Synthesis and biological evaluation of a potent salicylihalamide A lactam analogue

Article abstract of DOI:10.1039/c2ob26649k

The first synthesis of a lactam analogue 3 of salicylihalamide A (1) is reported. A key step in the approach was a photochemical acylation coupling between amine 10 and dioxinone 9 to form the amide 19. Acetylation followed by RCM with Grubbs 1st generation catalyst gave the desired E-lactam 23 (E:Z ratio 87:13) as the major compound. Conversion of macrolactam 23 into the vinyl iodide 26 followed by Cu catalysed cross coupling with the diene amide 7 gave aza-salicylihalamide analogue 3 in good yield. This compound demonstrated potent activity against several human leukaemia cell lines.

Full text of DOI:10.1039/c2ob26649k

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PAPER
Synthesis and biological evaluation of a potent salicylihalamide A lactam
analogue†
Dan Balan,a Christopher J. Burns,b,c Nicholas G. Fisk,d Helmut Hügel,*a David C. S. Huang,b,c David Segal,b,c
Charlotte White,d Jörg Waglera and Mark A. Rizzacasa*d
Received 15th June 2012, Accepted 24th August 2012
DOI: 10.1039/c2ob26649k
The first synthesis of a lactam analogue 3 of salicylihalamide A (1) is reported. A key step in the
approach was a photochemical acylation coupling between amine 10 and dioxinone 9 to form the
amide 19. Acetylation followed by RCM with Grubbs 1st generation catalyst gave the desired E-lactam
23 (E : Z ratio 87 : 13) as the major compound. Conversion of macrolactam 23 into the vinyl iodide 26
followed by Cu catalysed cross coupling with the diene amide 7 gave aza-salicylihalamide analogue 3
in good yield. This compound demonstrated potent activity against several human leukaemia cell lines.
Introduction
Salicylihalamide A (1)1 (Fig. 1) is a potent cytotoxic macrolide
salicylate2 isolated from a marine sponge of the genus Haliclona
(∼1 × 10−3% w/w yield from wet sponge) collected from waters
around Rottnest Island which is situated 18 km off the coast of
southern Western Australia. Testing of salicylihalamide A (1) in
the NCI 60-cell line human tumour screen gave a striking pattern
of differential toxicity which did not correlate with the profiles
of any known antitumor compounds in the standard database.1
These results suggest that compound 1 has a novel mechanism
of action and further biological evaluation revealed that com-
pound 1 was the first of a new class of mammalian specific
vacuolar-type (H+)-ATPase inhibitor.3
Salicylihalamide A (1) contains a 12-membered salicylate
macrolactone along with an enamide sidechain found in other
salicylate metabolites such as apicularen A.2 As would be
expected, this compound elicited the interest of the synthetic
community and a number of total4 and formal syntheses5 were
reported in a short time period as well as the production of a
number of potent analogues.2,4c,f,6 The majority of SAR studies
Fig. 1 Structures of salicylihalamide A, epothilone B and analogues.
have been conducted on salicylihalamide derivatives with
modified enamide sidechains and it has been shown that the
aSchool of Applied Sciences [Applied Chemistry], RMIT University,
presence of some form of enamide functionality is important for
Bowen Street, Melbourne, Vic. 3001, Australia
activity.6a,4d,f However, some small modifications in the macro-
lactone are tolerated without substantial loss of cytotoxic activity.
For example, Smith and Zheng4g reported that the simplified
analogue 2, which is devoid of the 12-methyl and 13-hydroxyl
groups, was a potent cytotoxin against solid tumour cells lines
with GI50 values ranging from 0.27 μM (SF268: CNS glioma) to
2.12 μM (DU-145: prostate cancer) which compared well to
those for the natural product 1 (GI50 = 0.18 μM for both SF268
bWalter and Eliza Hall Institute of Medical Research, 1G Royal Parade,
Parkville, Victoria 3052, Australia
cDepartment of Medical Biology, The University of Melbourne,
Parkville, Victoria, Australia
dSchool of Chemistry, the Bio21 Institute, The University of Melbourne,
VIC 3010, Australia. E-mail: masr@unimelb.edu.au
†Electronic supplementary information (ESI) available: Copies of the
1H and 13C NMR spectra of compounds 3, 10, 19, 20, 22–26 and crys-
tallographic data for compounds 20 and 25 in PDF and CIF format.
CCDC 886234 and 886235. For ESI and crystallographic data in CIF or
other electronic format see DOI: 10.1039/c2ob26649k
and DU-145 lines). However,
a
smaller 6-membered
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benzolactone analogue6c of 1 displayed only modest activity
(103 less active than 1), showing that the 12-membered ring may
be also important for activity.
precursor, salicylate 8 could be obtained via a photochemical
acylation13 of dioxinone 9 with optically pure amine 10.
We envisaged that a new modification of salicylihalamide (1)
would be to change the 12-membered macrolactone to a macro-
lactam as in the aza-salicylihalamide A analogue 3. This propo-
sal was inspired by the discovery of a potent macrolactam
analogue of the 15-membered macrolactone epothilone B (4).
