Synthesis and characterization of photoaffinity labelling reagents towards the Hsp90 C-terminal domain

Article abstract of DOI:10.1039/c6ob02097f

Glucosyl-novobiocin-based diazirine photoaffinity labelling reagents (PALs) were designed and synthesized to probe the Hsp90 C-terminal domain unknown binding pocket and the structure-activity relationship. Five PALs were successfully synthesized from novobiocin in six consecutive steps employing phase transfer catalytic glycosylation. Reactions were monitored and guided by analytical LC/MS which led to different strategies of adding either a PAL precursor or a sugar moiety first. The structures and bonding linkages of these compounds were characterised by various 2D-NMR spectroscopy and MS techniques. Synthetic techniques provide powerful probes for unknown protein binding pockets.

Full text of DOI:10.1039/c6ob02097f

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DOI: 10.1039/C6OB02097F
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Synthesis and Characterization of Photoaffinity Labelling reagents
Towards the Hsp90 C-terminal Domain
Binto Simon,a Xuexia Huang,a Huangxian Ju, b Guoxuan Suna and Min Yang*a
Received 00th January 20xx,
Accepted 00th January 20xx
Glucosyl-novobiocin-based diazirine photoaffinity labelling reagents (PALs) were designed and synthesized to probe the
Hsp90 C-terminal domain unknown binding pocket and structure-activity relationship. Five PALs were successfully
synthesized from novobiocin in six consecutive steps employing a phase transfer catalytic glycosylation. Reactions were
monitored and guided by analytical LC/MS which led to different strategies of adding either PAL precursor or sugar moiety
first. The structures and bonding linkages of these compounds were characterised by various 2D-NMR spectroscopy and
MS techniques. The synthetic technique provides powerful probes for unknown protein binding pocket.
DOI: 10.1039/x0xx00000x
www.rsc.org/
reported low resolution CTD: 2IOQ (E. coli.),15 2CG9 (Yeast),11
2O1U (Dog)16 and 3Q6N (Human, which has 18 amino acid
residues missing in the CTD).17 Previous research indicated a
Introduction
Heat Shock Protein 90 kDa (Hsp90) is a molecular chaperone
involved in folding up to 200 proteins with the assistance of 20
co-chaperones.1 It has been the target of research in genetics
and epigenetics,2 neurodegeneration3 and cell mobility.4
Hsp90 is particularly important for cancer cell survival. 5, 6 So far
seventeen Hsp90 inhibitors have been developed to various
stages of clinical trials, however, candidates were suspended
at different clinical stages.7,8 While these results validate
Hsp90 inhibition as a relevant anti-cancer strategy, there is no
FDA-approved drug.1, 9, 10, due to the high cytotoxicity from N-
terminal domain targeting inhibitors.
Hsp90 consists of three domains: an N-domain that contains
an ATP, drug-binding site and co-chaperone-interacting motifs;
an M-domain for client proteins and co-chaperones; and a C-
terminal domain (CTD) contains a dimerization MEEVD motif. 11
Inhibition of the Hsp90CTD decreases chaperone dimerization,
diminishes ATPase activity and impairs the formation of the
Hsp90 protein complex. Novobiocin based CTD inhibitors have
shown some superior activity against various cancer cell lines
in apoptosis12 or proliferation13 compared to N-terminal
inhibitors. Indeed, CTD inhibitors have attracted significant
attention recently.10, 14
19
few possible locations for the binding pockets18,
in CTD,
however, no confirmation has been obtained. Experimental-
based SAR can provide limited guidance in drug design (Fig.
1A), in-depth understanding of the exact binding of ligand
molecules to individual amino acid residues in the polypeptide
is urgently required.
Previously we demonstrated that the single glycosylation the 4'-
hydroxyl group of novobiocin (Fig. 1A) could increase its anticancer
21
activity20,
via binding to the Hsp90CTD22. Although protein
modelling data indicate the formation of strong hydrogen bonds
with Hsp90CTD peptides, the exact binding details is not clear.20 We
hereby designed and synthesized five glucosyl-novobiocin (Glc-Nov,
Fig. 1B) based trifluoromethyl diazirine-type photoaffinity labelling
reagents (PALs, 1-5) to probe the binding site of Hsp90CTD.
OH
OH
secondary amide
required for activity
Glycosylation
increases activity
A)
HO
amide is critical
O
5-substitution
decreases activity
HO
H
for activity
OH
H-bond acceptor
Aromatic side
chain critical
O
4'
H
N
6-O-propoxy
most active
H
O
O
7'
2'
O
O
SAR not clear
H3CO
CH3
O
Rigid carbon linker
increases activity
lactone
non-essential
O
OH
3-carbamate
O
8-methoxy
increases activity
Due to the lack of a high resolution crystal structure,
however, the binding site and structure-activity relationship
(SAR) of Hsp90CTD is still unclear. Most high resolution Hsp90
crystal structure does not contain CTD. Only 4 Hsp90 crystals
detrimental activity
H2N
OH
O
H
OH
B)
N
OR1
O
H
OH
N
O
O
O
HO
HO
O
O
O
O
R2O
HO
CH3
N
N
4
N
O
H3CO
=
N
CF3
a.
O
N
Department of Pharmaceutical
& Biological Chemistry, The School of
OH
O
R2
N
=
F3C
1 R1
=
3 R1 = H
R2
F3C
Pharmacy, University College London, London WC1N 1AX, UK Email:
min.yang@ucl.ac.uk
H2N
OH
O
b State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and
Chemical Engineering, Nanjing University, Nanjing 210023, P.R. China. Fax/Tel.: +86 25
89683593
N
N
R2 = H
R1
5
=
2
R1
=
R2
=
N
N
HO
HO
F3C
F3C
HO
Electronic Supplementary Information (ESI) available: NMR spectra & table, LC-MS
spectra. See DOI: 10.1039/x0xx00000x
Figure. 1 Experimental based Hsp90CTD SAR (A) and five PALs (B)
J. Name., 2013, 00, 1-3 | 1
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These five compounds were designed to probe the binding of
Synthesis of compound 1 was achiDeOveI:d10.b10y3V9ire/ewCa6AOcrttiBcilo0e2nO0n9loi7nFfe
sugars (4'-glucose or 7'-noviose) with Hsp90CTD. Compound 1 novobiocin with diazirine bromide (6) in a polar aprotic solvent
mimics Glc-Nov with the PAL in the 4'-OH position to probe the 4'- (DMF). Complete NMR assignment of all individual proton and
O-glucose moiety binding sites. Compound 2 mimics Glc-Nov with carbon resonances has been achieved using 1H, 13C-NMR,
the PAL in the 7'-OH and glucose in the 4'-OH positions respectively. DEPT-135, COSY, HSQC and HMBC (Fig. 2, S1a-c & Table S1).
Compound 3 mimics novobiocin with the PAL in the 7'- OH position The long range correlation between the H1''' and coumarin
to probe the 7'-O-noviose moiety binding sites in the absence of the ring C4' in the HMBC spectrum indicates that the C1''' was
4'-O-glucose moiety. Compound 4 tests the potential binding in the linked with the 4'-OH of novobiocin (Fig. 2).
