Diversity-oriented synthesis of pochonins and biological evaluation against a panel of kinases

Article abstract of DOI:10.1002/chem.200600553

Pochonins A-F were recently characterized as six new members of the naturally occurring family of 14membered resorcylic acid lactones. As there are a high number of ATPase and kinase inhibitors among natural resorcylic lactones, a library based on the poc

Full text of DOI:10.1002/chem.200600553

FULL PAPER
DOI: 10.1002/chem.200600553
Diversity-Oriented Synthesis of Pochonins and Biological Evaluation
against a Panelof Kinases
Emilie Moulin,[a] Sofia Barluenga,[a] Frank Totzke,[b] and Nicolas Winssinger*[a]
Abstract: Pochonins A–F were recently
characterized as six new members of
the naturally occurring family of 14-
membered resorcylic acid lactones. As
there are a high number of ATPase
and kinase inhibitors among natural re-
sorcylic lactones, a library based on the
pochonin scaffold, with five points of
diversity, was prepared which includes
diversity beyond that of the natural an-
alogues. The library was synthesized by
using solid-supported reagents amena-
ble to automation. Testing the library
for its inhibition against a panel of 24
kinases at 10 mm afforded a >14% hit
rate. These results demonstrate the po-
tential of the resorcylides towards the
inhibition of therapeutically relevant
kinases.
Keywords: Bergerat fold · combi-
natorial chemistry · kinase inhibi-
tors · organic synthesis · pochonin
Introduction
Modulation of protein activity through phosphorylation/de-
phosphorylation of a serine, threonine or tyrosine residue by
the action of kinases and phosphatases is at the center of
the majority of signal transduction mechanisms.[1] Small mol-
ecule inhibitors, such as 6-dimethylaminopurine and stauro-
sporine, were instrumental in understanding the importance
of such phosphorylation mechanisms and shed light on the
biological function of kinases. Kinases bind to ATP with a
Km of 0.1–10 mm, and transfer the g-phosphate group selec-
tively to a specific residue of a given protein. The core
domain of kinases, consisting of the ATP-binding site with
the residues involved in phosphotransfer reaction, are highly
conserved throughout the kinome.[2] This led to the specula-
tion that inhibitors targeting this highly conserved ATP-
binding pocket would not only have to compete with ATP
present at high concentration (mm), but would necessarily
lack selectivity. The discovery that modified purines, such as
(R)-roscovitine were potent and fairly selective inhibitors[3]
challenged that notion and inspired the synthesis of combi-
natorial libraries around the purine scaffold[4,5] yielding im-
portant leads.[6,7]
[a] E. Moulin, Dr. S. Barluenga, Prof. N. Winssinger
Organic and Bioorganic Chemistry Laboratory
Institut de Science et IngØnierie SupramolØculaires
UniversitØ Louis Pasteur–CNRS
Nevertheless, designing selective kinase inhibitors or tar-
geted libraries remains challenging. From a chemical biology
perspective, the ability to knock down a specific kinase ac-
tivity with a cell-permeable small molecule is an attractive
approach to deconvolute the role of specific kinases within
complex signaling networks. Furthermore, it has become in-
creasingly apparent that the biological function of kinases is
often regulated by their conformation, which is in turn dic-
8 allØe Gaspard Monge, 67000 Strasbourg (France)
Fax : (+33)390-245-112
E-mail: winssinger@isis.u-strasbg.fr
[b] F. Totzke
ProQinase GmbH, Breisacher Strasse 117
Freiburg (Germany)
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tated by their phosphorylation level and intra- as well as in-
termolecular associations. Small molecule inhibitors have
the potential to discriminate between the different confor-
mations of a given kinase which would provide a means to
dissect their respective functions. From a therapeutic per-
spective, imbalance in the phosphorylation/dephosphoryla-
tion equilibrium is at the source of numerous oncogenic
processes and inhibition of kinases has emerged as one of
the most promising avenues for chemotherapy.[8] Three
kinase inhibitors, gleevec, which inhibits Abl, iressa, and tar-
ceva, which both inhibit EGFR, have already been ap-
proved for treatment and herald a new era of highly target-
ed chemotherapy.
Given the challenge of developing selective kinase inhibi-
tors, radicicol caught our attention as it has been shown to
be a highly potent competitive ligand for the HSP90ꢁs ATP-
binding pocket while being highly selective for it.[9] HSP90 is
an ATPase rather than a kinase, and its ATP-binding pocket
has a Bergerat fold,[10,11] which is distinct from kinasesꢁ
ATP-binding pockets. However, literature search for other
resorcylic macrolides with the same skeletons as radicicol
revealed that closely related analogues are inhibitors of kin-
ases but not HSP90. Indeed, LL-Z1640-2 was found to be a
teraction, wherein radicicol substitutes the adenosine in the
ATP-binding pocket, it can be speculated that the kinase in-
hibitors amongst the resorcylides exert their effect also by
competing for the ATP-binding pocket. It was anticipated
that a library extending beyond the natural resorcylides
would lead to new kinase and ATPase inhibitors.[21–23]
Results and Discussion
The library was envisioned to stem from five points of diver-
sity around the resorcylic macrolide scaffold as shown in
Figure 1; modifications of the para phenol (R1, a number of
natural resorcylides bear a methyl group at that position),
Figure 1. General structure of the library.
the group on C17 (R2, both
stereochemistry are present in
natural resorcylides; however,
only with a methyl substitu-
ent), the C14–C15 olefin (R3),
the C9 carbonyl (R4), the
olefin C10–C11, and the meta
position on the aryl ring (R5, a
number of natural resorcylides
bear a chlorine at that posi-
tion). An important considera-
tion in the design of the chem-
istry used to elaborate the mo-
lecular diversity of this scaffold
was that a maximum number
of steps should be carried out
on the solid phase or by using
potent and selective inhibitor of TAK1 kinase for which rad-
icicol and other resorcylides were not active.[12–15] Closely re-
lated LL-783,227, in which one of the olefins has been re-
duced, is a potent inhibitor of MEK kinase.[15] Compound
F87-2509.04 was found to induce degradation of mRNA
containing AU-rich elements (ARE)[16] and hypothemycin
was found to inhibit the Ras-mediated cellular signaling.[17]
We have recently shown that aigialomycin D is a CDK in-
hibitor.[18] Interestingly, pochonin C which is closely related
to radicicol was found to inhibit Herpes Simplex Virus
(HSV) replication with a significant therapeutic window,
whereas it is a poor HSP90 inhibitor.[19] While radicicol and
pochonin C are structurally very similar, they have very dif-
ferent conformations in solution which can rationalize their
different biological activities.[20] Although structural infor-
mation has been reported only for the radicicol-HSP90 in-
polymer-bound reagents,[24,25] so as to minimize traditional
chromatography. The assembly of the macrocycle relied on
the chemistry developed for the synthesis of pochonin D.[26]
Thus, commercially available benzoic acid 1 (Scheme 1) and
its chlorinated analog 2 (the chlorine atom was introduced
on acid 1 prior to esterification by using HClO generated in
situ by the oxidation of acetaldehyde with NaClO2/sulfamic
acid)[26] were esterified with fifteen different homoallylic al-
cohols by using polymer-supported DEAD to yield esters 3
in excellent purity. The homoallylic alcohols that are not
commercially available were obtained in their enantiomeri-
cally pure form either by enzymatic resolution[27,28] of the
racemic alcohol or by means of Brown allylation of the cor-
responding aldehyde.[29] The products 3 were then protected
with ethoxymethylene chloride[30] (EOM-Cl) in the presence
of Hunigꢁs base to obtain the corresponding protected toluic
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Diversity-Oriented Synthesis of Pochonins
FULL PAPER
dition products 7. Nevertheless, the crude mixtures of these
reactions were used in the subsequent cyclization step. Un-
fortunately, compounds 4 bearing a furyl or pyridyl side
chain did not afford clean products in the acylation reaction.
The trienes then underwent ring-closing metathesis by using
Grubbs second-generation catalyst[31,32] under thermodynam-
ic conditions,[33] affording the desired 14-membered macro-
cycles. In the cases in which the metathesis reactions were
carried out with a mixture of 5 and 7, the corresponding 12-
membered-ring product 8 was obtained in addition to 6 as a
separable mixture. All successful reaction sequences were
purified by standard chromatography at this stage yielding
the macrocycles 6 and 8 in 30–60% and 8–10% overall
yield, respectively, from 1.
Macrocycles 6 were then used as the starting point for fur-
ther diversifications. Deprotection of the EOM groups of 6
by using sulfonic acid resin afforded compounds 9 in pure
form and excellent yields after simple filtration of the resin
and evaporation of the solvents (Scheme 2). The 12-mem-
bered-ring products 8 were deprotected as smoothly (not
shown). As expected, EOM deprotections under acid cataly-
sis were slower for the chlorinated analogues and had to be
monitored as prolonging the reaction times could lead to
conjugate additions (vide infra). Treatment of 6 with reduc-
ing agents led to either carbonyl reduction with DIBAL or
mixtures of carbonyl and 1,4-reduction with NaBH4. It is
known that using a noncoordinating counter ion for borohy-
Scheme 1. Synthesis of macrocyles
6 and 8: a) NaClO2 (5.0 equiv),
NH2SO3H (5.0 equiv), CH3CHO (1.0 equiv), THF/H2O 5:1, 08C, 0.5 h,
92%; b) PS-DEAD (2.5 equiv, 1.3 mmolgÀ1), alcohol (1.0 equiv),
PACHTREUNG(mClPh)3 (2.0 equiv), CH2Cl2, 238C, 0.5 h, 60–80%; c) iPr2EtN (4.0
equiv), EOMCl (4.0 equiv), TBAI (cat.), DMF, 808C, 5 h, 80–90%;
d) LDA (2.0 equiv), THF, À788C, Weinreb amide (1.0 eq), 10 min, Am-
berlite IRC-50 (20 equiv, 10.0 mmolgÀ1); e) Grubbs II (10% mol), tol-
uene (2 mm), 808C, 12 h, 60–85% after two steps. PS-DEAD=ethoxycar-
bonylazocarboxymethyl polystyrene, Grubbs II=rutheniumACHTER[UNG 1,3-bis(2,4,6-
trimethylphenyl)-2-imidazolidinylidene]dichloro(phenylmethylene)(tricy-
clohexylphosphine), LDA=lithium diisopropylamide, EOM=methoxy-
methylene; TBAI=tetrabutylammonium iodide.
esters 4, which could be used in the subsequent carbon-acy-
lation reaction without further purification.
Deprotonation of the toluic esters 4 by using two equiva-
lents of LDA, followed by addition of the a,b-unsaturated
Weinreb amide, previously prepared by solid-phase chemis-
try,[26] afforded acylation products 5. The reaction was
quenched with a polymer-bound acid which also sequestered
all the diisopropyl amine. We had previously noted that this
reaction could lead to some level of 1,4-conjugate addition
rather than desired 1,2-addition.[20] While the bulky chlorine
present in pochonin D suppresses this reaction, compounds
lacking the aryl chloride afforded 20% of the conjugate ad-
Scheme 2. Deprotection and synthesis of the reduced ketone analogues.
a) PS-TsOH (10.0 equiv, 3.2 mmolgÀ1), MeOH, 408C, 4 h, >90%;
b) BER resin (1.0 equiv, 2.5 mmolgÀ1), MeOH, 08C, 12 h, ~60%;
c) Ac2O (1.2 equiv), PS-NMM (1.2 equiv, 3.20 mmolgÀ1), DMAP
(0.05 equiv), DMF, 238C, 0.5 h, ~80%. BER resin=borohydride ex-
change resin, PS-TsOH=sulfonic acid resin MP, DIBAL=diisobutylalu-
minum hydride, DMAP=dimethylaminopyridine, PS-NMM=morpholi-
nomethyl polystyrene.
