Targeting the Hsp90 chaperone: Synthesis of novel resorcylic acid macrolactone inhibitors of Hsp90

Article abstract of DOI:10.1002/chem.200902766

A series of benzo-macrolactones has been prepared by chemical synthesis, and evaluated as inhibitors of heat shock protein 90 (Hsp90), an emerging attractive target for novel cancer therapeutic agents. A new synthesis of these resorcylic acid macrolactone analogues of the natural product radicicol is described in which the key steps are the acylation and ring opening of a homophthalic anhydride to give an isocoumarin, followed by a ring-closing metathesis to form the macrocycle. The methodology has been extended to a novel series of macrolactones incorporating a 1,2,3-triazole ring.

Full text of DOI:10.1002/chem.200902766

DOI: 10.1002/chem.200902766
Targeting the Hsp90 Chaperone: Synthesis of Novel Resorcylic Acid
Macrolactone Inhibitors of Hsp90
James E. H. Day,[a] Swee Y. Sharp,[b] Martin G. Rowlands,[b] Wynne Aherne,[b]
Paul Workman,[b] and Christopher J. Moody*[a]
Abstract: A series of benzo-macrolactones has been prepared by chemical synthe-
sis, and evaluated as inhibitors of heat shock protein 90 (Hsp90), an emerging at-
tractive target for novel cancer therapeutic agents. A new synthesis of these resor-
cylic acid macrolactone analogues of the natural product radicicol is described in
which the key steps are the acylation and ring opening of a homophthalic anhy-
dride to give an isocoumarin, followed by a ring-closing metathesis to form the
macrocycle. The methodology has been extended to a novel series of macrolac-
tones incorporating a 1,2,3-triazole ring.
Keywords:
cycloaddition · enzyme inhibitors ·
macrocyclic
metathesis
anticancer agents
·
compounds
·
Introduction
Heat shock protein 90 (Hsp90), one of the most abundant
proteins in eukaryotic cells, is an ATP-dependent chaperone
that plays a central role in regulating the stabilization, acti-
vation and degradation of a range of proteins.[1,2] These
“client” proteins include a number of known over-expressed
or mutant oncogenic proteins such as C-RAF, B-RAF,
ERBB2, AKT, telomerase and p53. Many of these proteins
are associated with the six hallmarks of cancer,[3,4] and
therefore Hsp90 has emerged as a very attractive target for
novel cancer therapeutic agents. Progress in this area has
been extensively reviewed in the last five years.[2,3,5–22] Sever-
al Hsp90 inhibitors have now entered clinical trial.[22]
The pioneering work on Hsp90 inhibition was done with
two natural products, radicicol (1) and geldanamycin (2)
(Figure 1), both of which bind to the ATP-binding pocket in
Figure 1. Structures of the naturally occurring Hsp90 inhibitors radicicol
1 and geldanamycin 2, Danishefskyꢀs cycloproparadicicol analogue 3, and
our synthetic radicicol analogue 4.
[a] Dr. J. E. H. Day, Prof. Dr. C. J. Moody
School of Chemistry, University of Nottingham
University Park, Nottingham, NG7 2RD (UK)
Fax : (+44)115-951-3564
the N-terminal domain of the protein.[23,24] The protein-
bound structures of both 1 and 2 have been studied by X-
ray crystallography[25] and in solution by NMR spectrosco-
py.[26]