The semisynthetic 15-aza-epothilone B (5)7 (ixabepilone®) is
currently in use for the treatment of aggressive metastatic taxol®
resistant breast cancer8 and was found to exhibit a better thera-
peutic range than epothilone B (4). For example, 15-aza-epothi-
lone B (5) (CCRF-CEM: leukemia IC50 = 2.1 nM) was ∼10-fold
lower in activity when compared with epothilone B (4)
(CCRF-CEM: leukemia IC50 = 0.2 nM) but displayed a 50-fold
decrease in toxicity.9 Key to the clinical success of ixabepilone
(5) is the stability of the macrolactam. The low in vivo activity
and toxicity of epothilone B (4) probably results from esterase-
mediated macrolactone hydrolysis to form the inactive and toxic
seco-acid.6 Following the above precedent, the salicylate macro-
lactam aza-salicylihalamide A analogue (3) possesses a more
stable macrocycle system and thus would be an interesting target
for synthesis and biological evaluation. To our knowledge, no
lactam analogues of salicylihalamide A (1) have been reported.
We now describe an efficient first synthesis of the novel salicyli-
halamide A lactam analogue (3) and its potent in vitro activity
against several leukemia cell lines.
Results and discussion
Synthesis of analogue 3
Our initial studies focused on amide formation in a simplified
system devoid of the C15 sidechain. We trialled a number of
traditional amine-acid coupling approaches to construct the
amide bond with very limited success so we then elected to
investigate the elegant photochemical method as reported by
De Brabander.13 This reaction can be utilised to form
hindered ortho-substituted salicylate amides under essentially
neutral conditions. As shown in Scheme 2, photolysis (300 nm)
of a CH2Cl2 solution of the benzo-1,3-dioxin-4-one 114b affords
the quinoneketene 12, which was captured by the simple
amine 1314 nucleophile to give the salicylate amide 14 in low
yield (12%).
When the 2,2-diphenyl 1,3-dioxin-4-one 913 was photolysed
in the presence of amine 13, the yield of 14 rose to 55%. These
results add support to the original proposal13 that the quinone-
ketene 12 is most likely formed by homolytic C–O bond fission
as shown since an intermediate diphenylmethyl radical would be
more stable than the dialkyl analogue.
The synthesis of the chiral amine required for the production
of 3 began with ring opening of the enantiopure epoxide 1515
and the cuprate derived from 16 as described by Smith and
Zhang4g to afford the alcohol (−)-17 in good yield (Scheme 3).
Mesylation of 17 followed by azide displacement gave the azide
18 which underwent Staudinger reduction to give the amine 10
in pure form after aqueous acid extraction. Irradiation of a sol-
ution of dioxinone 913 and amine 10 in CH2Cl2 at 300 nm gave
the amide 19 in a similar yield to that for the simpler system.
This reaction also gave similar yields on a 12 g scale demonstrat-
ing that this step is easily scalable. For the RCM reaction, pre-
liminary experiments indicated that Grubbs 1st generation
catalyst 2116 gave the best yields and E : Z selectivity and this
was also observed for the RCM based syntheses of the macrolac-
tone in salicylihalamide A (1).4f For the formation of the macro-
lactam, the highest yield and selectivity was obtained when the
phenol was protected as the acetate. Acetylation of phenol
19 gave crystalline acetate 20 and X-ray analysis confirmed
The synthetic approach to 3 is detailed in Scheme 1. We envi-
saged that the key enamide could be introduced by a Cu cata-
lysed cross coupling10 between the vinyliodide intermediate 6
and the known amide 7.4b,11 The critical macrolactam ring could
be secured by ring closing metathesis (RCM)12 of diene 8, hope-
fully with a degree of geometric control. The required diene
Scheme 1 Retrosynthetic analysis of aza-salicylihalamide analogue 3.
8148 | Org. Biomol. Chem., 2012, 10, 8147–8153
Scheme 2 Photochemical acylation studies.
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Scheme 4 Completion of the synthesis of analogue 3.
Table 1 Growth inhibitory activity of compound 3 and bafilomycin
Compound 3
IC50 (nM)
Bafilomycin A1 (27)
IC50 (nM)
Cell line
CCRF-CEM
HL-60
K562
371 39
116 14
506 47
30
17
43 15
1
1
Scheme 3 Synthesis of the E-macrolactam 23.
MOLT-4
RPMI-8226
277
117
9
4
23
13
1
1
the structure of this compound including the absolute
configuration as R.17 RCM using catalyst 21 gave the macrolac-
tams 22 and 23 in good yield favouring the desired E-isomer
23 (E : Z ratio 87 : 13). Compound 23 could be separated
from the small amount of Z-isomer 22 by flash chromatography.
Again, the RCM reaction can be conducted on a multigram
scale with little difference in yield and stereoselectivity.
Removal of the PMB group by treatment of 23 with DDQ
gave the alcohol 24. When the methyl ether or the free phenol
was used for the RCM reaction, yields and selectivities
were lower and the E and Z-isomers could only be separated
by HPLC.
The final steps to analogue 3 are shown in Scheme 4. Oxi-
dation of alcohol 24 gave the crystalline aldehyde 25 in
good yield (Scheme 4). This compound was also subjected to
X-ray crystallographic analysis18 which confirmed the
structure (see Scheme 4). A Takai homologation19 then
afforded the E-vinyl iodide ready for cross coupling. The
choice of acetate as protecting group in the RCM reaction was
also useful in the last step. Cross coupling of iodide 26
with known dienamide 7 was conducted under the conditions
developed by Buchwald10 and utilised in a recent total
synthesis of the related compound apicularen A.20 This reaction
formed the desired enamide and resulted in the concomitant clea-
vage of the phenolic acetate to provide the analogue (−)-3 in
64% yield.
