Synthesis of macrolactam analogues of radicicol and their binding to heat shock protein Hsp90

Article abstract of DOI:10.1039/c3ob42211a

A series of macrolactam analogues of the naturally occurring resorcylic acid lactone radicicol have been synthesised from methyl orsellinate in 7 steps, involving chlorination, protection of the two phenolic groups, and hydrolysis to the benzoic acid. Formation of the dianion and quenching with a Weinreb amide results in acylation of the toluene methyl group that is followed by amide formation and ring closing metathesis to form the macrocyclic lactam. Final deprotection of the phenolic groups gives the desired macrolactams whose binding to the N-terminal domain of yeast Hsp90 was studied by isothermal titration calorimetry and protein X-ray crystallography.

Full text of DOI:10.1039/c3ob42211a

Organic &
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PAPER
Synthesis of macrolactam analogues of radicicol
and their binding to heat shock protein Hsp90†
Cite this: Org. Biomol. Chem., 2014,
12, 1328
Bridie L. Dutton,a Russell R. A. Kitson,*a Sarah Parry-Morris,b S. Mark Roe,b
Chrisostomos Prodromoub and Christopher J. Moody*a
A series of macrolactam analogues of the naturally occurring resorcylic acid lactone radicicol have been
synthesised from methyl orsellinate in 7 steps, involving chlorination, protection of the two phenolic
groups, and hydrolysis to the benzoic acid. Formation of the dianion and quenching with a Weinreb
amide results in acylation of the toluene methyl group that is followed by amide formation and ring
closing metathesis to form the macrocyclic lactam. Final deprotection of the phenolic groups gives the
desired macrolactams whose binding to the N-terminal domain of yeast Hsp90 was studied by isothermal
titration calorimetry and protein X-ray crystallography.
Received 8th November 2013,
Accepted 9th January 2014
DOI: 10.1039/c3ob42211a
www.rsc.org/obc
readily metabolised. As a result, significant efforts have
focussed on synthesising radicicol analogues which retain the
Introduction
In recent years, the chaperone heat shock protein 90 (Hsp90) potency, yet are more metabolically stable (Fig. 1).2 Having
has become one of the most attractive and widely studied bio- established the importance of the epoxide stereochemistry for
logical targets for molecular cancer therapeutics.1,2 Ubiquitous good activity, Danishefsky and co-workers replaced the C5-6
in eukaryotic cells, Hsp90 utilises the energy released from epoxide with a cyclopropyl group in order to improve the stabi-
ATP-hydrolysis to effect the maturation or activation of client lity of the molecule. Although a drop in potency was observed
proteins and has been found to play a pivotal role in numerous (IC50 = 160 nM vs. IC50 = 20–23 nM for radicicol), cyclopropara-
oncogenic pathways. More recently, the significance of dicicol 3 was still a very potent Hsp90 inhibitor. It is note-
Hsp90 has increased further following the discovery of its rele- worthy that the stereochemistry of the cyclopropyl group was
vance in a number of other important medical conditions, also crucial for the activity. Additionally, the epoxide has been
ranging from neurodegenerative conditions,3–8 to HIV/ replaced with a thiirane,32 a cyclic carbonate,32 a diol32 and a
AIDS,9–11 and malaria.12–14 As a result, significant research
efforts have been devoted to discovering and synthesising
potent small molecule inhibitors of Hsp90, a topic which has
been the focus of many reviews.2,9,10,15–22 Seminal research in
this area was centred on the natural products geldanamycin
1,23,24 a benzoquinone ansamycin polyketide, which has been
the subject of previously published research from our
group,25–27 and radicicol 2, a resorcylic acid lactone (RAL) first
isolated in 1953,28 which is the most potent in vitro Hsp90
inhibitor found to date (IC50 = 20 – 23 nM).29–31 Unfortunately,
radicicol exhibits no in vivo activity, which may be due to the
highly sensitive functionality present in the molecule, includ-
ing an epoxide and a conjugated dienone, both of which are
aSchool of Chemistry, University of Nottingham, University Park, Nottingham NG7
2RD, UK. E-mail: c.j.moody@nottingham.ac.uk; Fax: (+44) (0)115 951 3564
bGenome Damage and Stability Centre, Science Park Road, University of Sussex,
Falmer, Brighton BN1 9RQ, UK. E-mail: chris.prodromou@sussex.ac.uk;
Fax: (+44) (0)1273 678121
†Electronic supplementary information (ESI) available: Copies of NMR spectra.
See DOI: 10.1039/c3ob42211a
Fig. 1 Radicicol analogue Hsp90 inhibitors.
1328 | Org. Biomol. Chem., 2014, 12, 1328–1340
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carbon chain (both saturated and unsaturated).33 Whilst the years.25–27,33,34,36,42,43 As previously mentioned, despite still
former three are significantly detrimental to the potency, a inhibiting Hsp90 effectively, replacement of the dienone with
carbon chain is tolerated with only a two-fold drop in perform- a C-11 ketone and the epoxide with a double bond or a satu-
ance.33 The replacement of the dienone with more stable func- rated hydrocarbon chain (compound 4) led to a 2-fold decrease
tionalities has also been investigated. Moody and Shinonaga in activity compared to radicicol itself,33 whilst the introduc-
have developed radicicol analogues with a C11 ketone and tion of extra H-bonding, either at the 6-position, or from a tria-
either a saturated carbon chain from C7–C10 or an enone zole in the macrocycle,34,36 gave a conformational change in
(4 and 5, respectively).32–34 Additionally, Danishefsky has the molecule and a loss in binding affinity. We also investi-
replaced the dienone of cycloproparadicicol with a triazole 635 gated the effect of varying the ring size and double bond posi-
and we have also incorporated a triazole into the macrocycle tion within the macrocycle and found that those analogues
(compound 7) to investigate the benefits of additional with 14 or 15-membered rings and the double bond located
H-bonding potential.36 Unfortunately, it appears that the con- γ,δ- with respect to the lactone ring oxygen to be the most
formational constraints of RALs is important for binding to potent Hsp90 inhibitors, whilst a 12,13 or 16-membered ring
the Hsp90 N-terminal domain and, as such, all of the ana- gave depleted activity.33 More recently, we investigated the syn-
logues showed a significant drop in activity compared to radi- thesis of a series of resorcylic acid lactams as more metaboli-
cicol itself, although potencies commensurate with other cally stable radicicol analogues,43 a strategy widely employed
important Hsp90 inhibitors were achievable.35
in drug development and also utilised in the development of
High throughput screening of simplified radicicol ana- similar RAL analogues by Danishefsky44 and Winssinger.45
logues, identified resorcinol-containing diaryl pyrazoles as Our original synthetic route is outlined in Scheme 1: addition
potent Hsp90 inhibitors.37 The hydrophobic group at C5 (alkyl of the malonate-derived acid chloride 13 to anhydride 12 gave
or halide), along with two free phenols were found to be essen- the acylated intermediate, which spontaneously underwent a
tial for a high binding affinity from SAR analysis, whilst the cyclisation/retrocyclisation, with the loss of CO2, giving the iso-
heterocycle was found to be variable, with isoxazoles, triazoles coumarin 14 in 75% yield. This was readily converted into
and benzisoxazoles also potent Hsp90 inhibitors.17,22,38,39 For both the NH and NMe macrolactams 16 (see Fig. 3 for bound
such compounds, the heterocycle mimics the radicicol lactone X-ray structure) and 17, respectively, and deprotection of the
unit, satisfying the same H-bonding interactions with the β-keto ester intermediate 15 also allowed for the incorporation
Hsp90 ATP-binding site. Lead optimisation gave compounds of amide and ester functional groups at C10 (18 and 19,
such as the resorcinol-pyrazole CCT018159 9 (IC50 = 7.1 μM), respectively), giving additional H-bonding potential. Pleas-
reported by Workman and colleagues.40,41 Presently, there are ingly, the macrolactams were indeed more metabolically stable
four closely related compounds in clinical trials.2 These (43% metabolism for macrolactam 17 vs. 84% for radicicol
include ganetespib 10, owned by Synta Pharmaceuticals, admi- after 15 minutes with human liver microsomes and NADH/
nistered intravenously, which has been evaluated in the NADPH) and significantly, were also often superior to the
clinic against non-small cell lung carcinoma and advanced corresponding macrolactones in terms of activity.43
solid tumours, and NVP-AU922 11, another intravenous drug
owned by Vernalis-Novartis, which is currently in phase 2 clini- pounds 18 and 19 showed by X-ray crystallography that the
cal trials (Fig. 2).2
C10 substituents displaced a loop between Leu93 and Lys98 in
Analysis of the Hsp90-bound protein structures of com-
Our laboratory has been interested in developing novel the ATP-binding site, allowing access to a further hydrophobic
Hsp90 inhibitors, including the synthesis of more potent and pocket, favourable for binding, and enhancing the biological
metabolically stable radicicol analogues, for some properties of these analogues.43 Since only one of these ana-
logues contained a substituted lactam nitrogen, we wanted to
extend our SAR and synthesise more of these analogues. We
herein report a new and improved synthetic route to N-methy-
lated resorcylic acid macrolactams, which is shorter and more
convergent and allows us to vary the ring size and the position
of the double bond. We also report the binding of these ana-
logues to Hsp90, as studied by protein crystallography.
