Function oriented synthesis: Preparation and initial biological evaluation of new A-ring-modified bryologs

Article abstract of DOI:10.1016/j.tet.2011.09.058

The synthesis and biological evaluation of the first members of a new series of designed bryostatin A-ring analogues (bryologs) are described. An advanced intermediate is produced that allows for step economical access to diverse analogs. The first of the

Full text of DOI:10.1016/j.tet.2011.09.058

Tetrahedron 67 (2011) 9998e10005
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Function oriented synthesis: preparation and initial biological evaluation of new
A-ring-modified bryologs
*
Paul A. Wender , Jenny Reuber
Department of Chemistry, Department of Chemical and Systems Biology, Stanford University, Stanford, CA 94305-5080, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 August 2011
Received in revised form 15 September 2011
Accepted 16 September 2011
Available online 22 September 2011
The synthesis and biological evaluation of the first members of a new series of designed bryostatin A-ring
analogues (bryologs) are described. An advanced intermediate is produced that allows for step eco-
nomical access to diverse analogs. The first of these analogues, bearing side chains of completely dif-
ferent polarities from alkyl to hydroxyl and carboxyl functionalities, were evaluated. All exhibit potent
protein kinase C binding (54.7e2.4 nM) with affinities increasing with decreasing side chain polarity.
This series of bryostatin analogues demonstrates that A-ring surrogates can indeed be used for tuning
pharmacophore and ADME characteristics as needed to improve bryolog function.
Keywords:
Bryostatin
PKC
Ó 2011 Elsevier Ltd. All rights reserved.
Bioactivity
Macrocycle
1. Introduction
The bryostatins are a structurally and biologically unique family
of macrocyclic lactones1 first collected from bryozoa2 in the Gulf of
Mexico in 1968 by Pettit and co-workers.3 In 1982, the structure of
bryostatin 1 (Fig.1), the lead member of this family, was established
by X-ray crystallography.4 Subsequent studies revealed that
bryostatin exhibits promising activity against several cancer cell
lines. Significantly, bryostatin promotes apoptosis,5 reverses mul-
tidrug resistance,6 and synergizes with other anticancer agents.7 In
contrast to many anticancer drugs, bryostatin stimulates the im-
mune system, thus providing the basis for a promising immuno-
therapeutic approach to cancer.8 Bryostatin has also been found to
facilitate learning and extend memory in animals9 and has recently
been entered into a clinical trial for treating Alzheimer’s disease.10
Remarkably, bryostatin also induces activation of latent HIV reser-
voirs, thus serving as a lead in efforts to eradicate HIV/AIDS.11
Bryostatin’s activities arise from binding to the C1 domain of
protein kinase C (PKC)12 and possibly other C1 domain targets.13
Unlike many kinase-targeting molecules that bind to the ATP
binding site and thus serve only to inhibit function, ligands tar-
geting the C1 domain can turn on or turn off function. Bryostatin
binds with high affinity (<10 nM) to conventional and novel PKC
isozymes.14 Since different isoforms are associated with various
therapeutic indications,15 bryostatin is a promising lead for drug
discovery.
Fig. 1. Bryostatin 1 and lead analogues (Ki¼binding affinity to rat brain PKC).
Despite this unique portfolio of biological activities, the study
and use of bryostatin has been impeded by its limited supply and
challenges associated with systematic modification of its complex
* Corresponding author. E-mail address: wenderp@stanford.edu (P.A. Wender).
0040-4020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2011.09.058

P.A. Wender, J. Reuber / Tetrahedron 67 (2011) 9998e10005
9999
structure. Low isolation yields (18 g are obtained from 14 tons of
bryozoa16) and environmental concerns about larger scale marine
harvesting make it impractical to obtain significant amounts from
natural sources. Early research by the groups of Masamune, Evans
and Yamamura led to impressive total syntheses of bryostatins 7, 2
and 3, respectively.17 These syntheses have, however, not been
further advanced and are currently too long (>70 steps) to impact
supply. Recent advances include a formal total synthesis of bryos-
tatin 7 by the Hale Group18 and a total synthesis of bryostatin 16 by
Trost and Dong.19 The Keck Group20 has also reported a notable
advance with an approximately 57-step synthesis of bryostatin 1
and recently the Krische Group21 described a 36-step synthesis of
bryostatin 7. Adam Schrier (Wender Group) recently reported
a scalable 42-step synthesis of bryostatin 9.22 Additional groups
have contributed substantially to this field, including those of
Thomas, Vandewalle, Roy, Burke, Hoffmann, and Yadav.23
Our A-ring analogues have been shown to translocate PKC
d
-GFP
from the cytosol to cellular membranes in RBL cells, an assay that
correlates with biological function.30 Analogue 4 in particular shows
a translocation profile similar to bryostatin.31 These significant
findings warranted further study of how this A-ring region could be
used to modulate activityand selectivity. It has been shown that non-
polar side chains are tolerated in this region,32 but little is known
about polar side chains. Polarity is however important, since bryos-
tatins1(OAc)and2(OH)differinPKC selectivity. Analogue 5 (Scheme
1) was chosen as a diversification node for accessing derivatives with
varying polarity in the A-ring region. Based on previous modeling
studies of 4 the meta-substitution of the phenyl-ring was expected to
favor contact with PKC in the vicinity of bryostatin’s C7-acetate.
2. Results and discussion
While efforts to synthesize natural bryostatins continue to im-
pressively advance, in the 1980s we24 took an alternative approach
to the goal of achieving more time- and step economical access to
molecules with bryostatin activity, that is, generally applicable to
many therapeutically promising complex molecules that are be-
yond the immediate reach of supply-impacting synthesis. Realizing
that the demand for bryostatin, not unlike that of many natural
products, is driven primarily by its function (i.e., activity) and not its
structure and that function is neither evolved nor optimized for
human therapy, we sought to use synthesis-informed design to
create simpler structures that would be more readily accessible and
yet exhibit similar if not superior activity to the natural lead. This
function oriented synthesis (FOS) approach25 has yielded to date
over 100 designed bryostatin analogues (bryologs), over 35 of
which have comparable or better affinities than bryostatin in
binding PKC and are synthetically accessible on scale in under 30
total steps.26 This FOS approach opens broad opportunities to ex-
plore the varied activities of tunable bryostatin-like compounds
and is expected to be a preferred strategy for those interested in its
function. Moreover, given supply and some off target clinical issues
with bryostatin, these analogs and related approaches could offer
more effective agents with fewer side effects. The potential of our
approach has indeed been recognized in recent clinical studies.7b
In computational studies of competitive PKC ligands, we pro-
posed that the affinity of bryostatin could arise in part from the
array of hydrogen bond donors and acceptors at C1, C19 and C26.26
Deletion of groups not in contact with PKC or that do not influence
conformational populations led to bryolog 2 (Fig. 1) with a PKC
affinity better than bryostatin and accessible in <30 steps.27 Further
simplification caused some loss in affinity (3) that was largely re-
stored in 4 by introduction of a conformation influencing phenyl
group at C9.28 Encouraged by the significant activity of 4, the cur-
rent study sought to create a diversifiable, advanced intermediate 5
(Scheme 1) that would allow systematic investigation of how var-
iations in this part of the molecule would affect PKC affinity with
the eventual goal of using this information to design selective PKC
regulators.29
Analogue 5 is synthesized in a highly convergent fashion by
coupling a top piece with a known bottom piece 727 in two steps
using a macrotransacetalization procedure (Scheme 1). The syn-
thesis of the C1-C13 spacer domain 6 (Scheme 2) began with
asymmetric allylation33 of 3-bromobenzaldehyde to give an alcohol
(92% enantiopurity, 76% yield) that on subsequent cyanoethylation
with acrylonitrile provided nitrile 8 in 98% yield. Blaise-type con-
ditions with Zn and catalytic Cp2TiCl2 converted the nitrile group in
8 into a b
-ketoester.34,35 Ozonolysis followed by addition of ethyl
diazoacetate and SnCl4 gave 9 via a Roskamp rearrangement.36
Asymmetric hydrogenation with (R)-BINOL/RuBr2 adjusted the
oxidation state of C3 and C11. HPLC analysis established that 10 was
obtained in 98% diastereomeric purity. Selective reduction of C13
was achieved with superhydride. Triol 10 was treated with excess
TBSOTf/2,6-lutidine, which silylated all three hydroxyl groups and
cleaved the tert-butyl ester functionality,37 affording acid 6 in 92%
yield. The synthesis of the new top piece 6 was thus achieved in
only 7 steps with an overall yield of 24%.