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N. Winssinger et al.
dride can favor the carbonyl reduction and this was most
conveniently achieved by using a polymer-supported quater-
nary ammonium borohydride known as borohydride ex-
change resin (BER).[34,35] Thus, ketones 6 could be reduced
with BER-resin to obtain both diastereoisomers of 10 in
~60% yield. Deprotection of the EOMs with sulfonic acid
resin under regular conditions afforded compounds 11. Ace-
tylation of the reduced intermediates 10 by using PS-NMM/
Ac2O yielded compounds 12 which led to elimination upon
deprotection to afford trienes 13 as a mixture of olefin geo-
metries.
As we observed that prolonged exposure of resorcylides 6
to methanol in the presence of sulfonic acid resin led to con-
jugate addition, we asked whether this reaction could be
driven to completion cleanly. We found that phenol 9
(Scheme 3) could be quantitatively converted to product 14
in 15 h. This product could obviously be obtained directly
from 6 under the same conditions.
Scheme 4. Derivatization of compounds 9. a) PS-TMABH3CN (2.0 equiv,
3.5 mmolgÀ1), CH2Cl2/AcOH (10:1), 238C, 4 h, ~50%; b) PPh3
(2.0 equiv), R2OH (2.0 equiv), PS-DEAD (2.0 equiv, 1.3 mmolgÀ1),
3
CH2Cl2, 238C, 8 h, ~60%; c) R X (0.9 equiv), PS-TBD (2.0 equiv,
Scheme 3. Synthesis of analogues 14 by conjugate addition. a) PS-TsOH
(10.0 equiv, 3.2 mmolgÀ1), MeOH, 408C, 15 h, 80%; PS-TsOH=sulfonic
acid resin.
2.9 mmolgÀ1), CH2Cl2, 238C, 3 h, ~90%; d) OsO4 (0.1 equiv), NMO
(1.0 equiv), acetone/H2O 10:1, 238C, 1 h, >70%; e) DMDO (1.2 equiv,
0.04m in acetone), CH3CN, 08C, 30 min, >90%. DMDO=dimethyldiox-
irane, NMO=4-methylmorpholine N-oxide, PS-TBD=TBD-methyl pol-
ystyrene, PS-TMABH3CN=(polystyrylmethyl)trimethylammonium cya-
noborohydride.
Compounds 9 were also used as starting points for further
diversifications (Scheme 4). Thus, treatment of 9 with poly-
mer-bound cyanoborohydride afforded the 1,4-reduction
products 15 in moderate yields. The more acidic para-hy-
droxyl groups of 9 were substituted by either Mitsunobu re-
action using polymer bound DEAD or alkylation using a
polymer-bound base to afford compounds with general
structure 16 and 17, respectively. Oxidation with OsO4 af-
forded the dihydroxylation products 18 as a mixture of iso-
mers as well as the products corresponding to the dihydrox-
ylation of the conjugate olefin (product not shown). Treat-
ment of 9 with freshly prepared dimethyldioxirane led to
the selective epoxidation of the nonconjugated olefin as a
mixture of diastereoisomers of pochonin A analogues 19.
We have recently shown[36] that higher diastereoselectivity
may be obtained for pochonin A if the phenols are protect-
ed with TBS; however, this was not found to be necessary
for the purpose at hand.
It is interesting to note that the conjugated olefin proved
to have different reactivity depending on the presence or
absence of the chlorine atom on the aryl ring. While the
EOM deprotection of compound 6 in which X=Cl and R=
Me could be carried out with HCl in dioxane, treatment of
the corresponding compound 6 in which X=H and R=Me
with the same conditions led to the conjugate addition of
the chlorine ion during the deprotections affording com-
pound 20 (Scheme 5). The difference in reactivity may be at-
tributed to the different conformation of these compounds.
It is conceivable that the differences in conformation affect
the level of conjugation between the olefin and the adjacent
carbonyl group. Nevertheless, the b-chlorine could be clean-
ly eliminated in the presence of polymer-bound base to re-
cover the conjugate compound 9.
While evaluating protecting groups for the phenols, we
noticed that dihydropyran, in the presence of a strong acid,
such as sulfonic acid, led to electrophilic aromatic substitu-
tion rather than phenol protection.[37] Applying these condi-
tions to compounds 9 (Scheme 5) afforded 21 as a separable
mixture of diastereoisomers.
The formation of oxime proved to be sluggish on com-
pounds with unprotected phenols. However, compounds 6
(X=H, Scheme 5) protected with EOM groups underwent
smooth oxime formation with nine different hydroxyl
amines to obtain compounds 22 as E/Z mixtures with varia-
ble ratios. EOM deprotection of 22 with sulfonic acid resin
in methanol followed by treatment with sulfonic acid resin
in dichloromethane in the presence of dihydropyran afford-
ed oximes 23 bearing a pyran substitution on the aromatic
ring as a mixture of diastereoisomers. For the case in which
the side chain contains an acid (R2X=OCH2COOH), the
deprotection of the EOM with the sulfonic acid resin in
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Diversity-Oriented Synthesis of Pochonins
FULL PAPER
Scheme 6. Oxime formation with compounds 9 (R=Me, X=Cl): a) TBS-
Cl (5.0 equiv), imidazole (5.0 equiv), DMF, 238C, 3 h, ~90%;
b) R2ONH2.HCl (5.0 equiv), Py/AcOH 5:1, 408C, 12 h, ~90%; c) TBAF
(2.5 equiv), THF, 238C, 2 h, ~80%. TBAF=tetrabutylammonium fluo-
ride, TBS-Cl=tert-butyldimethylsilyl chloride.
pounds) was tested for its inhibition in a panel of 24 kinase
(AKT1, ARK5, Aurora-A, Aurora-B, B-RAF-VE, CDK2/
CycA, CDK4/CycD1, CK2-a1, FAK, EPHB4, ERB2, EGF-
R, IGF1-R, SRC, VEGF-R2, VEGF-R3, FLT3, INS-R,
MET, PDGFR-b, PLK1, SAK, TIE2, COT) at 10 mm. Signif-
icantly, twelve compounds showed more than 50% inhibi-
tion which represents a >14% hit rate for a kinase. Interest-
ingly, pochonin D, A, and radicicol which have been shown
to be powerful inhibitors of HSP90 did not show significant
activity against this panel of kinases. Nine compounds were
selected to calculate IC50 against each of the 24 kinases
(Table 1). In this more detailed analysis, radicicol showed
only very mild activity against VGFR-R2 with no inhibition
for the twenty three other kinases. Several pochonin ana-
logues showed a well-defined pattern of activity against
therapeutically relevant enzymes, such as Src (8 mm for A2),
Aurora A (12 mm for A3), and IGF1-R (11 mm for A5). Im-
portantly, the compounds that were found to be kinase in-
hibitors were not inhibitors of HSP90 (data not shown) and
are not indiscriminate ATP-surrogates.
HSP90s ATPase pocket targeted by radicicol and pocho-
nin D has a specific fold that is present in a superfamily
which includes functionally diverse proteins, such as DNA
topoisomerase II, helicase, MutL, and histidine kinases (Ber-
gerat fold).[10,11] In fact, it has been shown that radicicol
does inhibit other members of this family albeit with lower
affinity.[43,44] While the pochonin library described herein
will certainly contain some compounds that are good inhibi-
tors of enzymes bearing a Bergerat fold, we wished to evalu-
ate whether modification around the pochonin scaffold
could retune the selectivity of these compounds from
HSP90 inhibitors to kinase inhibitors. The fact that more
than fourteen percent of the compounds showed a kinase in-
hibition of greater than 50% at 10 mm clearly supports the
hypothesis that resorcylides are a good scaffold for kinase
inhibition.
Scheme 5. Derivatization of macrocycles 6: a) 2.5% HCl/dioxane, 238C,
3 h, >75%; b) PS-TBD (0.5 equiv, 2.6mmolg À1), CH2Cl2, 238C, 8 h,
~90%; c) PS-TsOH (1.0 equiv, 3.2 mmolgÀ1), DHP (1.0 equiv), CH2Cl2,
238C, 5 h, ~80%; d) R2XNH2.HCl (5.0 equiv), pyridine/AcOH 5:1, 408C,
12 h, ~90%; e) PS-TsOH (10 equiv, 3.2 mmolgÀ1), MeOH, 408C, 4 h,
~80%; f) PS-TsOH (cat., 3.2 mmolgÀ1), DHP (1.0 equiv), CH2Cl2, 238C,
5 h, ~70%. DHP=dihydropyran.
methanol was accompanied by esterification of the carboxy-
late. Finally, all attempts of oxime formation on the chlori-
nated analogues with or without EOM-protecting groups
generated mostly the corresponding 1,4-addition of the hy-
droxylamines (Scheme 6). To our surprise, when pochonin D
was protected with a TBS groups, (24, Scheme 6) the forma-
tion of the desired oxime 25 was the only product observed
under the same reaction conditions. Deprotection of the
TBS groups was then achieved by using TBAF to obtain
oxime 26. This difference of reactivity is presumably due to
the level of conjugation between the olefin and the carboxy-
late which in turn is a product of the conformation of the
macrocycle. It is not so surprising that a bulky-protecting
group on the ortho phenol has a pronounced impact on the
macrocycle conformation as compared to the phenol which
forms a hydrogen bond to the carbonyl. Such effects have
already been documented to affect the outcome of ring-clos-
ing-metathesis reactions[38] and epoxidation diastereoselec-
tivity.[36] It should be noted that a number of oxime deriva-
tives of radicicol have been reported in the literature[39–41]
leading to the discovery that some oximes dramatically im-
prove the affinity of derivatized radicicol for HSP90.[42]
While not all permutations of the five points of diversity
were pursued, a total of 113 compounds were prepared
(Figure 2). A representative subset of the library (84 com-
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N. Winssinger et al.
Figure 2. Structures of the pochonin library.
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Diversity-Oriented Synthesis of Pochonins
FULL PAPER
Table 1. Inhibitory activity (IC50; mm) and structure of selected pochonin analogues in a panel of 24 kinase assays (blanks represent an IC50 >50 mm).
AKT1 ARK5 Aurora A Aurora B B-RAF-VE CDK2/CycA CDK4/CycD1 CK2-a1 FAK EPHB4 ERBB2 EGF-R
radicicol
A1
A2
A3
A4
A5
33
14
12
30
47
1650
14
3648
16
14
37
16
30
45
9
40
49
24
16
22
32
24
50
38
A61637
A7
34
40
16
A8
IGF1-R
SRC
VEGF-R2
49
VEGF-R3
FLT3
INS-R
MET
29
PDGFR-b
PLK1
SAK
TIE2
72
COT
radicicol
A1
A2
A3
A4
A5
A621
A7
23
168
19
13
11
20
11
19
14
12
12
40
20
30
23
19
31
3362
23
45
17
16
25
44
36
25
19
20
15
19
31
25
34
44
17
A8
light as visualizing agent and 10% ethanolic phosphomolybdic acid or va-
nillin solution and heat as developing agents. E. Merck silica gel (60, par-
ticle size 0.040–0.063 mm) was used for flash-column chromatography.
PTLC (preparative thin layer chromatography) was carried out on
0.25 mm E. Merck silica-gel plates. NMR spectra were recorded on
Bruker Advance-400 instrument and calibrated by using residual undeu-
terated solvent as an internal reference. The following abbreviations
were used to explain the multiplicities: s=singlet, d=doublet, t=triplet,
q=quartet, m=multiplet, and br=broad. LCMS were recorded by using
an Agilent 1100 HPLC with a Bruker micro-TOF instrument (ESI).