2'-O position and compound 5 tests the binding site with only the
PAL in the 4'-O position, but no substitution on the 7'-OH group.
Coumarin ring 7 (Table S2) and 4-hydroxy-3(3-methylbut--enyl)
benzoic acid 8 (Table S3) were obtained by acid hydrolysis of
Trifluoromethyl diazirine has an excellent chemical stability and is novobiocin following reported procedures, but with slight
highly resistant toward a number of factors such as temperature, modification as shown in the experimental section.25 Coupling was
nucleophiles, acidic and basic conditions and oxidizing/reducing performed using Steglich esterification reaction employing 1-Ethyl-
reagents. The activation by UV (350 nm) yields an extremely 3-(3dimethylaminopropyl) to obtain aminocoumarin 9 with 62% of
reactive flexible carbene, which can insert into C-H, N-H and O-H yield. The NMR spectrum of 9 showed 4'-OH (br), 7'-OH and NH at
bonds23 with low non-specific binding 24
.
11.73, 10.52 and 9.44 ppm respectively (Fig. S2, Table S4). The long-
range heteronuclear (H-C) correlations between 7'-OH and C7', and
C8' and C9' and NH coupled to C4' and C1 supported the assignment
and also indicated the formation of the amide bond (Fig. S2c).
Results & Discussion
Figure 2. Synthesis of compound 1. Top: Synthesis scheme. Bottom: HMBC NMR assignment of compound 1. Long range coupling
between H1''' and C4' (Red 4'). It also shows the coupling between C4'''/C6''' and C8''' (red 8''').
2 | J. Name., 2012, 00, 1-3
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DOI: 10.1039/C6OB02097F
Figure 3. Scheme to synthesis of compound 4 & 5 (inset, top left, analytical LC-MS spectrum of compound 10 &11).
Nucleophilic substitution reaction between compound 9 and the reactant ratios, catalyst equivalents, base concentrations, organic
photoaffinity labelling regent precursor resulted in two solvents and even solid phase transfer conditions (these
6
compounds (Fig. 3, inset, S3 & S4) with desired product masses (Fig. investigations will be reported in future paper). Phase transfer
S3d, S3e, S4e, S4f) of 4'- and 7'- linked products. Preparative HPLC glucosylation of compound 11 using Benzyl-tri-butyl ammonium
indeed separated those two compounds, the one with lower bromide (BTAB) gave 12 (Fig. S5, Table S7) with the glucose moiety
retention time (95 min, data not shown) was characterised to be linked with the 7'-OH of the coumarin ring. The disappearance of
the 4'-O compound 10 (Fig. 3, S3a-c and Table S5,) with the the 7'-OH signal and the coupling between H1''' and C7'
evidence of H1'' coupled to C4' in HMBC (Fig. S3b). However, the demonstrated the correct assignment (Fig. S5a-c). But, under the
fraction at 100 min was surprisingly not the expected 7'-O- same conditions, glucosylation of compound 10 gave two different
compound as the NMR did not show evidence of linkage to the 7'-C compounds (Fig. S6) which are probably due to early activation of
position (Fig. S4b). The 7'-OH and NH protons were still present in diazirine. Analytical LC/MS data showed that glucosylation
the 1H-NMR (Fig. S4, Table S6). The spectrum showed a shift for H1'' happened but under such conditions, the compound lost N2 to form
from 3.3 ppm to 5.5 ppm and coupling to a carbon atom in a very a radical, which led to either an insertion reaction (938, [M+H]+, Fig.
similar position to C4' (Fig. S4d), which was shown to be C2'. These S6b); or the removal of one additional acetyl group (896, [M+H]+,
results indicated that there is an equilibrium between the 4' and 2' Fig. S6c). Possible structures are proposed in Fig. S6b and S6c based
positions in the coumarin ring and that compound 11 is linked to on the MS data and mechanism.
the 2'-OH in the enol form.
Since directly coupling compound 6 to the 7'-OH position of
It is worth noting that conventional catalysts for glycosylation, such the coumarin was not possible, an alternative approach was
as Ag2O, Ag2CO3, AgOTf, TMSOTf and Hg(CN)2, did not work adopted by changing the sequence of reactions. Reaction of
efficiently with glycosyl acceptors 10 and 11 (data not shown). Here compound 9 with sugar donor 13 gave the expected product
we adopted
a different route employing phase transfer 14 (Fig. 4, S7, Table S8) and a di-glucosyl product (data not
glycosylation reaction. An optimised reaction condition was shown). Compound 14 reacted with PAL precursor 6 to give
obtained by conducting a number of experiments using varying compound 15 at moderate yield (Fig. S8, Table S9).
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DOI: 10.1039/C6OB02097F
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ARTICLE
Figure 4. Synthesis of compound 2 & 3
Deprotection of the acetyl groups using NaOMe was slow General experiment details
and step by step deacetylation was observed and confirmed by
Unless otherwise stated, all reactions were carried out in anhydrous
condition. Reactions were monitored by thin layer chromatography
(TLC) and/or a Shimadzu single quadrupole LC/MS. Flash column
chromatography was performed on Merck silica Gel 60 (particle size
40-63µm). 1HNMR and 13CNMR spectra were obtained using
Bruker 400 MHz or a Bruker 500 MHz spectrometers. Multiplicity is
abbreviated as follows (br = broad, s = singlet, d = doublet, dd =
double doublet, t = triplet, m = multiplet, etc.) and coupling
constants were obtained in Hertz. Assignments were aided by DEPT
90, DEPT 135, COSY, NOESY, HSQC and HMBC. High resolution mass
spectra were obtained using Water Q-Tof MS. Chemicals were
purchased from Sigma-Aldrich.
LC/MS, e.g. with one acetyl group (Fig. S9c&f) four acetyl
group (Fig. S9c&g) being observed. In addition, cleavage of the
glycoside bond was also observed (Fig. S9c&h) with the mass
of 592 in negative mode probably due to unavoidable basic
conditions caused by trace amount of water. The deprotection
of compound 15 gave the desired compound 2 (Fig. S9a-c,
Table S10) as well as compound 3 (Fig. S10, Table S11).
Compound 4 (Fig. S11, Table S12) and 5 (Fig. S12, Table S13)
were obtained by deprotecting compounds 12 and 10,
respectively.
Conclusions
General procedure for phase transfer glycosylation
In conclusion, the synthesis of five glucosyl-novobiocin based
PALs was achieved in six steps. This accentuates that the phase
transfer glycosylation is an effective way of for the
incorporation of sugar moieties to molecules of interest with
difficulties in conventional glycosylation methods. The
structures have been confirmed by different NMR technology.
This strategy of glucosyl-novobiocin modification provides a
valuable approach for further development of enhanced
glucosyl novobiocin mimetic. Preliminary data indicate the
binding between Hsp90 CTD with compound 1. and tandem
MS analysis indicated the exact peptide the PAL binds to. This
method provide an effective synthetic route for
multifunctional compounds, and a concise chemical biology
tool to probe protein unknown binding pocket SAR.