Results and discussion
The synthesis commenced from the methyl orsellinate 20,
readily prepared via a self-condensation of methyl acetoace-
tate.25 In our hands, the success of the previously reported46–48
sulfuryl chloride-mediated chlorination was found to be highly
temperature dependent; a reaction temperature of −30 °C was
Fig. 2 Pyrazole and isoxazole-containing Hsp90 inhibitors.
crucial for selective monochlorination, with the product 21
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Scheme 1 Original synthesis of 14-membered macrolactams, analogues of the RALs.43
Scheme 2 The synthesis of orsellinic acid 23 [EOM = ethoxymethyl].
initially performed a HATU-mediated amide coupling with
N-methylbut-3-en-1-amine and obtained the product in quanti-
tative yield. However, subsequent attempts to deprotonate and
acylate the methyl group were unsuccessful with a variety of
bases, additives and reaction conditions. We therefore turned
to dianion chemistry, described previously in similar synthetic
routes.50 Initial attempts to acylate the dianion formed from
the treatment of orsellinic acid 23 with two equivalents of base
(LDA or s-BuLi) were particularly low yielding, with only traces
Fig. 3 Hsp90-bound X-ray crystal structure of macrolactam 16.43
Image from the article by Day et al.,43 reprinted, with permission from
J. E. H. Day, S. Y. Sharp, M. G. Rowlands, W. Aherne, A. Hayes,
F. I. Raynaud, W. Lewis, S. M. Roe, C. Prodromou, L. H. Pearl,
P. Workman, C. J. Moody, ACS Chem. Biol., 2011, 6, 1339–1347. Copy-
right (2011) American Chemical Society.
isolated in 98% yield, whereas higher temperatures gave of product obtained and no purification possible without pro-
further chlorination. Protection of the resorcinol as the ethoxy- tecting group cleavage as described previously. Following sig-
methoxy ether proceeded smoothly, but saponification of the nificant investigative effort, it was established that the scale
ester proved to particularly sluggish, with lengthy reaction and concentration of the acylations were crucial for the
times at reflux with potassium hydroxide required. Addition- success of the reaction. Following optimisation studies, the
ally, the orsellinic acid product 2334,49 was incompatible with reactions were performed on scales in excess of 0.5 mmol of
chromatography; any attempts led to deprotection of the orsellinic acid, at a concentration of 0.2 M in THF at −78 °C,
hydroxyl group at the 6-position. Instead, a carefully controlled with the product 24 being coupled with the relevant amine salt
work-up procedure was performed, washing the basic solution without further purification. This protocol gave satisfactory
with organic solvent to remove any organic impurities, then yields over two steps (Scheme 3).
rapid acidification and extraction of the pre-cooled solution to
minimise any undesired deprotection. Satisfactory yields of lence in the proton NMR spectrum, exemplified by the split-
60–69% were achievable via this method (Scheme 2).
ting of the benzylic protons, observed as a pair of roofed
Interestingly, the products 25 exhibited magnetic inequiva-
With the key intermediate 23 in hand, our focus now doublets (J = 17 Hz). We postulated that this is a result of the
turned to the amide coupling and benzylic acylation steps. N-methyl group being sufficiently bulky to hinder the rotation
With the option of varying the order of the two steps, we past the lower chain, creating pairs of rotamers.
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Scheme 3 Acylation and coupling investigations.
Scheme 4 Formation of the radicicol analogues.
The macrocycles were formed via ring closing metathesis,
The radicicol analogues 27b–d were co-crystallised (major
utilising Grubbs’ 2nd generation catalyst, in moderate to good E/Z-isomer crystallised in all cases) with yeast Hsp90 in order
yield and as mixtures of E/Z-isomers. Deprotection of the EOM to probe the interaction of the compounds with the Hsp90
groups proceeded smoothly with trifluoroacetic acid, giving N-terminal domain (Fig. 4). The resorcylic acid macrolactams
the N-methyl resorcylic acid macrolactams 27 in up to 12% 27 bind to Hsp90 in a similar fashion to radicicol 2, with the
yield over four steps from the known precursor 23. The macro- same interactions exhibited by the resorcinol group in all
cycles were characterised as inseparable mixtures of E/Z- cases. However, there is a clear conformational change in the
isomers, with the major component being the E-isomer in all macrocycle from radicicol to the macrolactams, emphasising
cases with the exception of the 14-membered lactam 27d, the importance of the interaction between the radicicol
which was predominantly the Z-isomer (Scheme 4).
epoxide and the Hsp90 backbone. For radicicol the electron
Next, the thermodynamics of the binding of macrocycles density contoured at 1.5σ is clearly visible for the whole mole-
27b–d to Hsp90 was investigated through isothermal titration cule. In contrast, for radicicol analogues 27b–d, electron
calorimetry (ITC) (Table 1). The results show that there is a sig- density for the macrocycle was poor. Electron density con-
nificant enthalpic penalty compared to radicicol, although the toured at 1.5σ for compound 27b and 27d was lacking for
binding of the macrolactams was found to be superior to the carbon atoms 4 to 9 of the macrocycle ring. For compounds
macrolactone analogues we have previously described33 (Kd
=
27c the macrocycle was not represented by clear electron
210–1200 μM). It should be noted that compounds 27b–d are density for the carbon atoms 7 to 11. These observations are
mixtures of geometric isomers, albeit with heavy predomi- consistent with the idea that the epoxide group is important
nance of one isomer in each case, that makes interpretation of for radicicol binding. This is even more apparent on studying
the binding data less clear. Nevertheless the data suggest that the overlay of radicicol 2 with the corresponding NH and NMe
the interaction of the epoxide plays a significant role in the macrolactam analogues (17 and 27d, respectively), depicted
interaction with the protein.
in Fig. 5.
In conclusion we have devised a route to, and successfully
synthesised, a series of novel N-methyl macrolactam radicicol
analogues as Hsp90 inhibitors. Their binding to Hsp90 was
investigated and, despite diminished affinity compared to radi-
cicol itself, the binding was found to be superior to the corres-
ponding macrolactones. Importantly, details of the binding to
Hsp90 inhibitors through co-crystallisation with the protein,
with a change in the conformation of the macrocycle was
observed.
Table 1 Isothermal titration calorimetry for macrolactams 27b–d with
yeast Hsp90 N-terminal domain (N = stoichiometry of binding)
Ligand
N
Kd/μM
ΔH/kJ mol−1 ΔS/J mol−1 K−1
Radicicol 1.16
0.015 0.06 −6726
13.6
14.2
17.3
13.3
27b
27c
27d
1.0 (fixed)
0.92
1.1
144 25
41.8 8.7
110 7.9
−1012
−834.8
−1466
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Fig. 4 Structures of resorcylic acid macrolactams (yellow) bound in the ATP site of yeast Hsp90 as determined by protein X-ray crystallography,
showing the interactions of (a) radicicol 2 with Hsp90 (green and cyan residues); (b) macrolactam 27b (E-alkene) with Hsp90 (green and cyan resi-
dues); (c) macrolactam 27c (E-alkene) with Hsp90 (green and cyan residues); (d) macrolactam 27d (Z-alkene) with Hsp90 (green and cyan residues).
In general, the analogues exhibit a significant conformational change in the macrocycle. Dotted blue lines: hydrogen bonds, red-coloured spheres:
water molecules. The structures for radicicol and the macrolactam analogues were obtained at 2.5 (radicicol), 2.4 (27b), 2.0 (27c) and 2.3 Å (27d)
resolution and the atomic coordinates and structure factors deposited with the following PDB codes 4ce2, 4ce1 and 4ce3, respectively.
Thin layer chromatography (TLC) was carried out using
General experimental details
Merck Kieselgel 60GF254 pre-coated aluminium-backed plates.
Unless specified, all reagents were purchased from commercial The compounds were visualised using UV light (254 nm) and
sources and were used without further purification. Tetra- basic aqueous potassium permanganate solution, with
hydrofuran was distilled from sodium-benzophenone ketyl heating. Flash column chromatography was performed at
immediately before use. Water refers to deionised water, ether medium pressure using slurry packed Davisil silica gel
to diethyl ether, and light petroleum refers to the fraction with 35–70 μm, 60 Å with the specified eluant. Silver nitrate-impreg-
bp 40–60 °C.
nated silica TLC plates were prepared by soaking the Merck
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327.25 mmol, 0.95 eq.) was added via cannula over 1 h and the
solution was allowed to warm to room temperature over 16 h,
before being heated under reflux for 24 h. The mixture was re-
cooled to 0 °C and hydrochloric acid (1 M; 400 mL) was added
cautiously, the aqueous layer was acidified to pH 1 with con-
centrated hydrochloric acid and the mixture was warmed to
room temperature and stirred for 18 h. The layers were separ-
ated and the aqueous layer was washed with ethyl acetate (2 ×
400 mL). The combined organics were washed with water
(400 mL) and saturated brine (400 mL), before being dried
over magnesium sulfate and concentrated in vacuo. The
residue was purified by recrystallisation (ethyl acetate–light
petroleum) to give the title compound as a light brown, crystal-
line solid (16.31 g, 52%); δH (400 MHz; CDCl3) 11.41 (1H, s,
OH), 6.53 (1H, s, ArH), 6.08 (1H, s, ArH), 5.09 (1H, br. s, OH),
3.95 (3H, s, OMe), 2.63 (3H, s, CH3); δC (100 MHz; CDCl3)
171.4 (C), 162.9 (C), 156.0 (C), 139.5 (C), 107.1 (C), 102.0 (CH),
52.3 (Me), 30.9 (CH), 19.9 (Me); data consistent with those
reported in the literature.25
Fig. 5 Overlaid crystal structures showing the difference in the confor-
mation and binding of resorcylic acid macrolactams in comparison to
radicicol. The different conformations of radicicol 2 (PDB code 1BGQ;
green), NH macrolactam 16 (PDB code 2XX2; yellow)43 and NMe macro-
lactam 27d (PDB code 4ce3; cyan).
