Chemical synthesis and biological evaluation of second-generation palmerolide a analogues

Article abstract of DOI:10.1002/ejoc.201001562

In this communication, second-generation analogues of palmerolide A, a recently reported cytotoxic agent against melanoma cancer cells, were rationally designed, synthesized, and biologically evaluated. Structural variations of the enamide side chain and the C1-C8 segment of palmerolide A revealed a narrow structure-activity relationship window of the newly synthesized compounds. In addition, mechanistic and pharmacological studies were performed to assess the therapeutic potential of palmerolide A. Synthesis and biological evaluation of second-generation palmerolide A analogues are reported. This study focused on further modification of the enamide side chain, and the C1-C8 carbon backbone of palmerolide A. Structure-activity relationship, mechanistic, and pharmacological investigations of the palmerolide compounds pave the foundation for the ensuing in vivo studies.

Full text of DOI:10.1002/ejoc.201001562

SHORT COMMUNICATION
DOI: 10.1002/ejoc.201001562
Chemical Synthesis and Biological Evaluation of Second-Generation
Palmerolide A Analogues
Vengala Rao Ravu,[a] Gulice Y. C. Leung,[a] Chek Shik Lim,[a] Sin Yee Ng,[a] Rong Ji Sum,[a]
and David Y.-K. Chen*[a]
Keywords: Natural products / Total synthesis / Cytotoxicity / Antitumor agents
In this communication, second-generation analogues of pal-
merolide A, a recently reported cytotoxic agent against mela-
noma cancer cells, were rationally designed, synthesized,
and biologically evaluated. Structural variations of the en-
amide side chain and the C1–C8 segment of palmerolide A
revealed a narrow structure–activity relationship window of
the newly synthesized compounds. In addition, mechanistic
and pharmacological studies were performed to assess the
therapeutic potential of palmerolide A.
total synthesis of palmerolide A (1),[2b,2c] its stereochemical
congeners and analogues,[4] as outlined in Figure 1. In view
of the possible role of the enamide moiety involved in the
reversible inhibition of the V-ATPase,[6] palmerolide E (10)
and α,β-unsaturated methyl ester 11 could serve as negative
controls in the biological studies against the enamide-con-
taining compounds. As shown in Scheme 1, the preparation
of 10 and α,β-unsaturated methyl ester 11 began with re-
moval of the two MOM ether moieties from aldehyde
8[2b,2c] (HCl, 52% yield) to afford dihydroxy aldehyde 9,
which underwent smooth ring-closing metathesis[7] in the
presence of Grubbs II catalyst to furnish 10 in 62% yield.
Treatment of 10 with Ph3P=CHCO2Me smoothly delivered
enoate 11 in 55% yield as a single geometric isomer.
On the basis of our previous studies,[4] the biologically
inconsequential C7 hydroxy substituent of the palmerol-
ide A core structure prompted us to employ structurally
simplified 7-deoxyvinyl iodide 12[4] to pursue further SAR
investigations concerning the palmerolide enamide side
chain. At the same time, we previously concluded that a
lipophilic enamide side chain was necessary for retaining
the potent biological activity, that is, benzamide 2. There-
fore, under our carefully optimized conditions, application
of the Buchwald copper-catalyzed coupling (CuI, K2CO3
and N,NЈ-dimethylethylenediamine)[8] engaging macrocyclic
iodide 12 and primary amides 13–18 afforded palmerol-
ide A analogues 19–24 in moderate to average yields (26–
51%) as summarized in Table 1.
