The synthesis and evaluation of a solution phase indexed combinatorial library of non-natural polyenes for reversal of p-glycoprotein mediated multidrug resistance

Article abstract of DOI:10.1021/jo000453m

A combinatorial library of polyenes, based on (-)-stipiamide, has been constructed and evaluated for the discovery of new multidrug resistance reversal agents. A palladium coupling was used to react each individual vinyl iodide with a mixture of the seven acetylenes at near 1:1 stoichiometry. The coupling was also used to react each individual acetylene with the mixture of six vinyl iodides to create 13 pools indexed in two dimensions for a total of 42 compounds. Individual compounds were detected at equimolar concentration. The vinyl iodides, made initially using a crotylborane addition to generate the anti1,2-hydroxylmethyl products, were now made using a more efficient norephedrine propionate boron enolate aldol reaction. The indexed approach, ideally suited for cellular assays that involve membrane-bound targets, allowed for the rapid identification of reversal agents using assays with drug-resistant human breast cancer MCF7-adrR cells. Intersections of potent pools identified new compounds with promising activity. Aryl dimension pools showed R = ph and naphthyl as the most potent. The acetylene dimension had R' = phenylalaninol and alaninol as the most potent. Isolated individual compounds, both active and nonpotent, were assayed to confirm the library results. The most potent new compound was 4ek (R = naphthyl, R' = phenylaninol) at 1.45 μM. Other nonnatural individual naphthyl-amide compounds showed potent MDR reversal including the morpholino-amide 4ej (1.69 μM). Synergistic activities attributed to the two ends of the molecule were also identified. Direct interaction with Pgp was established by ATPase and photoaffinity displacement assays. The results indicate that both ends of the polyene reversal agent are involved in Pgp interaction and can be further modified for increased potency.

Full text of DOI:10.1021/jo000453m

J . Org. Chem. 2000, 65, 4973-4983
4973
Th e Syn th esis a n d Eva lu a tion of a Solu tion P h a se In d exed
Com bin a tor ia l Libr a r y of Non -Na tu r a l P olyen es for Rever sa l of
P -Glycop r otein Med ia ted Mu ltid r u g Resista n ce
Merritt B. Andrus,*,† Timothy M. Turner,† Zuben E. Sauna,‡ and Suresh V. Ambudkar‡
Brigham Young University, Department of Chemistry and Biochemistry, C100 BNSN,
Provo, Utah 84602-5700, and National Cancer Institute, NIH, Laboratory of Cell Biology, Building 37,
Room 1B-17 37 Convent Drive, MSC4255, Bethesda, Maryland 20878-4255
mbandrus@chemdept.byu.edu
Received March 27, 2000
A combinatorial library of polyenes, based on (-)-stipiamide, has been constructed and evaluated
for the discovery of new multidrug resistance reversal agents. A palladium coupling was used to
react each individual vinyl iodide with a mixture of the seven acetylenes at near 1:1 stoichiometry.
The coupling was also used to react each individual acetylene with the mixture of six vinyl iodides
to create 13 pools indexed in two dimensions for a total of 42 compounds. Individual compounds
were detected at equimolar concentration. The vinyl iodides, made initially using a crotylborane
addition to generate the anti1,2-hydroxylmethyl products, were now made using a more efficient
norephedrine propionate boron enolate aldol reaction. The indexed approach, ideally suited for
cellular assays that involve membrane-bound targets, allowed for the rapid identification of reversal
agents using assays with drug-resistant human breast cancer MCF7-adrR cells. Intersections of
potent pools identified new compounds with promising activity. Aryl dimension pools showed R )
ph and naphthyl as the most potent. The acetylene dimension had R′ ) phenylalaninol and alaninol
as the most potent. Isolated individual compounds, both active and nonpotent, were assayed to
confirm the library results. The most potent new compound was 4ek (R ) naphthyl, R′ )
phenylaninol) at 1.45 µM. Other nonnatural individual naphthyl-amide compounds showed potent
MDR reversal including the morpholino-amide 4ej (1.69 µM). Synergistic activities attributed to
the two ends of the molecule were also identified. Direct interaction with Pgp was established by
ATPase and photoaffinity displacement assays. The results indicate that both ends of the polyene
reversal agent are involved in Pgp interaction and can be further modified for increased potency.
While small molecule libraries have gained great
prominence as a means of identifying new inhibitors for
biological targets,1 unified approaches that allow for
efficient synthesis, direct screening, and facile individual
member identification are still needed. Solid-phase li-
braries that provide for synthetic efficiency are more
amenable to screening with isolable, gobular receptors
and are generally not applicable to cell assays where
membrane-bound targets are involved.2 In contrast to the
split-pool peptide approach, solid-phase small molecule
libraries have been more focused on spatially addressed,
serial methods. Solution-phase libraries3 offer the ad-
vantages of being able to use standard reagents, no linker
or support, and the ability to directly screen with isolated
receptors or cellular assays.4 Natural product-like librar-
ies, while more constrained with less total numbers
compared to peptides and nucleotides, are inherently
more diverse in that a great variety of functionality
can be explored within a core template.5 We now pro-
vide a full report of a solution-phase library,6 based
on the multidrug resistance (MDR) reversing polyene
(-)-stipiamide, that consists of mixtures indexed in two
dimensions that allows for efficient combinatorial syn-
thesis, direct screening with a cellular assay, together
with the isolation and testing of individual compounds.
Recently we showed that synthetic (-)-stipiamide 17
had only moderate reversal activity with colchicine
resistant cells,8 while 6,7-dehydrostipiamide (DHS) 2
(ED50 6.5 µM) and the truncated compound 3 (4 µM) were
potent using a variety of drugs with resistant MCF7-adrR
(human breast cancer) cells that express Pgp (P-glyco-
protein), the membrane-bound small molecule MDR
pump (Chart 1).9 Importantly, 2 and 3 were far less toxic
(4 and 14µM) compared to stipiamide 1 (0.01nM). Im-
portantly, 3 was further shown to directly bind Pgp by
monitoring ATPase activity (10 µm max. stimulation)10
and through direct displacement of a known photoaffinity
label iodoarylazidoprazosin (12 µm).10 Having explored
(5) Nicolaou, K. C.; Pastor, J .; Winssinger, N.; Murphy, F. J . Am.
Chem. Soc. 1998, 120, 5132. Tan, D. S.; Foley, M. A.; Shair, M. D.;
Schreiber, S. L. J . Am. Chem. Soc. 1998, 120, 8565.
(6) Andrus, M. B.; Turner, T. M.; Asgari, D.; Wenke, L. J . Org. Chem.
1999, 64, 2978.
(7) (a) Andrus, M. B.; Lepore, S. D. J . Am. Chem. Soc. 1997, 119,
2327. (b) Andrus, M. B.; Lepore, S. D.; Turner, T. M. J . Am. Chem.
Soc. 1997, 119, 12159.
(8) Kim, Y. J .; Furihata, K.; Yamanaka, S.; Fudo, R.; Seto, H. J .
Antibiot. 1991, 44, 553.
(9) Ambudkar, S. V.; Dey, S.; Hrycyna, C. A.; Ramachandra, M.
Pastan, I.; Gottesman, M. M. Annu. Rev. Pharmacol. Toxicol. 1999,
39, 361-398. Gottesman, M. M.; Pastan, I. Annu. Rev. Biochem. 1993,
62, 385-427.
† Brigham Young University.
‡ NIH.
(1) Thompson, L. A.; Ellman, J . A. Chem. Rev. 1996, 96, 555.
(2) Lam, K. S.; Salmon, S. E.; Hersh, E. M.; Hruby, V. J .; Kazmier-
ski, W. M.; Knapp, R. J . Nature 1991, 354, 82.
(3) Pirrrung, M. C.; Chen, J . J . Am. Chem. Soc. 1995, 117, 1240.
(4) Carell, T.; Wintner, E. A.; Rebek, J . J r. Angew. Chem., Int. Ed.
Engl. 1994, 33, 2061. Boger, D. L.; Chai, W.; Ozer, R. S.; Anderson, C.
Bioorg. Med. Chem. Lett. 1997, 7, 463.
10.1021/jo000453m CCC: $19.00 © 2000 American Chemical Society
Published on Web 07/20/2000

4974 J . Org. Chem., Vol. 65, No. 16, 2000
Andrus et al.
Ch a r t 1
Ch a r t 2
the length and conjugation as functions of MDR reversal
efficacy, our attention next turned to perturbing the end
groups of DHS 3 to improve binding to Pgp. Selection of
end groups for Pgp mediated MDR modulation is difficult
for several reasons. The structural features for small
molecule binding to Pgp are poorly understood since no
crystal structure for the protein has yet been reported.
Indirect methods, such as site directed mutagenesis
studies, have indicated that small molecule binding may
occur at several domains on along the protein. In
particular, the regions of transmembrane 5-6 and 11-
12 have been implicated in the binding of several
modulators.11 Additionally, it is believed that Pgp may
contain additional binding sites, thus complicating the
situation.12 Since highly refined three dimension data
regarding the structure of Pgp is not available, it is not
entirely clear at this time how these binding regions
interact with one another.13
heteroatom coupling procedures formed the mixtures in
reasonable yield and purity. Prior to our work a carbon-
carbon bond coupling had not been explored as the
diversity-generating step of a solution-phase library.
Exploration of the effect of the two end groups R and
R′ of 4 was thought to be ideally suited to this 2D
indexing approach (Chart 2). By reacting vinyliodides 5
with alkynyl amides 6 the library was to be constructed.
The new compounds formed would contain both elements
derived from the reacting ligands. Both R and R′ are basis
sets which possess the various structural features to be
explored. The reacting ligands were coupled in such a
way to produce mixtures of compounds having a specific
group at one terminus while being diversified at the
other. These pools of compounds were assayed to give an
average potency on either the x- or y-axis at the position
pertaining to the end group that was held constant. Once
evaluated, the intersection points of the most potent pools
determined the optimal end groups for potent activity.