Kieselgel 60GF254 pre-coated aluminium-backed plates in a
1 : 3 w/v solution of AgNO3 in MeCN for 5 min, before being
dried in an oven at 150 °C for 10 min. The TLC plates were
covered in aluminium foil and stored in the dark. Compounds
were visualised with basic aqueous potassium permanganate
solution and heating.
Methyl 3-chloro-4,6-dihydroxy-2-methylbenzoate 21
To a solution of methyl 2,4-dihydroxy-6-methylbenzoate 20
(5.73 g, 31.48 mmol, 1.0 eq.) in THF (315 mL) at −30 °C was
added sulfuryl chloride (2.78 mL, 34.62 mmol, 1.1 eq.) slowly
over 5 min, keeping the temperature between −20 and −30 °C
throughout. After stirring for a further 30 min, the reaction
mixture was quenched with ammonium chloride (200 mL) and
the layers were stirred and separated. The aqueous layer was
extracted with ether (2 × 200 mL) and the combined organics
were washed with saturated brine (2 × 200 mL), before being
dried over sodium sulfate and concentrated in vacuo to give a
yellow solid. The product was purified by recrystallisation
(dichloromethane–light petroleum) to give the title compound
as a colourless solid (6.651 g, 98%); mp 133–135 °C (lit.,51 mp
134 °C); δH (270 MHz, CDCl3) 11.41 (1H, s, 6-OH), 6.52 (1H, s,
H-5), 6.03 (1H, s, 4-OH), 3.31 (3H, s, OMe), 2.61 (3H, s, Me); δC
(100 MHz, CDCl3) 171.4 (C), 162.9 (C), 156.0 (C), 139.5 (C),
113.9 (C), 107.1 (C), 102.0 (CH), 52.3 (Me), 19.9 (Me). Data con-
sistent with those reported in the literature.46
Infrared spectra were obtained on a Perkin Elmer 1600
series FT-IR spectrometer using NaCl solution cells in chloro-
1
form or acetone. H NMR and 13C NMR spectra were obtained
using a JEOL EX270 operating at 270 MHz (13C 67.5 MHz), or
Bruker Avance III-400, Bruker Avance 400 or Bruker DPX 400
spectrometers operating at 400 MHz (13C 100 MHz). All spec-
troscopic data was recorded at 295 K. Chemical shifts are
quoted in parts per million (ppm), using the residual solvent
peak as an internal standard (7.26 ppm [1H NMR] and
77.2 ppm [13C NMR] for CDCl3 and 2.05 ppm [1H NMR] and
206.7, 29.9 ppm [13C NMR] for acetone-d6). Coupling constants
(J) are quoted in Hz. Multiplicity abbreviations used: s singlet, d
doublet, t triplet, q quartet, m multiplet. Signal assignment was
achieved via analysis of DEPT, COSY, HMBC and HSQC exper-
1
iments where needed. H NMR and 13C NMR spectra of final
compounds were performed on a Bruker AV(III)800 at 800 and
200 MHz, respectively. Low and high-resolution mass spectra
were performed for all novel compounds. Electrospray ionis-
ation (ESI) and high resolution mass spectrometric (HRMS) ana-
lyses were performed on a Bruker MicroTOF spectrometer.
Methyl 3-chloro-4,6-di(ethoxymethoxy)-2-methylbenzoate 22
Methyl 2,4-dihydroxy-6-methylbenzoate 2025
To a solution of methyl 3-chloro-4,6-dihydroxy-2-methylbenzo-
Sodium hydride (20.67 g, 0.52 mol, 1.5 eq.) was prewashed ate 21 (7.03 g, 32.52 mmol, 1.0 eq.) in dimethylformamide
with pentane (3 × 50 mL), before being suspended in THF (108 mL) at 0 °C under argon was added ethyl chloromethyl
(800 mL) and cooled to 0 °C under argon. Methyl acetoacetate ether (7.54 mL, 81.29 mmol, 2.5 eq.). N,N-Diisopropylethyl-
(40.00 g, 0.34 mol, 1.0 eq.) was added dropwise over 2 h and amine (16.99 mL, 97.55 mmol, 3.0 eq.) was added slowly and
the reaction mixture was warmed to room temperature over the reaction mixture was stirred for 30 min, before being
1 h. After cooling to −78 °C, n-butyllithium (130.90 mL, allowed to warm to room temperature and stirred for a further
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h. Saturated aqueous ammonium hydroxide solution an oven dried flask at −78 °C was added sec-butyllithium
(100 mL) was added and the reaction mixture was stirred for a (1.08 mL, 1.19 mmol, 2.2 eq.). The resulting red solution
further 10 min, before being extracted with ether (3 × 100 mL). was stirred for 40 min before a solution of N-methoxy-N-
The combined organics were washed with aqueous lithium methylhept-6-enamide (138 mg, 0.81 mmol, 1.5 eq.) in THF
chloride solution (5%; 3 × 100 mL), dried over magnesium (0.3 mL) was added. The reaction mixture was stirred for a
sulfate and concentrated in vacuo to give a yellow oil. The further 50 min at −78 °C. Water (5 mL) was added and the
product was purified by flash column chromatography, eluting solution was washed with ether (2 × 5 mL). The aqueous
with 9 : 1 light petroleum–ethyl acetate to give the title compound layer was acidified to pH 1 with hydrochloric acid (1 M;
as a colourless oil (10.37 g, 96%); (Found: M + Na+, 355.0916. 5 mL) and extracted with ethyl acetate (3 × 5 mL). The com-
C15H2135ClO6 + Na+, requires 355.0919); νmax (CHCl3)/cm−1 3526, bined organics were dried over magnesium sulfate and con-
2981, 2955, 1728, 1660, 1596, 1476, 1433, 1321, 1265, 1114, centrated in vacuo to give a yellow oil (171 mg). The crude
1042, 963, 932, 844; δH (400 MHz; CDCl3) 7.02 (1H, s, ArH), 5.32 material was used without further purification. 3-Chloro-4,6-
(2H, s, OCH2O), 5.22 (2H, s, OCH2O), 3.93 (3H, s, OMe), di(ethoxymethoxy)-2-(2-oxooct-7-en-1-yl)benzoic acid (171 mg,
3.82–3.76 (2H, q, J = 7.0, OCH2), 3.76–3.71 (2H, q, J = 7.0, OCH2), 0.40 mmol 1 eq.) was dissolved in DMF (2 mL). To the reac-
2.34 (3H, s, Me), 1.27–1.23 (3H, t, J = 7.0, CH3), 1.26–1.22 (3H, t, tion solution was added N-methylbut-3-en-1-amine hydro-
J = 7.0, CH3); δC (100 MHz; CDCl3) 167.9 (C), 154.3 (C), 153.1 (C), chloride
(73
mg,
0.60
mmol,
1.5
eq.),
N,N-
135.1 (C), 119.8 (C), 117.1 (C), 101.7 (CH), 93.8 (CH2), 93.8 diisopropylethylamine (2.15 mL, 1.60 mmol, 4.0 eq.) and
(CH2), 64.6 (CH2), 64.4 (CH2), 52.3 (Me), 17.5 (Me), 15.0 (Me), HATU (174 mg, 0.46 mmol, 1.15 eq.). The reaction mixture
15.0 (Me); m/z (ESI) 355 ([M(35Cl) + Na]+, 100%), 357 (33.0%).
was stirred at room temperature for 18 h. A further 0.2
equivalents of N-methylbut-3-en-1-amine hydrochloride
(10 mg) and 0.1 equivalents of HATU (15 mg) were added
and the reaction mixture was stirred for a further 3 h. The
solution was diluted with ethyl acetate (10 mL) and washed
with hydrochloric acid (1 M; 5 mL), saturated aqueous
sodium hydrogen carbonate (5 mL) and brine (5 mL). The
organic layer was dried over magnesium sulfate and concen-
trated in vacuo to give a yellow oil. The residue was purified
by flash column chromatography, eluting with 4 : 1, then
2 : 1 light petroleum–ethyl acetate to give the title compound
as a colourless oil (0.056 g, 33% over 2 steps) (3 : 1 mixture
3-Chloro-4,6-di(ethoxymethoxy)-2-methylbenzoic acid 23
To
a stirred solution of methyl 3-chloro-4, 6-di(ethoxy-
methoxy)-2-methylbenzoate 22 (6.26 g, 18.82 mmol, 1.0 eq.) in
1 : 1 methanol–water (60 mL) was added potassium hydroxide
(5.28 g, 94.09 mmol, 5.0 eq.) and the reaction mixture was
heated to 70 °C and stirred for 3 d. After cooling to room temp-
erature, water (50 mL) was added and the mixture was
extracted with ethyl acetate (50 mL). The aqueous layer was
cooled to 0 °C, acidified to pH 1 with concentrated hydro-
chloric acid and quickly extracted with ethyl acetate (3 ×
75 mL). The combined organic extracts were washed with
brine (100 mL), dried over magnesium sulfate and concen-
trated in vacuo to give the title compound as a colourless solid
(4.15 g, 69%); mp 78–80 °C; (Found: C, 52.8; H, 6.0%.