Introduction
Palmerolide A (1) is a cytotoxic marine macrolide re-
cently isolated from the circumpolar tunicate Synoicum ad-
areanum around Anvers Island on the Antarctic Penin-
sula.[1] Reported by the Baker team in April 2006, it was
found to exhibit potent and selective growth inhibitory
properties against melanoma cancer cells UACC-62 (LC50
= 18 nM), and its mechanism of action correlates well with
other enamide containing V-ATPase inhibitors.[1] Synthetic
efforts in the palmerolide A (1) arena have culminated its
total syntheses,[2] and approaches toward several key syn-
thetic intermediates.[3] Furthermore, structureϪactivity re-
lationship (SAR) studies[4] led to the identification of
benzamide derivative 2 and deoxygenated analogue 3 with
an eightfold increase in potency and comparable potency,
respectively, relative to parent natural product 1. Continu-
ing our chemical–biology investigations in this area, here
we report the synthesis and biological evaluation of palmer-
olide E (10),[5] and second-generation analogues including
side-chain analogues 19–24 and C1–C8 analogues 49a–d,
against a selected panel of cancer and normal cell lines.
Mechanistic and pharmacological studies of 1 and benz-
amide 2 were also carried out to assess the therapeutic po-
tential of the palmerolide family of antiproliferative agents.
Results and Discussion
The synthetic strategy towards the designed analogues is
in accordance to our previously described technology in the
Next, we returned our attention to the SAR exploration
in the C1–C8 domain of palmerolide A. These second-gen-
eration analogues were designed to probe (i) the importance
of the C2–C3 olefin in the α,β-unsaturated macrolactone
(i.e., 49a), (ii) the possibility of replacing the metabolically
labile aliphatic side chain with a conformationally rigid aro-
matic moiety (i.e., 49b), (iii) the effect of extending the C1–
[a] Chemical Synthesis Laboratory@Biopolis, Institute of Chemi-
cal and Engineering Sciences (ICES), Agency for Science, Tech-
nology and Research (A*STAR)
11 Biopolis Way, The Helios Block, #0308, Singapore 138667,
Singapore
E-mail: david_chen@ices.a-star.edu.sg
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201001562.
Eur. J. Org. Chem. 2011, 463–468
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463

D. Y.-K. Chen et al.
SHORT COMMUNICATION
Table 1. Preparation of palmerolide A analogues 19–24 through
copper-mediated coupling reactions.[a]
Figure 1. Structure of palmerolide A (1), benzamide (2), and 7-de-
oxypalmerolide A (3), and retrosynthetic analysis of palmerolide
structure 1a leading to building blocks 5, 6, 7a, 7b, 7c, and 7d. TBS
= tert-butyldimethylsilyl.
[a] Reagents and conditions: 13–18 (2.0 equiv.), CuI (1.5 equiv.),
K2CO3 (5.0 equiv.), N,NЈ-dimethylethylenediamine (3.0 equiv.),
DMF, 23 °C, 1 h. DMF = N,NЈ-dimethylformamide.
under the olefin metathesis conditions. The MOM ether
could be removed at a later stage under mild acid condi-
tions (HCl/MeOH or TMSCl/MeOH); however, this trans-
formation was routinely complicated with the formation of
the C10–C11 cyclic carbonate as a byproduct.[2b,2c] There-
fore, we thought to circumvent this late-stage counterpro-
ductive event by exchanging the MOM ether to a TBS ether
early in the synthetic sequence (Scheme 2). In this instance,
starting from MOM ether 25,[2b,2c] a two-step protecting
group interconversion (CBr4; TBSCl) smoothly delivered
Scheme 1. Synthesis of palmerolide E (10) and α,β-unsaturated
methyl ester analogue 11. Reagents and conditions: (a) HCl (3.0 n
aq., 20.0 equiv.), EtOH, 23 Ǟ 80 °C, 50 min, 52%; (b) Grubbs II
cat. (0.05 equiv.), CH2Cl2, 23 °C, 1 h, 62%; (c) Ph3P=CHCO2Me
(2.5 equiv.), CH2Cl2, 23 °C, 2 h, 55%. MOM = methoxymethyl.