Unless other wise stated, a Supelco C8 (5 cm”4.6mm, 5 mm particles)
column was used with a linear elution gradient from 100% H2O (0.5%
Conclusion
The synthesis of a library based on the pochonin macrolides
by using predominantly polymer-supported reagents has
been achieved. The synthetic methods that were developed
are amenable to automated synthesis. Screening of this li-
brary for kinase inhibition yielded a number of leads for
therapeutically important kinases including Src, EGFR, and
Aurora A and B. Importantly, the promising kinase leads
that were identified were not HSP90 inhibitors and showed
diverse selectivity profiles for kinase inhibition amongst 24
tested kinases. These results demonstrate the potential of re-
sorcylides for selective kinase inhibition.
HCO2H) to 100% MeCN in 13 min at a flow rate of 0.5 mLminÀ1
.
Generalprocedure for the synthesis of compounds 4 : A solution of acid
1 or 2 (1.0 equiv), homoallylic alcohol (1.0 equiv), and tris(3-chlorophe-
nyl)phosphine (2.0 equiv) in anhydrous dichloromethane (0.05m) was
treated at room temperature with PS-DEAD (2.5 equiv, 1.3 mmolgÀ1).
After stirring for 30 min, the reaction mixture was filtered on silica and
washed with hexane/EtOAc (10:1, 100 mL) and hexane/EtOAc (3:1,
100 mL). The 3:1 mixture was concentrated under reduce pressure to
yield compound 3 (60–80%). Without further purification, compound 3
(1.0 equiv) and tetrabutylammonium iodide (catalytic amount) were dis-
solved in DMF (0.15m) and treated with diisopropylethylamine
(4.0 equiv) and (chloromethyl)ethyl ether (4.0 equiv). After stirring over-
night at 808C, the reaction mixture was diluted with EtOAc and washed
several times with a saturated NH4Cl solution. The organic phase was
ExperimentalSection
Generaltechniques : All reactions were carried out under a nitrogen at-
mosphere with dry solvents under anhydrous conditions, unless otherwise
noted. Anhydrous solvents were obtained by passing them through a
commercially available alumina column (Innovative technology, VA).
Substituted polystyrene resins (100–200 mesh, 1% DVB) were purchased
from Novabiochem or Aldrich. Reactions were monitored by TLC car-
ried out on 0.25 mm E. Merck silica-gel plates (60F-254) by using UV
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N. Winssinger et al.
dried over MgSO4 and concentrated under reduce pressure to yield com-
pounds 4 (80–90%).
Selected examples of compounds 4: 1H NMR (CDCl3, 400 MHz, 258C):
d=7.04 (s, 1H), 5.89 (ddt, J=17.0, 10.5, 7.0 Hz, 1H), 5.31 (s, 2H), 5.21
(q, J=7.0 Hz, 2H), 3.69 (q, J=7.0 Hz, 2H), 2.51–2.46(m, 2H), 2.31 (s,
3H), 1.22–1.18 ppm (m, 6H); 13C NMR (CDCl3, 100 MHz, 258C): d=
167.3, 154.2, 153.0, 135.0, 133.9, 119.9, 117.2, 117.1, 101.7, 93.8, 93.6, 64.5,
64.3, 64.3, 33.0, 17.4, 14.9 ppm (”2); HRMS (ESI-TOF): m/z: calcd for
C18H26O6Cl: 373.1412 [M+H]+; found: 373.1364.
(s, 2H), 5.22–5.06(m, 3H), 3.79 (q, J=7.0 Hz, 2H), 3.72 (q, J=7.0 Hz,
2H), 2.48–2.44 (m, 2H), 2.36(s, 3H), 2.01 (qd, J=12.4, 7.0 Hz, 1H), 1.25
(t, J=7.0 Hz, 3H), 1.23 (t, J=7.0 Hz, 3H), 1.02 (d, J=6.4 Hz, 3H),
1.01 ppm (d, J=7.0 Hz, 3H); 13C NMR (CDCl3, 100 MHz, 258C): d=
167.3, 154.0, 152.9, 134.8, 134.1, 120.4, 117.5, 117.1, 101.5, 93.9, 93.4, 79.0,
64.6, 64.3, 35.6, 30.8, 18.4, 17.6, 17.5, 15.0 ppm (”2); HRMS (ESI-TOF):
m/z: calcd for C21H31O6ClNa: 437.1701 [M+Na]+; found: 437.1574.
1H NMR (CDCl3, 400 MHz, 258C): d=6.74 (d, J=1.2 Hz, 1H), 6.56 (s,
1H), 5.93–5.83 (m, 1H), 5.20 (ddt, J=16.9, 9.9, 7.0 Hz, 1H), 5.21 (s, 2H),
5.20 (s, 2H), 5.16–5.08 (m, 2H), 3.72 (q, J=7.0 Hz, 4H), 2.45 (t, J=
6.4 Hz, 2H), 2.31 (s, 3H), 1.68–1.39 (m, 4H), 1.23 (t, J=7.0 Hz, 3H), 1.22
(t, J=7.0 Hz, 3H), 0.96ppm (t, J=7.0 Hz, 3H); 13C NMR (CDCl3,
100 MHz, 258C): d=167.9, 158.6, 155.3, 137.4, 133.9, 118.9, 117.5, 110.4,
101.0, 93.2, 93.0, 73.9, 64.2 (x 2), 38.7, 35.7, 19.7, 18.5, 15.0, 15.0,
13.9 ppm; HRMS (ESI-TOF): m/z: calcd for C21H32O6Na: 403.2096
[M+Na]+; found: 403.2017.
1H NMR (CDCl3, 400 MHz, 258C): d=7.43 (d, J=1.8 Hz, 1H), 6.98 (s,
1H), 6.45 (d, J=2.9 Hz, 1H), 6.38 (dd, J=3.5, 1.8 Hz, 1H), 6.18 (t, J=
7.0 Hz, 1H), 5.83 (ddt, J=17.3, 10.5, 7.0 Hz, 1H), 5.30 (s, 2H), 5.20 (dd,
J=17.3, 1.6Hz, 1H), 5.14 (s, 2H), 5.11 (dd, J=10.5, 1.6Hz, 1H), 3.77 (q,
J=7.0 Hz, 2H), 3.65 (q, J=7.0 Hz, 2H), 2.88–2.82 (m, 2H), 2.26(s, 3H),
1.24 (t, J=7.0 Hz, 3H), 1.20 ppm (t, J=7.0 Hz, 3H); 13C NMR (CDCl3,
100 MHz, 258C): d=166.6, 154.2, 153.0, 151.9, 142.5, 135.1, 132.7, 118.3,
110.2, 109.0, 101.6, 101.1, 93.8, 93.4, 68.9, 64.6, 64.3, 36.7, 17.2, 15.0 ppm
(”2), one quartenary carbon is not visible; HRMS (ESI): m/z: calcd for
C22H27O7ClNa: 461.1338 [M+Na]+; found: 461.1215.
1H NMR (CDCl3, 400 MHz, 258C): d=7.34–7.25 (m, 5H), 6.75 (s, 1H),
6.54 (s, 1H), 5.94 (ddt, J=17.0, 9.9, 7.0 Hz, 1H), 5.48 (tt, J=6.4, 6.4 Hz,
1H), 5.22 (s, 2H), 5.18–5.14 (m, 4H), 3.74 (q, J=7.0 Hz, 2H), 3.70 (q, J=
7.0 Hz, 2H), 3.05 (dd, J=14.0, 7.0 Hz, 1H), 2.95 (dd, J=14.0, 6.4 Hz,
1H), 2.48–2.45 (m, 2H), 2.11 (s, 3H), 1.25 (t, J=7.3 Hz, 3H), 1.23 ppm
(t, J=7.3 Hz, 3H); 13C NMR (CDCl3, 100 MHz, 258C): d=167.8, 158.7,
155.4, 137.6, 137.5, 133.7, 129.5 (”2), 128.4 (”2), 126.5, 118.7, 117.9,
110.5, 101.1, 93.3, 93.0, 74.6, 64.3, 64.3, 39.8, 37.8, 19.4, 15.1, 15.0 ppm;
HRMS (ESI-TOF): m/z: calcd for C25H32O6ClNa: 451.2130 [M+Na]+;
found: 451.2084.
Generalprocedure for the synthesis of compounds 5 and 7 : A solution of
compound 4 (1.0 equiv) in anhydrous THF (0.2m) was treated at À788C
with freshly made LDA (2.0 equiv). Immediately after, the a,b-unsaturat-
ed Weinreb amide[21] was added to the cooled solution (1.0 equiv). The
resulting mixture was then stirred for 10 min at À788C and quenched by
addition of Amberlite resin (20 equiv). Upon warming up to room tem-
perature, the reaction was filtered through a pad of silica and washed
with EtOAc. Concentration under reduced pressure afforded the desired
compound 5. This compound was used directly in the metathesis reaction
without any further purification. When X=H, 20% of the corresponding
1,4-addition compound was observed and a fraction of the mixture was
purified for characterization of compounds 5 and 7 (silica gel, 0–20%
EtOAc/cyclohexane gradient).
Selected examples of compounds 5: 1H NMR (CDCl3, 400 MHz, 258C):
d=6.91 (dt, J=15.8, 6.7 Hz, 1H), 6.87 (d, J=1.8 Hz, 1H), 6.53 (d, J=
1.8 Hz, 1H), 6.19 (d, J=15.8 Hz, 1H), 5.91–5.77 (m, 2H), 5.23 (s, 2H),
5.22 (s, 2H), 5.15–5.00 (m, 5H), 3.87 (s, 2H), 3.73 (q, J=7.0 Hz, 4H),
2.45–2.40 (m, 2H), 2.35–2.29 (m, 2H), 2.25–2.20 (m, 2H), 2.00–1.92 (m,
1H NMR (CDCl3, 400 MHz, 258C): d=7.46–7.44 (m, 2H), 7.40–7.33 (m,
3H), 7.01 (s, 1H), 6.09 (dd, J=7.9, 5.6Hz, 1H), 5.83 (ddt, J=17.3, 10.2,
7.0 Hz, 1H), 5.31 (s, 2H), 5.18–5.10 (m, 2H), 5.11 (s, 2H), 3.79 (q, J=
7.0 Hz, 2H), 3.61 (q, J=7.0 Hz, 2H), 2.79 (ddd, J=15.0, 7.3, 7.3 Hz, 1H),
2.67 (ddd, J=15.1, 7.2, 7.2 Hz, 1H), 2.25 (s, 3H), 1.25 (t, J=7.0 Hz, 3H),
1.19 ppm (t, J=7.0 Hz, 3H); 13C NMR (CDCl3, 100 MHz, 258C): d=
166.7, 154.2, 153.1, 139.7, 135.1, 133.3, 128.3 (”2), 128.0, 126.8, 126.8
(”2), 119.9, 118.1, 101.6, 93.9, 93.5, 76.3, 64.6, 64.3, 40.5, 17.4, 15.0,
14.9 ppm; HRMS (ESI): m/z: C24H29O6ClNa: 471.1545 [M+Na]+; found:
471.1421.
1H NMR (CDCl3, 400 MHz, 258C): d=6.99 (s, 1H), 5.88–5.78 (m, 1H),
5.27 (s, 2H), 5.18 (s, 2H), 5.17–5.07 (m, 2H), 4.36(t, J=6.7 Hz, 2H), 3.74
8826
www.chemeurj.org
ꢀ 2006Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2006, 12, 8819 – 8834

Diversity-Oriented Synthesis of Pochonins
FULL PAPER
1H), 1.24 (t, J=7.0 Hz, 6H), 1.00 (d, J=2.3 Hz, 3H), 0.98 ppm (d, J=
3.0 Hz, 3H); 13C NMR (CDCl3, 100 MHz, 258C): d=196.3, 167.6, 159.0,
156.2, 147.1, 137.0, 135.0, 134.4, 129.5, 118.7, 117.2, 115.5, 111.0, 102.3,
93.3, 93.0, 78.8, 64.4, 64.3, 45.4, 35.8, 32.0, 31.7, 30.8, 18.5, 17.4, 15.0 ppm
(”2); HRMS (ESI-TOF): m/z: calcd for C28H40O7Na: 511.2666 [M+Na]+
; found: 511.2521.