C14H1935ClO6, requires C, 52.8; H, 6.0%); δH (400 MHz;
acetone-d6) 7.10 (1 H, s, ArH), 5.36 (2H, s, OCH2O), 5.27 (2 H,
s, OCH2O), 3.78 (2H, q, J 7.1, OCH2), 3.73 (2H, q, J 7.1, OCH2),
2.36 (3 H, s, Me), 1.21 (3H, t, J 7.1, Me), 1.19 (3H, t, J 7.1, Me);
δC (100 MHz; acetone-d6) 169.1 (C), 154.9 (C), 153.7 (C), 136.9
(C), 117.8 (C), 110.0 (C), 101.7 (CH), 94.3 (CH2), 93.8 (CH2),
64.8 (CH2), 64.8 (CH2), 17.9 (Me), 15.0 (Me), 15.0 (Me). Data
consistent with those reported in the literature.31
of isomers); (Found: M + Na+, 518.2287. C26H3835ClNO6
+
Na+, requires 518.2280); νmax (CHCl3)/cm−1 3665, 3079, 3001,
1759, 1628, 1590, 1486, 1453, 1327, 1244, 1054, 1036, 1029;
major isomer δH (400 MHz; CDCl3) 7.08 (1H, s, ArH),
5.89–5.73 (2H, m, CH), 5.29 (1H, d, J 12.8, OCH2O), 5.27
(1H, d, J 12.8, OCH2O), 5.18 (2H, s, OCH2O), 5.14–4.92 (4H,
m, vCH2), 3.98 (1H, d, J 17.6, CH2), 3.79 (1H, d, J 17.6,
CH2), 3.80–3.73 (2H, m, OCH2), 3.72–3.66 (2H, m, OCH2),
3.57 (1H, dt, J 13.1, 6.8, CH2), 2.83 (3H, s, NMe), 2.54–2.48
(2H, m, CH2), 2.39–2.34 (2H, m, CH2), 2.08–2.04 (2H, m,
CH2), 1.65–1.57 (3H, m, CH2), 1.43–1.36 (2H, m, CH2), 1.23
(6H, 2 × t, J 14.5, 7.1, Me); δC (100 MHz; CDCl3) 206.5 (C),
167.6 (C), 153.8 (C), 152.1 (C), 138.5 (CH), 135.4 (CH), 134.7
(C), 132.3 (C), 122.8 (C), 116.6 (CH2), 114.6 (CH2), 102.5
(CH), 94.0 (CH2), 93.7 (CH2), 64.6 (CH2), 64.4 (CH2), 46.6
(CH2), 44.9 (CH2), 42.5 (CH2), 36.1 (Me), 33.5 (CH2), 31.6
(CH2), 28.3 (CH2), 23.2 (CH2), 15.0 (Me), 15.0 (Me); for the
minor isomer, the following NMR signals are discernable,
the rest overlap with the major isomer: δH (400 MHz;
CDCl3) 7.12 (1H, s, ArH), 5.68–5.56 (2H, m, CH), 5.30 (1H,
d, J 15.1, O–CH2–O), 5.28 (1H, d, J 15.1, O–CH2–O), 3.87
(1H, d, J 17.5, CH2), 3.04 (3H, s, NMe), 2.26–2.21 (2H, m,
CH2); δC (100 MHz; CDCl3) 117.7 (CH2), 117.0 (CH2), 102.7
(CH), 64.5 (CH2), 50.2 (CH2), 45.3 (CH2), 42.2 (CH2), 32.6
N-(But-3-en-1-yl)-3-chloro-4,6-di(ethoxymethoxy)-N-methyl-2-
(2-oxooct-7-en-1-yl)benzamide 25a
To a solution of 3-chloro-4,6-di(ethoxymethoxy)-2-methylben- (CH2), 32.2 (CH2); m/z (ESI) 518 ([M(35Cl) + Na]+, 100%), 519
zoic acid 23 (170 mg, 0.54 mmol, 1.0 eq.) in THF (2.67 mL) in (27.0%), 520 (35.6%), 521 (9.5%).
1334 | Org. Biomol. Chem., 2014, 12, 1328–1340
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13-Chloro-14,16-di(ethoxymethoxy)-2-methyl-3,4,7,8,9,10-
hexahydrobenz[c][1]azacyclotetradecine-1,11(2H,12H)-dione
26a
dichloromethane (2 mL) was added trifluoroacetic acid
(0.32 mL, 0.04 mmol, 1.5 eq.) and the reaction mixture was
stirred at room temperature for 30 min. Saturated aqueous
sodium hydrogen carbonate (5 mL) was added and the mixture
was extracted with ethyl acetate (3 × 10 mL). The combined
organics were washed with saturated brine (10 mL), dried over
sodium sulfate and concentrated in vacuo to give a yellow oil.
The residue was purified by flash column chromatography,
eluting with 1% methanol–dichloromethane to give a colour-
less oil (7 mg, 72%) (9 : 1 mixture of isomers); major isomer δH
(400 MHz; CDCl3) 8.66 (1H, br. s, OH), 6.55 (1H, s, ArH), 5.51
(2H, dt, J 15.4, 9.0, CHvCH), 4.46 (1H, ddd, J 13.3, 9.3, 3.9,
CH2), 3.89 (2H, s, CH2), 2.94–2.83(1H, m, CH2), 2.81 (3H, s,
NMe), 2.77 (1H, ddd, J 13.6, 6.4, 3.8, CH2), 2.61 (1H, ddd, J
16.3, 8.1, 2.0, CH2), 2.52–2.33 (3H, m, CH2), 2.15–2.09 (1H, m,
CH2), 1.74 (1H, m, CH2), 1.68–1.58 (1H, m, CH2), 1.53–1.46
(2H, m, CH2); δC (100 MHz; CDCl3) 204.8 (C), 167.9 (C), 153.3
(C), 152.2 (C), 132.0 (CH), 129.1 (CH), 119.7 (C), 110.2 (C),
109.9 (C), 102.2 (CH), 46.8 (CH2), 44.2 (CH2), 40.2 (CH2) 35.1
(Me), 30.3 (CH2), 30.1 (CH2), 26.3 (CH2), 21.5 (CH2); for the
minor isomer, the following NMR signals are discernable, the
rest overlap with the major isomer: δH (400 MHz; CDCl3) 6.57
(1H, s, ArH), 5.58–5.44 (2H, m, CHvCH), 4.06 (1H, ddd, J 14.3,
8.05, 2.88, CH2), 3.97, (2H, d, J 9.3, CH2) 2.82 (3H, s, Me); δC
(100 MHz; CDCl3) 130.7 (CH), 44.1 (CH2). Data consistent with
those reported in the literature.1
A solution of N-(but-3-en-1-yl)-3-chloro-4,6-di(ethoxymethoxy)-
N-methyl-2-(2-oxooct-7-en-1-yl)benzamide
25a
(56
mg,
0.11 mmol, 1.0 eq.) dichloromethane (35 mL) was sparged
with nitrogen and Grubbs II catalyst (11 mg, 0.01 mmol, 0.11
eq.) was added. The reaction mixture was heated to reflux for
90 min. After cooling to room temperature, DMSO (0.1 mL)
was added and the solution was stirred for 18 h. The reaction
mixture was concentrated in vacuo and purified by flash
column chromatography, eluting with 4 : 1 light petroleum–
ethyl acetate to give the title compound as a colourless oil
(0.027 g, 51%) (4 : 1 mixture of E/Z isomers); (Found: M + Na+,
490.1956. C24H3435ClNO6
+
Na+, requires 490.1967);
νmax (CHCl3)/cm−1 3011, 2928, 2855, 1720, 1624, 1592, 1464,
1323, 1239, 1118, 1045, 929; major isomer δH (400 MHz; CDCl3)
7.10 (1H, s, ArH), 5.57–5.39 (2H, m, CHvCH), 5.30 (1H, d, J
15.1, OCH2O), 5.28 (1H, d, J 15.1, OCH2O), 5.22–5.18 (2H, m,
OCH2O), 4.56 (1H, ddd, J 19.1, 9.6, 5,6, CH2), 4.03 (1H, d, J
17.9, CH2), 3.85 (1H, d, J 17.9, CH2), 3.78 (2H, dq, J 14.2, 7.1,
OCH2), 3.70 (2H, dq, J 14.2, 4.1, OCH2), 2.77 (3H, s, NMe), 2.68
(1H, ddd, J 16.9, 7.9, 1.9, CH2), 2.59–2.50 (1H, m, CH2),
2.46–2.31 (3H, m, CH2), 2.20–2.11 (1H, m, CH2), 2.07–1.99 (1H,
m, CH2), 1.83–1.75 (1H, m, CH2), 1.68–1.59 (1H, m, CH2)
1.56–1.47 (2H, m, CH2), 1.23 (6H, 2 × t, J 8.8, 7.1, Me),
0.97–0.88 (1H, m, CH2); δC (100 MHz; CDCl3) 205.8 (C), 168.2
(C), 153.7 (C), 152.0 (C), 132.7 (CH), 132.0 (C), 128.4 (CH),
123.0 (C), 117.9 (C), 102.7 (CH), 93.9 (CH2), 93.7 (CH2), 64.5
(CH2), 64.4 (CH2), 46.3 (CH2), 44.8 (CH2), 40.4 (CH2), 36.1
(Me), 30.4 (CH2), 30.2 (CH2), 26.2 (CH2), 21.6 (CH2), 15.0 (Me),
15.0 (Me); for the minor isomer, the following NMR signals are
discernable, the rest overlap with the major isomer: δH
(400 MHz; CDCl3) 7.11 (1H, s, ArH), 4.06 (1H, d, J 18.1, CH2),
3.95 (1H, d, J 18.1, CH2), 2.82 (3H, s, NMe); δC (100 MHz;
CDCl3) 206.2 (C), 151.9 (C), 132.2 (CH), 131.2 (C), 129.3 (CH),
102.6 (CH), 47.3 (CH2), 44.6 (CH2), 41.6 (CH2), 37.1 (Me), 29.7
(CH2), 27.3 (CH2), 25.4 (CH2), 21.9 (CH2); m/z (ESI) 490
([M(35Cl) + Na]+, 100%), 492 (33.0%).