C8 aliphatic backbone (i.e., 49c), and (iv) the effect of the TBS ether 27 in 58% yield over the two steps. Further elab-
sterically demanding C5 gem-dimethyl substituents (i.e., oration of the alkyne terminus in 27 to key building block
49d). In accordance to our generalized retrosynthetic blue- vinyl stannane 6 was carried out by following our pre-
print for the preparation of the palmerolide family of com- viously reported palladium-catalyzed hydrostannation pro-
pounds (Figure 1), the synthesis of the required building cedure,[9] through intermediate bromide 28, in 69% overall
blocks and their unifications leading to analogues 49a–d are yield as a single geometric isomer. Stille[10] coupling be-
outlined in Schemes 2–4. In our previously reported synthe- tween vinyl stannane 6 and vinyl iodide 5[2b,2c] afforded
ses of palmerolide structures,[2b,2c] the C10 hydroxy group tetraene 29 in 79% yield, which serves as a common inter-
was protected as its MOM ether throughout the synthetic mediate for all ensuing synthetic investigations concerning
sequence and removed prior to the macrocyclic ring closure the variation of the C1–C8 segment of palmerolide A.
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Synthesis of Second-Generation Palmerolide A Analogues
Scheme 2. Synthesis of tetraene 29. Reagents and conditions:
(a) CBr4 (0.1 equiv.), iPrOH, 23 Ǟ 80 °C, 2 h, 62%; (b) TBSCl
(1.3 equiv.), imidazole (2.5 equiv.), 4-DMAP (cat.), DMF, 23 °C,
5 h, 94%; (c) NBS (1.1 equiv.), AgNO3 (0.1 equiv.), acetone, 23 °C,
1 h, 98%; (d) Pd(dba)2 (0.05 equiv.), PPh3 (0.22 equiv.), nBu3SnH
(2.2 equiv.), THF, 23 °C, 1 h, 70%; (e) Pd(PPh3)4 (0.1 equiv.), CuCl
(5.0 equiv.), LiCl (6.0 equiv.), DMSO, 23 °C, 8 h, 79%. 4-DMAP =
N,NЈ-dimethylaminopyridine, NBS = N-bromosuccinimide; dba =
dibenzylideneacetone.
Variation of the C1–C8 segment of palmerolide A also
called for the preparation of carboxylic acids 7a–d, and
their syntheses are shown in Scheme 3. Acid fragment 7a
was prepared through conjugate reduction of enoate 30
(NaBH4, CuCl), followed by saponification of resulting
ethyl ester 31 (KOH, 75% yield over the two steps). Aro-
matic acid 7b was synthesized from phenolic benzaldehyde
32 through intermediate triflate 33 (Tf2O, 46% yield), fol-
lowed by palladium-mediated cross-coupling with allyltri-
butyltin (34, 99% yield), homologation to enoate 35 (35%
yield), and saponification (LiOH, 95% yield). Two-carbon
homologated acid 7c was synthesized through sequential
Scheme 3. Synthesis of carboxylic acids 7a–d. Reagents and condi-
tions: (a) NaBH4 (10.0 equiv.), CuCl (0.75 equiv.), MeOH/THF
(4:7), 0 °C, 6 h, 91%; (b) KOH (5.0 equiv.), dioxane/H2O, (4:1),
23 °C, 12 h, 82%; (c) Tf2O (1.2 equiv.), pyridine (2.0 equiv.),
CH2Cl2, 0 Ǟ 23 °C, 3 h, 46%; (d) Pd(PPh3)4 (0.1 equiv.), LiCl
(3.0 equiv.), allyltributyltin (1.1 equiv.), 1,4-dioxane, 100 °C, 16 h,
99%; (e) trimethyl phosphonoacetate (1.2 equiv.), LiBr (4.0 equiv.),
oxidation of alcohol 36 (PCC; then NaClO2) in 64% yield Et3N (2.0 equiv.), THF, 23 °C, 16 h, 35%; (f) LiOH (3.0 equiv.),
THF/MeOH/H2O (2:1:1), 23 °C, 4 h, 95%; (g) PCC (1.6 equiv.),
CH2Cl2, 23 °C, 16 h, 88%; (h) NaClO2 (3.0 equiv.), 2-methyl-2-but-
ene (10 equiv.), NaH2PO4 (5.0 equiv.), tBuOH/H2O (1:1), 0 °C, 2 h,