2.08–2.03 (m, 2H), 1.19 (t, J=6.8 Hz, 6H), 1.01 ppm (t, J=6.5 Hz, 2H);
HRMS (ESI-TOF): m/z: calcd for C27H42O8N: 508.2905 [M+H]+; found:
508.2873.
Generalprocedure for the metathesis reaction : A solution of crude 5 (or
mixture of 5 and 7 in which X=Cl), in anhydrous toluene (2 mm) was
treated with Grubbs second-generation catalyst (0.10 equiv) and heated
at 808C for 12 h. The reaction was cooled down to room temperature
and the mixture was filtered through a pad of silica gel, washed with
CH2Cl2 followed by a mixture EtOAc/cyclohexane 1:1, and concentrated
under reduced pressure. Purification by flash chromatography (silica gel,
0–25% EtOAc/cyclohexane gradient) afforded compounds 6 or 8 (60–
85% over two steps):
Selected examples of compounds 6: [a]2D5 =+21.3 (c=1.00 in CHCl3);
1H NMR (CDCl3, 400 MHz, 258C): d=7.14 (s, 1H), 6.72–6.66 (m, 1H),
1H NMR (CDCl3, 400 MHz, 258C): d=6.89 (dt, J=15.4, 6.7 Hz, 1H),
6.83 (d, J=1.2 Hz, 1H), 6.51 (d, J=1.8 Hz, 1H), 6.17 (d, J=15.8 Hz,
1H), 5.90–5.73 (m, 2H), 5.20 (s, 2H), 5.19 (s, 2H), 5.16–5.12 (m, 2H),
5.06(2”d, J=11.1 Hz, 2H), 4.99 (d, J=11.1 Hz, 1H), 3.85 (s, 2H), 3.71
(q, J=7.0 Hz, 2H), 3.69 (q, J=7.0 Hz, 2H), 2.40 (dd, J=6.4, 6.4 Hz, 2H),
2.30 (ddd, J=7.2, 7.2, 7.0 Hz, 2H), 2.21 (ddd, J=6.9, 6.8, 6.6 Hz, 2H),
1.66–1.53 (m, 2H), 1.52–1.34 (m, 2H), 1.22 (t, J=6.4 Hz, 3H), 1.20 (t, J=
6.7 Hz, 3H), 0.92 ppm (t, J=7.3 Hz, 3H); 13C NMR (CDCl3, 100 MHz,
258C): d=196.1, 167.4, 159.0, 156.1, 147.1, 137.0, 134.9, 134.0, 129.5,
118.7, 117.5, 115.5, 111.0, 102.3, 93.3, 93.0, 74.2, 64.3, 64.3, 45.4, 38.6, 35.6,
32.0, 31.7, 18.4, 15.0, 15.0, 13.9 ppm; HRMS (ESI-TOF): m/z: calcd for
C28H40O7Na: 511.2672 [M+Na]+; found: 511.2714.
5.88 (d, J=15.2 Hz, 1H), 5.33–5.17 (m, 6H), 4.92–4.88 (m, 1H), 4.21 (d,
J=17.0 Hz, 1H), 3.92 (d, J=17.0 Hz, 1H), 3.79–3.67 (m, 4H), 2.33–2.17
(m, 5H), 2.07–1.96(m, 2H), 1.23 (t, J=7.0 Hz, 3H), 1.21 (t, J=7.0 Hz,
3H), 1.00 ppm (d, J=5.8 Hz, 6H); 13C NMR (CDCl3, 100 MHz, 258C):
d=195.7, 167.1, 154.7, 154.4, 147.4, 133.7, 131.2, 128.8, 128.4, 119.7, 118.0,
102.7, 93.9, 93.5, 80.0, 64.8, 64.5, 44.1, 32.3, 31.2, 30.7, 30.6, 18.3, 17.2,
15.0, 14.9 ppm; HRMS (ESI): m/z: calcd for C26H35O7ClNa: 517.1964
[M+Na]+; found: 517.1844.
1H NMR (CDCl3, 400 MHz, 258C): d=7.44–7.42 (m, 2H), 7.39–7.29 (m,
3H), 6.85 (d, J=1.8 Hz, 1H), 6.78 (dt, J=15.8, 6.4 Hz, 1H), 6.53 (s, 1H),
6.06 (d, J=16.4 Hz, 1H), 6.01 (t, J=7.0 Hz, 1H), 5.86–5.74 (m, 2H), 5.21
(s, 2H), 5.15 (s, 2H), 5.13–5.01 (m, 4H), 3.80 (d, J=16.4 Hz, 1H), 3.76
(d, J=16.4 Hz, 1H), 3.72 (q, J=7.0 Hz, 2H), 3.65 (q, J=7.0 Hz, 2H),
2.79 (ddd, J=14.0, 7.0, 7.0 Hz, 1H), 2.65 (ddd, J=14.6, 7.0, 7.0 Hz, 1H),
2.29–2.17 (m, 4H), 1.23 (t, J=7.0 Hz, 3H), 1.20 ppm (t, J=7.0 Hz, 3H);
13C NMR (CDCl3, 100 MHz, 258C): d=196.1, 167.2, 159.0, 156.2, 147.1,
137.0, 134.9, 133.9, 129.5, 128.2 (”2), 127.8, 126.9 (”2), 118.7, 117.6,
115.5, 111.2, 102.6, 93.4, 93.0, 71.1, 64.4, 64.3, 45.4, 40.2, 32.0, 31.7, 19.4,
15.0, 14.9 ppm; HRMS (ESI): m/z: calcd for C31H38O7Na: 545.2510
[M+Na]+; found: 545.2346.
[a]2D5 =À40.4 (c=0.79 in CHCl3); 1H NMR (CDCl3, 400 MHz, 258C): d=
7.49–7.47 (m, 2H), 7.40–7.29 (m, 3H), 7.10 (s, 1H), 6.84–6.77 (m, 1H),
5.98 (d, J=15.2 Hz, 1H), 5.78 (d, J=8.8 Hz, 1H), 5.44–5.30 (m, 4H), 5.15
(d, J=7.0 Hz, 1H), 5.05 (d, J=6.8 Hz, 1H), 4.07 (d, J=17.0 Hz, 1H),
3.90 (d, J=17.0 Hz, 1H), 3.80 (d, J=7.0 Hz, 2H), 3.60–3.51 (m, 2H),
2.68–2.62 (m, 1H), 2.50–2.47 (m, 1H), 2.38–2.29 (m, 2H), 2.14–2.02 (m,
2H), 1.25 (t, J=7.0 Hz, 3H), 1.17 ppm (t, J=7.0 Hz, 3H); 13C NMR
(CDCl3, 100 MHz, 258C): d=195.7, 166.7, 154.8, 154.2, 147.3, 140.7,
133.3, 132.1, 128.5, 128.3 (”2), 128.2, 127.9, 127.7, 126.7 (”2), 120.1,
118.1, 102.9, 93.9, 93.4, 77.4, 64.8, 64.4, 44.5, 40.5, 30.7, 15.0, 14.9 ppm;
HRMS (ESI): m/z: calcd for C29H33O7ClNa: 551.1680 [M+Na]+; found:
551.1807.
Selected example of compounds 7: 1H NMR (CDCl3, 400 MHz, 258C):
d=6.72 (s, 1H), 6.57 (s, 1H), 5.89–5.71 (m, 2H), 5.20–5.16 (m, 4H), 5.12–
[a]2D5 =À24.1 (c=0.33 in CHCl3); 1H NMR (CDCl3, 400 MHz, 258C): d=
7.39–7.33 (m, 4H), 7.31–7.27 (m, 1H), 6.82 (s, 1H), 6.82–6.75 (m, 1H),
6.63 (s, 1H), 6.02 (d, J=16.4 Hz, 1H), 5.35–5.29 (m, 2H), 5.27–5.20 (m,
5H), 4.16(d, J=14.6Hz, 1H), 3.79–3.70 (m, 4H), 3.52 (d, J=14.6Hz,
4.90 (m, 4H), 4.33 (t, J=6.8 Hz, 2H), 3.69 (2”q, J=7.0 Hz, 4H), 3.57 (s,
3H), 3.13 (s, 3H), 2.69–2.64 (m, 1H), 2.53–2.45 (m, 4H), 2.32 (m, 2H),
Chem. Eur. J. 2006, 12, 8819 – 8834
ꢀ 2006Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
8827

N. Winssinger et al.
1H), 3.37 (dd, J=13.4, 4.1 Hz, 1H), 2.78 (dd, J=13.5, 9.4 Hz, 1H), 2.37–
2.12 (m, 5H), 2.06–2.02 (m, 1H), 1.26 (t, J=7.0 Hz, 3H), 1.24 ppm (t, J=
7.0 Hz, 3H); 13C NMR (CDCl3, 100 MHz, 258C): d=197.6, 167.8, 159.2,
156.5, 149.0, 137.3, 135.5, 131.8, 129.9, 129.5 (”2), 128.6 (”2), 128.4,
126.7, 118.1, 109.9, 102.3, 93.5, 93.1, 75.8, 64.6, 64.4, 44.4, 41.0, 36.2, 31.0,
30.6, 15.0 ppm (”2); HRMS (ESI): m/z: calcd for C30H36O7Na: 531.2359
[M+Na]+; found: 531.2350.
Generalprocedure for EOM deprotection to generate deprotected com-
pounds 8 and 9: PS-TsOH (10.0 equiv, 3.2 mmolgÀ1) was added to a solu-
tion of the corresponding compound 6 or 8 (1.0 equiv) in MeOH (0.03m)
and the resulting suspension was shaken at 408C for 1 to 4 h. After this
time, the reaction mixture was filtered and the methanolic solution con-
centrated under reduced pressure. Purification by flash chromatography
(silica gel, 0–20% EtOAc/cyclohexane gradient) afforded the corre-
sponding deprotected compound 8 or 9 (>90%).
Selected example of deprotected compounds 8: Mixture of four diaster-
eoisomers: 1H NMR (CDCl3, 400 MHz, 258C): d=11.54 (s, 1H), 6.33 (d,
J=2.3 Hz, 1H), 6.25 (s, 1H), 5.53–5.51 (m, 1H), 5.44–5.41 (m, 1H), 5.11–
[a]2D5 =À108.3 (c=1.00 in CHCl3); 1H NMR (CDCl3, 400 MHz, 258C):
d=7.56–7.54 (m, 2H), 7.41–7.29 (m, 3H), 6.89–6.82 (m, 1H), 6.78 (d, J=
2.3 Hz, 1H), 6.61 (d, J=1.8 Hz, 1H), 6.06 (d, J=16.4 Hz, 1H), 5.98 (dd,
J=11.7, 2.4 Hz, 1H), 5.53–5.51 (m, 2H), 5.20 (d, J=7.0 Hz, 1H), 5.17 (d,
J=6.4 Hz, 1H), 5.07 (d, J=7.0 Hz, 1H), 4.96(d, J=7.0 Hz, 1H), 4.20 (d,
J=14.6 Hz, 1H), 3.73–3.68 (m, 2H), 3.54–3.45 (m, 3H), 2.71–2.66 (m,
1H), 2.55–2.51 (m, 1H), 2.38–2.32 (m, 2H), 2.23–2.06(m, 2H), 1.22 (t,
J=7.0 Hz, 3H), 1.14 ppm (t, J=7.0 Hz, 3H); 13C NMR (CDCl3,
100 MHz, 258C): d=197.6, 167.4, 159.3, 156.6, 149.0, 140.8, 135.6, 132.2,
129.9, 128.5, 128.2 (”2), 127.9, 126.9 (”2), 117.9, 109.9, 102.3, 93.2, 93.0,
76.6, 64.4, 64.3, 44.4, 40.5, 31.0, 30.6, 15.0, 14.9 ppm; HRMS (ESI): m/z:
calcd for C29H34O7Na: 517.2197 [M+Na]+; found: 517.2062.