It was anticipated that synergistic or antagonistic effects
involving a particular combination of compounds might
make it difficult to effectively determine optimal com-
pounds. However, the chance of finding potent new
compounds is significantly increased in that each com-
pound is represented twice in two entirely different
While solution phase libraries have centered on high-
throughput parallel synthesis and evaluation, there are
a few reports of solution phase libraries generated as
mixtures. Rebek generated amide diversity around the
periphery of a core dibenzopyran template to screen for
trypsin inhibitors.14 Smith and Pirrung utilized coupling
reactions to generate linear amides with diversity present
at the terminal positions to evaluate binding with the
NK3 receptor and acetylcholinesterase inhibition.15 Both
approaches used a two-dimensional indexing strategy
to evaluate the libraries with the receptor. Standard
(10) Ambudkar, S. V. Methods Enzymol. 1998, 292, 504-514. Dey,
S.; Ramachandra, M.; Pastan, I.; Gottesman, M. M.; Ambudkar, S. V.
Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 10594.
(11) Germann, U. A. Eur. J . Can. 1996, 32A, 927. Greenberger, L.
M. J . Biol. Chem. 1993, 268, 11417.
(12) (a) Loo, T. W.; Clarke, D. M. J . Biol. Chem. 1993, 268, 19965
(b) Loo, T. W.; Clarke, D. M. J . Biol. Chem. 1995, 270, 21839.
(13) Recently, a low resolution electron diffraction structure of Pgp
(25 Å) was reported: (a) Rosenburg, M. F.; Callaghan, R.; Ford, R. C.;
Higgins, C F. J . Biol. Chem. 1997, 272, 10685 (b) Higgins, C. F.;
Callaghan, R. Semin. Can. Biol. 1997, 8, 135.
(14) (a) Carell, T.; Wintner, E. A.; Bashir-Hashemi, A.; Rebek J .
Angew. Chem., Int. Ed. Engl. 1994, 33, 2059 (b) Carell, T.; Wintner,
E. A.; Sutherland, A. J .; Rebek, J .; Dunayevskiy, Y. M.; Vouros, P.
Chemistry & Biology 1995, 2, 171.
(15) (a) Smith, P. W.; Lai, J . Y. Q.; Whittington, A. R.; Cox, B.;
Houston, J . G. Bioorg. Med. Chem. Lett. 1994, 4, 2821 (b) Pirrung, M.
C.; Chen, J . J . Am. Chem. Soc. 1995, 117, 1240 (c) Pirrung, M. C.;
Chau, J . H-L.; Chen, J . Chemistry & Biology 1995, 2, 621. For other
recent examples of solution phase libraries (d) De´prez, B.; Williard,
X.; Bourel, L.; Coste, H.; Hyafil, F.; Tartar, A. J . Am. Chem. Soc. 1995,
117, 5405 (e) Han, H.; Wolfe, M. M.; Brenner, S.; J anda, K. D. Proc.
Natl. Acad. Sci. U.S.A. 1995, 92, 6419 (f) Edge, C. J .; Rademacher, T.
W.; Wormald, M. R.; Parekh, R. B.; Butters, T. D.; Wing, D. R.; Dwek,
R. A. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 6338 (g) Boger, D. L. Chai,
W.; J in, Q. J . Am. Chem. Soc. 1998, 120, 7220 (h) An example of a
C-C bond forming library was recently reported although no biological
evaluation was mention: Boger, D. L.; Goldberg, J .; Andersson, C.-M.
J . Org. Chem. 1999, 64, 2422.

Combinatorial Library of Non-Natural Polyenes
J . Org. Chem., Vol. 65, No. 16, 2000 4975
Sch em e 1
gave the homo-allylic alcohols 9a -f in modest yields
(34-76%).18 Low yields in some cases were attributed to
the difficulty in separating the desired alcohols from the
pinenol byproduct. Although the anti: syn selectivity was
low in some cases (4:1), the unwanted syn isomer could
be removed at a later stage of the synthetic sequence.
Protection of alcohols 9a -f followed by selective di-
hydroxylation of the terminal olefin gave the 1,2,-diols
10a -f.19 Analogous with the synthesis of 4,5-dehydro-
stipiamide, the TBS ether blocks dihydroxylation from
occurring at the internal olefin.20 The diols were cleaved
with sodium periodate to afford aldehydes which were
converted to the E-vinyl iodides using Takai reaction
conditions.21 Finally, removal of the silyl protecting group
gave the desired vinyl iodides 5a -f.
Although this route proved quite useful, a plan was
underway to generate the anti-hydroxy-methyl stereo-
centers in a more stereoselective manner. While numer-
ous alternatives abound for syn stereocontrol, only a
limited number of reliable methods for anti selective
additions to enals are known.22 Previous attempts to
generate this anti intermediate in the route to stipiamide
using Heathcock’s modification23 of the Evan’s aldol24
procedure resulted in poor yield and selectivity. Recently
Masamune has developed a norephedrine based chiral
propionate that generates the anti-aldol product in high
selectivity with a variety aldehydes including enals.25
Following this protocol addition of R,â-unsaturated al-
dehyde 11e to the boron enolate of the propionate
auxiliary afforded the anti-aldol product with 14:1 dias-
tereoselectivity (Scheme 2).26 No syn-aldol products were
observed. Protection with TBS-OTf gave 13 in 77%
overall yield for the aldol and protection steps. At this
point, concern was focused on removal of the bulky ester
auxiliary. LiEt3BH, LAH and DIBAL afforded alcohol in
low yield. Prolonged reaction times using these reagents
mixtures in opposite dimensions. An additional concern
was the requirement to achieve equimolar representation
of each member of a pool. The centrally located coupling
site remote from the ends was thought to ensure com-
parable reaction rates to provide adequate representation
of each compound. The ready identification, isolation, and
assay results of numerous individual compounds now
addresses this concern verifying the results from the
pools. The efficiency of this library is realized in that only
13 reactions and assays are required to screen 42
compounds. This represents an efficiency of 3.2 over a
serial approach. End groups were selected to provide a
range of steric and electronic variation. The large non-
polar adamantyl groups as well as the polar dimethoxy-
phenyl and bis-hydroxyethyl amides were convenient and
typical. Nonpolar aromatic functionality on the left side
was expected to be potent while other substituents, as
with removal of the amide hydroxyl group, were expected
to be ineffective. The library was restricted to 6 × 7 with
42 compounds to allow for rapid characterization of the
mixtures and easy separation of individual members.
The first objective was to synthesize the coupling
precursors 5 and 6. Six vinyl iodides 5a -f were prepared,
each containing a different end group remote from the
coupling site (Scheme 1). The starting materials were the
aldehydes 7a -f obtained commercially or formed using
standard protocols.16 The aldehydes 7a -f were subjected
to Wittig olefination to form the E- R,â-unsaturated
aldehydes 8a -f.17 E-Crotylborane addition to the alde-
hydes using Brown’s reagent derived from (-)-pinene
(18) Brown, H. C.; Bhat, K. S. J . Am. Chem. Soc. 1986, 108, 293.
(19) Kolb, H. C.; VanNieuwenhze, M. S.; Sharpless, K. B. Chem.
Rev. 1994, 94, 2483.
(20) Andrus, M. B.; Lepore, S. D.; Sclafani, J . A. Tetrahedron Lett.
1997, 38, 4043.
(21) Takai, K.; Nitta, K.; Utimoto, K. J . Am. Chem. Soc. 1986, 108,
7408.
(22) Crotylboron tartrates were also considered: Roush, W. R.; Ando,
K.; Powers, D. B.; Palkowitz, A. D.; Halterman, R. L. J . Am. Chem.
Soc. 1990, 112, 6339 and references therein. Aldol routes include:
Fleming, I. In Comprehensive Organic Synthesis, Vol. 2; Heathcock,
C. H., Ed.; Pergamon Press: Oxford, 1991; p 563. Also see: Myers, A.
G.; Widdowson, K. L J . Am. Chem. Soc. 1990, 112, 9672. Walker, M.
A.; Heathcock, C. H. J . Org. Chem. 1991, 56, 5747. Van Draanen, N.
A.; Arseniyadis, S.; Crimmins, M. T.; Heathcock, C. H. J . Org. Chem.
1991, 56, 2499. Braun, M.; Sacha, H.; Angew. Chem., Int. Ed. Engl.
1991, 30, 1318. Gennari, C.; Hewkin, C. T.; Molinari, F.; Bernardi, A.;
Comotti, A.; Goodman, J . M.; Paterson, I. J . Org. Chem. 1992, 57, 5173.
Gennari, C.; Moresca, D.; Vieth, S.; Vulpetti, A. Angew. Chem., Int.
Ed. Engl. 1993, 32, 1618. Oppolzer, W.; Lienard, P. Tetrahedron Lett.
1993, 34, 4321. Paterson, I.; Wallace, D. J .; Velazquez, S. M. Tetra-
hedron Lett. 1994, 35, 9083. Wang. Y.-C.; Hung, A.-W.; Chang, C. -S.;
Yan, T.-H.; J . Org. Chem. 1996, 61, 2038. Ghosh, A. K.; Ohnishi, M.
J . Am. Chem. Soc. 1996, 118, 2527. Ghosh, A. K.; Fidanze, S.; Onishi,
M.; Hussain, K. A. Tetrahedron Lett. 1997, 38, 7171. Li, Z.; Wu, R.;
Michalczyk, R.; Dunlap, R. B.; Odom, J . D.; Silks, L. A. III J . Am. Chem.
Soc. 2000, 122, 386.
(16) Aldehyde 7a was commercially available. 7b and 7c were
prepared via PCC oxidation of 3-phenylpropanol and 3-cyclohexylpro-
panol, respectively. 7d and 7e originated from 1-adamantylaldehyde
and 2-naphthaldehyde. Both of these aldehydes were treated with
triethylphosphonoacetate to form the E-R,â unsaturated esters. Sub-
sequent hydrogenation (Pd/C) followed by DIBAL reduction afforded
aldehydes 7d and 7e. 7f was made from 3,4-dimethoxypropanoic acid.
Methylation (MeI, K2CO3) followed by DIBAL reduction afforded the
desired aldehyde.
(23) (a) Danda, H.; Hansen, M. M.; Heathcock, C. H. J . Org. Chem.
1990, 55, 172 (b)Walker, M. A.; Heathcock, C. H. J . Org. Chem. 1991,
56, 4747.
(24) Evans, D. A.; Bartroli, J .; Shih, T. L. J . Am. Chem. Soc. 1981,
103, 2127.
(25) (a) Abiko, A.; Liu, J .-F.; Masamune, S. J . Am. Chem. Soc. 1997,
119, 2586 (b) Yoshimitsu, T.; Song, J . J .; Wang, G. Q.; Masamune, S.