73%; (i) 3-butenylmagnesium bromide (2.0 equiv.), CuCl
over the two steps. Finally, preparation of gem-dimethyl
acid 7d began with conjugate addition of 3-butenylmagne-
sium bromide to α,β-unsaturated diester 38 followed by
thermal decarboxylation to give ethyl ester 40 in 92% yield
over the two steps. Two-carbon homologation of ethyl ester
40 involved a reduction/oxidation/Wittig olefination se-
quence (DIBAL-H, DMP, Ph3P=CHCO2Me), through the
intermediacy of alcohol 41, to furnish α,β-unsaturated
(0.05 equiv.), THF, –30 Ǟ 23 °C, 1.5 h, 97%; (j) LiCl (2.0 equiv.),
H2O (1.0 equiv.), DMSO, reflux, 24 h, 95%; (k) DIBAL-H (1.4 m
in CH2Cl2, 2.5 equiv.), CH2Cl2, –78 °C, 1.5 h, 90%; (l) DMP,
(1.1 equiv.), NaHCO3 (5.0 equiv.), CH2Cl2, 23 °C, 0.5 h; (m) tri-
methyl phosphonoacetate, (1.1 equiv.), LiBr (4.0 equiv.), Et3N
(2.0 equiv.), 23 °C, 12 h, 78% over two steps; (n) LiOH (3.0 equiv.),
methyl ester 42 (70% overall yield). Finally, saponification THF/MeOH/H2O (2:1:1), 23 °C, 4 h, 95%. PCC = pyridinium
chlorochromate, Tf = trifluoromethanesulfonyl.
of methyl ester 42 (LiOH) afforded desired acid 7d in 95%
yield.
With building blocks 29 and 7a–d in hand, the next task
became their unification and further elaboration to targeted sponding aldehydes 45a (77%), 45b (68%), 45c (72%), and
analogues 49a–d, as summarized in Scheme 4. Thus, Yama- 45d (50%). Further elaboration of aldehydes 45a–d to
guchi esterification[11] (2,4,6-trichlorobenzoyl chloride, macrocyclic ring-closing metathesis precursors 47a–d in-
Et3N, 4-DMAP) between alcohol 29 and acid fragments volved Takai olefination[12] (CrCl2, CHI3; 46a–d) and TBS
7a–d afforded esters 43a (69%), 43b (70%), 43c (72%), and ether removal (HCl aq.; 47a: 76%, 47b: 40%, 47c: 42%,
43d (60%), respectively. Selective removal of the primary and 47d: 44% yield over the two steps). With allylic
TBS ether in 43a–d under the mild action of TFA, followed alcohols 47a–d in hand, their ring-closing-metatheses[7] un-
by oxidation (DMP) of resulting alcohols 44a–d gave corre- der the action of Grubbs II catalyst (CH2Cl2, 0.005 m,
Eur. J. Org. Chem. 2011, 463–468
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465

D. Y.-K. Chen et al.
SHORT COMMUNICATION
25 °C) proceeded smoothly to afford macrocycles 48a
The synthesized compounds were tested against a panel
(61%), 48b (60%), 48c (60%), and 48d (60%), respectively. of cancer cells, including breast (MCF-7), melanoma
Finally, installation of the enamide moiety onto vinyl io- (UACC-62), CNS (SF268), lung (NCI-H460), ovarian
dides 48a–d through the established copper-catalyzed cross- (1A9), Taxol®-resistant ovarian (PTX22),[13] epothilone-re-
coupling protocol[8] provided C1–C8 palmerolide A ana- sistant ovarian (A8),[14] and human melanocyte (HM2) cells
logues 49a (56%), 49b (28%), 49c (26%), and 49d (31%), using doxorubicin, Taxol®, and natural palmerolide A (1)
respectively.
as standards; the results are summarized in Table 2. As ex-
pected and on the basis of the hypothesized reversible V-
ATPase inhibition involving the enamide side chain,[6] both
palmerolide E (10) and methyl ester 11 showed complete
lost of activity up to 10 μm against all cells tested. Side-
chain analogues 19–24 revealed some interesting findings.
