7.21–7.14
(m, 3 H, H-6Bf, 2 × H-o-Phe-O), 5.48 (d, J1′-2′ = 2.9 Hz, 1 H, H-1′), 4.03
shock proteins · Molecular dynamics · Glycosylation · Medicinal
chemistry
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13598–13608.
(m, 1 H, H-2′), 3.93–3.86 (m, 2 H, H-3′, H-4′), 3.78 (br. dd, J5′eq-5′ax
=
11.4 Hz, J5′eq-4′ = 3.9 Hz, 1 H, H-5′eq), 3.59–3.50 (m, 1 H, H-5′ax),
2.39 (s, 3 H, CH3 Bf) ppm. 13C NMR (100 MHz, CD3OD): δ = 157.3
(Cq), 152.9 (Cq), 133.4 (Cq), 128.8 (C-m-Ph-O), 128.7 (Cq), 125.7 (Cq),
124.7 (C-6Bf), 119.3 (C-4Bf), 117.3 (C-o-Ph-O), 112.3 (C-7Bf), 110.4
(Cq), 99.3 (C-1′), 72.0 (C-4′), 70.7 (C-2′), 67.8 (C-3′), 64.3 (C-5′), 9.5
(CH3 Bf) ppm. ESI-MS: calcd. for C20H19ClNaO6 [M + Na]+ 413.1,
found 413.0. HR-MS: calcd. for C20H19ClNaO6 [M + Na]+ 413.07624,
found 413.07817.
[6] a) G. Morra, M. A. C. Neves, C. J. Plescia, S. Tsustsumi, L. Neckers, G.
Verkhivker, D. C. Altieri, G. Colombo, J. Chem. Theory Comput. 2010, 6,
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4-[5-Chloro-3-methylbenzofuran-2-yl]phenoxy ꢀ-
oside (47): An aliquot of 1 MeONa in MeOH (43 μL) was added
to a solution of 4-[5-chloro-3-methylbenzofuran-2-yl]phenoxy 2,3,4-
tribenzoyl-α- -lyxopyranoside (15 mg, 0.021 mmol) in MeOH
L-Lyxopyran-
M
L
(160 μL). The reaction was stirred under nitrogen atmosphere at
room temperature for 1 h (TLC 7:3 Hex/EtOAc) and then quenched
with Amberlite IR-120 H+. The resin was filtered off, the solvent was
evaporated in vacuo and the crude product (10.4 mg, quantitative
yield) was used without further purifications. 1H NMR (400 MHz,
CD3OD/CDCl3): δ = 7.74–7.70 (m, 2 H, 2 × H-m-Phe-O), 7.47 (d,
J4-6 = 2.0 Hz, 1 H, H-4Bf), 7.37 (d, J7-6 = 8.7 Hz, 1 H, H-7Bf), 7.23–
7.18 (m, 3 H, H-6Bf, 2 × H-o-Phe-O), 5.37 (d, J1′-2′ = 2.6 Hz, 1 H, H-1′
), 4.12–4.07 (m, 2 H, H-2′, H-5′eq), 3.89–3.84 (m, 1 H, H-4′), 3.79 (dd,
J3′-2′ = 6.2, J3′-4′ = 3.5 Hz, 1 H, H-3′), 3.43 (dd, J5′eq-5′ax = 11.9, J5′ax-
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= 5.5 Hz, 1 H, H-5′ax), 2.40 (s, 3 H, CH3 Bf ) ppm. 13C NMR
4′
(100 MHz, CD3OD/CDCl3): δ = 158.2 (Cq), 153.0, 153.0 (Cq), 133.6
(Cq), 128.9 (C-m-Ph-O), 128.8 (Cq), 126.0 (Cq), 124.8 (C-6Bf), 119.5 (C-
4Bf), 117.7 (C-o-Ph-O), 112.5 (C-7Bf), 110.6 (Cq), 99.2 (C-1′), 72.8 (C-
3′), 69.2 (C-4′), 68.3 (C-2′), 63.2 (C-5′), 9.4 (CH3 Bf) ppm. HRMS: calcd.
for C20H19ClNaO6 [M + Na]+ 413.07624, found 413.07813.
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4-[5-Chloro-3-methylbenzofuran-2-yl]phenoxy α-
D-Lyxopyrano-
side (48) and 4-[5-Chloro-3-methylbenzofuran-2-yl]phenoxy ꢀ-
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Received: April 4, 2016
Published Online: ■
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Allosteric Modulation
S. Sattin,* M. Panza, F. Vasile,
F. Berni, G. Goti, J. Tao,
E. Moroni, D. Agard, G. Colombo,
A. Bernardi ........................................ 1–17
Synthesis of Functionalized 2-(4-
Hydroxyphenyl)-3-methylbenzo-
furan Allosteric Modulators of
Hsp90 Activity
Hsp90 ATP-regulated internal dynam- chemical space at opposite ends of the
ics can be modulated in an allosteric benzofuran scaffold. Compound inter-
fashion, targeting the protein C-termi- actions with Hsc82, a yeast isoform of
nal domain (CTD) with a family of 2- full-length Hsp90, were explored using
phenylbenzofuran derivatives. Here we STD-NMR spectroscopy.
report 28 new derivatives that explore
DOI: 10.1002/ejoc.201600420
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