Biological assays
The analogue 3 was assayed against the NCI-60 leukaemia cell
lines [i.e. CCRF-CEM (acute lymphoblastic leukaemia), HL-60
(acute promyelocytic leukaemia), K562 (chronic myeloid leukae-
mia), MOLT-4 (acute lymphoblastic leukaemia) and RPMI 8266
(myeloma)] and exhibited antiproliferative effects at sub-micro-
molar concentrations (Table 1). For comparison, bafilomycin A1
(27) was also assayed against the same cells lines since both this
compound and salicylihalamide A (1) target vacuolar-type (H+)-
ATPase in the lysosome.3,21 Compound 3 was very active against
MOLT-4 (IC50 = 277 nM) and CCRF-CEM (IC50 = 371 nM) but
was most active against RPMI 8226 and HL-60 (IC50 = 117 and
116 nM for each respectively).
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that was either flame dried under an atmosphere of dry argon or
oven dried at 150 °C.
Amide (14)
A solution of the and amine 1314 (350 mg, 2.04 mmol) and the
benzodioxinone 913 (1.34 g, 4.08 mmol) in CH2C12 (8 mL)
under N2 was placed in a Rayonet photochemical reactor
equipped with 16 × 300 nm lamps and the solution was irradiated
for 4 h. The solvent was removed under reduced pressure and
the crude product purified by flash chromatography with 15%
EtOAc/petrol as eluent to afford the amide 14 (308 mg, 55%) as
a white solid. Rf (20% EtOAc/hexane) 0.39; νmax: 3258, 3077,
1747, 1631, 1588, 1533. δH 10.69 (1H, s) 7.24 (1H, t, J = 7.6
Hz), 6.85 (1H, d, J = 8.0 Hz), 6.68 (1H, d, J = 7.2 Hz), 6.59
(1H, br s), 6.14–6.23 (1H, m), 5.74–5.84 (1H, m), 5.28 (1H, dd,
J = 10.4, 1.6 Hz), 5.01 (1H, dd, J = 10.0, 2.0 Hz), 4.96 (1H, dd,
J = 10.0, 2.0 Hz), 4.94 (1H, dd, J = 9.6, 1.6 Hz), 3.52 (2H, m),
3.40 (2H, q, J = 5.6 Hz), 2.05 (2H, q, J = 6.8 Hz), 1.57 (2H, m),
1.31–1.47 (4H, m); δC 169.7, 159.6, 138.5, 138.4, 136.0, 132.0,
122.8, 118.5, 117.7, 115.9, 114.6, 39.8, 38.6, 33.5, 29.0, 28.4,
26.4; HRMS (ESI) calc for C17H23NO2Na [M + Na]+ 296.1621,
found 296.1621.
Fig. 2 Sensitivity of NCI-60 leukemia cells to bafilomycin A1 (27)
versus the aza-salicylihalamide analogue 3.
Bafilomycin A1 (27) was approximately 10 fold higher in
activity than the aza-salicylihalamide analogue 3 across all the
cell lines. In addition, the sensitivity profiles of compound 27
and analogue 3 were strongly correlated (Fig. 2) supporting a
similar mode of action against vacuolar-type (H+)-ATPase.
Conclusions
Azide (18)
The aza-salicylihalamide analogue 3 was synthesised for the first
time in a short sequence that is amenable to large scale. Key
steps include a photochemical acylation to form the amide
linkage, a E-selective ring closing metathesis to secure the
macrolactam and a Cu mediated cross coupling to introduce the
enamide and remove the phenolic acetate. Analogue 3 showed
potent activity against several leukemia cell lines correlating
strongly with bafilomycin A1 (27) and shows promise for further
development as an anti-cancer compound. The preliminary bio-
logical results demonstrate that the introduction of a stable
lactam linkage in the salicylihalamide structure affords a potent
simplified analogue 3 of this family of compounds which com-
pares well to other reported analogues and the natural product
itself. Further tests to determine the in vivo activity and stability
of this compound and derivatives are underway.
To a solution of alcohol 17 (2.4 g, 8.6 mmol) in Et2O (60 mL) at
0 °C was added triethylamine (4.23 mL, 30.2 mmol) and metha-
nesulfonyl chloride (2.33 mL, 30.2 mmol). After 15 min of stir-
ring at 0 °C the reaction mixture was warmed to room
temperature and the reaction was left to proceed for further 2 h.