Benzamide 19 exhibited the most potent activity, an obser-
vation similar to that previously reported for its C7 oxygen-
ated relative (compound 2). In stark contrast, biphenyl 20
and 2,4-dimethoxy 23 showed significantly lower activities,
whereas 4-fluoro 21, 4-trifluoromethyl 22, and 3,5-dimeth-
oxy 24 displayed modest activities. This seems to suggest a
very narrow SAR window concerning the steric environ-
ment of the aromatic side chain, even for 4-fluoro analogue
21. It is interesting to note that 2,4-dimethoxy and 3,5-di-
methoxy analogues 23 and 24 exhibited drastically different
cytotoxic activities, possibly attributed to the electron-do-
nating groups at the ortho and para positions of analogue
23, which shift the equilibrium towards the protonated en-
amide rather than the enamide/V-ATPase complex.[6] The
biological activities of C1–C8 analogues 49a–d also re-
vealed little SAR flexibility concerning this domain of the
palmerolide A core structure. In this instance, whereas C2–
C3 saturated analogue 49a displayed an approximately 4–
10-fold reduction in activity across all cell lines examined,
aromatic analogue 49b, two-carbon homologated analogue
49c, and gem-dimethyl analogue 49d all exhibited a dra-
matic reduction in their biological activities. Once again,
this indicates that a subtle increase in the steric environment
around the C1–C8 domain of palmerolide A cannot be tol-
erated at the active site of the enzyme. The therapeutic in-
dexes of the palmerolide family of compounds were also
determined by comparing their cytotoxicities against the
melanoma cancer cell line (UACC-62) and normal human
melanocyte (HM-2). Synthetic palmerolide A (1) and ana-
logues 2, 3, 19, 21, and 24 all exhibited excellent selectivity
towards UACC-62, with benzamide analogue 2 being the
most potent and most selective compound prepared to date.
Finally, mechanistic and pharmacological investigations
of palmerolide A (1) and benzamide analogue 2 were car-
ried out to further assess the therapeutic potential of the
palmerolide family of antitumor agents.[15] In the Apo-
Scheme 4. Synthesis of palmerolide A analogues 49a–d employing
acid fragments 7a–d, respectively. Reagents and conditions:
(a) 2,4,6-trichlorobenzoyl chloride (1.1 equiv.), Et3N (2.0 equiv.), 7a
or 7b or 7c or 7d (1.2 equiv.), 4-DMAP (1.0 equiv.), toluene, 23 °C,
12 h, 43a: 69%; 43b: 70%; 43c: 72%; 43d: 60%; (b) TFA (10% aq.,
1.0 equiv.), THF, 0 °C, 12 h, 44a: 91%; 44b: 90%; 44c: 90%; 44d: BrdU TUNEL assay (to detect the 3Ј-hydroxy ends of frag-
65%; (c) Dess–Martin periodinane (1.1 equiv.), NaHCO3
(5.0 equiv.), CH2Cl2, 23 °C, 20 min, 45a: 85%; 45b: 75%; 45c: 80%;
played cell killing effect resembling a late-apoptotic path-
45d: 75%; (d) CrCl2 (10.0 equiv.), CHI3 (3.0 equiv.), THF/dioxane
(1:6), 23 °C, 2 h, 46a: 87% (Ͼ95:5 E/Z); 46b: 55% (Ͼ95:5 E/Z);
mented DNA in apoptotic cells), palmerolide A (1) dis-
way. Furthermore, direct evidence in support of the V-AT-
Pase inhibitory properties of palmerolide A (1) was ob-
tained through LysoSensor studies (monitor pH level
changes of the lysosomes and acidic organelles), in com-
parison with concanamycin, a known V-ATPase inhibitor.