5.08 (m, 1H), 4.01 (d, J=11.7 Hz, 2H), 3.45 (s, 3H), 3.11 (s, 3H), 2.83–
2.73 (m, 1H), 2.68–2.59 (m, 1H), 2.27–2.20 (m, 1H), 2.10–1.87 (m, 6H),
1.82–1.72 (m, 1H), 1.01–0.94 ppm (m, 6H), para-phenol not detected;
HRMS (ESI-TOF): m/z: calcd for C22H31O6NNa: 428.2044 [M+Na]+;
found: 428.2109.
Selected examples of compounds 9: [a]2D5 =À35.6( c=0.52 in CHCl3);
1H NMR (C6D6, 400 MHz, 258C): d=12.31 (s, 1H), 6.83 (s, 1H), 6.74–
Selected examples of compounds 8: Mixture of four diastereoisomers;
1H NMR (CDCl3, 400 MHz, 258C): d=6.77 (s, 1H), 6.52 (s, 0.5H), 6.46
6.67 (m, 1H), 5.84 (brs, 1H), 5.82 (d, J=15.8 Hz, 1H), 5.03–4.95 (m,
1H), 4.88–4.86(m, 1H), 4.7–64.70 (m, 1H), 4.40 (d,
J=17.6Hz, 1H),
4.15 (d, J=17.5 Hz, 1H), 2.40–2.34 (m, 1H), 2.22–2.18 (m, 1H), 1.87–1.65
(m, 4H), 1.53–1.48 (m, 1H), 0.92 (d, J=6.4 Hz, 3H), 0.66 ppm (d, J=
7.0 Hz, 3H); 13C NMR (C6D6, 100 MHz, 258C): d=193.7, 164.2, 156.8,
145.8, 137.2, 131.8, 129.3, 126.3, 115.3, 107.9, 103.6, 82.1, 46.4, 33.3, 30.9,
30.7, 28.8, 20.1, 18.5, 18.3 ppm; HRMS (ESI-TOF): m/z: calcd for
C20H23ClO5Na: 401.1126[ M+Na]+; found: 401.1170.
(s, 0.5H), 5.59–5.37 (m, 2H), 5.21–5.18 (m, 4H), 5.09–4.92 (m, 1H), 3.75–
3.70 (m, 4H), 3.53–3.48 (m, 3H), 3.38–3.34 (m, 1H), 3.19–3.10 (m, 3H),
2.65–2.47 (m, 3H), 2.29–2.04 (m, 6H), 1.89–1.72 (m, 2H), 1.31–1.20 (m,
6H), 1.06–0.96ppm (m, 6H); HRMS (ESI-TOF): m/z: calcd for
C28H43O8NNa: 544.2881 [M+Na]+; found: 544.2907.
1
Mixture of four diastereoisomers; H NMR (CDCl3, 400 MHz, 258C): d=
7.51–7.42 (m, 2H), 7.38–7.31 (m, 3H), 6.73–6.70 (m, 1H), 6.60–6.49 (m,
1H), 6.45–6.31 (m, 1H), 5.73–5.39 (m, 2H), 5.23–5.00 (m, 4H), 3.75–3.69
(m, 2H), 3.56–3.34 (m, 6H), 3.19–3.09 (m, 3H), 2.66–2.08 (m, 8H), 1.31–
1.19 (m, 5H), 1.10–1.04 ppm (m, 3H); HRMS (ESI-TOF): m/z: calcd for
C31H41O8NNa: 578.2724 [M+Na]+; found: 578.2715.
[a]2D5 =À10.3 (c=0.25 in CHCl3); 1H NMR (C6D6, 400 MHz, 258C): d=
12.0 (brs, 1H), 7.32–7.29 (m, 3H), 7.19–7.15 (m, 2H), 6.86–6.79 (m, 1H),
6.51 (d, J=2.4 Hz, 1H), 6.27–6.25 (m, 1H), 6.11 (d, J=2.4 Hz, 1H), 6.02
(d, J=15.8 Hz, 1H), 5.49 (s, 1H), 5.17–5.10 (m, 1H), 4.97–4.90 (m, 1H),
4.40 (d, J=16.4 Hz, 1H), 3.97 (d, J=17.2 Hz, 1H), 2.83–2.76(m, 1H),
2.45–2.38 (m, 1H), 1.89–1.78 (m, 2H), 1.67–1.58 ppm (m, 2H); 13C NMR
(C6D6, 100 MHz, 258C): d=196.5, 169.6, 166.1, 161.3, 146.0, 140.5, 138.8,
132.1, 130.0, 128.6(”2), 127.3, 126.6(”2), 126.3, 112.2, 105.9, 103.0, 77.1,
48.6, 38.4, 30.9, 30.3 ppm; HRMS (ESI): m/z: calcd for C23H22O5Na:
401.1359 [M+Na]+; found: 401.1271.
8828
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ꢀ 2006Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2006, 12, 8819 – 8834

Diversity-Oriented Synthesis of Pochonins
FULL PAPER
1
[a]2D5 =+21.6( c=0.36in CHCl 3); H NMR (CDCl3, 400 MHz, 258C): d=
preparative TLC (silica gel, 25% EtOAc/cyclohexane) afforded 11
(~90%) as a mixture of two diastereoisomers 1:1.
12.43 (s, 1H), 6.74 (d, J=1.7 Hz, 1H), 6.73–6.65 (m, 1H), 6.48 (d, J=
1.7 Hz, 1H), 5.92 (d, J=15.8 Hz, 1H), 5.12–
1
Selected example of compounds 11: H NMR ((CD3)2CO, 400 MHz): d=
12.30 (s, 2H), 11.43 (s, 2H), 6.75 (s, 2H), 6.00 (brdd, J=6.4, 6.2 Hz, 1H),
5.00 (m, 2H), 4.91–4.80 (m, 1H), 4.19 (d, J=
17.0 Hz, 1H), 3.84 (d, J=16.4 Hz, 1H), 2.77
(m, 1H), 2.64–2.57 (m, 1H), 2.01–1.97 (m,
1H), 1.89–1.70 (m, 3H), 1.61–1.56 (m, 2H),
1.30–1.21 (m, 2H), 0.90 ppm (t, J=6.7 Hz,
3H), para-phenol not detected; 13C NMR
(CDCl3, 100 MHz, 258C): d=197.5, 169.9,
165.6, 160.6, 147.5, 140.2, 131.9, 129.5, 127.0,
112.8, 106.1, 102.9, 76.2, 48.7, 35.7, 34.3, 31.1, 29.7, 19.4, 13.8 ppm; HRMS
(ESI-TOF): m/z: calcd for C20H25O5: 345.1697 [M+H]+; found: 345.1739.
[a]2D5 =À45.1 (c=0.27 in CHCl3); 1H NMR (CD3OD, 400 MHz, 258C):
d=6.78–6.71 (m, 1H), 6.29 (d, J=2.4 Hz, 1H), 6.22 (d, J=2.0 Hz, 1H),
5.87 (d, J=15.5 Hz, 1H), 5.37–5.23 (m, 3H),
5.97 (brdd, J=6.4, 6.2 Hz, 1H), 5.97 (brd, J=6.7 Hz, 1H), 5.77 (brd, J=
6.7 Hz, 1H), 5.57–5.48 (m, 4H), 5.18–5.14 (m, 2H), 4.32–4.10 (m, 2H),
3.38–3.28 (m, 3H), 3.02 (dd, J=16.1, 10.5 Hz, 1H), 2.41–2.09 (m, 12H),
1.11 ppm (d, J=6.2 Hz, 6H), alcohols not detected; HRMS (ESI): m/z:
calcd for C18H21ClO5Na: 375.0970 [M+Na]+; found: 375.1029.
4.01 (d, J=17.2 Hz, 1H), 3.92 (d, J=17.0 Hz,
1H), 2.67–2.61 (m, 1H), 2.29–2.15 (m, 5H),
1.31 ppm (d, J=6.4 Hz, 3H), phenols not de-
tected; 13C NMR (CD3OD, 100 MHz, 258C):
Generalprocedure for the synthesis of compounds 12 : Ac2O (1.2 equiv),
morpholinomethyl polystyrene (1.2 equiv, 3.2 mmolgÀ1), and DMAP
(0.05 equiv) were added to a solution of the corresponding compound 10
(1.0 equiv) in DMF (0.02m) at 238C and the mixture was stirred for
30 min, followed by TLC until consumption of the starting material.
Then, the resin was filtered and the organic phase was concentrated
under reduced pressure. Purification by PTLC (silica gel, 20% EtOAc/
cyclohexane) afforded corresponding compound 12 (~80%) as a mixture
of two diastereoisomers 1:1.
d=198.5, 169.8, 164.2, 162.3, 148.4, 139.1,
131.6, 129.6, 127.3, 111.7, 101.7, 72.0, 47.7,
36.8, 30.8, 30.7, 17.4 ppm, one quartenary
carbon is not detected; HRMS (ESI-TOF): m/z: calcd for C18H20O5Na:
339.1203 [M+Na]+; found: 339.1141.
1H NMR (CD3OD, 400 MHz, 258C): d=6.74–6.68 (m, 1H), 6.48 (s, 1H),
5.86(d, J=15.2 Hz, 1H), 5.31–5.25 (m, 2H), 4.39 (t, J=5.3 Hz, 2H), 4.27
(s, 2H), 2.43–2.40 (m, 2H), 2.25 ppm (m,
4H), phenols not detected; 13C NMR
(CD3OD, 100 MHz, 258C): d=196.9, 170.1,
161.9, 158.1, 147.8, 135.9, 130.9, 130.2, 129.9,
115.2, 107.3, 102.4, 65.9, 46.2, 31.3, 30.9,
30.5 ppm; HRMS (ESI): m/z: calcd for
C17H18O5Cl: 337.0837 [M+H]+; found:
337.0797.
Selected examples of compounds 12: 1H NMR (CDCl3, 400 MHz, 258C):
d=7.04 (s, 1H), 7.01 (s, 1H), 5.86(dd, J=15.0, 6.9 Hz, 1H), 5.67 (dd, J=
Generalprocedure for the synthesis of compounds 10 : BER-resin (boro-
hydride on Amberlite, 1.0 equiv, 2.5 mmolgÀ1) was added to a solution of
the corresponding compound 6 (1.0 equiv) in MeOH (0.03m) at 08C and
the reaction was stirred over 12 h. The reaction was then filtered and
concentrated under reduced pressure. Purification by flash chromatogra-
phy (silica gel, 0–20% EtOAc/cyclohexane gradient) afforded 10 (~60%)
as a mixture of two diastereoisomers 1:1.
12.4, 6.2 Hz, 1H), 5.60–5.54 (m, 4H), 5.48 (dd, J=7.2, 7.2 Hz, 1H), 5.41–
5.34 (m, 3H), 5.32–5.30 (m, 4H), 5.28–5.23 (m, 2H), 5.21 (dd, J=11.0,
6.7 Hz, 2H), 5.17 (dd, J=11.8, 6.9 Hz, 2H), 3.81–3.69 (m, 8H), 3.43 (dd,
J=14.2, 7.5 Hz, 1H), 3.23–3.15 (m, 2H), 2.85 (dd, J=13.9, 5.4 Hz, 1H),
2.30–2.17 (m, 8H), 2.12 (s, 3H), 2.06(s, 3H), 1.95–2.00 (m, 4H), 1.39 (2”
d, J=5.6Hz, 6H), 1.24 ppm (m, 12H); HRMS (ESI): m/z: calcd for
C26H35ClO8Na: 533.1913 [M+Na]+; found. 533.1864.