Then the resulting mixture was quenched with water, the organic
layer was separated and the aqueous solution was extracted with
Et2O (3 × 50 mL). The combined organic extracts were washed
successively with 50 mL H2O and 50 mL sat. aq. NaCl, dried
over Na2SO4 and concentrated affording the crude mesylate
(2.88 g, 94%) directly used for the next step without further
purification. To
a solution of above mesylate (2.80 g,
7.86 mmol) in DMF (30 mL) was added NaN3 (1.12 g,
17.3 mmol) and the mixture was stirred at 72 °C for 20 h. After
cooling to room temperature the reaction mixture was quenched
with water and extracted with diethyl ether (3 × 50 mL). The
combined organic extracts were washed with water and brine and
dried over MgSO4, filtered and concentrated in vacuo to give the
crude product, which was purified by flash chromatography with
10% Et2O/hexane as eluent to give azide 18 (2.14 g, 90%) as a
colourless oil [α]D26 − 27.8 (c 1.12, CH2Cl2); Rf (10% Et2O/
hexane) 0.58; IR νmax: 2933, 2858, 2096, 1612, 1512, 1245,
1089, 1034, 910, 819 cm−1; δH 7.30 (2H, d, J = 8.5 Hz), 6.92
(2H, d, J = 8.5 Hz), 5.84 (1H, ddt, J = 17.1, 10.2, 6.6 Hz), 5.03
(2H, m), 4.48 (2H, ABq, J = 11.6 Hz), 3.83 (3H, s), 3.59 (2H,
m), 3.54 (1H, m) 2.10 (2H, m), 1.79 (2H, m) 1.50 (6H, m); δC
158.9, 138.3, 130.0, 129.0, 114.3, 113.5, 72.5, 66.2, 59.8, 54.9,
34.4, 34.2, 33.3, 28.3, 25.2; HRMS (ESI) calculated for
C17H25N3O2Na [M + Na]+ 326.18390, found 326.18396.
Experimental
General
Optical rotations were recorded in a 10 cm microcell. High resol-
ution mass spectra (HRMS) were run using electrospray ionis-
ation (ESI). Proton nuclear magnetic resonance (1H NMR,
300 MHz) and proton decoupled carbon nuclear magnetic reson-
ance spectra (13C NMR, 75.5 MHz) were recorded for deuterio-
chloroform solutions with residual chloroform as internal
standard unless otherwise stated. Analytical thin layer chromato-
graphy (TLC) was conducted on aluminium backed 2 mm thick
silica gel GF254. Compounds were visualized with solutions of
20% w/w phosphomolybdic acid in ethanol or under UV
(365 nm). Anhydrous tetrahydrofuran (THF) and diethyl ether
were dried using a Glass Contour cartridge solvent dispensing
system. All other commercial reagents were used as received. All
air and moisture sensitive reactions were performed in glassware
Amine (10)
To a solution of azide 18 (4.8 g, 15.8 mmol) and triphenylphos-
phine (16.9 g, 63.3 mmol) in THF (100 mL) was added water
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(3.13 mL, 174 mmol) and the mixture was stirred for 3 h at
70 °C. The reaction mixture was cooled to room temperature and
extracted into 0.5% aq. HCl. The aqueous phase was succes-
sively washed with Et2O (3 × 40 mL) and 5% aq. NaOH and
then extracted with CH2Cl2 (3 × 40 mL). The combined organic
extracts were dried over MgSO4, filtered and then concentrated
under reduced pressure to give pure amine 10 (4.08 g, 65%) as
an oil. [α]2D6 +3.13 (c 1.280, CH2Cl2); Rf (10% MeOH/CH2Cl2)
0.55; IR νmax: 3369, 2927, 2854, 1612, 1586, 1512, 1245, 1092,
1034, 908, 819, 721 cm−1; δH 7.25 (2H, d, J = 8.6 Hz), 6.86
(2H, d, 8.6 Hz), 5.79 (1H, ddt, J = 17.1, 10.2, 6.6 Hz), 4.99 (1H,
ddt, J = 17.1, 1.9, 1.6 Hz), 4.92 (1H, ddt, J = 10.2, 1.1, 1.1 Hz),
4.42 (2H, br s), 3.78 (3H, s), 3.54 (2H, m), 2.86 (1H, m), 2.04
(2H, m), 1.79 (2H, m), 1.50 (2H, m), 1.38 (4H, m); δC 159.3,
139.1, 130.8, 129.4, 114.5, 114.0, 72.8, 68.0, 55.4, 49.3, 38.4,
37.9, 33.9, 29.2, 25.8; HRMS (ESI) calculated for C17H28NO2
[M + H]+ 278.21146, found 278.21155.
acetate 20 as a colorless crystalline solid (915 mg, 90%). A
single-crystal X-ray diffraction analysis confirmed the identity of
this compound (see ESI† and CCDC-886235). mp 55 °C; Rf
(40%, EtOAc/CH2Cl2) 0.52; [α]2D3 +2.4 (c 0.91, CH2Cl2); IR
νmax: 3296, 3074, 2933, 2858, 1764, 1641, 1610, 1512, 1456,
1246, 1198, 1092, 1018, 911, 820, 733 cm−1; δH 7.33 (1H, t, J =
7.9 Hz), 7.19 (2H, d, J = 8.6 Hz), 7.12 (1H, d, J = 7.9 Hz), 6.95
(1H, d, J = 7.9 Hz), 6.82 (2H, d, J = 8.6 Hz), 6.07 (1H, d, J =
8.6 Hz), 5.93 (1H, ddt, J = 17.0, 10.2, 6.5 Hz), 5.80 (1H, ddt, J
= 17.1, 10.2, 6.6, Hz), 5.07 (1H, ddt, J = 10.2, 1.5, 1.3 Hz), 4.95
(1H, ddt, J = 17.0, 1.7, 1.5 Hz), 5.00 (1H, ddt, J = 17.0, 1.9,
1.5 Hz), 4.94 (1H, ddt, J = 10.0, 1.9, 1.1 Hz), 4.41 (2H, ABq, J
= 11.5 Hz), 4.23 (1H, m), 3.79 (3H, s), 3.56 (2H, m), 3.33 (2H,
d, J = 6.4 Hz), 2.27 (3H, s), 2.05 (2H, m), 1.82 (1H, m), 1.64
(1H, m), 1.43 (2H, m), 1.32 (4H, m); δH 170.0, 165.5, 159.1,
147.0, 138.8, 138.6, 136.6, 131.0, 130.1, 129.6, 129.3, 127.3,
120.5, 116.5, 114.5, 113.7, 72.8, 66.8, 55.2, 47.8, 37.0, 34.2,
34.0, 33.6, 28.8, 25.4, 21.0; HRMS (ESI) calculated for
C29H37NO5Na [M + Na]+ 502.25639, found 502.25646.