Palmerolide A (1) and benzamide analogue 2 were also
found to inhibit CYP3A4 metabolic activity (IC50 = 1.182
46c: 60% (Ͼ95:5 E/Z); 46d: 60% (Ͼ95:5 E/Z); (e) HCl (3.0 n aq.,
2.0 equiv.), EtOH, 23 °C, 12 h, 47a: 87%; 47b: 73%; 47c: 70%; 47d:
74%; (f) Grubbs II cat. (0.05 equiv.), CH2Cl2, 23 °C, 1 h, 48a: 61%,
48b: 60%; 48c: 60%; 48d: 60%; (g) 3-methyl-2-butenamide
(2.0 equiv.), CuI (1.5 equiv.), K2CO3 (6.0 equiv.), N,NЈ-dimethyl-
ethylenediamine (3.0 equiv.), DMF, 23 °C, 1 h, 49a: 56%; 49b:
28%; 49c: 26%; 49d: 31%. TFA = trifluoroacetic acid.
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Synthesis of Second-Generation Palmerolide A Analogues
Table 2. Cytotoxicity of natural and synthetic palmerolides against selected cancer and normal cell lines.
[a]
Entry
Cell line/
Compound
GI50 [μM]
UACC-62[b] MCF-7[b]
SF268[b]
NCI-H460[b]
IA9[c]
PTX22[c]
A8[c]
HM-2[d]
SI[e]
1
2
3
1
2
3
0.061
Ϯ0.011
0.009
Ϯ0.000
0.076
Ϯ0.010
Ͼ10
Ͼ10
0.068
Ϯ0.006
0.008
Ϯ0.001
0.153
Ϯ0.087
Ͼ10
9.061
0.040
Ϯ0.012
0.007
Ϯ0.001
0.069
Ϯ0.004
Ͼ10
Ͼ10
0.014
Ϯ0.004
0.007
Ϯ0.001
0.069
Ϯ0.004
Ͼ10
Ͼ10
0.070
Ϯ0.009
0.008
Ϯ0.001
0.087
Ϯ0.006
Ͼ10
Ͼ10
0.067
Ϯ0.004
0.029
Ϯ0.011
0.194
Ϯ0.078
Ͼ10
Ͼ10
0.059
Ϯ0.015
0.007
Ϯ0.001
0.091
Ϯ0.004
Ͼ10
Ͼ10
6.177
Ϯ1.272
2. 309
Ϯ1.269
14.006
Ϯ4.589
Ͼ10
101.3
256.6
184. 3
4
5
10
11
Ͼ10
Ϯ0.174
0.061
Ϯ0.004
6.320
Ϯ0.978
0.339
Ϯ0.093
0.864
Ϯ0.086
5.452
Ϯ0.659
0.855
Ϯ0.006
0.849
Ϯ0.083
2.357
6
7
19
20
0.055
Ϯ0.011
5.823
Ϯ0.625
0.227
Ϯ0.048
0.882
Ϯ0.041
9.026
Ϯ0.423
0.753
Ϯ0.113
0.750
Ϯ0.123
4.958
0.045
Ϯ0.008
8.181
Ϯ0.134
0.282
Ϯ0.074
0.936
Ϯ0.041
Ͼ10
0.027
Ϯ0.017
0.701
Ϯ0.028
0.069
Ϯ0.005
0.582
Ϯ0.072
5.093
Ϯ0.018
0.517
Ϯ0.048
0.465
Ϯ0.086
0.845
0.072
Ϯ0.004
7.309
Ϯ1.172
0.646
Ϯ0.098
3.004
Ϯ0.764
Ͼ10
0.075
Ϯ0.003
7.035
Ϯ0.858
0.631
Ϯ0.060
2.232
Ϯ0.491
9.206
Ϯ0.240
0.863
Ϯ0.161
0.723
Ϯ0.056
5.489
0.065
Ϯ0.003
6.217
Ϯ0.472
0.486
Ϯ0.122
0.785
Ϯ0.029
8.317
Ϯ0.299
0.710
Ϯ0.013
0.807
Ϯ0.075
4.269
8.256
Ϯ2.222
56.641
Ϯ0.989
27.186
Ϯ4.734
23.701
Ϯ1.541
42.201
Ϯ8.480
Ͼ100
150.1
9.7
8
21
119.8
26.9
4.7
9
22
10
11
12
13
14
23
24
0.789
Ϯ0.111
0.687
Ϯ0.121
2.686
0.869
Ϯ0.077
0.820
Ϯ0.066
7.084
Ͼ132.8
10.7
3.8
49a
49b
49c
8.020
Ϯ1.243
7.787
Ϯ0.889
8.619
Ϯ0.361
Ͼ10
0.061
6.857
Ϯ0.071
Ͼ10
5.708
6.793
5.41
8.102
6.66
5.626
1.2
15
16
49d
Taxol
Ͼ10
0.006
Ͼ10
0.024
Ͼ10
0.006
–
–
–
–
4.1
0.015
0.006
0.083
0.035
Ϯ0.007
0.207
Ϯ0.000
0.057
Ϯ0.008
0.160
Ϯ0.001
0.008
Ϯ0.000
0.035
Ϯ0.007
0.159
Ϯ0.016
0.063
Ϯ0.010
0.172
17
Doxorubicin
0.8
Ϯ0.091
Ϯ0.007
Ϯ0.053
Ϯ0.001
Ϯ0.010
Ϯ0.051
Ϯ0.013
Ϯ0.083
[a] Antiproliferative effects of tested compounds against human tumor cell lines and drug-resistant cell lines in a 48 h growth inhibition