J . Org. Chem. 1997, 62, 8968
(26) Aldehydes 11a and 11b gave similar diastereoselectivities
(10-13:1) for the anti-aldol product as determined by 1H NMR and
HPLC analysis. No syn-aldol products were observed. The diastereo-
merics were readily separated by flash chromatography.
(17) Alternatively, aldehydes 7a -f were converted to the unsatur-
ated esters, reduced to the allyl alcohol, and oxidized to the enals 8a -f
with TPAP. Griffith, W. P.; Ley, S. V. Aldrichim. Acta 1990, 23, 13.

4976 J . Org. Chem., Vol. 65, No. 16, 2000
Andrus et al.
Ta ble 1. 6 × 7 In d exed Libr a r y Syn th esis a n d MDR
Sch em e 2
Activity
yielda
(%)
ED50b
(µM)
R
R′
STDc
left side constant
right side constant
a
b
c
d
e
f
a-f
a-f
a-f
a-f
a-f
a-f
a-f
g-m
90
100
100
100
89
100
99
64
90
71
60
87
6.6
3.5
9.7
16
1.3
9.4
23
0.9
0.3
1.5
5
0.1
1.2
7
g-m
g-m
g-m
g-m
g-m
g
h
i
j
k
l
12
2
Sch em e 3
>70
2.3
1.6
0.9
1.8
0.9
0.1
0.1
0.2
m
42
a
Following silica gel filtration, TLC, NMR, MS, and HPLC
b
identified individual compounds. MCF7-adrR cells and 37 nM
adriamycin. c Standard deviation.
1:1.4 stoichiometry with 0.2 equiv of each acetylene
present in the mixture. This new set of conditions for this
reaction proved critical to the success of the library.7b
High yields were only obtained using ethyl acetate as
solvent and starting the reaction at -20 °C. Use of
benzene or THF as solvents resulted in low yields. The
scale (∼100 mg) allowed for multiple MDR assays (1-2
mg/run) and for subsequent isolation and testing of
selected individual compounds. The yields, following
filtration through silica gel to remove impurities, were
quantitative in many cases. The pools were purified using
radial chromatography with aggregate collection of the
UV-active enynes discarding higher and lower moving
impurities. Yields for the mixtures ranged from 42 to
100% and were based on an average molecular weight
for the set of compounds within the respective pools.
Individual compounds, in both high and lower yielding
reactions, were shown by TLC, NMR, and HPLC to be
present in approximately equal amounts in the pools.
Uniform coupling rates were essential to ensure that each
compound would have an effect in the assay. The reac-
tions resulted in six pools, constituting the left-hand
dimension of the library, where (R) was homogeneous
within each pool. Conversely, seven pools homogeneous
for the amide end group were made by reacting each of
the individual acetylenes 6g-m with a mixture of the
six vinyliodides to generate the right-hand dimension of
the library. In total, 13 couplings resulted in the forma-
tion of 42 compounds indexed in two dimensions. It is
important to note that each individual compound appears
in two distinct pools and is never found with another
compound twice.
often lead to removal of the TBS ether. Gratifyingly, use
of LiBH4 in THF at 90 °C for 9 h gave the desired product
in 77% yield along with the recovered auxiliary (92%).
The stereocenters were maintained in both the product
and recovered auxiliary as determined by 1NMR and
optical rotation analysis. Oxidation to the aldehyde 14
and ensuing Takai reaction and removal of the TBS
group afforded the desired iodide 5e. Although applica-
tion of the improved synthetic route shown concerns the
naphthyl vinyl iodide, the other aldehydes, 11a -d ,f work
equally well.
The synthesis of the alkynyl amides started with R,â-
unsaturated acid 15 derived from 4-pentyn-1-ol (Scheme
3). Coupling separately to the seven amines using PyBrop
afforded 6g-m .27 Primary and secondary amines varying
in ring structure as well as degree of polarity were
selected. Lipophilic moieties such as the dibenzyl 6h and
ethyl-stearate 6i were expected to partition into the lipid-
bilayer region adjacent to Pgp. Other more polar amides
were anticipated to bind more tightly to Pgp. The widely
varying yields (63-100%) with the various amines and
rates confirmed the decision not to use this reaction as
the key step for the library.
Iodides 5a -f were coupled with the acetylenes 6g-m
to give enynes 4 in an indexed manner using the
optimized Sonogashira coupling conditions (Table 1).28
This reaction proved ideal with short reaction times and
tolerance of the wide array of unprotected functionality
on the two ends. Each of the six left-hand iodides 5a -f
were reacted individually with a mixture of the seven
acetylenes 6g-m with (Ph3P)2PdCl2 (5 mol %), CuI (17
mol %) at low temperature (-20 °C) in ethyl acetate at
Characterization of the library mixtures for content
1
and purity included TLC, FAB-MS, HPLC, and H and
13C NMR. Not all tools gave full characterization for all
the library mixtures. Reverse phase HPLC worked nicely
to resolve all six compounds for the morpholino pool 4a -
f,j.29 This trace indicated that the desired enynes were
present at approximately 97% purity. Aside from the
naphthyl peak due to a greater response factor, the
(27) Coste, J .; Ferot, E.; J ouin, P.; Castro, B. Tetrahedron Lett. 1991,
32, 1967. The amine for 6i was obtained by EDCI coupling of
ethanolamine to stearic acid (86%).
(28) See ref 7b and: Sonogashira, K.; Tohda, Y.; Hagihara, N.
Tetrahedron Lett. 1975, 4467.
(29) See the Supporting Information.

Combinatorial Library of Non-Natural Polyenes
J . Org. Chem., Vol. 65, No. 16, 2000 4977
Ta ble 2. MDR Activity of In d ivid u a l Isola ted
Com p ou n d s 4
a
R(left)
b, Ph
R′(right)
l, ala-OH
m , bis-EtOH
g, adamantyl
h , Bn2
ED50 (µM)
STDb
1.85
8.8
12
0.04
0.1
2
b
c, c-hexyl
c
12
3
c
l, ala-OH
3.1
0.6
d , adamantyl
d
d
h , Bn2
i, ethyl stearate
l, ala-OH
>70
>70
2.6
0.5
1.1
11
0.02
0.08
0.05
0.02
1
F igu r e 1. Selected aromatic portion of H NMR (300 MHz)
e, 2-naphthyl
h , Bn2
6.6
46
1.69
1.45
1.73
1.48
of morpholino pool 4a -f,j shows compounds present in equi-
molar ratio as seen by integration of the ortho hydrogens.
e
e
e
e
e
i, ethyl stearate
j, morpholino
k , phe-OH
l, ala-OH
m , bis-EtOH
a
MCF7-adrR cells and 37 nM adriamycin. b Standard deviation.
dimensions of a 3D graph (Table 1 and Figure 3). As
illustrated, pools on the R dimension, blue bars, having
a naphthyl (4e) or phenyl (4b) group in common dem-
onstrated the most potent MDR reversal (3.5 and 1.3 µM).
In contrast, mixture having adamantyl (4d ) group held
constant showed the least potent ED50 value (16 µM).
Along the R′ axis, colored bars to the rear, pools with
hydroxylamine (4k -m ) groups along with the morpholine
(4j) group proved to be the most effective modulators
(2.3-0.9 µM). The nonpotent mixtures of the series
included those containing the adamantyl (4g) and ethyl
stearate (4i) groups at 23 and >70 µM, respectively.
From the ED50 values obtained from the pools, one can
then predict which individual compounds from the library
are most potent. The individual compound most likely
to be the most potent is the enyne 4e,l (naphthyl-
alaninol), which has the most potent groups in both
dimensions. Similarly, the most nonpotent of the com-
pounds should be 4d ,i (adamantyl-ethyl stearate). To
validate the viability of the approach, a number of
individual compounds were separated from the library
mixtures and tested individually for reversal efficacy in
the same MCF7adrR assay. Although HPLC was con-
sidered, it was found that radial chromatography was
most convenient for compound isolation. Based on the
observation of the pool potencies, selected compounds
were isolated and evaluated that were predicted to give
a wide range of potencies. The ED50 of each compound
was plotted on the 3D graph at the intersections of the
R and R′ groups which reflect the compound (Table 2,
Figure 3). The interior portion of the graph, colored bars,
now shows the ED50 values for selected individual
compounds.
F igu r e 2. Carbonyl region of the 13C NMR spectrum for the
adamantyl pool 4d ,g-m shows seven compounds present. The
eighth signal is due to the sterate ester in 4d ,i.
remaining compounds, are clearly present in roughly
equimolar quantities. FAB-MS also allowed for identifi-
cation of the compounds present within the library
pools.29 Direct injection of the library mixture and
subsequent high-resolution mass spectrometry (HRMS)
performed on the identified parent ion peaks allowed for
determination of the mass of the enynes 4a -f,j. This
method was particularly advantageous in that it was
applicable for most of the library pools.
Since the mixtures contained a relatively small number
1
of molecules (6 or 7), H and 13C was useful not only for
identifying the compounds present but also for determin-
ing their relative molar ratio. For example, 1H NMR
(6.7-7.8 ppm) of mixture 4a -f,j identified three resolv-
able splitting patterns for the compounds Re-R′j, Rb-
R′j, and Rf-R′j (Figure 1). Integration of the three sets
of peaks revealed a molar ratio of 0.99:1.0:0.97. Again it
was found that the coupling reaction occurred at equal
rates among the various coupling partners. 13C NMR was
most useful when examining the carbonyl region. Using
the same morpholino pool, 4a -f,j, one notices that the
diversity is present at a remote site from the carbonyl.
Thus, only one peak was observed in the carbonyl region
(172.5 ppm). However, in the case of the left-side ada-
mantyl pool 4d ,g-m , the diversity is now adjacent to the
carbonyl region (Figure 2). The resulting spectrum
displayed seven different carbonyl peaks. An additional
carbonyl peak was also observed which was attributed
to the ester present in the stearate compound 4d i.
The 13 pools were assayed for MDR reversal efficacy
using adriamycin resistant MCF7-adrR human breast
cancer cells with 37 nM adriamycin. ED50 MDR reversal
concentrations were determined for the mixtures, the
values were then placed on the respective R and R′
The 4e,k-naphthyl-phenylalaninol compound was found
to be the most potent (1.45 µM), while 4d ,h -adamantyl-
dibenzyl and 4d ,i-adamantyl-ethyl stearate were least
active (>70 µM). The discrepancy for predicting the most
potent group on the R′ dimension can be explained on
the basis that the four most potent pools (4e,j-m ) of the
library have very similar potencies. As shown (Table 1),
within two standard deviations, these four potencies
overlap one another. Thus, the library revealed a number
of potent compounds of similar MDR modulating activity.