N-(But-3-en-1-yl)-3-chloro-4,6-di(ethoxymethoxy)-N-methyl-2-
(2-oxonon-8-en-1-yl)benzamide 25b
As for the procedure for compound 25a, using 3-chloro-4,6-
di(ethoxymethoxy)-2-methylbenzoic acid 23 (200 mg, 0.63 mmol,
1.0 eq.), N-methoxy-N-methyloct-7-enamide (187 mg,
0.94 mmol, 1.5 eq.), N-methylbut-3-en-1-amine hydrochloride
(63 mg, 0.52 mmol, 1.5 eq.), N,N-diisopropylethylamine
(0.24 mL, 1.38 mmol, 4.0 eq.) and HATU (151 mg, 0.40 mmol,
1.15 eq.). The product was purified by flash column chromato-
graphy, eluting with 4 : 1 light petroleum–ethyl acetate to give
the title compound as a colourless oil (0.070 g, 25% over 2
steps) (3 : 1 mixture of isomers); (Found: M + Na+, 532.2442.
C27H4035ClNO6 + Na+, requires 532.2436); νmax (CHCl3)/cm−1
3685, 3001, 2930, 1719, 1630, 1596, 1466, 1407, 1321, 1274,
1264, 1151, 1120, 910; major isomer δH (400 MHz; CDCl3) 7.08
(1H, s, ArH), 5.93–5.74 (2H, m, CH), 5.29 (1H, d, J 13.0,
OCH2O), 5.27 (1H, d, J 13.0, OCH2O), 5.20–5.16 (2H, m,
OCH2O), 5.14–4.91 (4H, m, vCH2), 3.98 (1H, d, J 17.7, CH2),
3.79 (1H, d, J 17.7, CH2), 3.80–3.74 (2H, m, OCH2), 3.72–3.66
(2H, m, OCH2), 2.83 (3H, s, NMe), 2.50 (2H, dt, J 12.2, 7.2,
CH2), 2.39–2.34 (2H, m, CH2), 2.26–2.20 (1H, m, CH2),
13-Chloro-14,16-dihydroxy-2-methyl-3,4,7,8,9,10-
hexahydrobenzo[c][1]azacyclotetradecine-1,11(2H,12H)-dione
27a1
To a solution of 13-chloro-14,16-di(ethoxymethoxy)-2-methyl- 2.06–2.01 (3H, m, CH2), 1.64–1.57 (2H, m, CH2), 1.37 (2H, ddd,
3,4,7,8,9,10-hexahydrobenzo[c][1]azacyclotetradecine-1,11- J 14.9, 8.6, 2.7, CH2), 1.34–1.28 (2H, m, CH2), 1.23 (6H, 2 × t,
(2H,12H)-dione 26a (13 mg, 0.03 mmol, 1.0 eq.) in J 14.5, 7.3, Me); δC (100 MHz; CDCl3) 196.5 (C), 167.6 (C), 138.8
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(C), 138.7 (C), 135.4 (C), 135.0 (C), 134.8 (C), 116.6 (CH2), 114.4 14-Chloro-15,17-dihydroxy-2-methyl-3,4,7,8,9,10,11,13-
(CH2), 110.6 (CH), 102.6 (CH), 102.5 (CH) 94.0 (CH2), 93.7 octahydro-1H-benz[c][1]azacyclopentadecine-1,12(2H)-dione
(CH2), 64.6 (CH2), 64.4 (CH2), 46.5 (CH2), 44.8 (CH2), 42.6 27b
(CH2), 36.1 (Me), 33.6 (CH2), 31.6 (CH2), 28.7 (CH2), 28.6
(CH2), 23.6 (CH2), 15.0 (Me), 15.0 (Me); for the minor isomer,
the following NMR signals are discernable, the rest overlap
with the major isomer: δH (400 MHz; CDCl3) 6.98 (1H, s, ArH),
5.68–5.55 (2H, m, CH), 5.30 (1H, d, J 15.3, OCH2O), 5.28 (1H,
As for the procedure for compound 27a, using 14-chloro-15,17-
d, J 15.3, OCH2O), 3.88 (1H, d, J 17.5, CH2), 3.57 (2H, dq, J
di(ethoxymethoxy)-2-methyl-3,4,7,8,9,10,11,13-octahydro-1H-
13.6, 9.4, OCH2), 2.81 (3H, s, NMe), 1.27–1.16 (6H, m, Me); m/z
benzo[c][1]azacyclopentadecine-1,12(2H)-dione 26b (26 mg,
(ESI) 532 ([M(35Cl) + Na]+, 100%), 533 (34.6%), 534 (36.1%),
0.05 mmol, 1 eq.). Product purified by flash column chromato-
535 (10.5%).
graphy, eluting with 2% methanol–dichloromethane to give
the title compound as
a colourless solid (9 mg, 45%)
(3 : 1 mixture of isomers); mp 102–104 °C; (Found: M + Na+,
388.1296. C19H2435ClNO4 + Na+, requires 388.1286); νmax
(CHCl3)/cm−1 3438, 3274, 2859, 1732, 1728, 1701, 1623, 1455,
1445, 1391, 1343, 1312, 1251, 1100; major isomer δH (800 MHz;
acetone-d6) 8.69 (1H, br. s, OH), 7.97 (1H, s, OH), 6.55 (1H, s,
ArH), 5.46–5.41 (1H, m, CH), 5.40–5.34 (1H, m, CH), 4.51 (1H,
ddd, J 14.7, 11.9, 3.4, CH2), 3.98 (1H, d, J 17.9, CH2), 3.76 (1H,
d, J 17.9 CH2), 2.94 (3H, s, NMe), 2.75–2.71 (2H, m, CH2),
2.45–2.40 (1H, m, CH2), 2.38–2.33 (1H, m, CH2), 1.80–1.75 (1H,
m, CH2), 1.59–1.54 (1H, m, CH2), 1.42–1.36 (2H, m, CH2),
1.32–1.27 (3H, m, CH2), 1.24–1.15 (2H, m, CH2); δC (200 MHz;
acetone-d6) 208.0 (C), 170.2 (C), 163.7 (C), 154.2 (C), 134.9 (C),
133.3 (CH), 130.1 (CH), 122.3 (C) 114.2 (C), 104.1 (CH), 47.1
(CH2), 47.0 (CH2), 46.9 (CH2), 43.5 (CH2), 37.1 (Me), 35.6
(CH2), 28.6 (CH2), 27.6 (CH2), 25.2 (CH2); for the minor isomer,
the following NMR signals are discernable, the rest overlap
with the major isomer: δH (800 MHz; acetone-d6) 5.27–5.21
(1H, m, CH), 5.22–5.19 (1H, m, CH), 4.38 (1H, ddd, J 14.7,
11.9, 3.4, CH2), 4.16 (1H, d, J 18.8, CH2), 3.99 (1H, d, J 18.8
CH2), 2.88 (3H, s, NMe), 2.62–2.56 (2H, m, CH2), 1.94–1.88
(1H, m, CH2); δC (200 MHz; acetone-d6) 207.9 (C), 170.1 (C),
155.2 (C), 133.1 (CH), 131.2 (CH), 101.9 (CH), 49.3 (CH2), 47.8
(CH2), 42.6 (CH2), 32.4 (CH2), 28.1 (CH2), 26.8 (CH2), 23.6
(CH2); m/z (ESI) 388 ([M(35Cl) + Na]+, 100%), 389 (18.6%), 390
(31.6%), 391 (5.8%).