Brominated glycosyl donor (1.1-3.0 equiv), glycosyl acceptor (1
equiv.) and benzyl tributyl ammonium bromide (0.2 equiv.) in
dichloromethane (DCM, 10 mL) was stirred for 5 min at room
temperature. The 0.1 M NaHCO3 (6 mL) was then introduced to the
reaction mixture. The reaction mixture was stirred for specified
time at room temperature. Upon completion (as indicated by TLC
and LC/MS), the aqueous phase was washed with DCM (3 × 30 ml).
The dichloromethane extracts were combined and washed with
saturated NaHCO3 (2 × 30 ml), water (2 × 30 ml) and dried (MgSO4),
filtered and solvents removed in vacuo to give the corresponding
glycosides. The crude material was purified by column
chromatography (ethyl acetate: hexane 1:2 to 1:3 v:v depending on
the product) then by preparative HPLC using methods 1-3.
HPLC purification methods
Experimental Section
Waters 2555 pump and 2489 UV detector with Waters C-18 (25 ×
200 mm) column. Solvent A 0.1% TFA in Water, Solvent B 0.1% TFA
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in MeOH. Flow rate: 10 mL/min. Three methods, HPLC-1 (100 min), phase was decanted. The brown syrupDOwIa: 1s0.w10a3Vs9ihe/wCe6dAOrtiBcwl0ei2tO0hn9li7naFe
HPLC-2 (175 min) and HPLC-3 (255 min). small amount of diethyl ether (liquid kept for compound 8),
HPLC-1: t=0 min, 30% B, t=90 min, 95% B, t=100 min, 95% B, and later the precipitation of the crude product was collected.
t=101 min, 30% B, t=105 min, 30% B. The crude product was further washed by diethyl ether again
HPLC-2: t=0 min, 30% B, t=170 min, 95% B, t=175 min, 95% B, (liquid kept for compound 8) until getting the light grey
t=176 min, 30% B, t=177 min, 30% B. powder (9.5 g, 69%): Rf =0.13, petroleum ether/ethyl acetic 1:2
HPLC-3: t=0 min, 30% B, t=250 min, 95% B, t=255 min, 95% B, v/v. The above solid (7.0 g, 0.03 mol) was dissolved in
t=256 min, 30% B, t=257 min, 30% B.
anhydrous methanol (105 mL) and 10 % HCl/methanol (190
mL) and the mixture was refluxed for 2 hrs. The clear black
solution obtained was evaporated in vacuo until precipitation
Deprotection of acetate
o
started, then kept the reaction mixture at 4 C overnight. The
To a solution of the acetylated compound (1 mmol) in dry methanol
(10 ml vol. based on acetate groups) was added sodium methoxide
(0.1 mmol). The mixture was stirred at room temperature and
monitored by LC/MS. The reaction mixture was neutralised with a
drop of acetic acid, stirred for 0.5 hr. The reaction mixture was
filtered and concentrated under reduced pressure and then purified
by preparative HPLC.
precipitation was filtered and washed with ice cold methanol
to give the light grey powder. Then the filtrate was evaporated
again and the procedure was repeated twice, and afforded the
yellow solid (5.9 g, 95 %). Rf= 0.15, chloroform: methanol 4:1
1
v/v. HNMR (400 MHz, MeOD4): δ = 7.77 (1 H, d, J = 8.03 Hz,
H5), 6.95 (1 H, d, J = 8.56 Hz, H6), 2.28 (3 H, s, H11). 13CNMR
(125 MHz, MeOD4): δ = 164.2 (C2), 96.2 (C3), 161.7 (C4), 122.6
(C5), 113.7 (C6), 162.3 (C7), 113.5 (C8), 153.6 (C9), 108.1 (C10),
9.2 (C11). ESI+208 [M+H]+.
4-(4-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenyl)methanyl-
novobiocin (1)
Novobiocin sodium salt and 3-(4-(bromomethyl)phenyl)-3- Synthesis of 4-acetoxy-3-(3-methylbut-2-en-1-yl)benzoic acid (8)
(trifluoromethyl)-3H-diazirine (88 mg, 0.316 mmol) were dissolved
The filtrate from above procedure was concentrated to get
in 5 ml of anhydrous DMF under the protection of nitrogen. The
light brown syrup and was extracted using ethyl acetate (3 ×
mixture was protected from light and stirred vigorously at room
25 ml) and with dilute hydrochloric acid (3 × 20 ml of a 3%
temperature for 16h. The solvent were removed under vacuum and
aqueous solution). The organic extracts were combined and
syrup was purified by flash chromatography (ethyl acetate, Rf = 0.5)
washed sequentially with saturated aqueous solution of
to yield compound 1 as pale yellow solid (120mg, 47%). [α]D20: -
NaHCO3 (2 × 20 ml) and water (2 × 20 ml). The combined
1
130.5, (ACN). HNMR (400 MHz, MeOD4): δ = 7.55 (1 H, s, H3), 6.69
organic extracts were dried over magnesium sulphate, filtered
and concentrated in vacuo and purified via column
chromatography(petroleum ether:ethyl acetate 80:20) to
(1 H, d, J = 8.9 Hz, H6), 7.51 (1 H, d, J = 8.4 Hz, H7), 3.21 (2 H, m,
H8), 5.20 (1 H, t, J = 7.4 Hz, H9), 1.60 (6H, s, H11, H12), 7.50 (1 H, d,
afford white crystalline powder (1.2 g, 16 %). 1HNMR (500
J = 7.4 Hz, H5'), 6.88 (1 H, d, J = 8.4 Hz, H6'), 1.81 (3 H, s, H11'), 5.41
(1 H, s, H1''), 4.12 (1 H, s, H2''), 5.15 (1 H, d, J = 10.8 Hz, H3''), 3.46
MHz, CDCl3): δ = 8.03 (1H, s, H3), 7.17 (1H, d, J = 8.3 Hz, H6),
8.01 (1H, d, J = 8.3 Hz, H7), 3.31 (2H, d, J = 7.4 Hz, H8), 5.28
(1 H, d, J = 10.8 Hz, H4''), 1.18 (3 H, s, H6''), 0.93 (3 H, s, H7''); 3.42
(3 H, s, H8''); 3.33 (2 H, d, J = 11.8, 36.9 Hz, H1'''), 6.96 (2 H, d, J =
(1H, t, J = 7.1 Hz, H9), 1.79 (3H, s, H11), 1.75 (3H, s, H12), 2.34
7.9 Hz, H3''', H7'''), 6.81 (2 H, d, J = 7.9 Hz, H4''', H6'''). 13CNMR (125
(3H, s, H14). 13CNMR (125 MHz, CDCl3): δ = 171.8 (C1),
127.1(C2), 132.3 (C-3), 129.3 (C4), 163.4 (C5), 123.6 (C6), 129.5
MHz, MeOD4): δ = 169.70 (C1), 123.89 (C2), 130.91 (C3), 129.71
(C4), 160.6 (C5), 115.43 (C6), 128.51(C7), 29.33 (C8), 123.40 (C9),
133.50 (C10), 26.10(C11), 17.93 (C12), 191.50 (C2'), 69.34 (C3'),
170.30 (C4'), 126.60 (C5'), 111.25 (C6'), 162.50 (C7'), 115.33 (C8'),
154.10 (C9'), 116.21 (C10'), 8.30 (C11'), 100.07 (C1''), 71.01 (C2''),
(C7), 29.3 (C8), 121.0 (C9), 134.1 (C10), 26.3 (C11), 17.7 (C12)
168.9 (C13), 21.0 (C14). ESI+ 249 [M+H]+.