This finding is reasonable when considering that several

4978 J . Org. Chem., Vol. 65, No. 16, 2000
Andrus et al.
F igu r e 3. Bargraph of 6 × 7 pool MDR reversal results, R ) a-f left-side axis (blue bars) and R′ ) g-m right-side axis (colored
bars to the rear). Individual isolated compounds are also shown in the off-axis interior region (colored bars).
hydroxy amines were chosen to comprise part of the
right-side library R′ basis set. It is expected that a more
functionally diverse set would lead to larger difference
in potencies between the chosen groups. It is interesting
to note that the most active pool, 4a -f,l (0.9 µM) does
not contain the most active compound 4e,k (1.45 µM).
The alaninol pool is five standard deviations removed
from the phenylalaninol pool. Drug-drug interactions
may be the cause of the discrepancy, especially with
compounds of similar potency and a receptor with
multiple binding sites.
Upon close examination of the 3D ED50 profile, a
distinct division between potent and nonpotent individual
compounds is seen. The left half of the graph containing
R′g-i possesses the less potent enynes while the right half
R′j-m composes the potent region. Most interesting, the
presence of a potent group on either the left or right end
of the molecule can dramatically affect the activity
regardless of the functional group placed at the other
terminal site. For example, 4d -h ,i (adamantyl- dibenzyl,
ethyl stearate) were both found inactive (>70 µM) while
4d ,l (adamantyl-alaninol) gave a potent activity of 2.6
µM. Thus, even though the adamantyl group in the R
dimension is the least potent of the series, the addition
of the alaninol amine to the right-hand part of the
molecule produced a potent modulator. Alternatively,
4e,h (naphthyl- dibenzyl) with a poor group on the right
was moderately active at 6.6 uM while the above-
mentioned 4d ,h (adamantyl-dibenzyl), with both groups
poor, was not. It is interesting to note that from the left-
hand pool results for 4b, 4c, and 4d , it is difficult to
predict the relative high potency of the individual com-
pound with alaninol on the right, 4b,l, 4c,l, and 4d ,l.
Without isolating these compounds, the library results
may have missed these compounds. Yet the primary goals
of the approach of finding and varifying potent com-
pounds have been met, even while some hits may have
been missed. The most potent compound 4e,k was
identified as being present in the most potent left-hand
pool and the phenylalaninol component was shown to be
very similar to the most potent right-hand alaninol pool.
A somewhat surprising result is the potency of the
morpholino pool (2.3 µM) and the individual compound
4e,j. All others that lack a hydrogen bond donor were

Combinatorial Library of Non-Natural Polyenes
J . Org. Chem., Vol. 65, No. 16, 2000 4979
Ta ble 3. Com p a r ison of MDR Rever sa l, ATP a se Activity,
a n d In h ibition of IAAP Bin d in g
from that of the potent modulators. Compounds 4-naph-
thyl-alaninol, and several other potent modulators gave
sharp ATPase(max) peaks while 4-naphthyl, dibenzyl and
4-naphthyl-stearate did not.
It is also interesting to determine what affect might
occur by using the enantiomeric form of the reversal
agent. (11S, 10R)-4-naphthyl-morpholino (18.2 ED50)30
was an order of magnitude less active than (11R, 10S)-
4-naphthyl-morpholino (1.69 ED50) with stereochemistry
that corresponds to the natural product (-)-stipiamide.
The Ki for inhibition of IAAP binding was also weaker
but not to the same degree. This finding is parallel with
that of S-verapamil that was proven to be seven times
more potent than its R-counterpart.31
The approach has allowed for discovery of new potent
MDR modulators in a quick and resourceful manner. It
represents a unique example of a solution phase carbon-
carbon bond forming library targeted to a challenging
membrane bound receptor. The library has provided
valuable information regarding structure activity rela-
tionships of the truncated stipiamide structural variants.
Future libraries and SAR studies involving the template
can now be performed and characterized using the
information gathered herein to further increase potency.
a
b
4
R
R′
ED50
ATPase(max)
Kic
Rb-R′I
Re-R′I
Re-R′j
Re-R′h
Re-R′i
Ph
alaninol
alaninol
morph.
NBn2
1.85
1.73
1.69
6.6
57
57
90
12.0
10.5
2.68
28.3
83.1
Nap
Nap
Nap
Nap
stearate
46
a
MDR reversal concentration (µM), adriamycin present at 37
nm. Maximum stimulation of ATPasa (nmol Pi/min/mg protein).
Values are not normalized for Pgp concentration in membrane.
c Ki (µM) for the competition of 125IAAP binding to Pgp by the test
compound.
b
far less potent. While the approach does not unambigu-
ously identify the least potent compound, a wide range
of compounds, 4d (adamantyl) and 4h (dibenzyl) in
particular, can be safely disregarded as being nonpotent.
If the end groups were truly independent from each
other, we would expect to see a close correlation between
the potencies of the pools and the individual compounds.
As discussed, this seems to be the case. However, there
are some notable exceptions. For example, 4d ,h (ada-
mantyl, dibenzyl) is inactive (>70 µM) while the pools
corresponding to both of the subgroups Rd-R′g-m and
Ra-f-R′h gave activities of 23 and 12 µM, respectively.
The addition of two nonpolar groups to the enyne
dramatically lowered reversal ability. Synergistic activi-
ties attributed by the two ends are also seen in the
alaninol amides 4d ,l (2.6 µM) and naphthyl-stearate 4e,i
(46 µM). The more potent alaninol on the right can
override the poor binding of the left-side adamantyl group
pool (16 µM) and the naphthyl group can overcome to
some extent the poor stearate amide activity (>70 µM).
These results indicate that both ends of the polyene
reversal agent are involved in Pgp interaction. The
results indicate that polar endgroups on the right end of
the template combined with aromatic left end groups
leads to enhanced potency. Conversely, highly nonpolar
species on either end will reduce potency.
Exp er im en ta l Section
Gen er a l Meth od s. Air-sensitive reactions were performed
under a positive pressure of nitrogen. Air and moisture-
sensitive reagents were introduced by syringe or cannula
through rubber septa. All reaction solvents were of HPLC
grade quality or distilled prior to use from an appropriate
drying agent. Methylene chloride was distilled from CaH2.
THF and diethyl ether were distilled from sodium benzo-
phenone ketyl. Starting materials and reagents were pur-
chased from Aldrich, Sigma, or Novabiochem and used without
further purification. [125I]-Iodoarylazidoprazosin (2200 Ci/mmol)
was obtained from NEN. Purification by flash chromatography
was carried out in the indicated solvent system using 70-230
mesh silica gel. Purification by radial chromatography was
performed using 1, 2, and 4 mm plates loaded with 230-400
mesh PF-254 gypsum-bound silica. When filtering through a
silica plug, a buchner funnel was loaded with silica gel
(diameter x height) topped off with an Ottawa sand layer. TLC
analysis was conducted on silica gel 60 F254, 0.25 mm precoated
1
glass plates. All H NMR spectra were obtained using either
a 200, 300, or 500 MHz spectrometer employing chloroform
(7.26 ppm) or TMS (0.0 ppm) as an internal reference. Signals
are reported as: m (multiplet), s (singlet), d (doublet), t
(triplet), q (quartet), bs (broad singlet), bd (broad doublet), bt
(broad triplet), ABq (AB quartet); coupling constants (J ) are
reported in hertz (Hz). Carbon spectra were obtained at 50,
75, or 125 MHz and referenced against deuterated CDCl3.
Infrared spectra were obtained using a Varian FTIR spec-
trometer. Mass spectra were run by the Brigham Young mass
spectrometry facility. Generally, FAB or CI analysis was
performed. Optical rotations were obtained using a polarimeter
at rt employing the sodium D line. Concentrations are reported
in g/100 mL. Combustion analysis was run by M-H-W Labo-
ratories (Pheonix, AZ). Bicinchoninic acid MDR cytotoxicity
assays were performed by the Purdue Cancer Research Center
employing MCF-7-adrR human breast cancer cells.
Compounds isolated from the library were tested for
their ability to stimulate ATP hydrolysis10 as well as
displace 125I[IAAP], a photoaffinity analogue of prazosin,
a substrate of Pgp (Table 3).10 The new naphthyl com-
pounds 4-naphthyl-alaninol and 4-naphthyl-morpholino
had MDR reversal, ATPase stimulation and Ki values
which were similar or better than that for truncated-DHS
3, the original compound (4b,l). In particular, 4-naphthyl-
morpholino displayed significantly higher ATPase activ-
ity above any tested (90 nmolesPi/min/mg protein) and
a Ki for inhibition of IAAP binding at a low level of 2.7
µM. In contrast, the dibenzyl or stearate ester left end
groups had poor binding characteristics (Ki > 28 µM). The
comparison between MDR reversal efficacy and Ki bind-
ing show a good linear correlation. When compounds of
the naphthyl series were compared (R ) naphthyl, R′ )
varied), the correlation coefficient was 0.96.29 The cor-
relation dropped when all isolated compounds from the
(2E)-2-Meth yl-5-p h en yl-2-p en ten a l. To a stirring solution
of 3-phenylpropanal (4.3 g, 32 mmol) in toluene (160 mL, 0.2M)
was added 2-(triphenylphosphoranylidene)propionaldehyde
(13.7 g, 43 mmol). The solution was then heated at 90 °C for
24 h. After cooling, the toluene solvent was removed in vacuo
library had their ED50 and Ki values correlated (r2
0.76). Both 4-naphthyl, dibenzyl and 4-naphthyl-stearate
displayed distinct ATPase profiles which were different
)
(30) ent-4ej was produced in analogous fashion using the enantio-
meric form of the norephedrine ester. See supplementary data.
(31) Wilson, W. H.; J amis-Dow, C.; Bryant G. J . Clin. Oncol. 1995,
13, 1985 (b) Kayes, S. B. Br. Cancer 1993, 67, 641.