E-mail: c.j.moody@nottingham.ac.uk
[b] Dr. S. Y. Sharp, Dr. M. G. Rowlands, Dr. W. Aherne,
Prof. Dr. P. Workman
Radicicol (1) was originally isolated from the fungus
Monocillium nordinii over 50 years ago,[27] and subsequently
from both Nectria radicicola[28] and from the plant associated
fungus Chaetomium chiversii,[29] has attracted the interest of
synthetic chemists and has been the subject of three total
syntheses by the groups of Lett,[30–33] Danishefky,[34] and
Cancer Research UK Centre for Cancer Therapeutics
The Institute of Cancer Research, Haddow Laboratories
15 Cotswold Road, Sutton, Surrey, SM2 5NG (UK)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200902766.
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Winssinger.[35] It is one of the most potent Hsp90 inhibitors
in vitro,[36] although it has little or no activity in vivo,[37,38]
and is a member of a larger class of natural products known
as resorcylic acid lactones, many of which also exhibit
potent biological activity.[39–41] The lack of in vivo activity of
radicicol is presumably a result of its reactive epoxide and
dienone moieties, although its oxime derivative did possess
some in vivo activity.[42] In a search for further biologically
active analogues, Danishefsky and co-workers have devel-
oped a novel series of inhibitors based on cycloproparadici-
col,[43–45] including the recently reported triazole-cyclopro-
paradicicol 3.[46]
Our own work in this area started with a consideration of
the structure of 1 bound in the ATP pocket in the N-termi-
nal domain of yeast Hsp90 (Figure 2a),[25] together with pub-
lished biological data on radicicol analogues,[29,45,47–49] and re-
sulted in the synthesis and biological evaluation of a series
of novel resorcylic macrolactones of varying ring size and
conformation.[50] One of these analogues, NP261 (4), not
only showed the established molecular signature of Hsp90
inhibitors that bind to the N-terminus of the protein, that is,
depletion of client proteins with upregulation of Hsp70, but
also bound to the protein in a similar way to the structurally
more complex natural product (Figure 2b). Thus both com-
pounds adopt a similar folded conformation with the same
key hydrogen-bonding interactions involving the salicylate
ester and phenolic groups, the carboxylate side-chain of
Asp79, the main-chain amide group of Gly83, the hydroxyl
side-chain of Thr171, the main-chain carbonyl of Leu34, and
conserved water molecules (Figure 2). Although the radici-
col structure clearly shows the involvement of the epoxide
oxygen in hydrogen bonding to the e-amino side-chain of
Lys44, the relatively minimal two-fold loss of in vitro activi-
ty in the analogue 4 suggests that this interaction, although
useful, is not essential.
The aim of the present study was two-fold: firstly to de-
velop an improved chemical route to our radicicol analogue
4,[51] and secondly the application of the new synthetic
chemistry to the synthesis of novel resorcylic macrolactones
designed to incorporate heteroatoms with the potential to
bind to the side chains of Lys44, thereby increasing the po-
tential potency of the inhibitors.
Results and Discussion
Our original approach to the synthesis of radicicol analogues
such as 4 was based on Danishefsky and co-workersꢀ first-
generation synthesis of radicicol itself,[34] employing a ring-
closing metathesis (RCM) reaction to form the macrocycle,
a tactic commonly used in related syntheses.[35,44,49,52] We
have now developed a more versatile route to the RCM pre-
cursors that is based on isocoumarin chemistry and incorpo-
rates the necessary chlorine from an early stage, thereby
avoiding a late stage chlorination on every analogue. Inter-
estingly, isocoumarins have been postulated as biosynthetic
precursors to resorcylic lacones, a view supported by the
recent isolation of isocoumarins from the radicicol produc-
ing fungal strain Chaetomium chiversii,[53] and were also
used as intermediates in Lettꢀs first synthesis of radici-
col.[30,31] The starting point for the synthesis was the homo-
phthalate diester 5,[54] obtained in two steps from 4-chlorore-
sorcinol, using the addition of malonate anion to 3,5-dime-
thoxybenzyne and subsequent rearrangement to the homo-
phthalate ester 5, an elegant reaction used by Danishefsky
during a synthesis of dynemicin A.[55] Although both me-
thoxy groups of diester 5 could be removed using aluminium
chloride in dichloromethane on a small scale, this proved
difficult when increasing the scale of the reaction (>5.0 g).
However, using aluminium bromide (1.2 equiv) that initially
removes the methoxy adjacent to the ester, followed by a
large excess of aluminium chloride (8.0 equiv) to remove
the second methyl group, resulted in reproducible results
and a good yield of the resorcylic compound 6 (71%). The
chlorination of compound 6 was carried out at À308C with
a slight excess of sulfuryl chloride (1. equiv). At higher tem-
peratures (À10 and 08C), double chlorination was observed.