Scheme 2. Top piece synthesis. (a) (S)-BINOL/Ti(Oi-Pr)4 (10 mol %), B(OMe)3, Bu3Sn(allyl),
ꢀ
4 A MS, CH2Cl2, 76%; (b) acrylonitrile, Triton B in H2O, CH2Cl2, 98%; (c) t-Bu-bromoacetate,
Zn, cat. Cp2TiCl2, THF, 85%; (d) i. O3, PPh3 ii. ethyl diazoacetate, SnCl4, 63%; (e) ((R)-BINAP)
RuBr2, EtOH, H2, 80%; (f) Superhydride, THF, 81%; (g) TBSOTf, 2,6-lutidine, 92%.
The top and bottom pieces (6 and 7) were linked following
the Yamaguchi protocol via the mixed anhydride (Scheme 3).38
Subsequent addition of HF/pyridine cleaved the silyl protecting
groups and effected formation of the macrocyclic lactone 5 by
Scheme 3. Coupling and ring closure. (h) Et3N, 2,4,6-tribenzoyl chloride, then 7, DMAP,
toluene, 91%; (i) 70% HF/pyridine, 83%.
Scheme 1. Retrosynthetic analysis.

10000
P.A. Wender, J. Reuber / Tetrahedron 67 (2011) 9998e10005
Table 1
transacetalization. Analogue 5 was synthesized in 19 steps (lon-
gest linear sequence) and 2% overall yield.
Binding affinities for new A-ring bryologs (rat brain PKC)
The diversification of bryolog 5 was addressed next. While aryl
bromides are attractive groups for diversification, the presence in 5
of several sensitive groups including free alcohols, three esters, two
olefins (one a Michael acceptor), two acetals and one benzyl ether
puts severe constraints on reaction choice. Diversification re-
actions, such as palladium promoted carbonylation, alkynylation
and vinylation as well as radical allylations worked well for model
systems but did not transfer to reactions with analogue 5. Alke-
nylation was finally achieved using a Suzuki coupling protocol
employing the S-Phos ligand with Pd(OAc)2, CsF and trans-1-hexen-
1-ylboronic acid (Scheme 4). The resulting vinylated analogue 11 is
obtained in 70% yield.
Analogue
Ki nM
5
Br
2.4
11
4.0
13
14
15.5
54.7
3. Conclusion
These and previous studies show that simplified A-ring analogs
made in uniquely step economical sequences exhibit bryostatin like
binding and can be tuned as needed to improve function or suppress
undesired side effects. This work provides the shortest synthetic
sequence to natural or non-natural compounds with bryostatin-like
PKC affinities. This FOS approach provides quantities of diversifiable
intermediates that are being explored for preclinical candidacy.
Scheme 4. Suzuki coupling of analogue 5a (a) Pd(OAc)2, S-Phos, CsF, trans-hex-1-en-1-
4. Experimental section
ylboronic acid, dioxane, 60 ꢃC, 70%.
4.1. Synthesis of (S)-1-(3-bromophenyl)but-3-en-1-ol
Cleavage of the styrenyl double bond was accomplished che-
moselectively with 1 equiv of ozone or alternatively a dihydrox-
ylationddiol cleavage strategy. Subsequent reduction of the
resultant aldehyde gave the 9-(3-hydroxymethyl)aryl substituted
bryolog (13) in 72% yield (Scheme 5). Alternatively, the aldehyde
can be oxidized to carboxylic acid 14 upon treatment with m-CPBA.
Interestingly, the BaeyereVilliger oxidation only furnishes the 9-(3-
carboxy)-aryl substituted analogue (52% yield). The corresponding
aryl migration product was not detected by NMR or HPLC.
To a solution of 0.1 equiv (S)-BINOL (1.0 mmol, 288 mg) in 4.6 ml
CH2Cl2 4 g powdered 4 A MS were added followed by 0.05 equiv
Ti(Oi-Pr)4 (0.5 mmol, 150 ml). The resulting orange mixture was
ꢀ
refluxed for 1 h and then cooled in a water bath to rt. A solution of
1.0 equiv 3-bromo benzcarboxaldehyde (10.0 mmol, 1.17 ml) in 1 ml
CH2Cl2 followed by 1.2 equiv B(OMe)3 (12 mmol,1.34 ml) was added.
1.18 equivBu3SnCH2CHCH2 (11.8 mmol, 3.7 ml)wereadded dropwise
to the deep red suspension over 5 min. The resulting mixture was
stirred at rt for 14 h, then the orange mixture was filtered through
Celite into saturated NaHCO3 (50 ml). The Celite was washed with
Et2O and the biphasic mixture was stirred for 1 h. Afterwards the
aqueous layer was extracted with Et2O (3ꢀ30 ml) and the combined
organic layers were dried over Na2SO4 and concentrated to an orange
oil. The residue was purified by column chromatography (silica gel,
Et2O/pentane 1:4) to yield the (S)-1-(3-bromophenyl)but-3-en-1-ol
(1.723 g, 7.58 mmol, 76%) as a slightly yellow liquid. Rf¼0.55 (pen-
tane/EtOAc¼10:1)done blue spot with p-anisaldehyde stain IR (thin
film):3368, 3077, 3009, 2979, 2907,1642,1595,1570,1476,1429,1344,
1298, 1193, 1092, 1070,997, 919, 882, 783, 687, 666 cmꢁ1
.
1H NMR
Scheme 5. Late stage diversification. (a) OsO4, NaIO4, or O3 (b) NaBH4, MeOH, 72%
(over two steps); (c) m-CPBA, 52% (over two steps).
(500 MHz, CDCl3): 7.52 (mc, 1H, aryl-H); 7.42e7.37 (m, 1H, aryl-H);
d
7.29e7.18 (m, 2H, aryl-H); 5.78 (mc, 1H, H-2); 5.20e5.14 (m, 2H, H-
1); 4.69 (mc,1H, H-4); 2.54e2.40 (m, 2H, H-3); 2.21e2.16 (m,1H, OH).
A competitive binding assay was performed with the new A-
ring analogues 5, 11, 13 and 14 on a mixture of rat brain PKC iso-
zymes (Table 1) used as a preliminary benchmark assay to de-
termine whether further study is warranted. All compounds exhibit
potent nanomolar binding affinity to this PKC isozyme mixture.
Significantly, decreasing polarity in the side chain results in in-
creased affinity: the parent analogue 4 and its bromo variant 5 have
an affinity of 2.3e2.4 nM. The alkenyl substituted analogue 11
shows a similar (4.0 nM) binding affinity. Substitution of these non-
polar groups with oxygen-containing functional groups reduces the
binding affinity to 15.5 nM in case of the hydroxyl methyl analogue
13. Introduction of an even more polar carboxyl group still fur-
nishes a potent compound but with a further reduction in binding
affinity (54.7 nM). Although this number constitutes the lowest
binding affinity in the series it is striking that such drastic changes
in the polarity still led to highly potent compounds.