4980 J . Org. Chem., Vol. 65, No. 16, 2000
Andrus et al.
and the remaining oil was triturated with cold hexanes causing
a large fraction of the insoluble triphenylphosphine oxide to
precipitate. Following filtration (rinsing with hexanes), the
eluent was concentrated to give an oil. Purification was
performed using flash chromatography eluting with 250 mL
of 5, 10, and 15% EtOAc/hexanes. The product containing
fractions were concentrated to give 3.97g (71% yield) of the
title compound. 1H NMR shows >20:1 E/Z selectivity: Rf )
0.45 in 10% EtOAc/hexanes; 1H NMR (500 MHz, CDCl3) δ 7.38
- 7.16 (m, 5 H), 6.51 (t, J ) 7.3 Hz, 1 H), 2.81 (d, J ) 7.5 Hz,
2 H), 2.69 (t, J ) 7.5 Hz, 2 H), 1.68 (s, 3H); 13C (75 MHz, CDCl3)
δ 195.5, 153.5, 140.8, 128.8, 128.7, 128.5, 126.5, 34.6, 30.9, 9.4;
HRMS CI (M + H) calcd for C12H15O 175.1112, found 175.1101.
(3R,4R,5E)-4-Hyd r oxy-8-p h en yl-3,5-d im eth yl-1,5-octa -
d ien e 9b. To a stirring -78 °C solution of potassium tert-
butoxide (5.8 g, 51.7 mmol) in 150 mL of THF was added via
cannula trans-2-butene (10 mL, ∼91 mmol, condensed into a
graduated column at -78 °C). To the solution was added via
syringe n-butyllithium (2.5 M, 20.7 mL, 51.7 mmol) as a
hexane solution. The mixture was allowed to stir for 15 min
and (-)-Ipc2BOMe (diisopinylcampheylmethoxyborane derived
from (-)-R-pinene) was slowly added by syringe (2 M, 25.9 mL,
51.7 mmol) as a solution in ethyl ether. The solution was
stirred for 30 min and borontrifluoride etherate was slowly
added (8.6 mL, 70.0 mmol) by syringe. The mixture was stirred
for 5 min followed by the addition of (2E)-2-methyl-5-phenyl-
2-pentenal (4.24 g, 24.3 mmol) as an ether solution (5 mL) with
two 1 mL ether washings. After 15 h at -78 °C, 50 mL of 3 N
NaOH was added followed by 20 mL of 30% H2O2. The biphasic
mixture was then allowed to warm to rt and stir for 3 h. The
mixture was diluted with ether (15 mL) and washed with
brine. The separated aqueous layer was then washed twice
with methylene chloride (2 × 30 mL) and the combined organic
layers were dried over MgSO4. The solution was then concen-
trated and flash chromatographed eluting with 250 mL of 7,
9, 11, and 15% EtOAc/hexanes. The product containing frac-
tions were concentrated to give 4.4 g (79% yield) of the title
product as a clear oil: 1H NMR showed the product to be an
approximate 7:1 inseparable mixture of diastereomers: [R]D
at rt. The reaction was allowed to proceed for 24 h giving
predominantly terminal alkene dihydroxylation (Rf ) 0.41 in
35% EtOAc/hexanes). Occasionally, the purchased AD mix
reagent contained variable reactivity. It was often necessary
to add an additional portion of AD-mix-â (390 mg, 1.5 g/mmol)
following the initial 24 h. After another 24 h period of stirring,
the reaction mixture was then passed through a silica gel plug
(4 × 3 cm). The material was rinsed through with ether and
concentrated. Flash chromatography (10% EtOAc/hexanes)
afforded a small amount of the starting diene. Flash chroma-
tography with 35% EtOAc/hexanes gave 0.085 mg (86% yield)
of the desired diol product 10b.
The crude diol (2.14 g, 5.6 mmol) was dissolved in a mixture
of THF (11 mL) and water (11 mL). To this solution was added
sodium periodate (1.8 g, 8.5 mmol) and the mixture was
allowed to stir for 30 min giving aldehyde (Rf ) 0.38 in 10%
EtOAc/hexanes) exclusively. The reaction mixture was then
passed through a silica plug (8 × 4 cm). The material was
rinsed through with ether and concentrated giving 1.95 g of
crude aldehyde (one spot by TLC).
CrCl2 (660 mg, 5.1 mmol) was added to a 25 mL round
bottom flask and gently flame dried under high vacuum. Upon
cooling, the flask was released under nitrogen and charged
with 1.5 mL of THF and allowed to stir for 10 min at rt. The
slurry was cooled to 0 °C and allowed to stir for 10 min. To
another flask containing the crude aldehyde (252 mg, 0.73
mmol) was added 1 mL of THF followed by iodoform (660 mg,
1.68 mmol). This solution was then added dropwise by syringe
to the CrCl2 slurry which turned a brownish-red color. The
reaction was allowed to proceed for 4 h at 0 °C after which,
was poured in ice water (10 mL). The solution was extracted
with ether (3×), dried (MgSO4) and concentrated. Purification
via radial chromatography on a 1 mm plate using 2% EtOAc/
hexanes gave 194 mg (57%) of product (a clear oil) as an
mixture of diastereomers (E/Z, 20:1 by 200 MHz 1H NMR):
Rf ) 0.56 in 35% EtOAc/hexanes; 1H NMR (200 MHz, CDCl3)
δ 7.34-7.14 (m, 2 H), 6.47 (dd, J ) 14.8, 8.0 Hz, 1 H), 5.94 (d,
J ) 15.7 Hz, 1 H), 5.32 (t, J ) 14.4 Hz, 1 H), 3.61 (d, J ) 8.9
Hz, 1 H), 2.73-2.62 (m, 2 H), 2.44-2.19 (m, 3 H), 1.53 (s, 3
H), 0.87 (s, 9 H), 0.77 (d, J ) 7.4 Hz, 3 H), 0.02 (s, 3 H), -0.07
(s, 3 H); 13C NMR (50 MHz, CDCl3) δ 150.4, 142.4, 136.8, 128.9,
128.8, 127.5, 126.3, 82.8, 75.0, 45.5, 36.1, 29.8, 26.3, 19.3, 18.6,
11.6, -4.1, -4.4. Anal. Calcd for C22H35IOSi: C, 56.16; H, 7.50.
Found: C, 52.49; H, 7.49.
(1E,3R,4R,5E)-4-Hydr oxy-1-iodo-8-ph en yl-3,5-dim eth yl-
1,5-octa d ien e 5b. To a flask containing neat (1E,3R,4R,5E)-
4-tert-butylsilyloxy-1-iodo-8-phenyl-3,5-dimethyl-1,5-octadi-
ene (310 mg, 0.66 mmol) was added a THF solution of
tetrabutylammonium fluoride (5 mL, 9.9 mmol, 2 M) at 0 °C.
The reaction was allowed to warm to rt and to stir for 6 h.
Filtration of the reaction mixture through a plug of silica gel
(4 × 3 cm) with ether followed by concentration gave the crude
product. At this point the minor diastereomers from the
olefination and the crotyl boration reactions were separated
using radial chromatography (3-5% EtOAc/hexanes). Isolation
of the major diastereomer gave 172 mg (73%) of product. The
product was 70% enantiopure as determined by 1H NMR
analysis of the derivatized (R)-(+)-Mosher ester: [R]D +1.5 (c
1.22, CHCl3); Rf ) 0.20 in 10% EtOAc/hexanes; 1H NMR (200
MHz, CDCl3) δ 7.37-7.16 (m, 5 H), 6.52 (dd, J ) 15.0, 8.0 Hz,
1 H), 6.13 (d, J ) 15.0 Hz, 1 H), 5.43 (t, J ) 7.3 Hz, 1 H), 3.69
(d, J ) 8.5 Hz, 1 H), 2.77-2.64 (m, 2 H), 2.47-2.28 (m, 3 H),
1.67 (s, 1 H), 1.06 (s, 3 H), 0.95 (d, J ) 7.7 Hz, 3 H); 13C NMR
(50 MHz, CDCl3) δ 149.2, 142.3, 136.0, 128.9, 128.8, 128.6,
126.4, 81.5, 76.4, 44.9, 36.1, 29.9, 16.7, 11.6; HRMS (CI) calcd
for M + H - H2O for C16H21IO 339.0610, found 339.0596.
Nor ep h ed r in e Ester Ba sed Rou te to Vin yl Iod id e 9e.
(1′S,2′R,2S,3R,4E)-2′-(N-Ben zyl-N-m esit ylen esu lfon yl)-
a m in o-1′-p h en yl-1′-p r op yl-3-h yd r oxy-2,4-d im eth yl-7-(2′′-
n a p h th yl)-4-h ep ten oa te. To a stirred solution of (1S,2R)-2-
(N-benzyl-N-mesitylenesulfonyl)amino-1-phenyl-1-propyl pro-
pionate (0.111g, 0.23 mmol) in 2 mL of CH2Cl2 was added
triethylamine (0.08 mL, 0.56 mmol). The reaction flask was
then cooled to -78 °C. A solution of 1.0 M dicyclohexylboron-
triflate (0.7 mL, 0.70 mmol) in 2 mL CH2Cl2, precooled to -78
1
+21.2 (c 2.28, CHCl3); Rf ) 0.43 in 20% EtOAc/hexanes; H
NMR (200 MHz, CDCl3) δ 7.36-7.15 (m, 5 H), 5.85-5.64 (m,
1 H), 5.45 (t, J ) 7.0 Hz, 1 H), 5.24-5.07 (m, 2 H), 3.65 (d, J
) 8.9 Hz, 1 H), 2.77-2.64 (m, 2 H), 2.48 - 2.23 (m, 3 H), 1.82
(s, 1 H), 1.60 (s, 3 H), 0.85 (d, J ) 7.4 Hz, 3 H); 13C NMR (50
MHz, CDCl3) δ 142.5, 141.8, 136.1, 128.9, 128.8, 128.5, 126.3,
116.8, 81.8, 42.6, 36.2, 30.0, 17.2, 11.5. Anal. Calcd for C16H22O;
C, 83.40; H, 9.62. Found: C, 83.04, H, 9.92.