The protection of the chlororesorcinol (7) with two methox-
ymethyl (MOM) groups proceeded in excellent yield, and
this was followed by cleavage of both esters using sodium
hydroxide and dehydration with acetic anhydride to give the
Figure 2. Binding interactions of resorcylic macrolactone inhibitors with
yeast Hsp90; in all cases, the hydrogen-bonded interactions in the ATP-
binding site are represented as broken lines. a) Radicicol 1 (data taken
from Roe et al.[25] and available at PDB ID1BGQ); b) 14-membered lac-
tone NP261 (4) (data taken from Proisy et al.[50] and available at PDB
ID2CGF).
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C. J. Moody et al.
pivotal homophthalic anhydride intermediate 8 in 59%
yield over the last two steps (Scheme 1).
Scheme 1. Synthesis of the key homophthalic anhydride intermediate 8
from 4-chlororesorcinol. a) Me2SO4, aq NaOH, heptane (88%); b) di-
ethyl malonate, LDA, THF, À788C (57%); c) i) AlBr3, CH2Cl2, RT to
458C, ii) AlCl3, RT (71%); d) SO2Cl2, THF, À308C (81%); e) MOMCl,
iPr2NEt, DMF, 08C (85%); f) i) NaOH, THF/MeOH/H2O, 70 8C, ii)
Ac2O, PhMe, 1258C (59%).
Scheme 2. Synthesis of isocoumarins 13. a) KOH, EtOH, 08C to RT;
b) (COCl)2, DMF, CH2Cl2, 08C to RT; c) 8, TMG, MeCN, 08C, then
Et3N, 0 8C to RT.
The crucial component of our strategy involved the con-
version of anhydride 8 into an isocoumarin suitable for a
subsequent RCM reaction to give the macrolactone 4 (and
analogues thereof). This necessitated its acylation by an
ethyl malonyl chloride 10 bearing an appropriate alkenyl
side chain. The pentenyl derivative 10a was obtained by
mono-hydrolysis of the corresponding malonate 9a, itself
obtained by alkylation of triethyl methanetricarboxylate fol-
lowed by removal of one of the ester groups,[56] and conver-
sion into the acid chloride 10a using thionyl chloride. The
stage was now set for the coupling of anhydride 8 and acid
chloride 10a to give the isocoumarin 13a under the reaction
conditions for developed by Bauta et al. (Scheme 2),[54]
using tetramethylguanidine (TMG) to effect the initial acy-
lation. In this cascade process, the initial acylation product
11a undergoes further base-mediated cyclization to give
12a, ring opening of which followed by loss of carbon diox-
ide gives the desired isocoumarin 13a (Scheme 2). The pro-
cess is tolerant of the two MOM-protected phenols and the
chlorine adjacent to the reaction center, and a good yield of
the isocoumarin 13a was obtained (75%).
The conversion of isocoumarin 13a into radicicol ana-
logue 4 started with the hydrolysis of the ester-lactone 13a
using an excess of lithium hydroxide (20 equiv) (Scheme 3).
This resulted in hydrolysis of the lactone and ester moieties
with concomitant decarboxylation of the resulting b-ketoa-
cid fragment and delivered the desired ketoacid 14 directly
without the need for a separate decarboxylation step. The
ketoacid 14 was coupled directly with 3-butenol and its 2-
methyl derivative under the Mitsunobu protocol (diiso-
propyl azodicarboxylate (DIAD), Ph3P and toluene) that
gave the esters 15a and 15b (65–67%). The crucial ring-
closing metathesis using Grubbsꢀ second-generation catalyst
ed in good yield to give the macrolactones 16 isolated as the
E isomer after chromatography. Cleavage of the MOM
groups was achieved in good yield under acidic conditions
to give the previously synthesized 4 and its novel methyl an-
alogue, the resorcylic macrolactone 17 (Scheme 3), the struc-
ture of which was confirmed by X-ray crystallography.[51]
Thus, we have established a new route to the Hsp90 inhibi-
tor 4 that proceeds in a 11-step linear sequence in an overall
yield of 4%.