13C NMR (125 MHz, CDCl3):
d 146.1, 133.8, 130.5, 129.9, 128.9, 124.4,
122.5, 119.0, 72.4, 43.8.HRMS: calculated for C10H11OBr [M]þ:
225.9993. Found: 225.9990.½a D23
ꢁ44.5 (c 0.93in CHCl3); op¼87%.
ꢂ
4.2. Synthesis of nitrile 8
To a rt solution of 1.0 equiv (S)-1-(3-bromophenyl)but-3-en-1-ol
(7.39 mmol, 1.678 g) were added 5.0 equiv acrylonitrile
(36.95 mmol, 2.43 ml) followed by 0.1 equiv. Triton B (40% wt in H2O,
0.74 mmol, 0.29 ml), each via syringe. The reaction was monitored by
GC, since the starting material (tR¼12.15 min) and the product
(tR¼15.87 min)cospot. After3.5 htheyellowsolutionwaspouredinto
saturated aq NH4Cl solution (50 ml). The phases were separated and
the aqueous layer was extracted with CH2Cl2. The combined organic
phases were dried over Na2SO4 and concentrated in vacuo to furnish

P.A. Wender, J. Reuber / Tetrahedron 67 (2011) 9998e10005
10001
ayellowoil. The crudewaspurified bycolumn chromatography(silica
gel, pentane/Et2O 1:7/1:4) to give nitrile8 (2.023 g, 7.22 mmol, 98%)
as a colourless liquid. Rf¼0.24 (pentane/EtOAc¼6:1)done purple-
grey spot with p-anisaldehyde stain. IR (thin film): 3077, 3011, 2978,
2911, 2878, 2253,1642,1595,1571,1475,1429,1342,1279,1222,1193,
1106, 1071, 997, 921, 787, 699, 666 cmꢁ1. 1H NMR (500 MHz, CDCl3):
the reaction mixture was gradually warmed to rt and poured into
a mixture of 100 ml saturated aq NaHCO3 solution and 50 ml H2O.
The layers were separated and the aqueous layer was extracted
with CH2Cl2 (5ꢀ20 ml). The combined organic layers were dried
over Na2SO4, concentrated in vacuo and the remaining crude was
chromatographed on silica (pentane/EtOAc 4:1/ 3:1) to give the
d
7.17e7.40 (m, 2H, aryl-H);7.27e7.22 (m, 2H, aryl-H); 5.76 (mc,1H, H-
di-
b
-ketoester 9 (462 mg, 0.95 mmol, 63%). Rf¼0.57 (35%OAc in
2); 5.08e5.03 (m, 1H, H-1); 4.28 (dd, J¼6.0, 7.5 Hz, 1H, H-4); 3.53 (dt,
J¼6.5,9.5 Hz,1H, H-5);3.49(dt, J¼6.5,9.5 Hz,1H, H-50);2.60e2.53(m,
3H, H-6/H-30); 2.39 (dddt, J¼1.4, 6.0, 7.0, 13.0, 1H, H-3). 13C NMR
pentane)done red spot with p-anisaldehyde stain. IR (thin film):
3069, 2981, 2935, 1749, 1732, 1715, 1652, 1596, 1573, 1471, 1393,
1369, 1318, 1255, 1151, 1032, 999, 951, 840, 788, 697, 665 cmꢁ1. 1H
(125 MHz, CDCl3):
d
143.4, 133.6, 131.0, 130.2, 129.5, 125.1, 122.6,
NMR (500 MHz, CDCl3): d 7.48e7.40 (m, 2H, aryl-H); 7.28e7.20 (m,
117.70, 117.68, 82.0, 63.4, 42.2, 18.9. HRMS: calculated for C10H9NOBr
[MꢁCH2CHCH2]þ: 237.9868. Found: 237.9875. Elemental Analysis:
calculated for C13H14BrNO: C 55.73; H 5.04; N 5.00. Found: C 55.54; H
2H, aryl-H); 4.74 (dd, J¼7.5 Hz, 2H, Et); 4.18 (q, J¼7.5 Hz, 2H, Et);
3.61 (dt, J¼6.0, 9.5 Hz, 1H, H-6); 3.55 (dt, J¼6.0, 9.5 Hz, 1H, H-60);
3.45 (s, 2H, H-2 or H-9); 3.06 (dd, J¼9.0, 16.0 Hz, 1H, H-4); 2.75 (dt,
J¼1.9, 6.0 Hz, 2H, H-7); 2.68 (dd, J¼4.0, 16.0 Hz, 1H, H-40); 1.46 (s,
9H, t-Bu); 1.32 (t, J¼7.5 Hz, 3H, Et). Note: spectrum shows >16% of
enol tautomers present in solution. 13C NMR (125 MHz, CDCl3):
5.04; N 5.06. ½a 2D3
ꢂ
ꢁ34.5 (c 0.88 in CHCl3).
4.3. Synthesis of di-b-ketoester 9
d
201.4, 200.1, 166.8, 166.2, 143.1, 131.2, 130.3, 129.4, 125.1, 122.8,
In a dry flask 1.0 equiv nitrile 8 (5.25 mmol, 1.500 g) was dis-
solved in 53.5 ml dry THF under N2 before 15.0 equiv Zn
(80.25 mmol, 5.240 g), 0.03 equiv Cp2TiCl2 (0.16 mmol, 40 mg) and
tert-butyl-bromoacetate (1 drop) were added. The green suspension
was heated to 60 ꢃC and 10 equiv tert-butyl-bromoacetate
(53.5 mmol, 7.22 ml) were added in portions over 1 h. The reaction
was stirred for an additional 1.5 h and then allowed to cool to rt. The
mixture was filtered into 30 ml Et2O through a pad of Celite, which
was washed with Et2O (a white precipitate initially formed and then
dissolved). To the filtrate 70 ml 1 N HCl were added and the biphasic
mixture was stirred for 18 h. The layers were separated and the
aqueous phase was extracted with Et2O (3ꢀ30 ml). The combined
organic layers were dried over Na2SO4 and concentrated to give
a yellow oil. The crude residue was purified by silica gel column
81.9, 77.4, 63.8, 61.4, 50.9, 50.8, 50.0, 42.6, 27.9, 14.1. HRMS: calcu-
lated for C22H29O7NaBr [MþNa]þ: 507.0994. Found: 507.0982. El-
emental Analysis: calculated for C22H29BrO7: C 54.44; H 6.02.
Found: C 54.00; H 6.16. ½ ꢂD ꢁ36.9 (c 1.11 in CHCl3).
a 23
4.4. Synthesis of triol 10
The Ruthenium catalyst was prepared as described by Blanc and
co-workers.39 1.0 equiv (COD)Ru(2-methylallyl)2 (0.013 mmol,
4.21 mg, Acros) and 1.2 equiv (R)-BINAP (0.016 mmol, 10.21 mg)
were measured into a dry flask under N2 and a mixture of 0.9 ml
degassed anhydrous acetone and 0.09 ml degassed methanol were
added. Then 2.2 equiv HBr (48% aq HBr, 0.029 mmol, 3.16 ml) were
added and the brown solution was stirred for 1 h at rt. The solvent
was blown off with N2 gas and the brown residue dried under high
vacuum for 2.5 h. The flask was refilled with N2 and used directly
chromatography (pentane/Et2O 10:1), which furnished the
b-
ketoester (1.800 g, 4.53 mmol, 85%) as a colourless oil. Rf¼0.66
(pentane/EtOAc¼6:1)done brown spot with p-anisaldehyde stain.
IR (thin film): 3078, 2980, 2934, 2904, 2873, 1734, 1715, 1644, 1595,
1571,1476,1452,1394,1369,1316,1255,1149,1101, 997, 951.4, 918.28,
for the hydrogenation. To the prepared catalyst di-b-ketoester 9
(0.206 mmol, 100 mg) was added as a solution in degassed EtOH
(1.5 ml, 200 proof, Gold Shield Chemical Company) and the reaction
flask was placed in a high-pressure bomb, flushed with N2 and
sealed tightly. The apparatus was purged with H2 (3ꢀ20 bar) and
then finally pressurized to 40 bar H2. The apparatus was placed into
a 45 ꢃC oil bath on a stir plate and the mixture was stirred for 44 h.