(3R,4R,5E)-4-Dim eth yl-ter t-bu tylsilyloxy-8-p h en yl-3,5-
d im eth yl-1,5-octa d ien e. To a stirring solution of (3S,4R,5E)-
4-hydroxyl-8-phenyl-3,5-dimethyl-1,5-octadiene 9b (4.05 g, 17.6
mmol) in DMF (0.6 M, 30 mL) was added imidazole (3.6 g,
52.8 mmol) and tert-butyldimethylsilylchloride (5.3 g, 35.2
mmol) at rt. The reaction was stirred overnight, diluted with
50 mL of methylene chloride, and extracted with water (50
mL). The separated water layer was washed with methylene
chloride and the organic layers were combined. The solution
was dried, concentrated and flash chromatographed eluting
with 250 mL of 1, 2, 5% EtOAc/hexanes. Product containing
fractions were collected and concentrated to give 5.02 g (83%)
of a clear oil as an inseparable mixture of diastereomers (7:1
1
determined by H-formed during the previous reaction): Rf )
0.68 in 5% EtOAc/hexanes; 1H NMR (200 MHz, CDCl3) δ 7.38-
7.17 (m, 5 H), 5.99-5.81 (m, 1 H), 5.36 (t, J ) 7.5 Hz, 1 H),
5.13-4.97 (m, 2 H), 3.68 (d, J ) 8.7, 1 H), 2.77-2.66 (m, 2 H),
2.48-2.24 (m, 3 H), 1.60 (s, 3 H), 0.93 (s, 9 H), 0.84 (d, J ) 7.9
Hz, 3 H), 0.06 (s, 3 H), -0.01 (s, 3 H); 13C NMR (50 MHz,
CDCl3) δ 142.8, 137.6, 128.9, 128.8, 126.8, 126.3, 114.1, 83.6,
43.7, 42.6, 36.2, 29.8, 26.4, 18.9, 17.0, 11.8, -4.0, -4.5. Anal.
Calcd for C22H36OSi; C, 76.68; H, 10.53. Found: C, 76.32; H,
10.88.
(1E,3R,4R,5E)-4-ter t-Bu tylsilyloxy-1-iod o-8-p h en yl-3,5-
d im eth yl-1,5-octa d ien e. To a stirring solution of (3R,4R,5E)-
4-dimethyl-tert-butylsilyloxy-8-phenyl-3,5-dimethyl-1,5-octa-
diene (90 mg g, 0.26 mmol) in a mixture of tert-butanol (1 mL)
and water (1 mL) was added AD-mix-â (390 mg, 1.5 g/mmol)

Combinatorial Library of Non-Natural Polyenes
J . Org. Chem., Vol. 65, No. 16, 2000 4981
°C, was subsequently cannulated over to the reaction flask and
stirring continued for two and a half h at -78 °C. (2E)-2-
Methyl-5-(2′-naphthyl)-2-pentenal (0.065 g, 0.29 mmol) in 0.5
mL of CH2Cl2 was added dropwise to the reaction flask (with
0.5 mL CH2Cl2 rinse) and the reaction continued to stir for an
additional h at the same temperature. The mixture was
warmed to 0 °C and stirred for one h. The reaction was then
charged with pH 7 buffer solution (2 mL), MeOH (4 mL), and
30% H2O2 (0.5 mL). The mixture stirred vigorously overnight
at rt and was worked up with CH2Cl2 (3 extractions), dried
(MgSO4), and concentrated to give the crude oil. Purification
of the desired product with its accompanying minor diastereo-
mer was accomplished using radial chromatography (2 mm
plate, 7% EtOAc/hexanes) affording 0.175g of material. The
ratio of the two anti diastereomers (2′S, 3′R) and (2′R, 3′S)
was determined to be 14:1 using HPLC analysis. No syn
diastereomers were observed. Additional chromatography
allowed for separation and characterization of the two dia-
stereomers. Major isomer: [R]D -31.2 (c 2.25, CHCl3); Rf )
0.29 (20% EtOAc/hexanes); 1H NMR (300 MHz, CDCl3) 7.79-
7.72 (m, 3 H), 7.58 (s, 1 H), 7.45-7.12 (m, 11 H), 6.88 (s, 2 H),
7.82-6.79 (m, 2 H), 5.80 (d, J ) 3.6 Hz, 1 H), 5.46 (t, J ) 6.9
Hz, 1 H), 4.81 (A of ABq, J AB ) 16.8 Hz, 1 H), 4.60 (B of ABq,
J AB ) 16.8 Hz), 4.06-4.02 (m, 2 H), 2.85-2.67 (m, 2 H), 2.59-
2.36 (m, 4 H), 2.50 (s, 6 H), 2.27 (s, 3 H), 1.56 (s, 3 H), 1.12 (d,
J ) 7.5 Hz, 3 H), 0.80 (d, J ) 7.2 Hz, 3 H); 13C NMR (75.4
MHz, CDCl3) δ 174.9, 172.7, 140.4, 139.4, 139.1, 138.5, 134.8,
133.7, 133.7, 132.3, 132.1, 129.4, 128.5, 128.4, 128.0, 127.9,
127.8, 127.5, 127.4, 127.2, 126.6, 126.1, 125.9, 125.3, 80.3, 78.3,
57.0, 48.4, 43.4, 35.8, 29.5, 23.1, 21.0, 14.3, 14.2, 13.4, 10.7;
HRMS FAB (M + Na) calcd for C44H49O5NSNa 726.3223, found
726.3217.
(1′S,2′R,2S,3R,4E)-2′-(N-Ben zyl-N-m esitylen esu lfon yl)-
a m in o-1′-p h en yl-1′-p r op yl 3-ter t-bu tyld im eth ylsilyloxy-
2,4-dim eth yl-7-(2′′-n aph th yl)-4-h epten oate 11. 0.175 g (0.23
mmol assuming 100% theoretical yield) of the crude diastereo-
meric mixture isolated from the previous reaction was dis-
solved in 2 mL CH2Cl2 and cooled to 0 °C. This solution was
charged with 2,6-lutidine (0.12 mL, 1.0 mmol) and dropwise
addition of TBSOTf (0.17 mL, 0.74 mmol). The reaction was
complete after 20 min of stirring at this temperature. The
solution was diluted with brine and extracted with CH2Cl2
(3×), dried (MgSO4), filtered, and concentrated. Purification
by radial chromatography (3% EtOAc/hexanes) gave a white
solid (mp 63 °C) 0.146 g (77% yield for two steps overall): [R]D
-33.8 (c 1.85, CHCl3); Rf ) 0.63 (20% EtOAc/hexanes); 1H
NMR (300 MHz, CDCl3) 7.82-7.73 (m, 3 H), 7.59 (s, 1 H),
7.47-7.06 (m, 11 H), 6.90 (s, 2 H), 6.67 (d, J ) 7.2 Hz, 2 H),
5.66 (d, J ) 5.7 Hz, 1 H), 5.40 (t, J ) 6.3 Hz, 1 H), 4.95 (A of
ABq, J AB ) 15.9 Hz, 1 H), 4.41 (B of ABq, J AB ) 15.9 Hz), 4.09
(d, J ) 9.6 Hz, 1 H), 4.02-3.97 (m, 1 H), 2.85-2.78 (m, 2 H),
2.64-2.58 (m, 1 H), 2.48-2.38 (m, 1 H), 2.43 (s, 6 H), 2.33 (s,
3 H), 1.57 (s, 3 H), 1.14 (d, J ) 6.9 Hz, 3 H), 0.80 (s, 9 H), 0.66
(d, J ) 7.2 Hz, 3 H), -0.05 (d, J ) 9.9 Hz, 6 H); 13C NMR
(75.4 MHz, CDCl3) δ 174.0, 142.5, 140.6, 139.5, 138.9, 138.5,
135.3, 133.8, 133.3, 132.3, 128.9, 128.7, 128.5, 128.4, 128.1,
127.9, 127.8, 127.6, 127.4, 126.6, 126.5, 126.1, 125.3, 80.9, 77.7,
56.9, 48.5, 44.8, 35.8, 29.5, 26.1, 23.1, 21.1, 18.4, 15.0, 14.5,
10.7, -4.6, -4.7; HRMS FAB (M + Na) calcd for C50H63O5-
NSiSNa 840.4108, found 840.4122. Anal. Calcd for C50H63O5-
NSiS: C, 73.40; H, 7.76. Found: C, 73.14; H, 7.65.
7.83-7.76 (m, 3 H), 7.63 (s, 1 H), 7.48-7.40 (m, 2 H), 7.36 (d,
J ) 8.1 Hz, 1 H), 5.40 (t, J ) 6.9 Hz, 1 H), 3.81 (d, J ) 8.1 Hz,
1 H), 3.59 (d, J ) 5.7 Hz, 2 H), 2.85 (dd, J ) 7.5, 5.1 Hz, 2 H),
2.51-2.43 (m, 2 H), 1.89-1.75 (m, 1 H), 1.58 (s, 1 H), 0.89 (s,
9 H), 0.70 (d, J ) 7.2 Hz, 3 H), 0.06 (s, 3 H), -0.04 (s, 3 H);
13C NMR (75.4 MHz, CDCl3) δ 139.6, 136.7, 133.8, 132.2, 128.1,
127.8, 127.6, 127.4, 127.2, 126.6, 126.1, 125.3, 85.2, 67.3, 38.4,
35.8, 29.4, 26.0, 18.2, 14.4, 11.6, -4.2, -5.0; HRMS FAB
(M + Na) calcd for C25H38O2SiNa 421.2535, found 421.2531.
(1E,3R,4R,5E)-4-ter t-Bu tyld im eth ylsiloxy-1-iod o-8-(2′-
n a p h th yl)-3,5-d im eth yl-1,5-octa d ien e. To a stirring solu-
tion of (2R,3R,4E)-3-tert-butyldimethylsiloxy-2,4-dimethyl-7-
(2′′-naphthyl)-4-heptenol (1.142 g, 2.87 mmol) in 20 mL of
methylene chloride was added oven-dried, crushed 4 Å mo-
lecular sieves (1.14g, 400mg/mmol), NMO (0.50g, 4.3 mmol),
and TPAP (0.050g, 0.14 mmol); the latter was added at 0 °C.
The solution was warmed to rt and allowed to stir for 1 h. The
reaction mixture was then passed through a 4 cm diameter
course fritted funnel containing silica gel (2 in). The material
was rinsed through with ether. The filtered solution was
concentrated giving 1.00 g of crude aldehyde (89%, one spot
by TLC).