In line with our desire to use the above chemistry to
access novel resorcylic macrolactones with improved poten-
cy, we elected to incorporate a 1,2,3-triazole ring into the
macrocycle (cf. recent work by Lei and Danishefsky).[46] The
choice of the triazole heterocycle was determined by the
fact that it could be suitably positioned as to allow the addi-
tional heteroatoms the potential to bind to the side chains
of Lys44 and possibly Asn92, and also by its ready accessi-
bility using the copper-catalyzed Huisgen 1,3-dipolar cyclo-
addition between a terminal alkyne and an alkyl azide (click
chemistry).[57] Therefore, as described in Scheme 2, the ethyl
malonyl chlorides 10b–c, containing propargyl, 3-butynyl
and 4-pentynyl side chains, were reacted with the homo-
phthallic anhydride 8 to give the isocoumarins 13b–d bear-
ing terminal alkynyl side chains in good yield. The isocou-
marins 13b–d were ring opened under the conditions previ-
ously described, and the resulting ketoacids 18 were reacted
directly with either 2-azidoethanol or 3-azidopropanol under
Mitsunobu conditions (DIAD, Ph3P, toluene) to give a range
of five azides 19. The Mitsunobu products 19 could only be
partially purified due to the difficulty in removing all the
by-products—attempts to use alternative Mitsunobu proto-
cols resulted in poorer yields—and therefore they were di-
rectly subjected to the cycloaddition reaction. Initial at-
(benzylidineACHTUNGTRENNUNG[1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinyl-
idene]dichloro(tricyclohexylphosphine)ruthenium) proceed-
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Hsp90 Inhibitors
FULL PAPER
Scheme 4. Conversion of isocoumarins 13 into 1,2,3-triazole containing
resorcylic macrolactones 21 using click chemistry. a) LiOH, THF/MeOH/
H2O, RT; b) N3CH2CH2OH or N3CH2CH2CH2OH, Ph3P, DIAD, PhMe,
RT; c) CuSO4, sodium ascorbate, tBuOH/H2O, RT (8–33% over three
steps); d) TFA, CH2Cl2, RT (21–84%).
Table 1. Biological activity of the macrocyclic lactones 17 and 21a–e
compared to 1 and 4.
Entry
Compound
TR-Fret
FP IC50 [mm]
HCT116 SRB
IC50 [mm]
GI50 [mm]
1
2
3
4
5
6
7
8
radicicol (1)
NP261 (4)
17
21a
21b
21c
21d
21e
0.014
0.350
5.8
20%@10 mm
40%@10 mm
20%@10 mm
8.3
0.0043
0.0060
0.425
15, 21
8.6, 7.0
13, 11
2.7, 4.0
10, 13
0.00061
6.5
72.4
>100
>100
>100
>100
>100
Scheme 3. Conversion of isocoumarin 13a into resorcylic macrolactones 4
and 17. a) LiOH, THF/MeOH/H2O, RT; b) H2C=CHCHRCH2OH, Ph3P,
DIAD, PhMe, RT; c) Grubbs II catalyst (5 mol%), CH2Cl2, 458C;
d) HCl, dioxane, RT.
20%@10 mm
tempts to effect the macrocyclization using various copper-
catalyzed reaction conditions[58,59] only resulted in trace
amounts of triazoles being detected. However the optimal
conditions were found to be a large excess of both cop-
Conclusion
A
In summary, Hsp90 is an important drug target for cancer
therapeutics, and we have previously synthesized and evalu-
ated a simple analogue of the natural product radicicol
known as NP261 (4). We have now developed an improved
route to the resorcylic macrolactone 4 and its analogue 17,
and extended it to the synthesis of a series of novel 1,2,3-tri-
azole-containing macrocyclic lactones, although, the intro-
duction of a triazole ring into the macrocyclic lactone ap-
pears to be detrimental to Hsp90 inhibition. Further work is
ongoing to investigate the effects of ring substituents on
conformation, and therefore to optimize Hsp90 inhibition
and antitumor activity.
in tert-butyl alcohol/water (0.002m),[60] which gave the de-
sired macrocyclic triazoles 20a–e in 8-33% yield over three
steps. Cleavage of the MOM protecting groups was achieved
with TFA in dichloromethane to give the novel triazole–
macrocyclic lactones 21a–e in 21–84% yield (Scheme 4).