Selected example of compounds 10: 1H NMR (CDCl3, 400 MHz, 258C):
d=7.05 (s, 1H), 6.99 (s, 1H), 5.64–5.57 (m, 2H), 5.54–5.53 (m, 2H), 5.49–
Generalprocedure for the synthesis of compounds 13
: PS-TsOH
(10.0 equiv, 3.2 mmolgÀ1) was added to a solution of corresponding com-
pound 12 (1.0 equiv) in MeOH (0.02m) and the suspension was shaken at
408C for 4 h. After this time, the reaction mixture was filtered and the
methanolic solution concentrated under reduced pressure. Purification by
PTLC (silica gel, 20% EtOAc/cyclohexane) afforded compounds 13
(~60% yield).
Selected example of compounds 13: Mixture of diastereoisomers 2:1;
1H NMR (CDCl3, 400 MHz): d=12.6(s, 1H), 12.12 (s, 0.5H), 6.93 (d, J=
5.35 (m, 7H), 5.31–5.28 (m, 4H), 5.24–5.16(m, 4H), 5.13–5.08 (m, 1H),
4.68 (m, 1H), 4.56 (m, 1H), 3.81–3.69 (m, 8H), 3.25 (dd, J=13.9, 8.0 Hz,
1H), 3.19 (dd, J=13.7, 4.8 Hz, 1H), 3.11 (dd, J=13.5, 10.1 Hz, 1H), 2.90
(dd, J=13.9, 5.12 Hz, 1H, 35), 2.35 (m, 9H), 2.09–1.95 (m, 1H), 1.80–
1.70 (m, 2H), 1.39 (d, J=2.9 Hz, 3H), 1.37 (d, J=3.2 Hz, 3H), 1.24 ppm
(2”q, J=6.9, 5.0 Hz, 12H; 35+35’); HRMS (ESI): m/z: C24H33ClO7Na:
491.1807 [M+Na]+; found: 491.1729.
Generalprocedure for the synthesis of compounds 11
: PS-TsOH
(10.0 equiv, 3.2 mmolgÀ1) was added to a solution of the corresponding
compound 10 (1.0 equiv) in MeOH (0.02m) and the suspension was
shaken at 408C for 4 h. The reaction mixture was then filtered and the
methanolic solution concentrated under reduced pressure. Purification by
8.7 Hz, 0.5H), 6.66 (s, 1H), 6.64 (s, 0.5H), 6.62–6.60 (m, 1H), 6.10–6.05
(m, 3H), 5.47–5.33 (m, 5H), 2.60–2.53 (m, 1.5H), 2.26–2.02 (m, 7.5H),
1.44 (d, J=6.2 Hz, 1.5H), 1.43 ppm (d, J=6.4 Hz, 3H), para-phenol not
Chem. Eur. J. 2006, 12, 8819 – 8834
ꢀ 2006Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
8829

N. Winssinger et al.
detected; HRMS (ESI) m/z: calcd for C18H19ClO4Na: 357.0864 [M+Na]+
; found: 357.0898.
Generalprocedure for the preparation of compounds 14 : To a solution
of corresponding compound 6 (1.0 equiv) in methanol (0.03m) was added
sulfamic acid resin (10.0 equiv) and the suspension was stirred for 15 h at
408C. The reaction was then filtered and the resin washed several times
with CH2Cl2. Concentration under reduced pressure followed by purifica-
tion on PTLC (Hexane/EtOAc 1:1) afforded desired compounds 14 as a
mixture of diastereoisomers (2:1).
Selected example of compounds 14: 1H NMR (C6D6, 400 MHz, 258C):
d=12.28 (s, 0.4H), 11.91 (s, 0.6H), 7.21–7.11 (m, 5H), 6.62 (s, 1H), 6.03–
6.01 (m, 1H), 5.58 (brs, 1H), 5.38–5.33 (m, 1H), 5.27–5.20 (m, 1H), 4.76
2.76–2.69 (m, 1H), 2.38–2.05 (m, 11H), 1.42 (d, J=6.2 Hz, 3H), 1.35 ppm
(d, J=6.3 Hz, 3H); monoallylated compound: HRMS (ESI): m/z: calcd
for C21H23ClO5Na 413.1132 [M+Na]+; found: 413.1103; bisallylated com-
pound: HRMS (ESI): m/z: calcd for C24H27ClO5Na: 453.1449 [M+Na]+;
found: 453.1422.
Generalprocedure for the synthesis of compounds 17 : TBD-methyl poly-
styrene (2.0 equiv, 2.9 mmolgÀ1) and the corresponding alkyl bromide or
chloride (BrCH2COOtBu, EOMCl, 0.9 equiv) were added to a solution
of the corresponding compound 9 (1.0 equiv) in CH2Cl2 (0.05m) at 238C
and the mixture was shaken for 3 h. The resin was then filtered and the
filtrates were concentrated under reduced pressure. Purification by
PTLC (silica gel, 30% EtOAc/cyclohexane) afforded corresponding com-
pound 17 (>90%).
Selected examples of compounds 17: 1H NMR (CDCl3, 400 MHz): d=
11.84 (s, 1H), 6.69 (m, 1H), 6.41 (s, 1H), 5.76 (d, J=15.0 Hz, 1H), 5.43
(d, J=17.5 Hz, 0.6H), 4.02 (d, J=17.0 Hz, 0.4H), 4.18 (d, J=18.1 Hz,
0.6H), 4.09 (d, J=17.0 Hz, 0.4H), 3.87 (brs, 0.4H), 3.81 (brs, 0.6H), 3.15
(s, 1.8H), 3.12 (s, 1.2H), 2.83–2.78 (m, 1H), 2.45–2.30 (m, 2H), 2.18–2.16
(m, 1H), 2.02–1.97 (m, 2H), 1.79–1.72 ppm (m, 2H); HRMS (ESI-TOF):
m/z: calcd for C24H25O6ClNa: 467.1232 [M+Na]+; found: 467.1366.
Generalprocedure for the synthesis of compounds 15 : (Polystyrylme-
thyl)trimethylammonium cyanoborohydride (2.0 equiv, 3.5 mmolgÀ1) was
added to a solution of corresponding compound 9 (1.0 equiv) in CH2Cl2/
AcOH 10:1 (0.08m) at 238C and the reaction was monitored by TLC
until the starting material had been consumed (4 h). Then, the resin was
filtered and the organic phase was concentrated under reduced pressure.
Purification by PTLC (silica gel, 30% EtOAc/cyclohexane) afforded
compounds 15 (50–60%).
Selected example of compounds 15: 1H NMR (CDCl3, 400 MHz): d=
11.75 (s, 1H), 6.65 (s, 1H), 5.48 (m, 2H), 5.49 (ddt, J=6.1, 3.5, 2.9 Hz,
1H), 4.53 (d, J=17.5 Hz, 1H), 4.04 (d, J=17.7 Hz, 1H), 2.61–2.54 (m,
(m, 1H), 5,26(ddd, J=15.0, 9.1, 4.8 Hz, 1H), 5.18–5.11 (m, 1H), 4.65 (s,
2H), 4.33 (d, J=17.7 Hz, 1H), 4.16(d, J=17.5 Hz, 1H), 2.65–2.58 (m,
1H), 2.37–2.34 (m, 2H), 2.25–2.21 (m, 1H), 2.12–2.01 (m, 2H), 1.53 (s,
9H), 1.34 ppm (d, J=6.5 Hz, 3H); HRMS (ESI): m/z: calcd for
C24H29ClO7Na: 487.1494 [M+Na]+; found: 487.1498.
1H NMR (C6D6, 400 MHz, 258C): d=11.76 (s, 1H), 6.86 (s, 1H), 6.70 (dt,
J=14.9, 7.3 Hz, 1H), 5.77 (d, J=15.8 Hz, 1H), 5.46–5.42 (m, 1H), 5.37 (s,
2H), 5.30–5.19 (m, 2H), 4.34 (d, J=17.6Hz, 1H), 4.16 (d, J=18.1 Hz,
1H), 3.80 (q, J=7.0 Hz, 2H), 2.66–2.59 (m, 1H), 2.37–2.34 (m, 2H),
2.26–2.21 (m, 1H), 2.13–2.06 (m, 2H), 1.34 (d, J=6.4 Hz, 3H), 1.27 ppm
(t, J=7.0 Hz, 3H); HRMS (ESI): m/z: calcd for C21H25O6ClNa: 431.1237
[M+Na]+; found: 431.1257.
2H), 2.48–2.28 (m, 3H), 2.19–2.14 (m, 1H), 2.08–1.99 (m, 1H), 1.72–1.61
(m, 3H), 1.41 ppm (d, J=6.4 Hz, 3H), para-phenol not detected; HRMS
(ESI): m/z: calcd for C18H21ClO5Na: 375.0970 [M+Na]+; found:
375.1050.
Generalprocedure for the synthesis of compounds 16 : The corresponding
alcohol (2.0 equiv), triphenylphosphine (2.0 equiv), and ethoxycarbonyla-
zocarboxymethyl polystyrene (2.0 equiv, 1.3 mmolgÀ1) were added to a
solution of corresponding compound 9 (1.0 equiv) in THF (0.05m) in a
sequential manner. The reaction mixture was shaken at room tempera-
ture for 8 h, and then the resin was filtered and the filtrates were directly
purified by PTLC (silica gel, 10% EtOAc/cyclohexane) to afford a mix-
ture of compound 16 along with the bisallylated product (78%).
Generalprocedure for the synthesis of compounds 18 : OsO4 (0.1 equiv)
followed by NMO (1.0 equiv) was added to a solution of compound 9
(1.0 equiv) in acetone/H2O 10:1 (0.05m) at 238C and the mixture was stir-
red for 1 h. The crude mixture was filtered through a plug of silica, con-
centrated, and purified by PTLC (silica gel, 30% EtOAc/cyclohexane) to
afford 18 (>70%) as a mixture of two diastereoisomers 1:1.
Selected example of compounds 18: 1H NMR (CD3OD, 400 MHz): d=
7.19 (m, 1H), 6.89–6.81 (m, 1H), 6.52 (s, 1H), 6.47 (s, 1H), 6.20 (d, J=
16.1 Hz, 1H), 6.04 (d, J=15.6Hz, 1H), 5.54–5.49 (m, 1H), 5.43–5.36(m,
1H), 4.50 (d, J=17.7 Hz, 1H), 4.46(d, J=17.7 Hz, 1H), 4.39 (d, J=
17.2 Hz, 1H), 4.07 (d, J=17.2 Hz, 1H), 3.80–3.64 (m, 2H), 3.51–3.46 (m,
2H), 2.62–2.58 (m, 2H), 2.39–2.30 (m, 2H), 2.27–2.18 (m, 2H), 2.08–2.98
(m, 2H), 2.00–1.85 (m, 4H), 1.44 ppm (d, J=6.4 Hz, 6H), phenols and al-
Selected example of compounds 16: Mixture with the corresponding bi-
sallylated compound 1:1; 1H NMR (CDCl3, 400 MHz): d=11.83 (s, 1H),
6.82 (ddd, J=15.7, 8.2, 4.6 Hz, 1H), 6.72–6.65 (m, 1H), 6.46 (s, 1H), 6.41
(s, 1H), 6.09–5.98 (m, 3H), 5.82 (d, J=15.7 Hz, 1H), 5.46–5.16 (m, 8H),
4.57–4.54 (m, 3H), 4.51–4.49 (m, 3H), 4.19 (d, J=17.5 Hz, 1H), 4.11 (d,
J=14.6Hz, 1H), 3.78 (d, J=17.0 Hz, 1H), 3.51 (d, J=14.2 Hz, 1H),
8830
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Chem. Eur. J. 2006, 12, 8819 – 8834

Diversity-Oriented Synthesis of Pochonins
FULL PAPER
tected; HRMS (ESI): m/z: calcd for C23H21O6ClNa: 451.0919 [M+Na]+;
found: 451.1028.
cohol not detected; HRMS (ESI) m/z: calcd for C18H21ClO7Na: 407.0868
[M+Na]+; found: 407.1031.