Amide (19)
E- and Z-Macrolactams (23) and (22)
A solution of the amine 10 (116.4 mg, 0.42 mmol) and benzo-
dioxinone 913 (215.5 mg, 0.63 mmol) in CH2Cl2 (10 mL) under
N2 was placed in a Rayonet photochemical reactor equipped
with 16 × 300 nm lamps and the solution was irradiated for 6 h.
The solvent was evaporated under reduced pressure and the
crude product was purified by flash chromatography with 20%
EtOAc/hexane as eluent to afford amide 19 (151.4 mg, 55%) as
a colorless oil. Rf (30% EtOAc/hexane) 0.55; [α]2D3 −1.6 (c
0.790, CH2Cl2); IR νmax: 3280, 3240, 2932, 2857, 1631, 1517,
1462, 1245, 1091, 909, 817, 761 cm−1; δH 10.65 (1H, s), 7.25
(1H, t, J = 7.9 Hz), 7.19 (2H, d, J = 8.6 Hz), 6.87 (1H, dd, J =
8.2, 1.1 Hz), 6.81 (2H, d, J = 8.6 Hz), 6.66 (1H, dd, J = 7.5,
1.1 Hz), 6.61 (1H, d, J = 8.5 Hz), 6.05 (1H, ddt J = 17.2, 10.2,
5.1 Hz), 5.79 (1H, ddt, J = 17.1, 10.2, 6.6 Hz), 5.20 (1H, ddt,
J = 10.2, 1.7, 1.7 Hz), 5.02 (1H, ddt, J = 17.2, 1.9, 1.6 Hz), 4.94
(1H, ddt, J = 10.1, 1.2, 0.9 Hz), 4.90 (1H, ddt, J = 17.3, 1.8,
1.5 Hz), 4.40 (2H, s), 4.28 (1H, m), 3.78 (3H, s), 3.55 (2H, m),
3.43 (2H, m), 2.05 (2H, dt, J = 6.9, 6.9 Hz), 1.93 (1H, m), 1.72
(1H, m), 1.54 (2H, m), 1.39 (4H, m); δC 169.2, 159.3, 159.2,
138.6, 138.0, 136.4, 131.8, 130.0, 129.4, 122.6, 118.8, 117.6,
115.8, 114.6, 113.7, 72.9, 67.0, 55.2, 48.3, 38.4, 34.4, 34.2,
33.5, 28.6, 25.4; HRMS (ESI) calculated for C27H35NO4Na
[M + Na]+ 460.24583, found 460.24586.
Grubbs 1st generation catalyst (Cy3P)2Cl2Ru = CHPh 21
(66.9 mg, 0.081 mmol) in degassed CH2Cl2 was added to a sol-
ution of 20 (390 mg, 0.814 mmol) in CH2Cl2 (8 mL) and the
resulting solution was heated to reflux for 24 h. The solution was
cooled and evaporated until ca. 30 mL remained and then
filtered through a short plug of silica gel eluting with EtOAc.
The filtered solution was stirred with activated charcoal (2 g) for
12 h at room temperature, and the charcoal was removed by
filtration and the filtrate was concentrated under reduced
pressure. Purification of the crude product on silica gel with
EtOAc–hexane 1 : 20 → 1 : 4 → 1 : 1 as eluent afforded the
(E)-macrolactam 23 (258 mg, 66%) and (Z)-macrolactam 22
(41 mg, 10%; contaminated with ∼5% of the E-isomer) as
colourless solids.
(E)-macrolactam 23: mp 117 °C; Rf (50% EtOAc/hexane)
0.44; [α]2D3 −98.3 (c 0.720, CH2Cl2); IR νmax: 3293, 2926, 2855,
1765, 1644, 1512, 1455, 1246, 1200, 1089, 1020, 804,
733 cm−1; δH 7.30 (1H, t, J = 7.8 Hz), 7.26 (2H, d, J = 8.6 Hz),
7.11 (1H, d, J = 7.5 Hz), 6.92 (1H, d, J = 8.1 Hz), 6.88 (2H, d, J
= 8.6 Hz), 5.51 (1H, d, J = 8.6 Hz), 5.50 (1H, m), 5.26 (1H,
dddd, J = 14.9, 10.3, 3.3, 1.3 Hz), 4.46 (2H, ABq, J = 11.6 Hz),
4.11 (1H, m), 3.92 (1H, dd, 14.2, 3.8 Hz), 3.73 (3H, s), 3.58
(2H, app. t, J = 7.3 Hz), 3.21 (1H, dd, J = 14.2, 2.5 Hz), 2.16
(3H, s), 1.72 (4H, m), 1.41 (4H, m), 1.11 (2H, m); δC 170.8,
165.4, 159.2, 147.3, 140.6, 133.1, 130.7, 130.4, 129.9, 129.4,
128.5, 128.0, 120.8, 113.8, 72.8, 67.3, 55.2, 45.0, 37.9, 36.7,
32.8, 32.6, 24.7, 20.9, 20.7; HRMS (ESI) calculated for
C27H33NO5Na [M + Na]+ 474.22509, found 474.22504.