assay using the sulforhodamine B staining methods. Human cancer cell lines: melanoma (UACC-62); breast (MCF-7), CNS (SF268),
lung (NCI-H460), ovarian (IA9) and its drug resistant mutant PTX22 (taxol-resistant) and A8 (epithilone resistant), normal melanocyte
(HM-2). Growth inhibition of 50% (GI50) is calculated as the drug concentration, which caused a 50% reduction in the net protein
increase in control cells during the drug incubation. GI50 values for each compound represent mean of 2–5 independent experimentsϮSE.
[b] These cell lines were provided by the National Cancer Institute (NCI), Division of Cancer Treatment and Diagnosis (DCTD). [c] These
cell lines were provided by Professor Paraskevi Giannakakou, Weill Medical College of Cornell University. [d] These cells were purchased
from Cell Research Corporation (Singapore). [e] Selectivity index, SI (GI50 HM-2/GI50 UACC-62).
and 0.956 μm, respectively) and verapamil-stimulated Pgp- mechanistic and pharmacological findings of palmerolide A
ATPase activity (IC50 = 1.220 and 1.384 μm, respectively) (1) and benzamide 2, in vivo biological evaluations of se-
and inhibited hERG channel at 3.5 μm (with E4031 as the lected candidate(s) will be carried out, and the results of
positive control).
these studies will be reported in due course.
Supporting Information (see footnote on the first page of this arti-
cle): Experimental section; details of the cytotoxicity assay, APO-
BrdU TUNEL assay, pH-lysosensor assay, verapamil-stimulated
Pgp-ATPase assay, Vivid®CYP3A4 assay, and PredictorTM hERG
Conclusions
1
fluorescence polarization assay; H and 13C NMR spectra of com-
In conclusion, the design and synthesis of second-genera-
tion palmerolide A analogues with structural variations in
the enamide side chain and the C1–C8 aliphatic backbone
were accomplished. The synthesized compounds were
evaluated against a panel of cancer and normal cell lines,
revealing a narrow SAR window concerning both of these
structural domains. The beneficial effect of the benzamide
group was once again confirmed through C7 deoxygenated
analogue 19. The most active members of the palmerolide
analogues also exhibited promising therapeutic indexes
when compared between cancerous (UACC-62) and normal
(HM-2) cell lines. In conjunction with the preliminary
pounds.
Acknowledgments
We thank Ms. Doris Tan (ICES) for high-resolution mass spec-
trometry (HRMS) assistance. Financial support for this work was
provided by Agency for Science, Technology and Research, Singa-
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Received: November 19, 2010
Published Online: December 13, 2010
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