Afterwards the apparatus was removed from the bath, allowed to
cool to rt and slowly depressurized. The solvent was blown off with
N2 and the black residue was purified by silica gel chromatography
(silica gel, pentane/EtOAc 3:2/1:1) to the diol (80.5 mg,
840, 786, 699, 666 cmꢁ1. 1H NMR (500 MHz, CDCl3):
d 7.45e7.37 (m,
2H, aryl-H); 7.23e7.17 (m, 2H, aryl-H); 5.71 (mc,1H, H-2); 5.05e4.98
(m, 2H, H-1); 4.22 (dd, J¼6.0, 7.5 Hz,1H, H-4); 3.57 (dt, J¼3.1, 6.0 Hz,
2H, H-5); 3.39 (s, 2H, H-8); 2.80 (dt, J¼6.5,17.0 Hz,1H, H-6); 2.73 (dt,
J¼6.0,17.0 Hz,1H, H-60); 2.49 (ddd, J¼7.0, 7.5, 14.5 Hz,1H, H-3); 2.34
(ddd, J¼6.0, 6.5, 14.5 Hz, 1H, H-30); 1.47 (s, 9H, t-Bu-H). Note: spec-
trum shows 9% of the enol tautomer present in solution. 13C NMR
(125 MHz, CDCl3):
d 201.6, 166.2, 144.1, 134.0, 130.6, 129.9, 129.5,
125.1, 122.4, 117.3, 81.8, 81.7, 63.7, 51.0, 42.8, 42.2, 27.8 (3C). HRMS:
calculated for C16H20O4Br [MꢁCH2CHCH2]þ: 355.0545. Found:
355.0553. Elemental Analysis: calculated for C19H25BrO4: C 57.44; H
0.164 mmol, 80%) as
a
pale yellow oil. Rf¼0.23 (pentane/
EtOAc¼3:2)done black-blue spot with p-anisaldehyde stain. IR
(thin film): 3445, 2980, 2931, 2873, 1732, 1715, 1595, 1571, 1471,
1428, 1394, 1369, 1300, 1258, 1155, 1098, 952, 845, 787, 700,
6.34. Found: C 57.77; H 6.22. ½a D23
ꢁ25.8 (c 1.38 in CHCl3).
ꢂ
b
-Ketoester (1.0 equiv, 1.51 mmol, 600 mg) was dissolved in
666 cmꢁ1. 1H NMR (500 MHz, CDCl3):
d 7.48e7.39 (m, 2H, aryl-H);
15 ml CH2Cl2. Ozone was bubbled through the solution at ꢁ78 ꢃC
(bath temperature) until the blue colour persisted (1 min). Then
oxygen was bubbled through until the blue colour disappeared
(3 min). To the cooled solution 1.5 equiv solid triphenylphosphine
(2.29 mmol, 600 mg) were added in one portion and the mixture
was allowed to warm to rt. The mixture was stirred for 2 h at rt
before another 0.48 equiv solid triphenylphosphine (0.73 mmol,
191 mg) were added and the reaction was stirred another 1 h to
complete reduction of the ozonide. The reaction mixture was
recooled to ꢁ78 C and 3.0 equiv ethyl diazoacetate (4.53 mmol,
0.65 ml) were added in one portion via syringe followed by
1.5 equiv. SnCl4 (1 M in CH2Cl2, 2.27 mmol, 2.3 ml). The mixture
was allowed to slowly warm up. At ꢁ35 ꢃC the formation of bubbles
in the solution was observed. At this temperature another 1.5 equiv.
SnCl4 (1 M in CH2Cl2, 2.27 mmol, 2.3 ml) was added in small por-
tions over 1 h until a bubble-free solution was observed. Afterwards
7.28e7.20 (m, 2H, aryl-H); 4.49 (dd, J¼5.5, 8.5 Hz, 1H, H-5); 4.15
(dq, J¼1.2, 7.0, 2H, Et); 4.11 (mc,1 h, H-8); 4.08 (mc,1H, H-3); 3.72 (d,
J¼2.3 Hz, 1H, OH); 3.49 (ddd, J¼6.0, 6.5, 9.5, 1H, H-6); 3.44 (dt,
J¼6.0, 9.5 Hz, 1H, H-60); 3.40 (d, J¼3.6 Hz, 1H, OH), 2.51 (dd, J¼8.0,
16.0 Hz, 1H, H-2); 2.42 (dd, J¼4.8, 16.0 Hz, 1H, H-20); 2.38 (dd, J¼4.5,
16.5, 1H, H-9); 2.33 (dd, J¼8.0, 16.5 Hz, 1H, H-90); 2.01 (ddd, J¼3.5,
9.0, 14.0, 1H, H-4); 1.82e1.66 (m, 3H, H-40/H-7); 1.46 (s, 9H, t-Bu);
1.26 (t, J¼7.0 Hz, 3H, Et). Note: The spectrum shows 5% of a second
diastereomer. 13C NMR (125 MHz, CDCl3):
d 172.2,172.1,144.0,131.0,
130.2, 129.6, 125.2, 122.8, 81.3, 81.1, 66.8, 66.0, 65.9, 60.7, 44.3, 42.2,
41.6, 36.1, 28.8 (3C), 14.1. HRMS: calculated for C22H33O7NaBr
[MþNa]þ: 511.1307. Found: 511.1301. ½a 2D3
ꢁ33.2 (c 0.78 in CHCl3).
ꢂ
Superhydride (7.0 equiv, 1.0 M solution of lithium triethylbor-
ohydride in THF, 0.147 mmol, 147 ml) was added dropwise via 2 min
to a solution of 1.0 equiv of the diol (0.02 mmol, 9.8 mg) at 0 ꢃC. The
reaction was stirred for 1 h at 0 ꢃC, gradually warmed to rt and then

10002
P.A. Wender, J. Reuber / Tetrahedron 67 (2011) 9998e10005
stirred additional 1.5 h at rt. Afterwards the reaction was recooled
to 0 ꢃC followed by addition of H2O2 (30% in H2O, 50
l) and aq
chloride. The reaction mixture was allowed to stir at rt for 6 h.
DMAP (5.0 equiv, 0.111 mmol, 13.7 mg) and 1.1 equiv alcohol 7
(0.024 mmol, 14.3 mg) were dissolved in a second flask in 0.3 ml
toluene and added dropwise over 5 min to the reaction mixture via
syringe. The flask and the syringe were washed three times with
0.3 ml toluene. The cloudy mixture was stirred for 1.5 h at rt. Af-
terwards the solution was concentrated to w30% by blowing with
N2 gas and the concentrated mixture was directly purified by
chromatography on silica (pentane/EtOAc 80:20). The enal
(26.1 mg, 0.020 mmol, 91%) was furnished as a colourless oil.