14-Chloro-15,17-di(ethoxymethoxy)-2-methyl-3,4,7,8,9,10,11,13-
octahydro-1H-benzo[c][1]azacyclopentadecine-1,12(2H)-dione
26b
As for the procedure for compound 26a, using N-(but-3-en-1-
yl)-3-chloro-4,6-di(ethoxymethoxy)-N-methyl-2-(2-oxonon-8-en-
1-yl)benzamide 25b (70 mg, 0.14 mmol, 1.0 eq.). The product
was purified by flash column chromatography, eluting with
2 : 1 light petroleum–ethyl acetate to give the title compound as
a colourless oil (0.026 g, 39%) (2 : 1 mixture of isomers);
(Found: M + Na+, 504.2124. C25H3635ClNO6 + Na+, requires
504.2123); νmax (CHCl3)/cm−1 3686, 3600, 2928, 2855, 1717,
1605, 1465, 1286, 1269, 1044, 910; major isomer δH (400 MHz;
CDCl3) 7.10 (1H, s, ArH), 5.48–5.35 (2H, m, CHvCH), 5.30
(1H, d, J 16.6, OCH2O), 5.28 (1H, d, J 16.6, OCH2O), 5.22–5.18
(2H, m, OCH2O), 4.64 (1H, ddd, J 14.9, 9.4, 5.9, CH2), 4.13 (1H,
d, J 18.0, CH2), 3.83–3.74 (2H, m, OCH2), 3.73–3.67 (2H, m,
OCH2), 3.69 (1H, d, J 18.0, CH2), 2.83 (3H, s, NMe), 2.82–2.73
(2H, m, CH2), 2.48–2.33 (3H, m, CH2), 2.09–2.00 (2H, m, CH2),
1.87–1.75 (1H, m, CH2), 1.63–1.52 (1H, m, CH2), 1.46–1.32 (4H,
m, CH2), 1.24 (6H, 2 × t, J 13.1, 7.1, Me); δC (100 MHz; CDCl3)
207.5 (C), 168.8 (C), 153.7 (C), 152.2 (C), 132.5 (C), 132.3 (CH),
127.7 (CH), 123.7 (C), 117.4 (C), 102.7 (CH), 94.0 (CH2), 93.8
(CH2), 64.6 (CH2), 64.5 (CH2), 45.7 (CH2), 45.5 (CH2), 42.2
(CH2), 34.6 (Me), 30.8 (CH2), 29.9 (CH2), 26.7 (CH2), 25.9
(CH2), 23.4 (CH2), 15.1 (Me), 15.0 (Me); for the minor isomer,
the following NMR signals are discernable, the rest overlap
with the major isomer: δH (400 MHz; CDCl3) 7.11 (1H, s, ArH),
5.31 (1H, d, J 17.3, OCH2O), 5.29 (1H, d, J 17.3, OCH2O), 4.14
N-(But-3-en-1-yl)-3-chloro-4,6-di(ethoxymethoxy)-N-methyl-2-
(2-oxodec-9-en-1-yl)benzamide 25c
(1H, d, J 18.8, CH2), 3.27–3.19 (2H, m, CH2), 3.11–3.06 (1H, m, As for the procedure for compound 25a, using 3-chloro-4,6-
CH2), 2.64–2.56 (1H, m, CH2), 2.18–2.09 (2H, m, CH2), bis(ethoxymethoxy)-2-methylbenzoic acid 23 (200 mg,
1.99–1.91 (2H, m, CH2); δC (100 MHz; CDCl3) 207.0 (C), 154.1 0.63 mmol, 1.0 eq.), N-methoxy-N-methylnon-8-enamide
(C), 152.7 (C), 133.3 (C), 131.8 (CH), 128.4 (CH), 122.4 (C), (174 mg, 0.94 mmol, 1.5 eq.), N-methylbut-3-en-1-amine hydro-
118.2 (C), 102.4 (CH), 94.1 (CH2), 94.0 (CH2), 48.0 (CH2), 46.8 chloride (74 mg, 0.61 mmol, 1.5 eq.). The product was purified
(CH2), 41.2 (CH2), 31.1 (CH2), 29.0 (CH2), 26.0 (CH2), 25.2 by flash column chromatography, eluting with 4 : 1 light pet-
(CH2), 22.0 (CH2); m/z (ESI) 504 ([M(35Cl) + Na]+, 100%), 505 roleum–ethyl acetate to give the title compound as a colourless
(23.4%), 506 (32.4%), 507 (8.4%).
oil (0.051 g, 16% over 2 steps) (4 : 1 mixture of isomers);
1336 | Org. Biomol. Chem., 2014, 12, 1328–1340
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(Found: M + Na+, 546.2590 C28H4235ClNO6 + Na+, requires 128.0 (CH), 123.6 (C), 117.6 (C), 102.7 (CH), 94.0 (CH2), 93.8
546.2593); νmax (CHCl3)/cm−1 3742, 3678, 3050, 2999, 2872, (CH2), 64.6 (CH2), 64.5 (CH2), 48.2 (CH2), 45.3 (CH2), 40.9
1631, 1464, 1380, 1366, 1276, 1259, 1172, 1149, 1126, 1039, (CH2), 37.8 (Me), 31.1 (CH2), 30.3 (CH2), 27.0 (CH2), 26.6
851; major isomer δH (400 MHz; CDCl3) 7.11 (1H, s, ArH), (CH2), 25.0 (CH2), 21.7 (CH2), 15.1 (Me), 15.0 (Me); for the
5.92–5.77 (2H, m, CH), 5.32 (1H, d, J 13.0, OCH2O), 5.30 (1H, minor isomer, the following NMR signals are discernable, the
d, J 13.0, OCH2O), 5.23–5.19 (2H, m, OCH2O), 5.16–4.94 (4H, rest overlap with the major isomer: δH (400 MHz; CDCl3) 4.48
m, vCH2), 4.02 (1H, d, J 17.7, CH2), 3.82 (1H, d, J 17.7, CH2), (1H, dt, J 13.3, 7.6, CH2), 3.98 (1H, d, J 17.7, CH2), 3.87 (1H, d,
3.83–3.76 (2H, m, OCH2), 3.75–3.68 (2H, m, OCH2), 3.64–3.53 J 17.7, CH2), 3.23–3.14 (1H, m, CH2), 3.10–3.04 (1H, m, CH2),
(1H, dt, J 15.6, 9.4, CH2), 2.86 (3H, s, NMe), 2.52 (2H, ddd, J 3.02 (3H, s, NMe), 2.46–2.37 (1H, m, CH2), 1.14–1.03 (1H, m,
15.1, 7.1, 1.1, CH2), 2.42–2.37 (1H, m, CH2), 2.35–2.24 (2H, m, CH2); δC (100 MHz; CDCl3) 206.9 (C), 151.9 (C), 131.9 (CH),
CH2), 2.09–2.03 (2H, m, CH2), 1.67–1.56 (2H, m, CH2), 102.7 (CH), 64.5 (CH2), 45.0 (CH2), 30.6 (CH2), 30.6 (CH2), 27.7
1.44–1.37 (3H, m, CH2), 1.36–1.30 (3H, m, CH2), 1.23 (6H, 2 × (CH2), 26.3 (CH2); m/z (ESI) 518 ([M(35Cl) + Na]+, 100%), 519
t, J 14.5, 7.3, Me); δC (100 MHz; CDCl3) 206.6 (C), 167.5 (C), (28.0%), 520 (33.5%), 521 (8.0%).
153.8 (C), 152.0 (C), 138.9 (CH), 135.4 (CH), 134.6 (C), 132.3
(C), 122.8 (C), 116.6 (CH2), 114.2 (CH2), 102.5 (CH), 93.9 (CH2),
93.6 (CH2), 64.5 (CH2), 64.4 (CH2), 46.5 (CH2), 44.8 (CH2), 42.6
(CH2), 36.1 (Me), 33.6 (CH2), 31.5 (CH2), 28.9 (CH2), 28.8
(CH2), 28.6 (CH2), 23.7 (CH2), 15.0 (Me), 15.0 (Me); for the
minor isomer, the following NMR signals are discernable, the
15-Chloro-16,18-dihydroxy-2-methyl-3,4,7,8,9,10,11,12-
octahydrobenz[c][1]azacyclohexadecine-1,13(2H,14H)-dione
27c
rest overlap with the major isomer: δH (400 MHz; CDCl3) 7.14
(1H, s, ArH), 5.71–5.76 (2H, m, CH), 5.31 (1H, d, J 15.4,
OCH2O), 3.92 (1H, d, J 17.6, CH2), 3.10 (3H, s, NMe); δC
(100 MHz; CDCl3) 206.3 (C), 153.9 (C), 152.4 (C), 135.5 (CH),
134.2 (C), 132.4 (C), 122.3 (C), 117.8 (CH2), 117.6 (CH2), 102.7 As for the procedure for compound 27a, using 15-chloro-16,18-
(CH), 93.7 (CH2), 93.5 (CH2), 64.4 (CH2), 46.3 (CH2), 45.2 di(ethoxymethoxy)-2-methyl-3,4,7,8,9,10,11,12-octahydrobenzo-
(CH2), 42.4 (CH2), 32.5 (CH2), 32.1 (CH2), 29.3 (CH2), 28.8 [c][1]azacyclohexadecine-1,13(2H,14H)-dione 26c (15 mg,
(CH2); m/z (ESI) 546 ([M(35Cl) + Na]+, 100%), 547 (28.8%), 548 0.33 mmol, 1 eq.). The product was purified by flash column
(34.9%), 549 (7.7%).