CrCl2 was added to a 50 mL round bottom flask and gently
flame dried under high vacuum. Upon cooling, the flask was
released under nitrogen and charged with 13 mL of THF. The
slurry was cooled to 0 °C and allowed to stir. To another flask
containing the crude aldehyde (1.00g, 2.5mmol) stirring in 7
mL of THF was added iodoform (2.3 g, 5.9 mmol). This solution
was then cooled to 0 °C and stirred for 10 min. This aldehyde/
iodoform solution was then taken up in a syringe and added
dropwise to the CrCl2 slurry (also at 0 °C) which turned a
brownish-red color. The reaction was allowed to proceed for
3 h at 0 °C and was then poured into 50 mL H2O. The mixture
was extracted with ether (3×), dried, and concentrated. The
residue was purified by flash chromatography using 2% EtOAc/
hexanes to give the desired product as a clear oil (0.99 g, 75%
yield). Upon characterization, only the E-isomer was ob-
served: [R]D -1.0 (c 2.0, CHCl3); Rf ) 0.69 (10% EtOAc/
1
hexanes); H NMR (300 MHz, CDCl3) δ 7.82-7.76 (m, 3 H),
7.63 (s, 1 H), 7.46-7.37 (m, 2 H), 7.36 (d, J ) 8.4 Hz, 1 H),
6.47 (dd, J ) 14.1, 8.1 Hz, 1 H), 5.92 (d, J ) 14.4 Hz, 1 H),
5.35 (t, J ) 6.9 Hz, 1 H), 3.61 (d, J ) 8.4 Hz, 1 H), 2.87-2.80
(m, 2 H), 2.50-2.40 (m, 2 H), 2.32-2.22 (m, 1 H), 1.55 (s, 3
H), 0.87 (s, 9 H), 0.76 (d, J ) 6.9 Hz, 3 H), 0.01 (s, 3 H), -0.08
(s, 3 H); 13C NMR (75.4 MHz, CDCl3) δ 150.1, 139.7, 136.6,
133.8, 132.2, 128.1, 127.8, 127.6, 127.4, 127.1, 126.6, 126.1,
125.3, 82.6, 74.8, 45.2, 35.9, 29.4, 26.0, 18.3, 16.2, 11.3, -4.4,
-4.7; HRMS FAB (M + Na) calcd for C26H37OSiNaI 543.1564,
found 543.1572.
(1E,3S,4R,5E)-4-Hydr oxy-1-iodo-8-(2′-n aph th yl)-3,5-dim -
eth yl-1,5-octa d ien e 5e. To a flask containing (1E,3S,4R,5E)-
4-tert-butylsilyloxy-1-iodo-8-phenyl-3,5-dimethyl-1,5-octadi-
ene (0.230 g, 0.44mmol) 14 was added a 1 M THF solution of
tert-butylammonium fluoride (6.6 mL, 6.6 mmol) at 0 °C. The
reaction was allowed to warm to rt and stirred for 90 min.
Filtration of the reaction mixture through a plug of silica gel
(2 in) with ether was followed by concentration to give a crude
oil. Purification via radial chromatography (5% EtOAc/
hexanes) gave the desired product (0.174 g, 97% yield) which
gave a light yellow solid upon freezing. Full characterization
for this compound was described previously: [R]D +2.4 (c 1.73,
CHCl3). The 5e enantiomer was also prepared (97% enantio-
pure-Mosher ester analysis): [R]D -2.7 (c 1.47, CHCl3).
(2E)-Eth yl 2-Meth yl-2-h epten -6-yn oate.Pyridinium chloro-
chromate (0.971 g, 4.50 mmol) was added at 0 °C to a stirred
solution of 4-pentyn-1-ol (0.252, 3.0 mmol), sodium acetate
(0.737 g, 9.0 mmol), and 4 Å sieves (0.367 g). The mixture was
warmed to rt and allowed to stir for 5 h. The solution was then
filtered through silica gel with ether and carefully concentrated
to give crude 4-pentyn-1-al as a volatile liquid: Rf ) 0.58 (20%
(2R,3R,4E)-3-ter t-Bu tyl-d im eth ylsilyloxy-2,4-d im eth yl-
7-(2′′-n a p h th yl)-4-h ep ten ol. (1′S,2′R,2S,3R,4E)-2′-(N-Benzyl-
N-mesitylenesulfonyl)amino-1′-phenyl-1′-propyl 3-tert-butyl-
dimethylsilyloxy-2,4-dimethyl-7-(2′′-naphthyl)-4-hepten-oate (2.5
g, 3.1 mmol), was dissolved in 22 mL of THF and charged with
solid LiBH4 (0.20 g, 9.2 mmol) at 0 °C. The stirred solution
was removed from the ice bath and heated under reflux for
9 h at 90 °C. The mixture was then cooled and extract with
CH2Cl2 (3×), dried, and concentrated. Purification via radial
chromatography (4-10% EtOAc/hexanes) gave the recovered
hydroxy auxiliary (1S,2R)-2-(N-benzyl-N-mesitylenesulfonyl)-
amino-1-phenyl-1-propanol (0.94 g, 92%), along with the title
compound (0.941 g, 77% yield): [R]D +9.23 (c 2.17, CHCl3);
1
EtOAc/hexanes); H NMR (200 MHz, CDCl3) δ ) 9.8 (s, 1 H,
CHO). The crude aldehyde was dissolved in toluene (15 mL),
and (carbethoxyethylidene) triphenylphosphorane (1.50 g, 4.14
mmol) was added and the reaction mixture was heated to
90 °C for 22 h. The solution was filtered through silica gel
1
Rf ) 0.28 (10% EtOAc/hexanes); H NMR (300 MHz, CDCl3)

4982 J . Org. Chem., Vol. 65, No. 16, 2000
Andrus et al.
(20% Et2O/pentane) and concentrated to give the crude vinyl
ester: 1H NMR showed E/Z selectivity of 15:1; (200 MHz,
CDCl3) δ ) 6.75 (t, J ) 7.1 Hz, E-vinyl-H), 6.0 (t, J ) 8.0 Hz,
Z-vinyl-H). Purification of the crude mixture via radial chro-
matography (5% Et2O/pentane) gave the E vinyl ester as a
clear oil (0.226 g, 54% yield): Rf ) 0.60 (20% EtOAc/hexane);
calcd for C24H39O3NNa 412.2827, found 412.2808; C25H39O3-
NNa 424.2827, found 424.2817; C25H41O4NNa 442.2933, found
442.2918; C30H43O3NNa 488.3140, found 488.3141; C31H47O2-
NNa 488.3505, found 488.3490; C35H45O2NNa 534.3348, found
534.3334; C41H71O4NNa 664.5280, found 664.5276.
(2E,8E,10R,11R,12E)-N-1′-Ad a m a n tyl 11-Hyd r oxy-15-
su bstitu ted Ra-f-2,10,12-tr im eth yl-2,8,12-pen tadecatr ien -
6-yn a m id es 4a -f,g. To a solution of (2E)-N-1′-adamantyl
2-methyl-2-hepten-6-ynamide 6g (67.5 mg, 0.25 mmol) was
added (1E,3R,4R,5E)-4-hydroxy-1-iodo-3,5-dimethyl-1,5-undeca-
diene 5a (9.7 mg, 0.030 mmol), (1E,3R,4R,5E)-4-hydroxy-1-
iodo-8-phenyl-3,5-dimethyl-1,5-octadiene 5b (10.8 mg, 0.030
mmol), (1E,3R,4R,5E)-4-hydroxy-1-iodo-8-cyclohexyl-3,5-dimeth-
yl-1,5-octadiene 5c (10.7 mg, 0.030 mmol), (1E,3R,4R,5E)-4-
hydroxy-1-iodo-8-(1′-adamantyl)-3,5-dimethyl-1,5-octadiene 5d
(12.2 mg, 0.029 mmol), (1E,3R,4R,5E)-4-hydroxy-1-iodo-8-(2′-
naphthyl)-3,5-dimethyl-1,5-octadiene 5e (12.0 mg, 0.030 mmol),
and (1E,3R,4R,5E)-4-hydroxy-1-iodo-8-(3′,4′-dimethoxyphenyl)-
3,5-dimethyl-1,5-octadiene 5f (12.3 mg, 0.030 mmol) all as a
solution in ethyl acetate (12 mL). The mixture was cooled to
-20 °C and (Ph3P)2PdCl2 (6.5 mg, 0.009 mmol), CuI (5.9 mg,
0.030 mmol), and i-Pr2NH (0.9 mL) were sequentially added.
The reaction was immediately removed from the cool bath,
protected from light, and warmed to rt with continued stirring
for 3.5 h. TLC showed disappearance of the alkynyl amide and
three new uν spots appeared which stained intensely using
Ce/Mo staining solution. The brown reaction mixture was
filtered through a plug of silica gel (ca. 3 g) followed by
purification using radial chromatography to collect the uν
bands as a mixture of six compounds (92.3 mg, 99% yield):
Rf ) 0.57, 0.46, 0.46 in 35% EtOAc/hexanes; 13C NMR (125
MHz, CDCl3) δ 168.7; MS (FAB) for (M - H2O)H+ 448, 482,
488, 532, 540, 542.
Bicin ch on in ic Acid (BCA) Assa y for th e Deter m in a -
tion of Ad r ia m ycin Cytotoxicity in th e P r esen ce of MDR
Rever sa l Agen ts. MCF-7 adriamycin-resistant (MCF-7/ADR)
cells were seeded into 96-well microtiter dishes (precoated with
poly-D-lysine) at a concentration of 1000 cells/well (5000 cells/
mL). The cells were then subsequently seeded in 200 µl of
RPMI medium (containing 10% FCSH I and PS) per well and
incubated for 24 h at 37 °C. The MDR reversal agent (pool or
individual compound) was first dissolved in DMSO to give a
concentration of less than 0.1% DMSO in the well and was
then further diluted with media. Twelve aliquots of the
reversal compound(s) varying in concentration ranging from
0.005 to 50 µg/mL were then added to the resistant cells
followed by incubation at 37 °C for 30 min. At this point,
adriamycin at a concentration of 2 × 10-2 µg/mL, was added
to each well containing reversal agent. After a 6 d incubation
period, the media was removed from the wells followed by
washing of the wells with phosphate-buffered saline (PBS).