Rf¼0.84 (pentane/EtOAc¼80:20)done black spot with p-anisalde-
hyde stain. IR (thin film): 3479 (w), 2954, 2920, 2857, 1724, 1692,
1435, 1387, 1361, 1255, 1155, 1104, 1036, 836, 776, 669, 665 cmꢁ1. 1H
NMR (600 MHz, CDCl3): 9.55 (d, J¼6.5 Hz 1H, H-11); 7.29 (d,
J¼13.5 Hz, 1H, H-13); 7.28e7.32 (m, 2H, aryl-H); 7.19e7.14 (m, 2H,
aryl-H); 6.00 (d, J¼1.6 Hz, 1H, H-23); 5.94 (dd, J¼6.5, 13.5 Hz, 1H, H-
12); 5.19 (ddt, J¼1.0, 4.7, 9.0 Hz, 1H, H-21), 5.12 (s, 1H, H-16); 4.25
(dd, 4.7, 6.5 Hz, 1H, H-6); 4.22 (quint, J¼5.5 Hz, H-3); 3.85 (quint,
J¼4.7 Hz, 1H, H-8); 3.79 (tt, J¼1.5, 9.5 Hz, 1H, H-19); 3.67 (s, 3H,
OMe); 3.66e3.60 (m, 5H, H-10/H-18/H-22); 3.25 (dt, J¼5.5, 7.5 Hz,
1H, H-5); 3.21 (dt, J¼7.5, 8.7 Hz, 1H, H-50); 3.07 (s, 1H, OH(C-15));
2.35 (dd, J¼4.8, 5.5 Hz, 1H, H-2); 2.11e1.88 (m, 5H, H-70/H-18/H-
20/H-24/H-240); 1.77e1.63 (m, 7H, H-4/H-7/H-9/H-200); 1.47 (dt,
J¼6.5, 12.5 Hz, 1H, H-25); 1.30e1.16 (m, 9H, H-12dH-29); 1.20 (s,
3H, CH3); 1.14 (s, 3H, CH3); 0.88 (s, 9H, OTBDMS); 0.87 (s, 9H,
OTBDMS); 0.85 (t, J¼6.0 Hz, 3H, H-30); 0.84 (s, 9H, OTBDMS); 0.78
(s, 9H, OTBDMS); 0.048 (s, 3H, OTBDMS); 0.044 (s, 3H, OTBDMS);
0.026 (s, 3H, OTBDMS); 0.014 (s, 3H, OTBDMS); 0.005 (s, 3H,
OTBDMS); ꢁ0.012 (s, 3H, OTBDMS); ꢁ0.017 (s, 3H, OTBDMS);
m
NaOH (1 M, 1 drop). The mixture was allowed to stir for 45 min
before 2 ml NH4Cl and 1 ml H2O were added at 0 ꢃC. After warming
the mixture to rt the layers were separated and the aqueous layer
was extracted with EtOAc (3ꢀ5 ml). The combined organic layers
were dried over Na2SO4 and concentrated in vacuo. The crude
residue was purified by chromatography on silica (pentane/EtOAc
70:30/EtOAc 100%) to give triol 10 (7.2 mg, 0.016 mmol, 81%) as
a colourless oil. Rf¼0.54 (EtOAc 100%)done bright blue spot with p-
anisaldehyde stain. IR (thin film): 3387, 2974, 2922, 2872, 1724,
1595, 1571, 1473, 1427, 1368, 1258, 1155, 1099, 997, 955, 885, 786,
700, 665 cmꢁ1. 1H NMR (500 MHz, CDCl3): 7.46e7.40 (m, 2H, aryl-
H); 7.27e7.22 (m, 2H, aryl-H); 4.48 (dd, J¼3.3, 10.0 Hz, 1H, H-5);
4.13 (m, 2H, H-3/H-8); 3.99 (s, 1H, OH); 3.83 (mc, 2H, H-1);
3.53e3.40 (m, 3H, H-6/OH), 2.80 (mc, 1H, OH(H-1)); 2.39 (dd, J¼4.0,
16.5 Hz); 2.34 (dd, J¼8.5,16.5 Hz,1H, H-90); 1.99 (dt, J¼10.0,15.0 Hz,
1H, H-4); 1.73e1.65 (m, 5H, H-40/H-2/H-7); 1.46 (s, 9H, t-Bu). 13C
NMR (125 MHz, CDCl3):
d 172.2, 144.1, 131.0, 130.3, 129.4, 124.9,
122.8, 82.6, 81.5, 71.6, 65.9, 65.5, 61.3, 45.3, 42.2, 38.6, 36.0, 28.1.
HRMS: calculated for C20H31O6NaBr [MþNa]þ: 469.1202. Found:
a 23
469.1201. ½ ꢂD ꢁ38.5 (c 0.66 in CHCl3).
4.5. Synthesis of carboxylic acid 6
2,6-Lutidine (30.0 equiv, 1.95 mmol, 230 ml) was added to a solu-
tion of 1.0 equiv triol 10 (0.065 mmol, 29 mg) in 1.5 ml CH2Cl2 under
N2 atmosphere. The solution was cooled to 0 ꢃC and 10.0 equiv.
TBSOTf (0.65 mmol, 149 ml) were added dropwise via 30 s. The ice
bath was removed and the solution was slowly concentrated by
blowing with N2 gas until w0.3 M solution was obtained. The con-
centrated mixture was allowed to stir for 22 h at rt. Afterwards first
1.0 ml H2O and 1.0 ml CH2Cl2 were added to the reaction followed by
NaOH (1 N in MeOH/THF/H2O 3:1:1, 0.3 ml). The slightly yellow bi-
phasic mixture was stirred for 2 h and solid KHSO4 was added until
a pH of 7 of the aqueous phase was reached. The organic phase was
extracted with EtOAc (4ꢀ10 ml) and the combined organic layers
were dried over Na2SO4. After concentration in vacuo the crude res-
iduewaspurified bychromatographyonsilica(pentane/EtOAc 80:20)
to give the carboxylic acid 6 (43.7 mg, 0.06 mmol, 92%) as a colourless
oil. Note: The product is acid sensitive; chromatography on silicawith
1% AcOH in the solvent mixture leads to decomposition! Rf¼0.30
(pentane/EtOAc¼80:20)done black spot with p-anisaldehyde stain.
Note: The Rf valueisstrongly dependenton the concentration. IR(thin
film): 2955 (br), 2929, 2856, 1712, 1594, 1571, 1471, 1429, 1361, 1256,
1185, 1097, 940, 836, 776, 699, 664 cmꢁ1. 1H NMR (500 MHz, CDCl3):
7.39e7.35 (m, 2H, aryl-H); 7.21e7.15 (m, 2H, aryl-H); 4.26 (dd, J¼5.0,
8.5 Hz, 1H, H-5); 4.24 (quit, J¼5.5 Hz,1H, H-8); 3.87 (tt, J¼5.5, 6.5 Hz,
1H, H-3); 3.65 (quint, J¼6.5 Hz,1H, H-1); 3.64 (quint, J¼6.5 Hz,1H, H-
10); 3.26 (mc, 2H, H-6); 2.49 (dd, J¼5.0, 15.5 Hz, 1H, H-9); 2.39 (dd,
J¼6.0, 15.5 Hz, 1H, H-90); 1.94 (ddd, J¼5.5, 8.5, 13.5 Hz, 1H, H-4);
1.85e1.63 (m, 5H, H-2/H-40/H-7); 0.87 (s, 9H, OTBDMS); 0.84 (s, 9H,
OTBDMS); 0.83 (s, 9H, OTBDMS); 0.07 (s, 3H, OTBDMS); 0.06 (s, 3H,
OTBDMS); 0.02 (s, 3H, OTBDMS); 0.01 (s, 3H, OTBDMS); 0.00 (s, 3H,
ꢁ0.021 (s, 3H, OTBDMS). 13C NMR (125 MHz, CDCl3):
d 194.6, 172.6,
171.7, 166.6, 166.4, 150.4, 145.3, 130.6, 130.2, 129.8, 127.4, 125.1,
122.6, 120.8, 99.5, 78.9, 72.5, 71.5, 66.52, 66.46, 66.2, 65.0, 64.9,
59.6, 51.2, 45.7 (ꢀ2), 42.2, 40.0, 37.5, 37.3, 34.5, 31.6, 30.9, 28.88,
28.86, 25.92 (3C), 25.90 (3C), 25.8 (3C), 25.7 (3C), 24.4, 23.0, 22.5,
20.0, 18.3, 18.2, 18.1, 18.0, 14.1, ꢁ4.2, ꢁ4.4, ꢁ4.7, ꢁ4.8, ꢁ5.30, ꢁ5.32,
ꢁ5.4 (2C). HRMS: calculated for C65H117O14NaSi4Br [MþNa]þ:
1335.6602. Found: 1335.6584. ½a D23
ꢁ27.3 (c 1.22 in CHCl3).