Synthesis of 4-((4,7-dihydroxy-8-methyl-2-oxo-2H-chromen-3-
yl)carbamoyl)-2-(3-methylbut-2-en-1-yl)phenyl acetate (9)
72.96 (C3''), 82.55 (C4''), 80.34 (C5''), 29.34 (C6''), 22.92 (C7''), 62.08
(C8''), 159.17 (C9''), 43.90 (C1'''), 127.34 (C2'''), 131.94 (C3'''),
129.72 (C4''')135.31 (C5'''), 127.34 (C6'''), 131.94 (C7'''), 29.10 Compound 7 (1.0 g, 4.8 mmol) was dissolved in a mixture of
(C8'''), 123.20 (C9'''). ESI+811 [M+H]+, HRMS: 811.2767, calculated anhydrous dichloromethane and pyridine (7:3). Then the N-(3-
for C40H42F3N4O11: 811.2802.
dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDCI,
1.0 g, 5.2 mmol) and the compound 8 was added dropwise
dissolved in a mixture of DCM:pyridine (84:36) and stirred at room
temperature under nitrogen. After 12 hrs of reaction was washed
with aq. NaHCO3 (30 ml) and water (30 mL × 2), the organic phase
was obtained and the excess solvent was removed in vacuo and the
residue was purified via solid chromatography (petroleum ether:
ethyl acetate, 3:1) to afford a yellow solid (1.3 g, 62%). 1HNMR(500
MHz, DMSO-d6): δ = 7.90 (1H, s, H3), 7.22 (1H, d, J = 8.1 Hz, H6),
7.89 (1H, d, J = 8.1 Hz, H7), 3.28 (2H, d, J = 7.1 Hz, H8), 5.22 (1H, t, J
= 6.8 Hz, H9), 1.71 (6H, s, H11, H12), 2.33 (3H, s, H14), 7.61 (1H, d, J
3-amino-4,7-dihydroxy-8-methyl-2H-chromen-2-one (7)
Novobiocin sodium salt (20 g, 0.0315 mol) was dissolved into a
mixture of pyridine and acetic anhydride (5:1, 240 mL), and
heated under reflux for 4 hours. After being cooled to room
temperature, the mixture was then acidified with 5N-HCl drop
by drop, the pH was monitored by the pH paper (pH=1) and
the temperature of the reaction mixture was kept below 25°C
by ice bath. Then the brown syrup precipitated, the aqueous
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= 8.1 Hz, H5'), 6.90 (1H, d, J = 8.1 Hz, H6'), 2.19 (3H, s, H11'), 11.73 DMF was added drop wise to the reaction flask. The reaction
(1H, br, 4'-OH), 10.52 (1H, s, 7'-OH), 9.44 (1H, s, NH). 13CNMR(125 was allowed to slowly warm up to roomDteOmI: 1p0e.1r0a3t9u/rCe6OanBd02w09a7sF
MHz, DMSO-d6): δ =165.94 (C1), 133.13 (C2), 129.96 (C3), 132.50 stirred for 72 h). The reaction mixture was then extracted with
(C4), 150.99 (C5), 122.44 (C6), 126.94 (C7), 28.99 (C8), 121.55 (C9), ethyl acetate (2 × 20 ml) and water. The combined organic
131.75 (C10), 26.36 (C11), 18.51 (C12), 168.89 (C13), 21.26 (C14), extracts was dried over magnesium sulfate. The drying agent
160.57 (C2'), 99.72 (C3'), 160.85 (C4'), 121.52 (C5'), 111.83 (C6'), was filtered off and the organic extract was concentrated in
159.10 (C7'), 110.43 (C8'), 151.47 (C9'), 108.07 (C10'), 8.88 (C11'). vacuo to give the compound as yellow foam. The desired
HMBC: NH linked with C4' and C1. ESI+ 438 ([M+H]+). HRMS products was isolated via preparative HPLC-1 with a retention
438.1552, calculated 438.1553 (C24H24NO7).
time of 100 min and freeze dried to obtain a light yellow solid
(0.09 g, 31 %). HNMR (500 MHz, DMSO-d6): δ = 7.83 (1H, s,
1
H3), 7.22 (1H, d, J = 8.8 Hz, H6), 7.82 (1H, d, J = 5.8 Hz, H7),
3.27 (2H, d, J = 7.4 Hz, H8), 5.20 (1H, t, J = 5.7 Hz, H9), 1.69
(6H, s, H11, H12), 2.33 (3H, s, H14), 7.50 (1H, d, J = 8.9 Hz,
H5'), 6.90 (1H, d, J = 8.8 Hz, H6'), 2.19 (3H, s, H11'), 5.46 (2H, s,
H1''), 7.55 (2H, d, J = 8.1 Hz, H3'', H7''), 7.27 (2H, d, J = 7.5 Hz,
H4'', H6''). 13CNMR(125 MHz, DMSO-d6): δ = 166.23 (C1),
131.17 (C2), 129.70 (C3), 133.49 (C4), 151.38 (C5), 122.76 (C6),
126.62 (C7), 28.39 (C8), 121.34 (C9), 132.63 (C10), 25.36 (C11),
17.59 (C12), 168.90 (C13), 20.81 (C14), 160.97 (C2'), 48.71
(C3'), 161.16 (C4'), 121.75 (C5'), 112.27 (C6'), 159.37 (C7'),
108.52 (C8'), 150.83 (C9'), 110.66 (C10'), 8.12 (C11'). 73.10
(C1''), 127.50 (C2''), 128.52 (C3''), 126.62 (C4''), 138.46 (C5''),
126.62 (C6''), 128.52 (C7''), 28.56 (C8''), 124.0 (C9''). HMBC:
H1'' liked with C2'. ESI+ 636 [M+H]+. HRMS: 636.1963,
calculated for C33H29F3N3O7: 636.1958.