chromatography, eluting with 2% methanol–dichloromethane
to give the title compound as a colourless solid (4 mg, 33%)
(5 : 1 mixture of isomers); mp 204–206 °C; (Found: M + Na+,
402.1442. C20H2635ClNO4 + Na+, requires 402.1443); νmax
(CHCl3)/cm−1 3440, 3280, 2613, 1710, 1720, 1702, 1630, 1450,
1430, 1413, 1369, 1334, 1252, 1096; major isomer δH (800 MHz;
acetone-d6) 8.68 (1H, s, OH), 8.65 (1H, br. s, OH), 6.56 (1H, s,
ArH), 5.44–5.41 (2H, m, CHvCH), 4.32–4.28 (1H, m, CH2),
3.88 (1H, d, J 17.1, CH2), 3.67 (1H, d, J 17.1, CH2), 2.94 (3H, s,
NMe), 2.77–2.72 (1H, m, CH2), 2.67 (1H, ddd, J 17.5, 9.4, 2.6,
15-Chloro-16,18-di(ethoxymethoxy)-2-methyl-
3,4,7,8,9,10,11,12-octahydrobenz[c][1]azacyclohexadecine-1,13
(2H,14H)-dione 26c
As for the procedure for compound 26a, using N-(but-3-en-1- CH2), 2.59–2.53 (1H, m, CH2), 2.53–2.45 (1H, m, CH2),
yl)-3-chloro-4,6-di(ethoxymethoxy)-N-methyl-2-(2-oxodec-9-en- 2.32–2.28 (1H, m, CH2), 2.02–1.98 (1H, m, CH2), 1.84–1.77 (1H,
1-yl)benzamide 25c (60 mg, 0.11 mmol, 1.0 eq.). The product m, CH2), 1.54–1.48 (1H, m, CH2), 1.48–1.42 (1H, m, CH2),
was purified by flash column chromatography, eluting with 1.41–1.34 (2H, m, CH2), 1.32–1.25 (2H, m, CH2), 1.25–1.21 (1H,
2 : 1 light petroleum–ethyl acetate to give the title compound as m, CH2), 1.17–1.10 (1H, m, CH2); δC (200 MHz; acetone-d6)
a colourless oil (19 mg, 34%) (4 : 1 mixture of isomers); 207.4 (C), 169.8 (C), 155.3 (C), 154.3 (C), 134.8 (C), 132.8 (C),
(Found: M + Na+, 518.2283. C26H3835ClNO6 + Na+, requires 130.7 (CH), 129.4 (CH) 114.4 (C), 104.2 (CH), 49.5 (CH2), 47.0
518.2280); νmax (CHCl3)/cm−1 3686, 2929, 2856, 1720, 1627, (CH2), 41.7 (CH2), 38.6 (Me), 32.9 (CH2), 32.1 (CH2), 28.8
1461, 1324, 1278, 1273, 1250, 1151, 1118, 940; major isomer δH (CH2), 28.3 (CH2), 26.6 (CH2), 23.3 (CH2); for the minor isomer,
(400 MHz; CDCl3) 7.08 (1H, s, ArH), 5.49–5.31 (2H, m, the following NMR signals are discernable, the rest overlap
CHvCH), 5.30–5.24 (2H, m, OCH2O), 5.19 (2H, s, OCH2O), with the major isomer: δH (800 MHz; acetone-d6) 8.70 (1H, s,
4.40 (1H, ddd, J 13.2, 6.8, 3.2, CH2), 4.03 (1H, d, J 17.3, CH2), OH), 6.55 (1H, s, ArH), 3.96 (1H, d, J 17.8, CH2), 2.91 (3H, s,
3.75 (4H, dq, J 14.3, 7.6, OCH2), 3.71–3.66 (2H, m, CH2), 3.59 NMe), 2.63–2.55 (1H, m, CH2), 2.47–2.40 (1H, m, CH2),
(1H, d, J 17.3, CH2), 2.92 (3H, s, NMe), 2.79 (1H, ddd, J 17.8, 2.40–2.35 (1H, m, CH2), 2.20–2.14 (1H, m, CH2), 1.80–1.72 (1H,
15.4, 2.6, CH2), 2.71–2.60 (2H, m, CH2), 2.55–2.46 (1H, m, m, CH2), 1.64–1.57 (1H, m, CH2), 1.36–1.28 (1H, m, CH2); δC
CH2), 2.37–2.25 (2H, m, CH2), 2.09–1.98 (2H, m, CH2), (200 MHz; acetone-d6) 122.2 (CH), 104.1 (CH), 101.9 (C), 56.0
1.97–1.83 (1H, m, CH2), 1.56–1.43 (2H, m, CH2), 1.41–1.33 (2H, (CH2), 31.6 (CH2), 31.3 (CH2), 27.9 (CH2), 26.8 (CH2); m/z (ESI)
m, CH2), 1.22 (6H, dt, J 13.2, 7.0, Me); δC (100 MHz; CDCl3) 402 ([M(35Cl) + Na]+, 100%), 403 (21.9%), 404 (32.4%), 405
206.7 (C), 168.2 (C), 153.7 (C), 152.1 (C), 132.6 (C), 131.7 (CH), (6.8%).
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Organic & Biomolecular Chemistry
N-(Pent-4-en-1-yl)-3-chloro-4,6-di(ethoxymethoxy)-N-methyl-2-
(2-oxohept-6-en-1-yl)benzamide 25d
Na+, 490.1965. C24H3435ClNO6 + Na+, requires 490.1967); νmax
(CHCl3)/cm−1 3688, 3005, 2930, 2361, 2341, 1719, 1626, 1597,
1456, 1422, 1319, 1150, 1051, 941, 909; major isomer δH
(400 MHz; CDCl3) 7.10 (1H, s, ArH), 5.52 (2H, dt, J 9.9, 5.8,
CHvCH), 5.28 (1H, d, J 15.8 OCH2O), 5.27 (1H, d, J 15.8
OCH2O), 5.20 (2H, s, OCH2O), 4.45 (1H, ddd, J 13.3, 6.9, 3.7,
CH2), 4.08 (1H, d, J 18.6, CH2), 3.90 (1H, d, J 18.6, CH2), 3.76
(2H, q, J 7.1, OCH2), 3.69 (2H, q, J 7.1, OCH2), 2.86 (3H, s,
NMe), 2.69–2.61 (2H, m, CH2), 2.45 (1H, ddd, J 18.5, 6.0, 2.2
CH2), 2.36–2.27 (1H, m, CH2), 2.11–2.00 (2H, m, CH2),
1.93–1.77 (2H, m, CH2), 1.73–1.61 (3H, m, CH2), 1.22 (6H, dt, J
8.3, 7.1, Me); δC (100 MHz; CDCl3) 205.6 (C), 167.9 (C), 153.7
(C), 151.8 (C), 131.9 (C), 130.7 (CH), 128.7 (CH), 123.0 (C),
118.0 (C), 102.6 (CH), 93.9 (CH2), 93.7 (CH2), 64.5 (CH2), 64.4
(CH2), 47.4 (CH2), 44.8 (CH2), 38.5 (CH2), 37.5 (Me), 28.5
(CH2), 25.1 (CH2), 24.6 (CH2), 21.6 (CH2), 15.0 (Me), 15.0 (Me);
for the minor isomer, the following NMR signals are discern-
able, the rest overlap with the major isomer: δH (400 MHz;
CDCl3) 7.08 (1H, s, ArH), 5.32–5.25 (2H, m, OCH2O), 5.19 (2H,
s, OCH2O), 4.05 (1H, d, J 18.0, CH2), 2.88 (3H, s, NMe); m/z
(ESI) 490 ([M(35Cl) + Na]+, 100%), 491 (23.5%), 493 (33.3%),
494 (6.6%).
As for the procedure for compound 25a, using 3-chloro-4,6-
di(ethoxymethoxy)-2-methylbenzoic acid 23 (200 mg, 0.63 mmol,
1
eq.),
N-methoxy-N-methylhex-5-enamide
(148
mg,
0.94 mmol, 1.5 eq.) and N-methylpent-4-en-1-amine (91 mg,
0.68 mmol, 1.5 eq.). The product was purified by flash column
chromatography, eluting with 4 : 1 light petroleum–ethyl
acetate to give the title compound as a yellow oil (81 mg, 28%
over 2 steps) (3 : 1 mixture of isomers); (Found: M + Na+,
518.2281 C26H3835ClNO6
+
Na+, requires 518.2280); νmax
(CHCl3)/cm−1 3686, 3600, 3058, 2996, 1719, 1628, 1597, 1458,
1408, 1319, 1278, 1265, 1152, 1119, 1042, 941, 896; major
isomer δH (400 MHz; CDCl3) 7.11 (1H, s, ArH), 5.92–5.72 (2H,
m, CH), 5.32 (1H, d, J 13.2, OCH2O), 5.30 (1H, d, J 13.2,
OCH2O), 5.22 (2H, s, OCH2O), 5.10–4.93 (4H, m, vCH2), 4.03
(1H, d, J 17.7, CH2), 3.86 (1H, d, J 17.7, CH2), 3.82–3.77 (2H, m,
OCH2), 3.76–3.66 (2H, m, OCH2), 2.85 (3H, s, NMe), 2.54 (2H,
ddd, J 15.0, 7.4, 3.0 CH2), 2.35–2.29 (1H, m, CH2), 2.20–2.06
(6H, m, CH2), 1.77–1.68 (6H, m, CH2), 1.23 (6H, dt, J 8.3, 7.1,
Me); δC (100 MHz; CDCl3) 206.4 (C), 167.5 (C), 153.8 (C), 152.1
(C), 137.9 (CH), 137.8 (CH), 132.3 (C), 122.9 (C), 117.7 (C),
115.3 (CH2), 115.1 (CH2), 102.6 (CH), 94.0 (CH2), 93.7 (CH2),
64.6 (CH2), 64.5 (CH2), 46.5 (CH2), 44.8 (CH2), 41.8 (CH2), 35.9
(Me), 33.0 (CH2), 30.9 (CH2), 26.2 (CH2), 22.9 (CH2), 15.0 (Me),
15.0 (Me); for the minor isomer, the following NMR signals are
discernable, the rest overlap with the major isomer: δH
(400 MHz; CDCl3) 7.15 (1H, s, ArH), 5.33 (1H, d, J 13.8, O–
CH2–O), 5.31 (1H, d, J 13.8, O–CH2–O), 5.20 (2H, s, O–CH2–O),
3.91 (1H, d, J 17.0, CH2), 3.64–3.55 (2H, m, O–CH2), 3.50–3.40
(2H, m, O–CH2), 3.07 (3H, s, NMe), 2.54 (2H, ddd, J 15.0, 7.4,
3.0 CH2), 1.98–1.91 (1H, m, CH2); δC (100 MHz; CDCl3) 206.6
(C), 138.0 (CH), 137.4 (CH), 115.2 (CH2), 102.8 (CH), 41.5
(CH2), 30.8 (CH2), 22.8 (CH2); m/z (ESI) 518 ([M(35Cl) + Na]+,
100%), 519 (24.9%), 520 (32.6%), 521 (7.6%).