Generalprocedure for the synthesis of compounds 19 : Freshly made
DMDO (1.2 equiv, 0.04m in acetone) was added to a solution of com-
pound 9 (1.0 equiv) in CH3CN (0.03m) at 08C and the mixture was stir-
red for 30 min. After evaporation of the solvents under reduced pressure,
purification by PTLC (silica gel, 30% EtOAc/cyclohexane) afforded ep-
oxides 19 (>90%) as a mixture of two diastereoisomers (1:1 to 3:1).
Major isomer; 1H NMR (C6D6, 400 MHz, 258C): d=11.94 (s, 1H), 7.36–
7.28 (m, 5H), 6.95–6.88 (m, 1H), 6.42 (s, 1H), 6.22 (s, 1H), 6.11 (d, J=
15.8 Hz, 1H), 5.47 (m, 1H), 5.41 (brs, 1H), 4.43 (d, J=17.5 Hz, 1H), 3.56
(d, J=17.6Hz, 1H), 3.19 (dd, J=13.7, 6.0 Hz, 1H), 3.03 (dd, J=13.7,
7.9 Hz, 1H), 2.87 (brs, 1H), 2.70–2.28 (m, 4H), 2.03–1.93 ppm (m, 2H),
para-phenol not detected; HRMS (ESI): m/z: calcd for C24H24O6Na:
431.1465 [M+Na]+; found: 431.1578.
Selected example of compounds 19: 1H NMR (CDCl3, 400 MHz): d=
11.84 (s, 2H), 6.94–6.82 (m, 2H), 6.69 (s, 1H), 6.66 (s, 1H), 6.23 (d, J=
17.1 Hz, 1H), 6.11 (dd, J=13.2, 1.6Hz, 1H), 5.39 (tdd, J=7.5, 3.2,
2.7 Hz, 1H), 5.32 (m, 1H), 4.53 (d, J=17.7 Hz, 2H), 4.27 (d, J=17.7 Hz,
2H), 2.79–2.76(m, 1H), 2.74–2.69 (m, 1H), 2.58 (m, 1H), 2.56(m, 1H),
2.47–2.24 (m, 8H), 2.13–2.08 (m, 1H), 2.05–2.03 (m, 1H), 1.91 (dd, J=
4.3, 4.3 Hz, 1H), 1.87 (dd, J=4.3, 4.3 Hz, 1H), 1.51 (d, J=6.4 Hz, 3H),
1.35 ppm (d, J=6.4 Hz, 3H), para-phenol not detected; HRMS (ESI):
m/z: calcd for C18H19ClO6Na: 389.0762 [M+Na]+; found: 389.0724.
1H NMR (C6D6, 400 MHz, 258C): d=11.98 (s, 1H), 6.91–6.83 (m, 1H),
6.43 (d, J=2.3 Hz, 1H), 6.24 (d, J=2.4 Hz, 1H), 6.11 (d, J=15.8 Hz,
1H), 5.35 (brs, 1H), 5.29 (m, 1H), 4.52 (d, J=17.5 Hz, 1H), 3.63 (d, J=
17.5 Hz, 1H), 2.77 (m, 2H), 2.57–2.52 (m, 2H), 2.46–2.27 (m, 2H), 2.14–
2.10 (m, 1H), 1.93–1.88 (m, 1H), 1.48 ppm (d, J=6.4 Hz, 3H); other
1
isomer: H NMR (C6D6, 400 MHz, 258C): d=11.67 (s, 1H), 6.89–6.83 (m,
1H), 6.40 (d, J=2.4 Hz, 1H), 6.24 (d, J=2.9 Hz, 1H), 6.21 (d, J=
16.4 Hz, 1H), 5.37 (brs, 1H), 5.22 (m, 1H), 4.20 (d, J=17.0 Hz, 1H), 4.06
(d, J=17.0 Hz, 1H), 2.74 (m, 2H), 2.57–2.20 (m, 4H), 1.80–1.76(m, 1H),
1.68–1.60 (m, 1H), 1.37 ppm (d, J=6.4 Hz, 3H), para-phenol not detect-
ed; HRMS (ESI): m/z: calcd for C18H20O6Na: 355.1152 [M+Na]+; found:
355.1249.
Generalprocedure for the preparation of macrocycels 20 : HCl (concd,
20 equiv) was added to a solution of compound 6 (1.0 equiv) in dioxane
(0.05m) at 238C, and the mixture was stirred for 3 h. After this time, the
reaction was filtered through a plug of silica gel, the solvents evaporated
under reduced pressure, and purified by PTLC (silica gel, 30% EtOAc/
cyclohexane) to afford compound 20 (>75%) as a mixture of two dia-
stereoisomers 1:1.
Selected examples of compounds 20: 1H NMR (CDCl3, 400 MHz): d=
12.11 (s, 1H), 11.78 (s, 1H), 6.51 (s, 1H), 6.43 (s, 1H), 6.41 (d, J=2.4 Hz,
1H), 6.37 (d, J=2.7 Hz, 1H), 6.21 (d, J=2.4 Hz, 1H), 6.11 (d, J=2.4 Hz,
1H NMR (C6D6, 400 MHz, 258C): d=11.56 (2”s, 2H), 6.92–6.82 (m,
2H), 6.71 (s, 1H), 6.67 (s, 1H), 6.20 (m, 3H), 6.06 (d, J=15.8 Hz, 1H),
5.11 (brs, 1H), 5.94 (m, 1H), 4.46(2”d, J=18.1 Hz, 2H), 4.20 (2”d, J=
18.1 Hz, 2H), 2.72–2.70 (m, 2H), 2.53–2.48 (m, 4H), 2.38–2.35 (m, 3H),
2.25–2.13 (m, 5H), 1.84–1.77 (m, 2H), 1.05–1.01 (m, 6H), 0.91–0.88 (m,
3H), 0.86–0.84 ppm (m, 3H), para-phenol not detected; HRMS (ESI):
m/z: calcd for C20H23O6ClNa 417.1075 [M+Na]+; found: 417.1128.
1H NMR (C6D6, 400 MHz, 258C): d=11.80 (2”s, 2H), 7.43–7.18 (m,
10H), 7.03–6.95 (m, 2H), 6.69 (s, 1H), 6.61 (s, 1H), 6.30 (d, J=16.4 Hz,
1H), 6.21 (d, J=15.8 Hz, 1H), 6.15–6.10 (m, 1H), 6.03 (d, J=11.1 Hz,
1H), 4.84 (2”d, J=18.1 Hz, 2H), 4.41 (2”d, J=17.6 Hz, 2H), 2.68–2.60
(m, 4H), 2.41–2.27 (m, 8H), 1.83–1.76ppm (m, 4H), para-phenol not de-
1H), 5.59–5.51 (m, 3H), 5.40–5.32 (m, 3H), 4.54 (d, J=17.2 Hz, 1H),
4.42 (d, J=17.2 Hz, 1H), 3.60 (d, J=17.2 Hz, 1H), 3.45 (d, J=17.0 Hz,
1H), 3.28 (dd, J=18.5, 9.4 Hz, 1H), 3.11 (dd, J=13.7, 6.2 Hz, 1H), 3.07
(dd, J=13.4, 4.6Hz, 1H), 2.76 (dd, J=19.0, 6.2 Hz, 1H), 2.62 (ddd, J=
15.5, 8.8, 4.0 Hz, 1H), 2.54 (ddd, J=15.3, 6.2, 3.2 Hz, 1H), 2.40–2.26 (m,
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4H), 2.25–2.13 (m, 4H), 2.03–1.91 (m, 2H), 1.42 (d, J=6.4 Hz, 3H),
1.40 ppm (d, J=6.4 Hz, 3H), para-phenol not detected; HRMS (ESI):
m/z: calcd for C18H21ClO5Na: 375.0970 [M+Na]+; found: 375.0928.
14H), 4.50 (d, J=17.2 Hz, 1H), 3.38–3.65 (m, 8H), 3.60 (d, J=17.1 Hz,
1H), 3.54 (d, J=17.1 Hz, 1H), 3.24 (d, J=17.2 Hz, 1H), 2.48–2.36(m,
4H), 2.17–2.21 (m, 2H), 2.04–2.11 (m, 2H), 1.95–1.83 (m, 2H), 1.62–1.51
(m, 2H), 1.49 (d, J=6.4 Hz, 6H), 1.20–1.32 ppm (m, 12H); 13C NMR
(CDCl3, 100 MHz): d=168.02, 167.85, 159.08, 158.83, 157.23, 155.55,
155.36, 154.19, 140.75, 138.23, 138.19, 137.75, 136.93, 136.74, 132.32,
132.28, 128.34 (”2), 128.31 (”2), 128.18, 128.09 (”2), 127.99 (”2), 127.71,
127.63, 125.50, 118.82, 118.56, 118.34, 108.84, 108.50, 101.72, 101.68, 93.49,
93.44, 93.12 (”2), 77.21, 76.02, 75.88, 71.18, 70.99, 64.47, 64.45, 64.33,
64.31, 39.99, 39.96, 34.87, 32.42, 32.31, 31.63, 31.09, 28.86, 20.25, 20.19,
15.04 (”2), 14.98 ppm (”2); HRMS (ESI): m/z: calcd for C31H39NO7Na:
560.2619 [M+Na]+; found: 560.2627.
1H NMR (C6D6, 400 MHz, 258C): d=11.76(s, 0.5H), 11.36(s, 0.5H),
7.40–7.29 (m, 5H), 6.65 (s, 0.5H), 6.62 (s, 0.5H), 6.18 (t, J=5.8 Hz, 1H),
6.14 (s, 0.5H), 6.12 (s, 0.5H), 5.67–5.62 (m, 1H), 5.55–5.49 (m, 1H), 4.93
(d, J=18.1 Hz, 0.5H), 4.80 (d, J=17.1 Hz, 0.5H), 4.58–4.56(m, 1H), 4.38
(d, J=18.1 Hz, 0.5H), 4.18 (d, J=17.1 Hz, 0.5H), 3.33–3.27 (m, 1H), 3.10
(dd, J=18.4, 3.8 Hz, 0.5H), 2.84–2.68 (m, 2.5H), 2.42–2.32 (m, 2H), 2.23–
2.17 (m, 1H), 2.13–2.04 ppm (m, 1H), para-phenol not detected; HRMS
(ESI-TOF): m/z: calcd for C23H22O5Cl2Na: 471.0737 [M+Na]+; found:
471.0754.
Generalprocedure for the synthesis of compounds 23
: PS-TsOH
(10.0 equiv, 3.2 mmolgÀ1
)
was added to solution of compound 22
a
(1.0 equiv) in MeOH (0.02m) and the suspension was shaken at 408C for
4 h. After this time, the reaction mixture was filtered and the methanolic
solution concentrated under reduced pressure. The crude product ob-
tained was submitted without further purification to the next step. Thus
DHP (1.0 equiv) and PS-TsOH (cat, 3.2 mmolgÀ1) were added to a solu-
tion of this crude in CH2Cl2 (0.02m) at 238C, and the mixture was stirred
for 5 h. After this time, the mixture was filtered, the solvents were evapo-
rated under reduced pressure, and the remaining residue was purified by
PTLC (silica gel, 30% EtOAc/cyclohexane) to afford two different dia-
stereoisomers 1:1 of 23 (~65%).
Elimination of b-Clfrom compound 20 : PS-TBD (51 mg, 2.6mmolg À1
)
was added to a solution of compound 20 (95 mg, 270 mmol) in CH2Cl2
(5 mL) at 238C, and the mixture was stirred for 8 h. After that time, the
reaction was filtered, the solvents were evaporated under reduced pres-
sure, and the remaining residue was purified by flash chromatography
(silica gel, 0–30% EtOAc/cyclohexane gradient) to afford 9 (X=Cl, R=
Me, 84 mg, 98%).