4-((7-hydroxy-8-methyl-2-oxo-4-((4-(3-(trifluoromethyl)-3H-
diazirin-3-yl)benzyl)oxy)-2H-chromen-3-yl)carbamoyl)-2-(3-
methylbut-2-en-1-yl)phenyl acetate (10)
To an oven dried flask equipped with magnetic stirrer bar,
rubber septa and a nitrogen inlet, compound 9 (0.2 g, 0.45
mmol) and 10 mL of anhydrous DMF were added. The solution
was cooled to 0oC and sodium hydride (4.2 mg of 60% mineral
oil dispersion) was added, allowed the reaction mixture to stir
for 30 min at 0oC. Compound 6(0.14 g, 0.49 mmol) dissolved in
DMF was added drop wise to the reaction flask. The reaction
was allowed to slowly warm up to room temperature and was
stirred for 72 h). The reaction mixture was then extracted with
ethyl acetate (2 × 20 ml) and water. The combined organic
extracts was dried over magnesium sulfate. The drying agent
was filtered off and the organic extract was concentrated in
vacuo to give the compound as yellow foam. The desired
products was isolated via preparative HPLC-1 with retention
time of 95 min and freeze dried to obtain a light yellow solid
(2S,3R,5R,6R)-2-((3-(4-acetoxy-3-(3-methylbut-2-en-1-
yl)benzamido)-8-methyl-4-oxo-2-((4-(3-(trifluoromethyl)-3H-
diazirin-3-yl)benzyl)oxy)-4H-chromen-7-yl)oxy)-6-
1
(0.12 g, 42 %). HNMR (500 MHz, DMSO-d6): δ = 7.86 (1H, s,
(acetoxymethyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate
H3), 7.34 (1H, d, J = 8.2 Hz, H6), 7.92 (1H, d, J = 8.8 Hz, H7),
3.34 (2H, d, J = 7.6 Hz, H8), 5.28 (1H, t, J = 7.1 Hz, H9), 1.79 (12)
(3H, s, H11), 1.77(3H, s, H12), 2.42 (3H, s, H14), 7.51 (1H, d, J =
Compound 11 (0.05 g, 0.07 mmol) was reacted with glucosyl
9.5 Hz, H5'), 6.77 (1H, d, J = 8.9 Hz, H6'), 1.90 (3H, s, H11'), 3.4
(2H, m, H1''), 7.21 (2H, d, J = 7.6 Hz, H3'', H7''), 7.09 (2H, d, J =
7.6 Hz, H4'', H6''), 10.96 (1H, s, 7'-OH), 9.93 (1H, s, NH).
13CNMR(125 MHz, DMSO-d6): δ = 166.05 (C1), 129.46 (C2),
129.82 (C3), 133.61 (C4), 151.56 (C5), 122.84 (C6), 126.72 (C7),
28.92 (C8), 121.24 (C9), 132.80 (C10), 26.06 (C11), 18.45 (C12),
169.90 (C13), 8.19 (C14), 188.40 (C2'), 67.69 (C3'), 167.57 (C4'),
125.24 (C5'), 112.05 (C6'), 163.17 (C7'), 111.78 (C8'), 152.69
(C9'), 110.68 (C10'), 7.49 (C11'). 42.4 (C1''), 126.84 (C2''),
130.78 (C3''), 125.87 (C4''), 134.30 (C5''), 125.87 (C6''), 130.78
(C7''), 28.43(C8''), 123.76 (C9''). HMBC: NH linked with C4' and
C1. ESI+ 636 [M+H]+. HRMS: 636.1959, calculated for
C33H29F3N3O7: 636.1958.
bromide (compound 13, 0.12 g, 0.30 mmol, 3 equiv.) using the
general phase transfer glycosylation procedure (in DCM) for 13
days. The product was purified by preparative HPLC-2 with a
retention time of 96 min afford compound 12 as a colourless
white powder (LCMS yield : 52 %, isolated yield, 0.02 g, 31 %).
20
1
[α]D : -22.41, (CHCl3). HNMR (500 MHz, CDCl3): δ = 7.88 (1H,
d, J = 2.33 Hz, H3), 7.20 (1H, d, J = 8.33 Hz, H6), 7.82 (1H, dd, J
= 2.46, 9.49 Hz, H7), 3.34 (2H, d, J = 7.02 Hz, H8), 5.25 (1H, m,
H9), 1.79 (3H, s, H11), 1.75 (3H, s, H12), 2.38 (3H, s, H14), 7.70
(1H, d, J = 9.02 Hz, H5'), 7.01 (1H, d, J = 9.07 Hz, H6'), 2.29
(3H, s, H11'), 5.38 (2H, s, H1''), 7.50 (2H, d, J = 8.60 Hz, H3'',
H7''), 7.21 (2H, d, J = 8.21 Hz, H4'', H6''), 5.15 (1H, d, J = 7.68
Hz, H1'''), 5.39 (1H, m, H2'''), 5.36 (1H, m, H3'''), 5.24 (1H, m,
H4'''), 3.93 (1H, m, H5'''), 4.34 (1H, m, H6a'''), 4.22 (1H, m,
H6b'''), 2.09-2.11 (12 H, s, 4 * CH3CO), 7.75 (1H, s, NH).
13CNMR(125 MHz, CDCl3): δ = 166.62 (C1), 130.82 (C2), 129.97
(C3), 134.78 (C4), 152.50 (C5), 123.11 (C6), 126.38 (C7), 28.92
(C8), 120.78 (C9), 134.27 (C10), 25.71 (C11), 17.90 (C12),
168.98 (C13), 20.82 (C14), 157.78 (C2'), 104.95 (C3'), 162.43
(C4'), 122.03 (C5'), 111.62 (C6'), 157.33 (C7'), 116.11 (C8'),
150.00 (C9'), 113.19 (C10'), 8.4 (C11'). 72.49 (C1''), 129.64
(C2''), 128.26 (C3''), 126.82 (C4''), 137.73 (C5''), 126.82 (C6''),
128.26 (C7''), 28.19 (C8''), 122.6 (C9''), 99.22 (C1'''), 70.89
4-((7-hydroxy-8-methyl-4-oxo-2-((4-(3-(trifluoromethyl)-3H-
diazirin-3-yl)benzyl)oxy)-4H-chromen-3-yl)carbamoyl)-2-(3-
methylbut-2-en-1-yl)phenyl acetate (11)
To an oven dried flask equipped with magnetic stirrer bar,
rubber septa and a nitrogen inlet, compound 9 (0.2 g, 0.45
mmol) and 10 mL of anhydrous DMF were added. The solution
was cooled to 0oC and sodium hydride (4.2 mg of 60% mineral
oil dispersion) was added, allowed the reaction mixture to stir
for 30 min at 0oC. Compound 6(0.14 g, 0.49 mmol) dissolved in
6 | J. Name., 2012, 00, 1-3
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Organic & Biomolecular Chemistry
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(C2'''), 72.18 (C3'''), 68.22 (C4'''), 72.23 (C5''') 62.01 (C6a'''), stirred for 20 h. The reaction mixture was then extracted with
DOI: 10.1039/C6OB02097F
62.01 (C6b'''), 20.69 (4 * CH3CO), 170.51 (CH3CO), 170.22 ethyl acetate (2 × 20 ml) and water. The combined organic
(CH3CO), 169.41 (CH3CO), 169.21 (CH3CO).HMBC: H1''' linked extracts was dried over magnesium sulfate. The drying agent
with C7' and H1'' lined with C2'. ESI+ 966 [M+H]+. HRMS: was filtered off and the organic extract was concentrated in
7966.2873, calculated for C47H47F3N3O16: 966.2873.
vacuo to give the compound as yellow foam. The desired
products was isolated via preparative HPLC-2 with a retention
of 160 min and freeze dried to obtain a light yellow solid
(LCMS yield : 35 %, isolated yield, 0.02 g, 34 %). [α]D20: -43.73,
Glycosylation of compound 10
The same procedure was followed in the synthesis of
compound 12, i.e. Compound 10 (0.05 g, 0.07 mmol) was
reacted with glucosyl bromide (compound 13, 0.12 g, 0.30
mmol, 3 equiv.) using the general phase transfer glycosylation
procedure (in DCM) for 13 days. However, no desired
glycosylation product formed but new reactions with N 2
removal and possible insert reactions were discovered by
LC/MS.