13-Chloro-14,16-dihydroxy-2-methyl-2,3,4,5,9,10-
hexahydrobenz[c][1]azacyclotetradecine-1,11(8H,12H)-dione
27d
As for the procedure for compound 27a, using 13-chloro-14,16-
di(ethoxymethoxy)-2-methyl-2,3,4,5,9,10-hexahydrobenz[c][1]-
azacyclotetradecine-1,11(8H,12H)-dione
26d
(52
mg,
0.111 mmol, 1 eq.). The product was purified by flash column
chromatography, eluting with 2% methanol–dichloromethane
to give the title compound as a yellow solid (11 mg, 28%)
(4 : 1 mixture of isomers); mp 178–180 °C; (Found: M + Na+,
374.1138. C18H2235ClNO4 + Na+, requires 374.1130); νmax
(CHCl3)/cm−1 3483, 3280, 1730, 1723, 1708, 1630, 1435, 1419,
1396, 1376, 1334, 1249, 12191, 1094; major isomer δH
(800 MHz; acetone-d6) 8.75 (1H, s, OH), 8.66 (1H, s, OH), 6.58
(1H, s, ArH), 5.55 (1H, dt, J 10.6, 5.6, CH), 5.33 (1H, dt, J 10.6,
5.6, CH), 4.35–4.31 (1H, m, CH2), 4.05 (1H, d, J 18.6, CH2), 3.97
(1H, d, J 18.6, CH2), 2.91 (3H, s, NMe), 2.72–2.64 (2H, m, CH2),
2.57–2.52 (1H, m, CH2), 2.39–2.34 (1H, dt, J 11.4, 9.9, CH2),
2.10–2.05 (1H, m, CH2), 2.02–1.96 (1H, m, CH2), 1.92–1.85 (2H,
m, CH2), 1.71–1.66 (1H, m, CH2), 1.64–1.58 (1H, m, CH2),
1.34–1.29 (1H, m, CH2); δC (200 MHz; acetone-d6) 205.8 (C),
168.6 (C), 154.2 (C), 152.8 (C), 133.3 (C), 131.9 (CH), 129.3
(CH), 120.9 (C), 113.9 (C), 103.1 (CH), 47.9 (CH2), 45.1 (CH2),
13-Chloro-14,16-di(ethoxymethoxy)-2-methyl-2,3,4,5,9,10-
hexahydrobenz[c][1]azacyclotetradecine-1,11(8H,12H)-dione
26d
As for the procedure for compound 26a, using N-(pent-4-en-1- 38.9 (CH2), 37.5 (Me), 29.5 (CH2), 25.9 (CH2), 25.5 (CH2), 22.5
yl)-3-chloro-4,6-di(ethoxymethoxy)-N-methyl-2-(2-oxohept-6-en-1- (CH2); for the minor isomer, the following NMR signals are dis-
yl)benzamide 25d (0.081 g, 0.176 mmol, 1 eq.). The product cernable, the rest overlap with the major isomer: δH (800 MHz;
was purified by flash column chromatography, eluting with acetone-d6) 8.86 (1H, s, OH), 8.57 (1H, s, OH), 6.82 (1H, s,
1% methanol–dichloromethane to give the title compound as a ArH), 5.42–5.36 (1H, m, CH), 5.30–5.22 (1H, m, CH), 4.08
yellow oil (0.056 g, 68%) (9 : 1 mixture of isomers); (Found: M + (1H, d, J 18.6, CH2), 2.51–2.46 (1H, m, CH2), 2.23–2.19 (1H, m,
1338 | Org. Biomol. Chem., 2014, 12, 1328–1340
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CH2), 2.16–2.13 (1H, m, CH2); δC (200 MHz; acetone-d6) 154.3 dimensions 0.3 mm × 0.2 mm × 0.2 mm. These were flash
(C), 152.6 (C), 133.4 (C), 103.2 (CH), 30.6 (CH2), 21.5 (CH2); m/z frozen after stepwise addition of glycerol to 30%, and the data
(ESI) 374 ([M(35Cl) + Na]+, 100%), 375 (17.7%), 376 (30.6%), were collected on station I02 at Diamond Light Source. The
377 (6.2%).
data were integrated and scaled using the automatic Xia253,54
processing at Diamond.
Isothermal titration calorimetry (ITC)
The complexes were initially solved by isomorphous re-
placement using a previously determined N-terminal structure
ITC was carried out as previously described by Proisy and co-
workers.16 Briefly, heats of the interaction were measured at
30 °C on an ITC200 (GE healthcare), with a cell volume of
200 μL. Ten injections of 3.8 μL of 600 μM of radicicol or radi-
cicol analogue, in 20 mM Tris, 5 mM NaCl, 1 mM EDTA and
2% DMSO, were injected into 40 μM yeast Hsp90 N-terminal
domain in the same buffer. Heats of dilution were determined
in a separate experiment by injecting compound into buffer
containing 2% DMSO, and the corrected data were fit with a
nonlinear least square curve-fitting algorithm (Microcal
Origin) with three floating variables: stoichiometry, binding
constant, and change in enthalpy of interaction.
(PDB 1̲A̲H̲6̲) in the usual space group, P4322. The model was
refined using REFMAC55 and rebuilt using COOT.56 The inhibi-
tors were built using the PRODRG server.57 The inhibitor mole-
cule and the waters were added in the final stages. Various
programs from the CCP458 suite were used during the refine-
ment. All refinements proceeded smoothly. The crystallo-
graphic statistics are given in Table 2.
Acknowledgements
We thank the Parkinson’s UK for their generous support
(R.R.A.K. and C.J.M.). We thank Mr Shazad Aslam and
Mr Kevin Butler for help with NMR studies. We are also grate-
ful to Dr Mick Cooper, Mr Graham Coxhill and Mr Ben
Pointer-Gleadhill (University of Nottingham Mass Spec-
trometry) for technical assistance.
Protein co-crystallisation experimental
The expression, purification and co-crystallisation of the
N-terminal domain of yeast Hsp90 have been previously
described.52 Single crystals appeared overnight of approximate
Table 2 Crystallographic data
References
Data set (highest shell
in parentheses)
27b
27c
27d
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a (Å)
b (Å)
c (Å)
α (°)
β (°)
γ (°)
74.32
74.32
110.46
90
90
90
P4322
74.16
74.16
110.43
90
90
90
74.18
74.18
110.49
90
90
90
P4322
0.97961
52.5–2.31
(2.37–2.31)
Space group
Wavelength (Å)
Resolution limit (Å)
P4322
0.97961
52.4–2.01
(2.06–2.01)
21192 (1511) 13834 (1002)
99.9 (99.8)
8.5 (9.0)
0.97961
47.5–2.38
(2.45–2.38)
12939 (927)
99.9 (100.0)
8.5 (9.0)
0.111 (0.803) 0.065 (0.720) 0.071 (0.676)
0.043 (0.301) 0.024 (0.263) 0.035 (0.318)
14.2 (2.8)
Number of obs.
Completeness (%)
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Rpim(I)
I/σI
98.8 (98.3)
3.8 (3.9)
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20.4 (3.5)
17.5 (2.5)
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Resolution range (Å)
Rcryst
Rfree
47.5–2.38
0.1930
0.2394
52.4–2.01
0.2058
0.2660
52.5–2.32
0.1933
0.2613
Number of protein
atoms
Number of ligand
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Number of solvent
atoms
1691
25
1690
26
1675
24
126
115
113
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Rmsd bond
lengths (Å)
42.4
0.016
36.5
0.018
39.7
0.016
Rmsd bond angles (°) 1.848
1.935
1.802
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Organic & Biomolecular Chemistry
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