The NP261 analogue 17 and the five novel triazole-macro-
cyclic lactones 21a–e were evaluated for Hsp90 inhibition in
two Hsp90 binding assays: the fluorescence polarization
(FP) assay,[61,62] and the TR-Fret assay.[63] Their growth in-
hibitory potency in HCT116 human colon cancer cell line, as
measured by the SRB assay, was also determined (Table 1).
As can be seen in Table 1, introduction of a methyl group
(analogue 17) results in a loss of potency compared to 4
itself. With the exception of compound 21d that is about 20
times less potent than NP261 (4), the triazole–macrocyclic
lactones 21 only showed weak Hsp90 inhibition, suggesting
that introduction of the triazole in the macrocyclic lactone
ring is detrimental to the binding of these compounds to
Hsp90. None of the compounds show significant growth in-
hibition of the HCT116 cell line in comparison with radici-
col.
Experimental Section
Full experimental procedures and spectroscopic characterization data are
given in the Supporting Information. Data for the compounds that were
evaluated in biological assays are given below.
(E)-1-Chloro-2,4-dihydroxy-7,8,11,12,13,14-hexahydro-8-methyl-6-oxa-
16H-benzocyclotetradecene-5,15-dione (17): colorless solid; m.p. 163–
1648C; 1H NMR (400 MHz; CDCl3): d=11.78 (s, 1H, OH), 6.63 (s, 1H,
ArH), 6.00 (brs, 1H, OH), 5.46 (dt, 1H, J = 15.6, 7.5 Hz, =CH), 5.34 (dt,
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C. J. Moody et al.
1H, J = 15.6, 7.3 Hz, =CH), 4.27 (dd, 1H, J = 10.7, 3.0 Hz, CO2CH),
4.28 (s, 2H, CH2), 3.99 (t, 1H, J = 10.7 Hz, CO2CH), 2.58 (m, 3H, CH2,
CH), 2.13 (m, 3H, CH2, CH), 1.72 (m, 3H, CH2, CH), 1.04 ppm (d, 3H,
J = 7.0 Hz, CH3); 13C NMR (125 MHz; CDCl3): d=205.7 (C), 171.8 (C),
163.8 (C), 158.9 (C), 138.3 (C), 134.3 (CH), 132.5 (CH), 115.9 (C), 107.8
(C), 103.6 (CH), 71.1 (CH2), 47.1 (CH2), 41.2 (CH2), 36.7 (CH), 32.7
(CH2), 26.6 (CH2), 23.0 (CH2), 17.4 ppm (CH3); IR (dichloromethane):
nmax =3686, 3512, 1720, 1658, 1604 cmÀ1; MS (ESI): m/z (%): 377/375 (33/
100) [M+Na]+, 355/353 (8/23), 335/337 (31/10); HRMS (ESI): m/z: calcd
for C18H2135ClNaO5: 375.0970; found: 375.0959 [M+Na]+.
Macrocyclic triazole 21a: colorless solid; m.p. >2308C; 1H NMR
(400 MHz, [D6]DMSO): d=10.6 (brs, 1H, OH), 10.0 (brs, 1H, OH), 7.26
(s, 1H, triazole-H), 6.54 (brs, 1H, ArH), 4.63 (t, 2H, J = 4.7 Hz, CH2),
4.53 (brs, 2H, CH2), 3.48 (brs, 2H, CH2), 2.87 (t, 2H, J = 6.6 Hz, CH2),
2.62 ppm (t, 2H, J = 6.6 Hz, CH2); 13C NMR (125 MHz, [D6]DMSO):
d=206.7 (C), 167.0 (C), 155.5 (C), 155.0 (C), 144.6 (C), 132.8 (C), 125.0
(CH), 114.0 (C), 113.0 (C), 102.7 (CH), 63.0 (CH2), 48.6 (CH2), 47.9
(CH2), 41.6 (CH2), 22.0 ppm (CH2); IR (solid): nmax =1715, 1604,
1581 cmÀ1; MS (ESI): m/z (%): 376/374 (37/100) [M+Na]+, 354/352 (17/
50) [M+H]+; HRMS (ESI): m/z: calcd for C15H1435ClNaN3O5: 374.0514;
found: 374.0516 [M+Na]+; HPLC: tR = 6.32 min, purity (AUC) 95%.