1
(CHCl3). HNMR (500 MHz, CDCl3): δ = 7.89 (1H, s, H3), 7.20
(1H, d, J = 8.42 Hz, H6), 7.83 (1H, d, J = 8.91 Hz, H7), 3.34 (2H,
d, J = 7.02 Hz, H8), 5.26 (1H, m, H9), 1.78 (3H, s, H11), 1.75
(3H, s, H12), 2.34 (3H, s, H14), 7.63 (1H, d, J = 9.22 Hz, H5'),
6.90 (1H, d, J = 8.76 Hz, H6'), 2.29 (3H, s, H11'), 5.23 (2H, s,
H1''), 7.50 (2H, d, J = 8.64 Hz, H3'', H7''), 7.26 (2H, d, J = 8.21
Hz, H4'', H6''), 5.40 (1H, d, J = 8.63Hz, H1'''), 5.36 (1H, t, J = 7.90
Hz, H2'''), 5.22 (1H, m, H3'''), 5.13 (1H, t, J = 8.77 Hz, H4'''), 3.64
(1H, m, H5'''), 4.13 (1H, dd, J = 5.69, 12.8 Hz, H6a'''), 3.90 (1H,
dd, J = 2.13, 12.09 Hz, H6b'''), 1.96-2.03 (12 H, s, 4 * CH 3CO).
13CNMR(125 MHz, CDCl3): δ = 166.50 (C1), 130.84 (C2), 129.95
(C3), 134.77 (C4), 152.29 (C5), 122.98 (C6), 126.32 (C7), 28.93
(2S,3R,5R,6R)-2-((3-(4-acetoxy-3-(3-methylbut-2-en-1-
yl)benzamido)-7-hydroxy-8-methyl-2-oxo-2H-chromen-4-yl)oxy)-6-
(acetoxymethyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (14)
Compound 9 (0.3 g, 0.68 mmol) was reacted with glucosyl (C8), 120.77 (C9), 134.12 (C10), 25.75 (C11), 18.00 (C12),
bromide (13, 0.84 g, 2.0 mmol) using the general phase 168.84 (C13), 20.89 (C14), 161.29 (C2'), 106.98 (C3'), 156.61
transfer glycosylation procedure (in DCM) for 7 days. The (C4'), 122.08 (C5'), 108.63 (C6'), 159.49 (C7'), 114.54 (C8'),
product was purified by preparative HPLC-2 with a retention 150.58 (C9'), 110.65 (C10'), 8.48 (C11'). 69.67 (C1''), 129.03
time of 112 min to afford compound 14 as a white powder (C2''), 127.42 (C3''), 126.89 (C4''), 138.08 (C5''), 126.89 (C6''),
(0.10 g, 19%). [α]D20: -66.03, (CHCl3). 1HNMR (500 MHz, 127.42 (C7''), 28.56 (C8''), 117.37 (C9''), 98.57 (C1'''), 71.15
CDCl3): δ = 7.95 (1H, s, H3), 7.23 (1H, d, J = 9.15 Hz, H6), 7.87 (C2'''), 72.34 (C3'''), 67.86 (C4'''), 72.46 (C5'''), 61.34 (C6'''),
(1H, d, J = 8.56 Hz, H7), 3.37 (2H, d, J = 7.85 Hz, H8), 5.27 (1H, 20.53-20.62 (4 * CH3CO), 170.34 (CH3CO), 169.98 (CH3CO),
m, H9), 1.79 (3H, s, H11), 1.76 (3H, s, H12), 2.38 (3H, s, H14), 169.52 (CH3CO), 169.33 (CH3CO). HMBC: H1''' linked with C4'
7.35 (1H, d, J = 9.18 Hz, H5'), 6.75 (1H, d, J = 9.21 Hz, H6'), and H1'' lined with C7'. ESI+ 1953 [2M+Na+]+. HRMS: 966.2930,
2.21 (3H, s, H11'), 5.36 (1H, m, H1''), 5.36 (1H, m, H2''), 5.24 calculated for C47H47F3N3O16: 966.2908.
(1H, m, H3''), 5.12 (1H, t, J = 9.39 Hz, H4''), 3.64 (1H, m, H5''),
4.17 (1H, m, H6a''), 4.24 (1H, m, H6b''), 2.00-2.07 (12 H, s, 4 *
4-hydroxy-N-(8-methyl-2-oxo-7-((4-(3-(trifluoromethyl)-3H-
diazirin-3-yl)benzyl)oxy)-4-(((3R,5S,6R)-3,4,5-trihydroxy-6-
(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-2H-chromen-3-yl)-
3-(3-methylbut-2-en-1-yl)benzamide (2) and 4-hydroxy-N-(4-
hydroxy-8-methyl-2-oxo-7-((4-(3-(trifluoromethyl)-3H-diazirin-3-
yl)benzyl)oxy)-2H-chromen-3-yl)-3-(3-methylbut-2-en-1-
yl)benzamide (3)
CH3CO), 7.81 (1H, s, NH). 13CNMR(125 MHz, CDCl3): δ = 167.51
(C1), 130.57 (C2), 130.19 (C3), 134.87 (C4), 152.49 (C5), 123.00
(C6), 126.49 (C7), 29.05 (C8), 120.79 (C9), 134.06 (C10), 25.76
(C11), 17.96 (C12), 168.85 (C13), 20.55 (C14), 157.55 (C2'),
107.02 (C3'), 160.98 (C4'), 121.41 (C5'), 112.64 (C6'), 158.71
(C7'), 109.01 (C8'), 151.16 (C9'), 112.10 (C10'), 8.08 (C11').
98.95 (C1''), 71.20 (C2''), 72.35 (C3''), 67.98 (C4''), 72.57 (C5'')
Compound 15 (0.02 g, 0.02 mmol) was reacted with sodium
61.49 (C6''), 20.54-20.63 (4 * CH3CO), 170.56 (CH3CO), 170.05
(CH3CO), 169.39 (CH3CO), 169.33 (CH3CO). ESI- 766 [M-H]-.
methoxide (catalytic percentage) using the general acetate
deprotection procedure (in CH3OH) for 26 h. The product was
HRMS: 768.2504, calculated for C38H42NO16: 768.2504.
purified by preparative HPLC-3 with a retention of 201 min to afford
compound 2 as a white powder (1.2mg, 7.9 %) and 256 min for
(3R,5R,6R)-2-((3-(4-acetoxy-3-(3-methylbut-2-en-1-yl)benzamido)-
8-methyl-2-oxo-7-((4-(3-(trifluoromethyl)-3H-diazirin-3-
yl)benzyl)oxy)-2H-chromen-4-yl)oxy)-6-
(acetoxymethyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (15)
compound 3 as white powder (3 mg, 11%).