Acetate (20)
To a solution of phenol 19 (0.909 g, 2.07 mmol) and 4-
(dimethylamino) pyridine (DMAP, 378 mg) in CH2Cl2 (10 mL)
was added pyridine (0.84 mL, 10.3 mmol) and freshly distilled
acetic anhydride (0.29 mL, 3 mmol). After stirring for 12 h at
room temperature, the mixture was diluted with Et2O (20 mL),
and aqueous NaHCO3 (40 mL) was added and the mixture
stirred at rt for 10 min. The organic layer was separated and the
aqueous layer was extracted with Et2O (3 × 40 mL). The com-
bined organic layers were washed successively with CuSO4
solution, water, NaHCO3 solution and sat. aq. NaCl, dried over
Na2SO4 and evaporated. The residue was purified by flash
chromatography with 30%, EtOAc/CH2Cl2 as eluent to give
(Z)-Macrolactam 22: mp 119–120 °C; Rf (50% EtOAc/
hexane) 0.43; [α]D27 −8.82 (CH2Cl2); IR νmax: 3289, 2928, 2855,
1764, 1642, 1512, 1455, 1245, 1200, 1094, 1023, 818,
733 cm−1; δH 7.35 (1H, t, J = 7.8 Hz), 7.24 (2H, d, J = 8.6 Hz),
7.20 (1H, d, J = 7.8 Hz), 6.93 (1H, d, J = 8.1 Hz), 6.87 (2H, d,
J = 8.5 Hz), 5.80 (1H, d, J = 8.1 Hz), 5.46 (1H, app. t, J =
10.3 Hz), 5.18 (1H, app. t, J = 10.3 H), 4.44 (2H, ABq, J =
11.9 Hz), 4.28 (1H, m), 3.81 (3H, s), 3.70 (1H, dd, J = 14.9,
4.8 Hz), 3.52 (2H, m, H17), 3.22 (1H, br d, J = 14.9 Hz), 2.18
This journal is © The Royal Society of Chemistry 2012
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(3H, s), 1.94 (2H, m), 1.73 (2H, m), 1.42 (2H, m), 1.02 (2H, m);
δC 170.4, 166.1, 159.5, 147.1, 140.6, 131.3, 130.7, 130.5, 130.3,
130.2, 129.5, 128.1, 120.5, 114.1, 73.0, 67.4, 55.6, 47.6, 33.1,
31.8, 30.3, 26.6, 24.5, 21.2, 19.0; HRMS (ESI) calculated for
C27H33NO5Na [M + Na]+ 474.22509, found 474.22515.
130.2, 130.1, 128.4, 128.3, 120.8, 50.1, 43.0, 37.8, 32.9, 32.7,
24.6, 21.0, 20.8; HRMS (ESI) calculated for C19H23NO4Na
[M + Na]+ 352.15193, found 352.15199.
Vinyl iodide (26)
To a slurry of chromium(II) chloride (112.95 mg, 0.919 mmol) in
dry THF (5 mL) under a N2 atmosphere was added via cannula a
solution of aldehyde 25 (43.2 mg, 0.13 mmol) and iodoform
(155.1 mg, 0.39 mmol) in dry THF (4 mL) and the initial grey
suspension turned almost immediately reddish-brown. After stir-
ring at room temperature for 8 h, the reaction mixture was
diluted with water and extracted in Et2O (3 × 20 mL). The com-
bined organic layers were washed with aqueous solution of
Na2S2O3, brine, dried over Na2SO4, filtered and concentrated in
vacuo. The product was purified by chromatography with 30%
EtOAc/hexane as eluent to afford the (E)-iodoalkene 26 (38 mg,
72%) as a white solid. mp 154–156 °C; Rf (80% Et2O/hexane)
0.72; [α]2D3 −96.4 (c 0.805, CH2Cl2); IR νmax: 3255, 2924, 1760,
1633, 1546, 1456, 1207, 1019, 966, 873, 708 cm−1; δH 7.32
(1H, t, J = 7.9 Hz), 7.16 (1H, d, J = 7.6 Hz), 6.91 (1H, dd, J =
8.08, 0.93 Hz), 6.37 (1H, dtd, J = 14.3, 7.3, 7.1 Hz), 6.09 (1H,
app. dt, J = 14.3, 1.2 Hz), 5.92 (1H, d, J = 8.1 Hz), 5.42 (1H,
app. t, J = 10.0 Hz), 5.13 (1H, app. t, J = 10.5 Hz), 4.10 (1H,
m), 3.61 (1H, dd, 15.1, 4.7 Hz), 3.17 (1H, br dd, J = 14.9,
2.5 Hz), 2.38 (1H, m), 2.29 (3H, m), 2.17 (2H, m), 1.94 (1H,
m), 1.83 (1H, m), 1.48 (1H, m), 1.37 (2H, m), 1.16 (2H, m); δC
169.8, 165.8, 146.6, 142.0, 139.9, 130.5, 130.2, 129.76, 129.71,
127.4, 119.9, 77.2, 48.8, 39.0, 31.2, 29.8, 26.0, 24.0, 21.2, 18.2;
Alcohol (24)
To a solution of PMB ether 23 (120 mg, 0.266 mmol) in CH2Cl2
(40 mL) at 0 °C was added pH 7 buffer (6 mL) followed by
DDQ (302 mg, 1.33 mmol). After being stirred at 0 °C for 5 min