Macrocycle 21: DHP (3.7 mL, 40.8 mmol) and PS-TsOH (12.7 mg,
40.8 mmol, 3.2 mmolgÀ1) were added to a solution of compound 9 (X=
Cl, R=Me, 12.9 mg, 40.8 mmol) in CH2Cl2 (1 mL) at 238C, and the mix-
ture was stirred for 5 h. After this time, the reaction was filtered and the
solvents were evaporated under reduced pressure. Purification by PTLC
(silica gel, 30% EtOAc/cyclohexane) afforded 21 (13.8 mg, 85%) as a
mixture of two diastereoisomers.
Selected examples of compounds 23: Less polar diastereoisomers;
1H NMR (CDCl3, 400 MHz, 258C): d=9.25 (s, 1H), 9.24 (s, 1H), 7.46–
1H NMR (CDCl3, 400 MHz): d=12.33 (s, 1H), 12.11 (s, 1H), 9.45 (s,
1H), 9.40 (s, 1H), 6.67, (m, 2H), 6.28 (2”s, 2H), 5.83 (d, J=13.2 Hz,
1H), 5.79 (d, J=12.9 Hz, 1H), 5.35–5.30 (m, 3H), 5.27–5.22 (m, 3H),
5.06(brd, J=8.2 Hz, 2H), 4.10 (d, J=17.5 Hz, 2H), 3.90–3.85 (m, 1H),
3.80–3.76(m, 1H), 3.65 (d, J=17.7 Hz, 2H), 3.57–3.52 (m, 2H), 3.46–3.41
(m, 2H), 2.77–2.71 (m, 3H), 2.53–2.49 (m, 3H), 2.36–2.29 (m, 4H), 2.24–
1.56(m, 12H), 1.31 (d, J=6.4 Hz, 3H), 1.28 ppm (d, J=6.4 Hz, 3H);
HRMS (ESI): m/z: C23H28O6Na: 423.1778 [M+Na]+; found: 423.1778.
7.33 (m, 10H), 6.29 (s, 1H), 6.26 (s, 1H), 6.07–6.02 (m, 2H), 5.75 (d, J=
15.8 Hz, 1H), 5.69 (d, J=15.8 Hz, 1H), 5.44–5.38 (m, 6H), 5.23 (s, 4H),
5.03 (d, J=8.8 Hz, 2H), 4.34–4.13 (m, 6H), 3.69–3.63 (m, 2H), 2.70–2.67
(m, 2H), 2.30–2.16(m, 6H), 2.08–1.94 (m, 8H), 1.73–1.65 (m, 8H), 1.42
(t, J=6.4 Hz, 3H), 1.39 ppm (t, J=7.0 Hz, 3H); HRMS (ESI): m/z: calcd
for C30H35NO6Na: 528.2357 [M+Na]+; found: 528.2562.
More polar diastereoisomers: 1H NMR (CDCl3, 400 MHz, 258C): d=
11.61 (s, 1H), 9.27 (s, 1H), 7.41–7.33 (m, 5H), 6.62 (d, J=16.4 Hz, 1H),
6.47 (s, 1H), 6.15–6.07 (m, 1H), 5.50–5.38 (m, 3H), 5.16 (s, 2H), 5.04 (d,
J=10.5 Hz, 1H), 4.30 (d, J=15.2 Hz, 1H), 4.24 (d, J=10.5 Hz, 1H), 3.84
(d, J=15.2 Hz, 1H), 3.66 (t, J=11.4 Hz, 1H), 2.71–2.65 (m, 1H), 2.28–
2.08 (m, 6H), 1.73–1.64 (m, 5H), 1.38 ppm (t, J=7.0 Hz, 3H); HRMS
(ESI): m/z: calcd for C30H35NO6Na: 528.2357 [M+Na]+; found: 528.2494.
Generalprocedure for the synthesis of compounds 22 : The corresponding
hydroxylamine (5.0 equiv) was added to a solution of corresponding com-
pound 6 (1.0 equiv) in pyridine/AcOH (5:1, 0.03m) and the mixture was
heated up to 408C. After stirring overnight, the solvents were evaporated
under reduced pressure with silica gel. Elution of the compound over a
short path of silica gel with a mixture of 30% EtOAc/cyclohexane afford-
ed after evaporation 22 (~99%) as a mixture of two diastereoisomers cis/
trans).
Selected example of compounds 22: 1H NMR (CDCl3, 400 MHz): d=
7.50–7.25 (m, 10H), 6.82 (s, 1H), 6.75 (s, 1H), 6.66 (s, 1H), 6.48 (s, 1H),
6.24–6.11 (m, 2H), 6.11–6.05 (m, 2H), 5.45–5.38 (m, 4H), 5.34–5.31 (m,
Macrocycle 24: TBSCl (53.6mg, 356 mmol) and imidazole (23.6mg, 356
mmol) were added to a solution of pochonin D (9, X=Cl and R=Me,
25 mg, 71.2 mmol) in DMF (5 mL) and the mixture was stirred for 3 h at
room temperature. Purification by column chromatography (silica gel, 0–
30% EtOAc/cyclohexane gradient) afforded after evaporation 24 (40 mg,
98%).
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Diversity-Oriented Synthesis of Pochonins
FULL PAPER
J=6.4 Hz, 3H); HRMS (ESI): m/z: calcd for C25H26ClNO5Na: 478.1392;
found: 478.1522 [M+Na]+.
Kinase assay: All protein kinases were expressed in Sf9 insect cells as
human recombinant GST-fusion proteins or His-tagged proteins by
means of the baculovirus expression system. Kinases were purified by af-
finity chromatography using either GSH-agarose (Sigma) or Ni-NTH-
agarose (Qiagen). The purity of each kinase was checked by SDS-PAGE/
silver staining and the identity of each kinase was verified by western
blot analysis with kinase-specific antibodies or by mass spectroscopy. For
measuring the enzymatic activity of the protein kinases a proprietary pro-
tein kinase assay (33PanQinaseꢂ Activity Assay) was used. All kinase
assays were performed in 96-well FlashPlates from Perkin–Elmer/NEN
(Boston, MA, USA) in a 50 mL reaction volume by using a Beckman-
Coulter/Sagian robotic system. The reaction cocktail was pipetted in four
steps in the following order: 1) 20 mL of assay buffer, 2) 5 mL of ATP sol-
ution (in H2O), 3) 5 mL of test compound (in 10% DMSO), and 4) 10 mL
of substrate/10 mL of enzyme solution (premixed). The assay for all kinas-
es contained HEPES-NaOH (60 mm), MgCl2 (pH 7.5, 3 mm), MnCl2
(3 mm), Na-orthovanadate (3 mm), DTT (1.2 mm), PEG20000 (50 mgmLÀ1),
[g-33P]-ATP (1 mm, ca. 5”105 cpm per well). The final DMSO concentra-
tion was 1% in all assays. The reaction cocktails were incubated at 308C
for 80 minutes. The reaction was stopped with H3PO4 (50 mL, 2% v/v).
Plates were aspirated and washed two times with H2O (200 mL) and
NaCl (200 mL, 0.9% w/v). Incorporation of 33Pi was determined with a
microplate scintillation counter (Microbeta, Wallac).
1H NMR (CDCl3, 400 MHz, 258C): d=6.71 (dt, J=15.3, 7.3 Hz, 1H),
6.45 (s, 1H), 5.81 (d, J=15.3 Hz, 1H), 5.25 (s, 2H), 5.04–5.03 (m, 1H),
3.89 (d, J=17.4 Hz, 1H), 3.57 (d, J=17.4 Hz, 1H), 2.31–2.04 (m, 6H),
1.35 (d, J=6.4 Hz, 3H), 1.03 (s, 9H), 0.99 (s, 9H), 0.28–0.24 ppm (m,
12H); 13C NMR (CDCl3, 100 MHz, 258C): d=195.8, 166.8, 152.9, 151.7,
146.5, 132.7, 131.9, 128.6, 126.8, 122.8, 119.7, 110.7, 71.9, 45.6, 38.5, 30.9,
25.7 (”4), 25.6(”4), 18.7, 18.3, À4.1 (”2), À4.4 ppm (”2); HRMS (ESI):
m/z: calcd for C30H47ClO5Si2Na: 601.2543 [M+Na]+; found: 601.2568.
Generalprocedure for compounds 25 : The corresponding hydroxylamine
(5.0 equiv) was added to a solution of compound 24 (1.0 equiv) in pyri-
dine/AcOH (5:1, 250 mL), and the mixture was heated up to 408C. After
stirring overnight, the solvents were evaporated under reduced pressure,
and filtration through silica gel with a mixture of 30% EtOAc/cyclohex-
ane afforded after evaporation two isomers of 25 (~90%).
Selected example of compounds 25: cis Oxime: 1H NMR (CDCl3,
400 MHz): d=7.42 (brd, J=6.4 Hz, 2H), 7.36 (brdd, J=7.5, 6.9 Hz, 2H),
For each concentration of the test compounds residual activities (in %)
were calculated relative to control values without test compounds for
each kinase assay. With the set of residual activities (in %) obtained for
each test compound in a particular kinase assay, IC50 values were calcu-
lated by using Quattro Workflow V2.0.1.3 (Quattro Research GmbH,
Munich, Germany; www.quattro-research.com). The mathematical model
used was “sigmoidal response (variable slope)” with parameters “top”
fixed at 100% and “bottom” at 0%.
Acknowledgements
7.34–7.32 (m, 1H), 6.52 (d, J=16.1 Hz, 1H), 6.38 (s, 1H), 6.18–6.10 (m,
1H), 5.36–5.32 (m, 2H), 5.16 (brs, 2H), 4.99–4.95 (m, 1H), 3.79–3.76 (m,
2H), 2.40–1.99 (m, 6H), 1.45 (d, J=6.2 Hz, 3H), 1.03 (s, 9H), 0.99 (s,
9H), 0.28 (s, 3H), 0.26(s, 3H), 0.20 ppm (s, 6H); trans oxime: 1H NMR
(CDCl3, 400 MHz): d=7.44 (brd, J=6.5 Hz, 2H), 7.37 (brdd, J=7.6,
6.9 Hz, 2H), 7.33–7.31 (m, 1H), 6.41 (s, 1H), 6.04–5.97 (m, 1H), 5.48
(brd, J=15.0 Hz, 1H), 5.29–5.27 (m, 1H), 5.22 (brs, 2H), 5.00–4.95 (m,
1H), 3.98–3.89 (m, 2H), 2.39–2.02 (m, 6H), 1.37 (d, J=5.9 Hz, 3H), 1.04
(s, 9H), 0.99 (s, 9H), 0.28 (s, 3H), 0.27 (s, 3H), 0.23 (s, 3H), 0.22 ppm (s,
3H).
A BDI fellowship from the CNRS and the region Alsace to E.M. is
gratefully acknowledged. This work was partly supported by a grant from
the European Commission (MIRG-CT-2004-505317), the Association
pour la Recherche sur le Cancer (ARC), Agence Nationale de la Recher-
che (ANR) part of the COST network D28. The authors thank Pilar
Lopez, Pierre-Yves Dakas and Lise Brethous for their preliminary contri-
butions to this project.
Generalprocedure for compounds 26 : TBAF (2.5 equiv, 1m solution in
THF) was added to a solution of corresponding compound 25 (1.0 equiv)
in THF, and the mixture was stirred at room temperature for 2 h. The
solvents were then evaporated under reduced pressure, and filtration on
silica gel with a mixture of 30% EtOAc/cyclohexane afforded after evap-
oration, compounds 26 in >85% yield.
cis: 1H NMR (CDCl3, 400 MHz, 258C): d=11.52 (s, 1H), 7.45–7.34 (m,
5H), 6.64 (s, 1H), 6.09–6.02 (m, 2H), 5.34–5.25 (m, 4H), 5.18–5.08 (m,
2H), 4.33 (d, J=17.0 Hz, 1H), 4.15 (d, J=
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Received: April 19, 2006
Published online: September 5, 2006
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Chem. Eur. J. 2006, 12, 8819 – 8834