compound 2: [α]D20: -66.7, (CHCl3). 1HNMR (500 MHz, MeOD4): δ =
7.82 (1H, s, H3), 6.87 (1H, d, J = 8.63 Hz, H6), 7.77 (1H, d, J = 8.61
Hz, H7), 3.42 (2H, m, H8), 5.38 (1H, m, H9), 1.77 (6H, s, H11, H12),
To an oven dried flask equipped with magnetic stirrer bar, 7.97 (1H, d, J = 8.61 Hz, H5'), 7.12 (1H, d, J = 7.75 Hz, H6'), 2.38
rubber septa and a nitrogen inlet, compound 14 (0.05 g, 0.07 (3H, s, H11'), 5.33 (2H, s, H1''), 7.63 (2H, d, J = 7.75 Hz, H3'', H7''),
mmol) and 10 mL of anhydrous DMF were added. The solution 7.33 (2H, d, J = 8.62 Hz, H4'', H6''), 5.22 (1H, d, J = 8.82 Hz, H1'''),
was cooled to 0oC and sodium hydride (2.1 mg of 60% mineral 3.49 (1H, m, H2'''), 3.30 (1H, m, H3'''), 3.36 (1H, m, H4'''), 3.12 (1H,
oil dispersion) was added, allowed the reaction mixture to stir m, H5'''), 3.68 (1H, d, J = 11.8 Hz, H6a'''), 3.52 (1H, m, H6b''').
for 30 min at 0oC. Compound 6 (0.02g, 0.08mmol) dissolved in 13CNMR(125 MHz, MeOD4): δ = 170.34 (C1), 125.26 (C2), 130.98
DMF was added drop wise to the reaction flask. The reaction (C3), 129.96 (C4), 160.66 (C5), 115.53 (C6), 128.52 (C7), 29.33 (C8),
was allowed to slowly warm up to room temperature and was 123.41 (C9), 133.57 (C10), 26.1 (C11), 18.0 (C12), 168.84 (C13),
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163.09 (C2'), 108.87 (C3'), 161.14 (C4'), 124.12 (C5'), 110.06 (C6'), Compound 10(0.01 g, 0.02 mmol) wasDrOeaI:c1t0e.1d03w9/iCth6OsBo0d2i0u9m7F
160.88 (C7'), 114.92 (C8'), 152.38 (C9'), 111.92 (C10'), 8.4 (C11'). methoxide (catalytic percentage) using the general acetate
70.82 (C1''), 129.71 (C2''), 129.04 (C3''), 127.92 (C4''), 140.59 (C5''), deprotection procedure (in CH3OH) for 15 h. The product was
127.92 (C6''), 129.04 (C7''), 29.41 (C8''), 123.75 (C9''), 103.76 (C1'''), purified by preparative HPLC using 30-95 % methanol water in
75.35 (C2'''), 77.74 (C3'''), 70.82 (C4'''), 78.76 (C5'''), 61.96 (C6'''). 180 min gradient elution to afford compound 5 as a white
1
HMBC: H1''' linked with C4' and H1'' lined with C7'. ESI- 754 [M-H]-. powder (6.6 mg, 50 %). HNMR (500 MHz, DMSO-d6): δ = 7.63
HRMS:756.2365, calculated for C37H37F3N3O11: 756.2380.
Compound 3:
(1H, d, J = 2.01Hz, H3), 6.83 (1H, d, J = 8.01 Hz, H6), 7.57 (1H,
m, H7), 3.38 (2H, d, J = 7.51 Hz, H8), 5.31 (1H, m, H9), 1.80
1HNMR (500 MHz, MeOD4): δ = 7.61 (1H, s, H3), 6.78 (1H, d, J = (3H, s, H11), 1.70 (3H, s, H12), 2.42 (3H, s, H14), 7.57 (1H, m,
8.99 Hz, H6), 7.61 (1H, d, m, H7), 3.30 (2H, d, 7.64, H8), 5.17 (1H, s, H5'), 6.53 (1H, d, J = 8.61 Hz, H6'), 2.01 (3H, s, H11'), 3.44 (2H,
H9), 1.68 (6H, s, H11, H12), 7.70 (1H, d, J = 9.13 Hz, H5'), 6.79 (1H, m, H1''), 7.13 (2H, d, J = 8.03 Hz, H3'', H7''), 7.00 (2H, d, J =
d, J = 8.67 Hz, H6'), 2.25 (3H, s, H11'), 5.07 (2H, s, H1''), 7.36 (2H, d, 8.03 Hz, H4'', H6''). 13CNMR(125 MHz, DMSO-d6): δ = 162.3
J = 9.18 Hz, H3'', H7''), 7.11 (2H, d, J = 9.18 Hz, H4'', H6''), 13.89 (C1), 127.5 (C2), 130.0 (C3), 127.5 (C4), 158.4 (C5), 115.7 (C6),
(1H, s, 4'-OH), 8.60 (1H, s, NH). 13CNMR(125 MHz, MeOD4): δ = 127.4 (C7), 25.8 (C8), 120.9 (C9), 135.7 (C10), 29.5 (C11), 18.1
166.95 (C1), 123.81 (C2), 129.92 (C3), 127.58 (C4), 158.97 (C5), (C12), 169.90 (C13), 8.19 (C14), 188.9 (C2'), 67.4 (C3'), 168.0
116.22 (C6), 127.64 (C7), 29.77 (C8), 120.59 (C9), 136.27 (C10), (C4'), 127.5 (C5'), 112.1 (C6'), 162.1 (C7'), 112.3 (C8'), 153.4
25.82 (C11), 18.11 (C12), 161.76 (C2'), 103.06 (C3'), 153.40 (C4'), (C9'), 126.0 (C10'), 8.0 (C11'). 43.3 (C1''), 129.2 (C2''), 130.5
122.57 (C5'), 108.67 (C6'), 159.12 (C7'), 114.15 (C8'), 149.81 (C9'), (C3''), 126.5 (C4''), 132.8 (C5''), 126.5 (C6''), 130.5 (C7''), 43.0
111.07 (C10'), 8.50 (C11'). 69.91 (C1''), 129.02 (C2''), 127.41 (C3''), (C8''), 120.6 (C9''). HMBC: 1'' lined with C4'. ESI+ 594 [M+H]+;
126.85 (C4''), 138.29 (C5''), 126.85 (C6''), 127.41 (C7''), 28.70 (C8''), HRMS: 594.1863, calculated for C31H27F3N3O6: 594.1852.
122.10 (C9''). HMBC: H1'' lined with C7'. ESI- 592 [M-H]-. HRMS:
594.1842, calculated for C31H27F3N3O6: 594.1852.
Acknowledgements
We thank EPSRC (EP/K023071/1) for the financial support.
4-hydroxy-N-(8-methyl-4-oxo-2-((4-(3-(trifluoromethyl)-3H-
diazirin-3-yl)benzyl)oxy)-7-(((2S,3R,5S,6R)-3,4,5-trihydroxy-6-
(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-4H-chromen-3-yl)-
3-(3-methylbut-2-en-1-yl)benzamide (4)
References
1. J. Trepel, M. Mollapour, G. Giaccone and L. Neckers, Nat. Rev.
Cancer, 2010, 10, 537-549.