Nonidet P-41 1% (10 µl) was added to each well of cells followed
by 200 µl of a premixed bicinchoninic acid (BCA)-4% copper
sulfate (50:1) solution. The 96-well plate was again incubated
at 37 °C for 30 min. whereupon the purple-tinted wells
indicated the presence of cellular protein and viable cells. The
optical density of each well was read at 570 nm using a
microplate reader. The correlation already established by
Chang between cell number and optical density allowed for
determination of the ED50 of adriamycin in the presence of
reversal agent(s). The cell assays were done in quadruplicate
allowing for calculation of the ED50 value (determined by linear
interpolation) and standard deviation.
1
IR (neat) 3300, 2983, 2916, 2119, 1712, 1652 cm-1; H NMR
(200 MHz, CDCl3) δ 6.75 (t, J ) 7.1 Hz, 1 H), 4.15 (q, J ) 2.9
Hz, 2 H), 2.45-2.20 (m, 4 H), 1.96 (t, J ) 2.5Hz, 1 H), 1.83 (s,
3 H), 1.24 (t, J ) 7.1Hz, 3 H); 13C NMR (50 MHz, CDCl3) δ
168.3, 139.8, 129.7, 83.6, 69.5, 60.9, 28.2, 18.2, 14.7, 13.0; MS
(CI) 167 (M + H, 100). Anal. Calcd for C10H14O2: C, 72.26; H,
8.49. Found: C, 71.88; H, 8.83.
(2E)-2-Met h yl-2-h ep t en -6-yn oic Acid 15. (2E)-Ethyl
2-methyl-2-hepten-6-ynoate (5.28 g, 31.8 mmol) was added to
a solution containing THF (110 mL), MeOH (32 mL), and H2O
(32 mL) The mixture was cooled to 0 °C and LiOH (2.29 g,
95.4 mmol) was added followed by stirring at rt for 1 d upon
which TLC showed disappearance of the ester. The solution
was filtered through silica gel (25% EtOAc/MeOH) to give the
yellow acid residue. The acid was then recrystallized in
hexanes/EtOAc giving 3.32 g of a white solid (76% yield): mp
1
46 °C; H NMR (200 MHz, CDCl3) δ 11.1 (bs, 1 H), 6.97 (t, J
) 7.4 Hz, 1 H), 2.51-2.34 (m, 4 H), 2.02 (t, J ) 2.6 Hz, 1 H),
1.90 (s, 3 H); 13C NMR (50 MHz, CDCl3) δ 173.6, 142.4, 128.6,
83.0, 69.1, 27.8, 17.6, 12.1.
(1′S,2E)-(-)-N-(2′-Hyd r oxy-1′-m eth yleth yl) 2-Meth yl-2-
h ep ten -6-yn a m id e 6l. (2E)-Ethyl 2-methyl-2-hepten-6-ynoic
acid 15 (0.30 g, 2.2 mmol) was then dissolved in CH2Cl2 (11
mL). PyBrop (1.2 g, 2.6 mmol) and (S)-(+)-2-amino-1-propanol
(0.20 g, 2.66 mmol) were sequentially added followed by
addition of DIEA (1.2 mL, 6.9 mmol) at 0 °C. The reaction
mixture stirred for 12 h at rt. The solution was filtered through
silica gel (10% MeOH/EtOAc) and purified via radial chroma-
tography (gradient elution, 70% EtOAc/hexanes to 100%
EtOAc) giving a yellow white solid (0.367g, 87%): mp ) 38-
40 °C; Rf ) 0.263 (EtOAc); [R]D -4.08 (c 3.56, EtOAc); IR (neat)
3297, 2974, 2918, 2877, 2117, 1661, 1617, 1530 cm-1; 1H NMR
(200 MHz, CDCL3) δ 6.35 (t, J ) 6.4Hz, 1 H), 6.0 (bd, 1 H),
4.15-4.0 (m, 1 H), 3.71-3.64 (dd, J ) 11.0, 3.75 Hz, 1 H),
3.59-3.50 (dd, J ) 11.0, 6.0 Hz, 1 H), 3.18 (bs, 1 H), 2.41-
2.26 (m, 4 H), 1.97 (t, J ) 2.4 Hz, 1 H), 1.85 (s, 3 H), 1.2 (d,
J ) 6.8 Hz, 3 H); 13C NMR (50 MHz, CDCl3) δ 170.4, 134.4,
132.6, 83.8, 69.6, 67.5, 48.4, 27.9, 18.4, 17.6, 13.4. MS (CI) 196
(M + H, 100). Anal. Calcd for C11H17O2N: C, 67.66; H, 8.78.
Found: C, 67.31; H, 8.92.
P r ep a r a tion of Libr a r y Mixtu r es. (2E,8E,10R,11R,12E)-
N-Su b st it u t ed
2,8,12-oct a d eca t r ien -6-yn a m id es 4a ,g-m . To
R ′g-m -11-h yd r oxy-2,10,12-t r im et h yl-
a
flask
containing (1E,3R,4R,5E)-4-hydroxy-1-iodo-3,5-dimethyl-1,5-un-
decadiene 5a (69.5 mg, 0.22 mmol) was added (2E)-N-1′-
adamantyl 2-methyl-2-hepten-6-ynamide 6g (11.8 mg, 0.044
mmol), (2E)-N,N-dibenzyl 2-methyl-2-hepten-6-ynamide 6h
(13.8 mg, 0.044 mmol), (2E)-N-(2′-stearoyl)ethyl 2-methyl-2-
hepten-6-ynamide 6i (19.2 mg, 0.043 mmol), (2E)-N-4′-mor-
pholinyl 2-methyl-2-hepten-6-ynamide 6j (8.9 mg, 0.043 mmol),
(1S′,2E)-N-(2′-hydroxy-1′-benzyl)ethyl 2-methyl-2-hepten-6-
ynamide 6k (11.6 mg, 0.043 mmol), (1S′,2E)-(-)-N-(2’-hydroxy-
1′-methyl)ethyl 2-methyl-2-hepten-6-ynamide 6l (8.3 mg, 0.043
mmol), and (2E)-N,N-diethanol 2-methyl-2-hepten-6-ynamide
6m (9.9 mg, 0.44 mmol) all as a solution in ethyl acetate (8
mL). The mixture was cooled to -20 °C and (Ph3P)2PdCl2 (7.5
mg, 0.011 mmol), CuI (6.0 mg, 0.031 mmol), and i-Pr2NH (1.1
mL) were sequentially added. The reaction was immediately
removed from the cool bath, protected from light, and allowed
to warm to rt with continued stirring for 2 h. At this point,
TLC showed disappearance of the alkynyl amides and seven
new UV spots appeared which stained intensely using Ce/Mo
staining solution. The brown reaction mixture was filtered
through a plug of silica gel (ca. 3 g) followed by purification
using radial chromatography to collect the uν bands as a
mixture of seven compounds (90.1 mg, 90% yield): Rf ) 0.53,
0.38, 0.21 in 35% EtOAc/hexanes; Rf ) 0.72, 0.59, 0.46, 0.13
in 100% EtOAc; 13C NMR (125 MHz, CDCl3) δ 175.9, 174.4,
174.3, 172.4, 170.02, 169.96, 169.4, 168.8; HRMS (FAB + Na)
Mea su r em en t of ATP a se Activity. ATPase activity of Pgp
was measured by the endpoint, inorganic phosphate (Pi) assay.
Pgp-specific activity was recorded as the vanadate (Vi) (0.3
mM)-sensitive ATPase activity. The compounds being tested
were added from 100× stock solutions in DMSO so that the
DMSO concentration was no greater than 1%; this concentra-
tion of DMSO had no effect on the activity of Pgp. The Pi assay
measured the amount of inorganic phosphate released over
20 min at 37 °C (100 µL assay volume). Pgp was incubated in
ATPase buffer (50 mM MES-Tris, pH 6.8, 50 mM KCl, 5 mM
NaN3, 2 mM EGTA, 1 mM ouabain, 5 mM MgCl2 and 2 mM
DTT) and the compounds being tested were incubated for 5

Combinatorial Library of Non-Natural Polyenes
J . Org. Chem., Vol. 65, No. 16, 2000 4983
min at 37 °C, both in the presence and absence of 0.3 mM
Vanadate. The ATPase reaction was initiated by the addition
of ATP and quenched with SDS (2.5% final concentration); the
amount of inorganic phosphate released was quantitated using
colorimetric method as previously described (Ramachandra,
M.; Ambudkar, S. V.; Gottesman, M. M.; Pastan, I.; Hrycyna,
C. A. Mol. Cell Biol. 1996, 7, 1485-1498).
P h otoa ffin ity La belin g w ith IAAP . Crude plasma mem-
branes of High Five insect cells containing Pgp were used for
this experiment. The crude plasma membrane (10-20 µg
protein) was labeled in 50 mM Tris-HCl, pH 7.4 and was
incubated with the MDR modulator for 3 min at rt in plastic
eppendorf vials. 125IAAP was added in the dark and incubated
at rt for five min. Usually, between 3-6 nM of IAAP was added
per sample. The membranes were then exposed to a UV lamp
(G.E. no. F15T8-BLB) at 365 nm for 10 min at rt. 5× SDS
PAGE sample buffer was added to each of the samples and
these were subsequently incubated at rt for 30 min. Samples
were then run on an 8% Tris-glycine gel at constant voltage.
Gels were dried and exposed to Biomax MR (Kodak) auto-
radiography film.
centration of the MDR modulators (0-250 µM) and incubated
at rt for 3 min. IAAP (3-6 nM) was added to each test tube
and incubated for another 3 min. The membranes were photo-
crosslinked as described in the previous section and run on
an 8% Tris-glycine gel. Gels were then dried and the radio-
activity of IAAP bound Pgp was estimated using the STORM
860 phospholimager system with Image Quant software. The
radioactivity in the Pgp band was obtained as arbitrary units.
The data was fitted to a one phase experimental decay using
GraphPad Prism 2.0 software for Macintosh.
Ack n ow led gm en t. We wish to thank the National
Institutes of Health (GM57275), The American Cancer
Society, and Brigham Young University for funding, Dr.
Bruce J ackson for mass spectrometry, and Vicki Croy
(Purdue U.) for the MDR assays.
Su p p or tin g In for m a tion Ava ila ble: Experimental de-
tails, analytical data, and MDR, ATPase, and displacement
protocols for selected compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
Deter m in a tion of Ki for th e MDR Mod u la tor s. The
crude plasma membranes (20 µg protein in 40 mL of 50 mM
Tris-HCl, pH 7.4) were placed in vials containing varied con-
J O000453M