(ESI): m/z: calcd for C18H2035ClN3O5: 416.0989; found: 416.0991
[M+Na]+; HPLC: tR = 5.6 min, purity (AUC) 92%.
Biology
Compounds were assayed for their ability to inhibit Hsp90 using two
assays:
FP assay: This is a measurement of binding competition with a fluores-
cent probe as described previously.[61,62]
TR-Fret assay: A highly robust time-resolved fluorescence energy trans-
fer (TR-FRET) assay was used to measure the binding of biotinylated
geldanamycin (600 nm) to the full length human Hsp90 His-tagged pro-
tein (40 nm), as described previously.[63] Briefly, the europium labelled
anti-His-tagged protein antibody (Perkin–Elmer Prod.No.AD0110) was
added at 1 nm and the streptavidin Surelight APC (Perkin–Elmer Prod.
No.AD0201) at 90 nm. Compounds were tested across a ten point concen-
tration range up to 10 mm and the IC50 determined.
Growth inhibition assay: The colorimetric sulforhodamine B assay (SRB)
was used to measure growth inhibition studies as described previously.[64]
The IC50 was calculated as the drug concentration that inhibits cell
growth by 50% compared with control growth.
Macrocyclic triazole 21b: colorless solid; m.p. 207–2098C; 1H NMR
(400 MHz, [D6]DMSO): d=10.39 (brs, 1H, OH), 7.66 (s, 1H, triazole-
H), 6.27 (brs, 1H, ArH), 4.42 (t, 2H, J = 5.9 Hz, CH2), 4.11 (t, 2H, J =
4.9 Hz, CH2), 3.67 (s, 2H, CH2), 2.86–2.83 (m, 2H, CH2), 2.64–2.61 (m,
2H, CH2), 2.18 ppm (quin, 2H, J = 5.9 Hz, CH2); 13C NMR (125 MHz,
[D6]DMSO): d=206.7 (C), 168.6 (C), 158.2 (C), 158.0 (C), 146.1 (C),
132.8 (C), 123.2 (CH), 119.1 (C), 116.7 (C), 102.9 (CH), 63.9 (CH2), 48.5
(CH2), 47.3 (CH2), 40.8 (CH2), 28.6 (CH2), 21.0 ppm (CH2); MS (ESI):
m/z (%): 390/388 (31/100) [M+Na]+, 366 (42) [M+H]+; HRMS (ESI):
m/z: calcd for C16H1535ClNaN3O5: 388.0676; found: 388.0674 [M+Na]+;
HPLC: tR = 5.9 min, purity (AUC)>95%.
Macrocyclic triazole 21c: colorless solid; m.p. 226–2278C; 1H NMR
(400 MHz, [D6]DMSO): d=10.42 (s, 1H, OH), 10.07 (s, 1H, OH), 7.69
(s, 1H, triazole-H), 6.47 (s, 1H, ArH), 4.63 (brs, 4H, 2ꢂCH2), 2.65 (brs,
2H, CH2), 1.92 ppm (brs, 2H, CH2); 2ꢂCH2 hidden under DMSO and
water peaks; 13C NMR (100 MHz, [D6]DMSO): d=204.6 (C), 167.34 (C),
155.4 (C), 155.0 (C), 145.2 (C), 133.3 (C), 123.4 (CH), 114.8 (C), 112.1
(C), 102.5 (CH), 63.1 (CH2), 48.2 (CH2), 45.3 (CH2), 25.3 (CH2),
21.0 ppm (CH2); CH2 hidden under solvent peak; IR (solid): nmax =1708,
1656, 1610, 1579 cmÀ1; MS (ESI): m/z (%): 390/388 (13/38) [M+Na]+,
368/366 (34/100) [M+H]+; HRMS (ESI): m/z: calcd for C16H1735ClN3O5:
366.0851; found: 366.0844 [M+H]; HPLC: tR = 6.4 min, purity (AUC)
>95%.