2. K. S. K. Wong and W. A. Houry, Cell Res., 2006, 16, 742-749.
3. W. Luo, W. Sun, T. Taldone, A. Rodina and G. Chiosis, Mol.
Neurodegener., 2010, 5, 24.
4. S. Tsutsumi and L. Neckers, Cancer Sci., 2007, 98, 1536-1539.
5. D. J. Stravopodis, L. H. Margaritis and G. E. Voutsinas, Curr. Med.
Chem., 2007, 14, 3122-3138.
Compound 12 (0.01 g, 0.01 mmol) was reacted with sodium
methoxide (catalytic percentage) using the general acetate
deprotection procedure (in CH3OH) for 22 h. The product was
purified by preparative HPLC-3 with a retention time of 136 min to
afford compound 4 as a white powder (1.2mg,7.9 %).[α]D20: -41.9
(ACN).1HNMR (500 MHz, MeOD4): δ = 7.79 (1H, s, H3), 6.87 (1H, d, J
= 6.4 Hz, H6), 7.70 (1H, m, H7), 3.4 (2H, m, H8), 5.38 (1H, m, H9),
1.76 (6H, s, H11, H12), 7.97 (1H, d, J = 8.61 Hz, H5'), 6.85 (1H, d, J =
6.3 Hz, H6'), 2.39 (3H, s, H11'), 5.51 (2H, d, J = 3.3 Hz, H1''), 7.48
(2H, m, H3'', H7''), 7.23 (2H, d, J = 7.23 Hz, H4'', H6''), 5.06 (1H, d, J
= 7.9 Hz, H1'''), 3.56 (1H, m, H2'''), 3.49 (1H, m, H3'''), 3.46 (1H, m,
H4'''), 3.41 (1H, m, H5'''), 3.92 (1H, d, J = 12.15 Hz, H6a'''), 3.72 (1H,
dd, J = 5.5, 12.15 Hz, H6b'''), 7.71 (1H, s, NH). 13CNMR(125 MHz,
MeOD4): δ = 164.0 (C1), 131.2 (C2), 131.0 (C3), 133.7 (C4), 148.6
(C5), 123.40 (C6), 128.47 (C7), 29.32 (C8), 123.39 (C9), 135.5 (C10),
26.00 (C11), 17.96 (C12), 163.81 (C2'), 105.9 (C3'), 164.2 (C4'),
127.99 (C5'), 115.53 (C6'), 160.53 (C7'), 116.3 (C8'), 154.4 (C9'),
112.9 (C10'), 8.63 (C11'). 75.20 (C1''), 130.4 (C2''), 129.33 (C3''),
112.97 (C4''), 139.8 (C5''), 112.97 (C6''), 129.33 (C7''), 28.19 (C8''),
126 (C9''), 102.21 (C1'''), 74.86 (C2'''), 78.17 (C3'''), 71.26 (C4'''),
78.39 (C5'''), 62.52 (C6'''). HMBC: H1''' linked with C7' and H1'' lined
with C2'. ESI- 755 [M-H]-. HRMS: 756.2365, calculated for
C37H37F3N3O11: 756.2380.
6. L. Neckers, J. Biosci., 2007, 32, 517-530.
7. T. M. B. Staff, Bristol-Myers Squibb Halts Development of
Tanespimycin.
8. Information
about
Retaspimycin
hydrochloride
http://gisttrials.fmgateway.com/iLRG/drug_detail.php?drug=74
, Accessed 25/09/2015, 2015.
9. K. Jhaveri, T. Taldone, S. Modi and G. Chiosis, Biochim. Biophys.
Acta, 2012, 1823, 742-755.
10. A. S. Duerfeldt and B. S. J. Blagg, Bioorg. Med. Chem. Lett., 2010,
20, 4983-4987.
11. M. M. Ali, S. M. Roe, C. K. Vaughan, P. Meyer, B. Panaretou, P.
W. Piper, C. Prodromou and L. H. Pearl, Nature, 2006, 440,
1013-1017.
12. S. B. Matthews, G. A. Vielhauer, C. A. Manthe, V. K. Chaguturu,
K. Szabla, R. L. Matts, A. C. Donnelly, B. S. J. Blagg and J. M.
Holzbeierlein, Prostate, 2010, 70, 27-36.
13. S. N. Shelton, M. E. Shawgo, S. B. Matthews, Y. Lu, A. C.
Donnelly, K. Szabla, M. Tanol, G. A. Vielhauer, R. A. Rajewski, R.
L. Matts, B. S. Blagg and J. D. Robertson, Mol. Pharmacol., 2009,
76, 1314-1322.
14. B. Boppa, E. Cigliab, A. Ouald-Chaibb, G. Grothc, H. Gohlkeb and
J. Josea, Biochim. Biophys. Acta, 2016, 1860, 1043-1055.
15. A. K. Shiau, S. F. Harris, D. R. Southworth and D. A. Agard, Cell,
2006, 127, 329-340.
4-hydroxy-N-(7-hydroxy-8-methyl-2-oxo-4-((4-(3-(trifluoromethyl)-
3H-diazirin-3-yl)benzyl)oxy)-2H-chromen-3-yl)-3-(3-methylbut-2-
en-1-yl)benzamide (5)
8 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx

Page 9 of 9
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C6OB02097F
16. D. E. Dollins, J. J. Warren, R. M. Immormino and D. T. Gewirth,
Mol. Cell, 2007, 28, 41-56.
17. C. C. Lee, T. W. Lin, T. P. Ko and A. H. J. Wang, PLoS ONE, 2011,
6, e19961.
18. R. L. Matts, A. Dixit, L. B. Peterson, L. Sun, S. Voruganti, P.
Kalyanaraman, S. D. Hartson, G. M. Verkhivker and B. S. J. Blagg,
ACS Chem. Biol. , 2011, 6, 800-807.
19. M. Sgobba, R. Forestiero, G. Degliesposti and G. Rastelli, J.
Chem. Inf. Model., 2010, 50, 1522-1528.
20. S. M. Patel, M. Fuente, S. Ke, A. Guimaraes, A. O. Oliyide, X. Ji, P.
Stapleton, A. Osbourn, Y. Pan, D. J. Bowles, B. G. Davis, A.
Schatzlein and M. Yang, Chem. Commun., 2011, 47, 10569-
10571.
21. M. Yang, UK, 2013, pp. US9045517, EP2627661.
22. I. N. Cruz, Y. Zhang, M. de la Fuente, A. Schatzlein and M. Yang,
Anal. Biochem., 2013, 428, 107-109.
23. L. Dubinskya, B. P. Kromb and M. M. Meijler, Bioorg. Med.
Chem., 2012, 20, 554-570.
24. E. Smith and I. Collins, Future Med. Chem., 2015, 7, 159-183.
25. J. W. Hinman, E. L. Caron and H. Horeksema, J. Am. Chem. Soc.,
1957, 79.
Table of content
Synthesis of diazirine type of photoaffinity labelling reagents to
probe Hsp90 C-terminal Domain binding pocket and structure-
activity relationship. Structure to illustrate probe positions only.
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