ꢂ
Enal (1.0 equiv, 0.02 mmol, 26 mg) was dissolved in 5.5 ml
anhydrous THF in a polypropylene vial under N2 at 0 ꢃC. Then
2.8 equiv HF$pyridine (0.055 mmol, 1.07 ml) were added via sy-
ringe and the reaction was stirred at 0 ꢃC for 1 h. Afterwards the
bath was removed and the reaction allowed to stir additional 4 h.
The reaction mixture was transferred to a separating funnel
containing 8 ml H2O using 5 ml EtOAc. Saturated aq NaHCO3
(w50 ml) was added in portions until the vigorous bubbling
stopped. The layers were separated and the aqueous layer was
extracted using Et2O (3ꢀ15 ml). The combined organic layers were
dried over Na2SO4, filtered and concentrated in vacuo. The crude
was purified by column chromatography on silica using pentane/
EtOAc 40:60/20:80 as eluents. Purification yielded the bryosta-
tin analogue 5 (14 mg, 0.017 mmol, 83%) as a white solid. Rf¼0.48
(pentane/EtOAc¼20:80)done black spot with p-anisaldehyde
stain. IR (thin film): 3465, 3333, 2926, 2856, 1731, 1715, 1667, 1595,
1573, 1469, 1434, 1403, 1378, 1362, 1285, 1259, 1230, 1159, 1136,
1105, 980, 940, 918, 879, 851, 807, 791, 791, 736, 705 cmꢁ1. 1H NMR
(600 MHz, CDCl3): 7.44e7.40 (m, 2H, aryl-H); 7.24e7.18 (m, 2H,
aryl-H); 6.01 (d, J¼16.2 Hz, 1H, H-13); 6.01 (d, J¼1.7 Hz, 1H, H-23);
5.44 (dd, J¼7.4,16.2 Hz,1H, H-12); 5.43e5.38 (m,1H, H-21); 5.16 (s,
1H H-16 or C15eOH); 5.11 (d, J¼7.6 Hz, 1H, H-11); 5.08 (s, 1H, H-16
or C15eOH); 4.35 (d, J¼12.0 Hz, 1H, C3eOH); 4.32 (dd, J¼2.6,
11.4 Hz,1H, H-6); 4.32e4.26 (m,1H, H-3); 4.12e4.01 (m, 3H, H-8/H-
10/H-19); 3.90 (dt, J¼1.9, 12.0 Hz, 1H, H-10); 3.87 (mc, 1H, H-22);
3.72 (dd, J¼2.2,13.8 Hz,1H, H-18); 3.68 (s, 3H, OMe); 3.70e3.63 (m,
1H, H-220); 3.45 (ddd, J¼1.1, 5.0, 9.6 Hz, 1H, H-5); 3.33 (ddd, J¼2.2,
9.6, 10.2 Hz, 1H, H-50); 2.56 (dd, J¼10.8, 12.6 Hz, 1H, H-2); 2.52 (dd,
J¼3.3, 12.6 Hz, 1H, H-20); 2.30 (mc, 2H, H-24); 2.24 (ddd, J¼2.6, 5.2,
OTBDMS); ꢁ0.02 (s, 3H, OTBDMS).13C NMR (125 MHz, CDCl3):
d 174.1,
145.1, 130.7, 130.2, 129.7, 125.2, 122.7, 79.0, 66.9, 66.5, 64.9, 59.7, 45.8,
41.5, 39.9, 37.1, 25.9 (ꢀ6), 25.7 (3C), 18.2, 18.0, 17.9, ꢁ0.01, ꢁ4.4, ꢁ4.7,
ꢁ4.9, ꢁ5.4 (2C). HRMS: calculated for C34H65O6BrSi3Na [MþNa]þ:
755.3170. Found: 755.3167. ½a D23
ꢁ24.1 (c 0.82 in CHCl3).
ꢂ
4.6. Synthesis of analogue 5
To a solution of 1.0 equiv carboxylic acid 6 (0.022 mmol,
16.0 mg) in 1.5 ml toluene were added 4.0 equiv. Et3N (0.088 mmol,
12.5
ml) in one portion at rt under N2 followed by addition of a so-
lution of 1.1 equiv (0.024 mmol, 3.8
ml) 2,4,6-trichlorobenzoyl

P.A. Wender, J. Reuber / Tetrahedron 67 (2011) 9998e10005
10003
10.2, 15.6 Hz, 1H, H-4); 2.14e1.96 (m, 3H, H-7/H-20/H-180); 1.81
(ddd, J¼2.6,11.4,13.8 Hz,1H, H-200); 1.76 (dq, J¼5.0,12.6 Hz,1H, H-
9); 1.65e1.53 (m, 3H, H-70/H-25); 1.51 (d (br), J¼15.6 Hz, 1H, H-40);
1.39 (d (br), J¼12.6 Hz,1H, H-90); 1.32e1.21 (m, 8H, H-26e29); 1.20
(s, 3H, -C(CH3)2); 1.06 (s, 3H, eC(CH3)2); 0.87 (t, J¼6.6 Hz, 3H, H-
via syringe followed by addition of 6.0 equiv of solid NaIO4
(0.0222 mmol, 4.7 mg). The reaction mixture was stirred for 15 min
at rt before the product was extracted from the solution using ethyl
acetate (3ꢀ5 ml). The combined organic layers were dried over
Na2SO4, filtered and concentrated in vacuo. The crude was dried in
vacuum for 1 h and was then dissolved in 1.0 ml anhydrous
methanol and 0.2 ml anhydrous CH2Cl2. Afterwards the solution
was cooled to 0 ꢃC via ice bath before 8.0 equiv NaBH4
(0.0296 mmol, 1.2 mg) were added as a solid. The reaction mixture
was stirred at 0 ꢃC for 30 min, then the ice bath was removed and
the mixture was allowed to warm to rt and stirred for 1 h at rt. The
reaction mixture was transferred to a separating funnel, which
contained 5.0 ml concentrated NH4Cl solution in water. The layers
were separated and the aqueous layer was extracted using CH2Cl2
(4ꢀ10 ml). The combined organic layers were dried over Na2SO4,
filtered and concentrated in vacuo. The crude was filtered through
a pad of silica gel using EtOAc/pentane 80:20 as eluent. All fractions
containing product were further purified by reverse phase HPLC
(see below for conditions) to give 2.1 mg (0.0027 mmol, 72%) 13 as
a white amorphous solid. Rf¼0.23 (pentane/EtOAc¼20:80)done
blue spot with p-anisaldehyde stain. IR (thin film): 3459, 3359,
3199, 2922, 2852, 1738, 1731, 1715, 1667, 1660, 1463, 1455, 1434,
1409, 1378, 1361, 1280, 1260, 1231, 1157, 1133, 1103, 1063, 1025, 980,
881, 861, 800, 737, 709 cmꢁ1. 1H NMR (600 MHz, CDCl3): 7.36e7.33