and at room temperature for 2 h, the reaction was quenched with
saturate aqueous NaHCO3 and saturated aqueous Na2S2O3, and
extracted with CH2Cl2 (3 × 20 mL). The combined organic
extracts were washed with brine, dried over MgSO4, filtered and
then concentrated under reduced pressure. The residue was
purified by flash chromatography with 30% EtOAc/hexane as
eluent to afford alcohol 24 (81 mg, 84%) as a white solid. mp
156–157 °C; Rf (40% EtOAc/hexane) 0.47; [α]2D3 −169.5 (c
0.710, CH2Cl2); IR νmax: 3369, 3264, 2944, 2926, 2862, 1759,
1548, 1456, 1215, 966, 750 cm−1; δH 7.33 (1H, t, J = 7.9 Hz),
7.13 (1H, d, J = 7.5 Hz), 6.94 (1H, d, J = 8.2), 5.47 (1H, d, J =
8.7 Hz), 5.48 (1H, m), 5.27 (1H, m), 4.15 (1H, m), 3.89 (1H,
dd, J = 14.0, 3.3 Hz), 3.72 (2H, m), 3.20 (1H, app. dd, J = 14.0,
2.3 Hz), 2.26 (3H, s), 1.81 (2H, m), 1.61 (2H, m), 1.48 (2H, m),
1.33 (2H, m), 1.13 (2H, m); δC 170.9, 167.6, 147.2, 140.4,
133.3, 130.3, 130.1, 128.5, 128.1, 120.9, 58.3, 43.6, 40.0, 37.9,
33.8, 32.5, 24.4, 20.9, 20.8; HRMS (ESI) calculated for
C19H25NO4Na [M + Na]+ 354.16758, found 354.16763.
HRMS (ESI) calculated for C20H24INO3Na [M
476.06931, found 476.06950.
+
Na]+
Aldehyde (25)
Dess–Martin periodinane (1.53 g, 3.625 mmol) was added to a
cooled (0 °C) solution of alcohol 24 (300 mg, 0.906 mmol) in
CH2Cl2 (50 mL) and the resultant mixture was stirred at rt for
45 min. The white suspension was then cooled to 0 °C and satu-
rated aqueous Na2S2O3 (100 mL) and saturated aqueous solution
of NaHCO3 (100 mL) were added and the resulting mixture
stirred for 30 min until it became clear. The mixture was diluted
with CH2Cl2 (50 mL), the organic layer was separated and the
aqueous layer was extracted with CH2Cl2 (3 × 50 mL). The com-
bined organic extracts were washed with a 40% aqueous solution
of sodium thiosulfate (200 mL) and brine, dried over MgSO4
and concentrated. Flash chromatography provided aldehyde 25
(250.5 mg, 90%) as a white solid, which was recrystallized from
EtOAc affording colourless crystals suitable for X-ray crystallo-
graphic analysis. A single-crystal X-ray diffraction analysis
confirmed the identity of this compound (see ESI† and CCDC-
886234) mp 168 °C; Rf (80% EtOAc/hexane) 0.78; [α]2D3 −112.5
(c 1.03, CH2Cl2); IR νmax: 3254, 2930, 2852, 2737, 1763, 1724,
1634, 1546, 1458, 1202, 1019, 973, 756 cm−1; δH 9.81 (1H, t, J
= 2.1 Hz), 7.28 (1H, t, J = 7.8 Hz), 7.08 (1H d, J = 7.4 Hz),
6.90 (1H, d, J = 8.1 Hz), 5.74 (1H, d, J = 7.8 Hz), 5.47 (1H,
dddd, J = 14.9, 10.1, 4.5, 1.6 Hz), 5.26 (1H, dddd, J = 14.9,
10.1, 3.4, 1.3 Hz), 4.44 (1H, m), 3.82 (1H, dd, J = 14.1, 3.9 Hz),
3.21 (1H, dq, J = 14.1, 2.7 Hz), 2.57, (2H, m), 2.29 (3H, s),
2.18 (1H, m, H14), 1.69 (1H, m), 1.56 (2H, m), 1.43 (2H, m),
1.11 (2H, m); δC 200.5, 170.9, 165.7, 147.2, 140.3, 132.7,
Aza-salicylihalamide analogue (3)
A degassed solution of iodoalkene 26 (34 mg, 0.075 mmol),
Cs2CO3 (132 mg, 0.375 mmol), CuI (29 mg, 0.151 mmol),
N,N′-dimethylethylenediamine (41 μL, 0.38 mmol) and dienamide
74b (49 mg, 0.38 mmol) in dry DMF (2 mL) was stirred under
nitrogen at rt for 2 h. The reaction mixture was then diluted with
water and the aqueous layer was extracted with EtOAc (3 ×
15 mL). The combined organic extracts were washed with water
and brine, dried over MgSO4 and concentrated. Flash chromato-
graphy with 40% EtOAc/hexane as eluent provided the aza-sali-
cylihalamide analogue 3 (19.7 mg, 64%) as a colourless solid.