Acknowledgements
This work was supported by Cancer Research UK [CUK] grant numbers
C215 A7606 (C.J.M.) and CA309 A8274 (P.W.). P.W. is a Cancer Re-
search UK Life Fellow. We also acknowledge NHS funding to the NIHR
Biomedical Research Centre.
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Macrocyclic triazole 21d: colorless solid; m.p. 228–2308C; 1H NMR
(300 MHz, [D6]DMSO): d=10.36 (brs, 2H, 2ꢂOH), 7.87 (s, 1H, triazole-
H), 6.44 (s, 1H, ArH), 4.67 (s, 4H, 2ꢂCH2), 3.31 (s, 2H, CH2), 2.67 (m,
2H, CH2), 2.15 (t, 2H, J
1.55 ppm (quin, 2H,
=
6.4 Hz, CH2), 1.68–1.66 (m, 2H, CH2),
J
=
6.1 Hz, CH2); 13C NMR (125 MHz,
[D6]DMSO): d=205.5 (C), 167.4 (C), 156.4 (C), 156.0 (C), 147.7 (C),
133.6 (C), 123.0 (CH), 113.2 (C), 112.9 (C), 102.7 (CH), 63.6 (CH2), 48.8
(CH2), 44.9 (CH2), 26.7 (CH2), 24.9 (CH2), 22.4 ppm (CH2); CH2 hidden
under solvent peak; IR (solid): nmax =1708, 1679, 1609, 1582 cmÀ1; MS
(ESI): m/z (%): 404/402 (32/100) [M+Na]+, 382/380 (28/90) [M+H]+;
HRMS (ESI): m/z: calcd for C17H1935ClNaN3O5: 402.0827; found:
402.0836 [M+H]+; HPLC: tR = 6.9 min, purity (AUC) >95%.
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Macrocyclic triazole 21e: colorless solid; m.p. 200–2028C; 1H NMR
(400 MHz, [D6]DMSO): d=10.6 (brs, 1H, OH), 10.1 (brs, 1H, OH), 7.94
(s, 1H, triazole-H), 6.53 (brs, 1H, ArH), 4.42 (t, 2H, J = 5.5 Hz, CH2),
3.81 (s, 2H, CH2), 3.76 (t, 2H, J = 6.4 Hz, CH2), 2.70 (t, 2H, J = 5.8 Hz,
CH2), 2.28–2.21 (m, 4H, 2ꢂCH2), 1.66 (quin, 2H, J = 6.1 Hz, CH2),
1.27 ppm (quin, 2H,
J
=
7.5 Hz, CH2); 13C NMR (125 MHz,
[D6]DMSO): d=206.2 (C), 167.7 (C), 155.7 (C), 155.3 (C), 146.3 (C),
133.6 (C), 124.1 (CH), 113.8 (C), 112.9 (C), 102.72 (CH), 61.0 (CH2), 45.9
(CH2), 45.0 (CH2), 40.8 (CH2), 28.5 (CH2), 27.3 (CH2), 23.8 (CH2),
22.7 ppm (CH2); IR (solid): nmax =1706, 1655, 1609, 1576 cmÀ1; MS (ESI):
m/z (%): 418/416 (33/100) [M+Na]+, 394/396 (33/100) [M+H]+; HRMS
2762
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Chem. Eur. J. 2010, 16, 2758 – 2763

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Received: October 6, 2009
Published online: January 19, 2010
Chem. Eur. J. 2010, 16, 2758 – 2763
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
2763