(m, 1H, aryl-H); 7.31e7.26 (m, 2H, aryl-H); 7.21e7.19 (m, 1H, aryl-
H); 6.01 (d, J¼15.6 Hz, 1H, H-13); 6.01 (d, J¼1.9 Hz, 1H, H-23);
5.45 (dd, J¼7.2, 15.6 Hz, 1H, H-12); 5.43e5.38 (m, 1H, H-21); 5.16 (s,
1H, H-16 or C15eOH); 5.11 (d, J¼7.2 Hz, 1H, H-11); 5.11 (s, 1H, H-16
or C15eOH); 4.70 (s, 2H, H-31); 4.42 (d, J¼12.0 Hz, 1H, C3eOH);
4.37 (dd, J¼2.8, 12.0 Hz, 1H, H-6); 4.32e4.26 (m, 1H, H-3);
4.12e4.02 (m, 3H, H-8/H-10/H-19); 3.91 (dt, J¼2.3, 12.0 Hz, 1H, H-
100); 3.87 (dd, J¼3.1, 12.0 Hz, 1H, H-22); 3.72 (dd, J¼2.0, 13.8 Hz, 1H,
H-18); 3.68 (s, 3H, OMe); 3.68e3.63 (m, 1H, H-220); 3.44 (ddd,
J¼1.2, 4.7, 9.6 Hz, 1H, H-5); 3.32 (ddd, J¼2.5, 11.4, 15.6 Hz, 1H, H-50);
2.56 (dd, J¼12.0, 12.6 Hz, 1H, H-2); 2.52 (dd, J¼2.7, 12.6 Hz, 1H, H-
20); 2.30 (dt, J¼4.3, 7.2 Hz, 2H, H-24); 2.27e2.20 (m, 1H, H-4); 2.14
(mc, 1H, H-7); 2.05 (mc, 2H, H-20/H-180); 1.81 (ddd, J¼2.6, 11.4,
14.4 Hz, 1H, H-200); 1.73 (dq, J¼4.9, 12.6 Hz, 1H, H-9); 1.67e1.53 (m,
3H, H-70/H-25); 1.48 (d (br), J¼15.6 Hz, 1H, H-40); 1.39 (d (br),
J¼13.8 Hz, 1H, H-90); 1.32e1.22 (m, 8H, H-26e29); 1.20 (s, 3H,
-C(CH3)2); 1.06 (s, 3H, -C(CH3)2); 0.87 (t, J¼6.6 Hz, 3H, H-30). 13C
30). 13C NMR (125 MHz, CDCl3):
d 172.3, 172.1, 167.0, 151.6, 143.6,
142.6,131.2,130.4,129.5,125.9,124.9,122.8,119.9,102.2, 98.9, 83.3,
75.6, 74.1, 71.6, 68.7, 66.2, 66.0, 65.7, 64.5, 51.1, 45.5, 45.1, 42.1, 35.8,
34.6, 33.6, 32.3, 31.6, 31.0, 29.0, 28.9, 24.7, 24.3, 22.6, 19.4, 14.1.
HRMS: calculated for C41H59O13NaBr [MþNa]þ: 861.3037. Found:
861.3024. ½a 2D3
ꢁ72.7 (c 0.43 in CDCl3).
ꢂ
4.7. Synthesis of analogue 11
Aryl bromide 5 (1.0 equiv, 0.012 mmol, 10.0 mg) was measured
in a dry vial, which was evacuated and refilled with N2. Then
7.0 equiv CsF (0.083 mmol, 12.6 mg), which was dried in a 200 ꢃC
vacuum oven, were added and the mixture was placed under high
vacuum for 30 min. The vial was refilled with N2 and kept under
N2 atmosphere throughout the reaction. The mixture was dis-
solved in 0.1 ml dioxane. Then 1.25 equiv Pd(OAc)2 (0.015 mmol,
3.3 mg), 1.8 equiv S-Phos (0.021 mmol, 8.8 mg) and 6.0 equiv
trans-1-hexen-1-ylboronic acid were added via a stock solution in
dioxane (0.1 ml solution). The orange-red reaction mixture was
sealed with a 2-ply Teflon tape, followed by a plastic screw cap
and finally the vial was sealed with parafilm. Afterwards the vial
was vortexed and placed in a 60 ꢃC oil bath and stirred for 2 h at
60 ꢃC. The black reaction mixture was filtered through a plug of
silica using ethyl acetate and concentrated under a stream of N2.
Purification of the yellow crude by preparative HPLC (MeCN/H2O
65%/90% at 20 ml/min) furnished the coupling product 11 as
a white solid in 70% yield. Rf¼0.59 (pentane/EtOAc¼30:70)done
blue spot with p-anisaldehyde stain. IR (thin film): 3460, 3333,
2925, 2854, 1737, 1731, 1667, 1463, 1434, 1404, 1378, 1362, 1287,
1259, 1230, 1159, 1134, 1105, 1063, 1006, 979, 918, 887, 806,
737 cmꢁ1 1H NMR (600 MHz, CDCl3): 7.26e7.21 (m, 3H, aryl-H);
.
7.07e7.02 (m, 1H, aryl-H); 6.33 (d, J¼16.0 Hz, 1H, H-31); 6.23 (d,
J¼6.5 Hz, 1H, H-32); 5.99 (d, J¼16.0 Hz, 1H, H-13); 5.99 (d,
J¼1.4 Hz, 1H, H-23); 5.44 (dd, J¼7.5, 16.0 Hz, 1H, H-12); 5.41e5.36
(m, 1H, H-21); 5.14 (s, 1H, H-16 or C15eOH); 5.11 (s, 1H, H-16 or
C15eOH); 5.09 (d, J¼7.5 Hz, 1H, H-11); 4.42 (d, J¼12.0 Hz, 1H,
C3eOH); 4.31 (dd, J¼2.6, 12.0 Hz, 1H, H-6); 4.30e4.22 (m, 1H, H-
3); 4.11e3.98 (m, 3H, H-8/H-10/H-19); 3.89 (mc, 1H, H-10); 3.85
(md, 1H, H-22); 3.70 (dd, J¼1.7, 13.5 Hz, 1H, H-18); 3.668 (s, 3H,
OMe); 3.68e3.62 (m, 1H, H-220); 3.44 (ddd, J¼1.4, 4.7, 9.5 Hz, 1H,
H-5); 3.29 (ddd, J¼1.5, 9.5, 12.0 Hz, 1H, H-50); 2.56 (t, J¼12.5 Hz,
1H, H-2); 2.50 (dd, J¼2.4, 12.5 Hz, 1H, H-20); 2.28 (dt, J¼3.7, 7.2 Hz,
2H, H-24); 2.26e2.21 (m, 2H, H-33); 2.19 (mc, 1H, H-4); 2.12 (mc,
1H, H-7); 2.05e1.95 (m, 2H, H-20/H-180); 1.79 (dt, J¼2.8, 12.0 Hz,
1H, H-200); 1.73 (dq, J¼4.7, 12.5 Hz, 1H, H-9); 1.62e1.55 (m, 3H, H-
70/H-25); 1.49e1.41 (m, J¼15.6 Hz, 1H, H-40); 1.36 (mc, 1H, H-90);
1.30e1.19 (m, 12H, H-26e29/H-34/H-35); 1.18 (s, 3H, eC(CH3)2);
1.03 (s, 3H, eC(CH3)2); 0.91 (t, J¼7.5 Hz, H-36); 0.85 (t, J¼6.5 Hz,
NMR (125 MHz, CDCl3):
d 172.4, 172.1, 167.0, 151.6, 142.5, 141.6,
141.3, 129.0, 126.6, 125.9, 125.6, 124.8, 119.9, 102.3, 98.9, 83.9, 75.8,
74.1, 71.6, 68.8, 66.2, 65.8, 65.7, 65.1, 64.6, 51.1, 45.6, 45.1, 42.1, 35.9,
34.6, 33.7, 32.3, 31.6, 31.0, 29.0, 28.9, 24.7, 24.3, 22.6, 19.4, 14.1.
HRMS: calculated for C42H62O14Na [MþNa]þ: 813.4037. Found:
813.4028. ½a 2D3
ꢁ38.9 (c 0.22.in CHCl3).