mp 151–152 °C; Rf (55% EtOAc/hexane) 0.41; [α]2D3 −13.4 (c
0.21, CH2Cl2); IR νmax: 3348, 2931, 2471, 2243, 2214, 2141,
2071, 1633, 1595, 1461, 1121, 1090, 972, 821 cm−1; δH (d4-
methanol) 7.32 (1H, dt, J = 10.8, 1.2 Hz), 7.08 (1H, t, J =
7.9 Hz), 6.87 (1H, dd, J = 11.6, 1.1 Hz), 6.80 (1H, d, J =
14.4 Hz), 6.73 (1H, d, J = 7.9 Hz), 6.6 (1H, d, J = 7.7 Hz), 5.83
(1H, dddd, J = 10.8, 7.7, 7.5, 1.3 Hz), 5.70 (1H, d, J = 11.6 Hz),
5.41 (2H, m), 5.26 (1H, m), 3.97 (1H, m), 3.76 (1H, dd, J =
14.3, 4.1 Hz), 3.13 (1H, br dd, J = 14.3, 2.3 Hz) 2.31 (2H,
dquin, J = 7.5, 1.5 Hz), 2.25 (2H, m), 2.16 (1H, m), 1.69 (1H,
m), 1.56 (2H, m), 1.42 (2H, m), 1.20 (2H, m), 1.03 (3H, t, J =
7.5 Hz); δC (d4-methanol) 170.0, 165.9, 155.9, 142.6, 141.0,
137.7, 133.4, 131.0, 130.0, 126.3, 125.7, 125.5, 122.5, 120.6,
115.2, 112.1, 49.6, 39.3, 37.0, 33.9, 32.9, 26.3, 22.2, 21.6, 14.5;
8152 | Org. Biomol. Chem., 2012, 10, 8147–8153
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+
Na]+
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and G. I. Georg, Org. Lett., 2003, 5, 4007; (b) G. A. Holloway,
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(c) C. Herb and M. E. Maier, J. Org. Chem., 2003, 68, 8129;
(d) K. Yang, T. Haack, B. Blackman, W. E. Diederich, S. Roy, S. Pusuluri
and G. I. Georg, Org. Lett., 2003, 5, 4007; (e) K. Yang, B. Blackman,
W. Diederich, P. T. Flaherty, C. J. Mossman, S. Roy, Y. M. Ahn and
G. I. Georg, J. Org. Chem., 2003, 68, 10030; (f) J. S. Yadav and
P. Srihari, Tetrahedron: Asymmetry, 2004, 15, 81; (g) C. Herb, A. Bayer
and M. E. Maier, Chem.–Eur. J., 2004, 10, 5649; (h) T. Haack, K. Haack,
W. E. Diederich, B. Blackman, S. Roy, S. Pusuluri and G. I. Georg,
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P. Purushothama Rao, M. Sridhar Reddy and A. R. Prasad, Lett. Org.
Chem., 2010, 7, 457.
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4241; (b) S. Lebreton, X.-S. Xie, D. Ferguson and J. K. De Brabander,
Tetrahedron, 2004, 60, 9635; (c) S. Tang and K. L. Erickson, J. Nat.
Prod., 2008, 71, 898; (d) S. Lebreton, J. Jaunbergs, M. G. Roth,
D. A. Ferguson and J. K. De Brabander, Bioorg. Med. Chem. Lett., 2008,
18, 5879; (e) Y. Sugimoto, K. Konoki, M. Murata, M. Matsushita,
H. Kanazawa and T. Oishi, J. Med. Chem., 2009, 52, 798.
7 R. M. Borzilleri, X. Zheng, R. J. Schmidt, J. A. Johnson, S.-H. Kim,
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and G. D. Vite, J. Am. Chem. Soc., 2000, 122, 8890.
HRMS (ESI) calculated for C25H32N2O3Na [M
431.23051, found 431.23053.
Biological assays
Serial dilutions of compound 3 were incubated with leukemia
cell lines for 72 h and proliferation was assessed using the Cell
Titre Glo assay (Promega). Data was analysed using nonlinear
regression algorithms in Prism Graphpad software to calculate
IC50 values. Data shown represents the mean IC50 SEM of n =
6 (aza-salicylihalamide analogue) and n = 3 (bafilomycin A1)
independent experiments.
Acknowledgements
We thank the Australian Research Council Discovery Grants
Scheme (DP0878279 to M.A.R. and H.H.) and the National
Health and Medical Research Council (Fellowships and Program
Grant to C.J.B., D.C.S.H. and D.S.) for financial support. J.W. is
grateful to Prof. Suresh K. Bhargava and the Advanced Materials
and Industrial Chemistry (AMIC) Group at RMIT University for
financial support of a visiting scholarship.
8 F. Y. F. Lee, R. Borzilleri, C. R. Fairchild, A. Kamath, R. Smykla,
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This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 8147–8153 | 8153