ꢂ
4.9. Synthesis of analogue 12
Analogue 11 (1.0 equiv, 0.008 mmol, 6.8 mg) was dissolved in
500
m
l THF and 100
ml deionized H2O. Then 0.1 equiv of OsO4
(0.0008 mmol, 5.4
m
l) were added as a 4% stock solution in water
3H, H-30). 13C NMR (125 MHz, CDCl3):
d
172.5, 172.1, 167.0, 151.6,
via syringe followed by addition of 6.0 equiv of solid NaIO4
(0.048 mmol, 10.3 mg). The reaction mixture was stirred for
15 min at rt before the product was extracted from the solution
using ethyl acetate (3ꢀ5 ml). The combined organic layers were
dried over Na2SO4, filtered and concentrated in vacuo. The crude
was purified by column chromatography on silica gel using ethyl
acetate/pentane 90:10 as eluent. The purification yielded the
bryostatin analogue 12 (4.8 mg, 0.006 mmol, 75%) as a white
solid. Rf¼0.45 (pentane/EtOAc¼20:80)done blue spot with p-
anisaldehyde stain. IR (thin film): 3460, 3359, 2960, 2922, 2851,
1715, 1737, 1731, 1667, 1633, 1463, 1455, 1434, 1403, 1378, 1362,
1280, 1260, 1230, 1157, 1133, 1102, 1063, 1022, 980, 863, 801, 722,
142.5, 141.4, 138.4, 131.9, 129.2, 128.8, 126.0, 125.7, 124.8, 123.5,
119.9, 102.3, 98.9, 84.1, 76.0, 74.1, 71.6, 68.8, 66.3, 65.7 (2C), 64.5,
51.1, 45.5, 45.1, 42.1, 35.8, 34.6, 33.6, 32.7, 32.3, 31.6, 31.4, 31.0, 29.0,
28.9, 24.7, 24.3, 22.6, 22.3, 19.4, 14.1, 14.0. HRMS: calculated for
C47H70O13Na [MþNa]þ: 865.4714. Found: 865.4701. ½a 2D3
ꢁ74.3 (c
ꢂ
0.42 in CHCl3).
4.8. Synthesis of analogue 13
Analogue 11 (1.0 equiv, 0.0037 mmol, 3.1 mg) was dissolved in
300 l THF and 60 l deionized H2O. Then 0.1 equiv of OsO4
(0.0004 mmol, 2.5 l) were added as a 4% stock solution in water
m
m
m
704, 665 cmꢁ1 1H NMR (500 MHz, CDCl3): 10.02 (s, 1H, H-31);
.

10004
P.A. Wender, J. Reuber / Tetrahedron 67 (2011) 9998e10005
7.84.7.76 (m, 2H, aryl-H); 7.60e7.52 (m, 2H, aryl-H); 6.02 (d,
J¼16.0 Hz, 1H, H-13); 6.01 (d, J¼1.9 Hz, 1H, H-23); 5.45 (dd, J¼7.5,
16.0 Hz, 1H, H-12); 5.44e5.38 (m, 1H, H-21); 5.16 (s, 1H, H-16 or
C15eOH); 5.12 (d, J¼7.0 Hz, 1H, H-11); 5.09 (s, 1H, H-16 or
C15eOH); 4.46 (dd, J¼2.8, 12.0 Hz, 1H, H-6); 4.37 (d, J¼12.0 Hz,
1H, C3eOH); 4.34e4.26 (m, 1H, H-3); 4.14e4.03 (m, 3H, H-8/H-
10/H-19); 3.92 (dt, J¼1.8, 11.5 Hz, 1H, H-100); 3.87 (dt, J¼3.7,
11.5 Hz, 1H, H-22); 3.72 (dd, J¼2.5, 14.0 Hz, 1H, H-18); 3.70e3.64
(m, 1H, H-220); 3.68 (s, 3H, OMe); 3.44e3.39 (m, 1H, H-5);
3.39e3.33 (m, 1H, H-50); 2.57 (dd, J¼10.5, 12.5 Hz, 1H, H-2); 2.53
(dd, J¼3.4, 12.5 Hz, 1H, H-20); 2.30 (dt, J¼3.4, 7.5 Hz, 2H, H-24);
2.24 (m, 1H, H-4); 2.13 (mc, 1H, H-7); 2.09e1.98 (m, 2H, H-20/H-
180); 1.81 (ddd, J¼3.0, 10.0, 14.0 Hz, 1H, H-200); 1.76 (dq, J¼4.2,
12.0 Hz, 1H, H-9); 1.66e1.56 (m, 3H, H-70/H-25); 1.51 (d (br),
J¼15.0 Hz, 1H, H-40); 1.41 (d (br), J¼13.0 Hz, 1H, H-90); 1.35e1.17
(m, 8H, H-26e29); 1.20 (s, 3H, eC(CH3)2); 1.06 (s, 3H, eC(CH3)2);
31.0, 29.0, 28.9, 24.7, 24.3, 22.6, 19.4, 14.1. HRMS: calculated for
C42H60O15Na [MþNa]þ: 827.3830. Found: 827.3828. ½a 2D3
ꢁ100.8 (c
ꢂ
0.15 in CHCl3).
Acknowledgements
This manuscript is dedicated to Gilbert Stork, whose towering
contributions to science and education has enriched us all. J.R.
gratefully acknowledges support by a Feodor-Lynen Fellowship
(Humboldt foundation). We thank the Mochly-Rosen group (Stan-
ford University) for access to assay materials. HRMS analysis for
some compounds was performed at UCSF.
References and notes
0.87 (t, J¼7.0 Hz, 3H, H-30). 13C NMR (125 MHz, CDCl3):
d 191.9,
1. For pertinent reviews, see: (a) Wender, P. A.; Baryza, J. L.; Hilinski, M. K.; Horan,
J. C.; Kan, C.; Verma, V. A. In Drug Discovery Research: New Frontiers in the Post-
Genomic Era; Huang, Z., Ed.; Wiley: Hoboken, 2007; pp 127e162; (b) Mutter, R.;
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Chem. Soc. 1982, 104, 6846e6848.
172.3, 172.1, 166.9, 151.6, 142.6, 142.5, 136.8, 132.1, 129.7 (2C),
127.4, 125.9, 119.9, 102.2, 98.9, 83.4, 75.6, 74.1, 71.6, 68.7, 66.2,
66.1, 65.7, 64.6, 51.1, 45.5, 45.1, 42.2, 35.8, 34.6, 33.7, 32.3, 31.6,
31.0, 29.0, 28.9, 24.7,þ24.3, 22.6, 19.4, 14.1. HRMS: calculated for
a 23
C42H60O14Na [MþNa] : 811.3881. Found: 811.3874. ½ ꢂD ꢁ68.8 (c
0.40 in CDCl3).
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4.10. Synthesis of analogue 14
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Analogue 12 (1.0 equiv, 0.0036 mmol, 2.8 mg) was dissolved
in 100 ml acetonitrile (HPLC grade). Then 10 equiv m-CPBA
(0.035 mmol, 6 mg) were added as a solid in three single portions
over 5 h. The conversion of the reaction was observed by Maldi-
MS. The reaction was stirred at rt. After 7 h 0.1 ml of a saturated
solution of sodium thiosulfate in water were added to the reaction
mixture and the mixture was further diluted with 1.0 ml water
and 1.0 ml acetonitrile. The layers were separated and the aqueous
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The crude was filtered through a pad of silica gel using ethyl ac-
etate as eluent. All fractions containing product were further
purified by reverse phase HPLC to give 1.5 mg (0.002 mmol, 52%)
14 as a white amorphous solid. Rf¼0.11 (pentane/EtOAc¼20:80)d
one blue spot with p-anisaldehyde stain; Rf shows a strong con-
centration dependency. IR (thin film): 3460, 3424, 2925, 2855,
1722, 1715, 1434, 1403, 1378, 1362, 1288, 1259, 1231, 1160, 1136,
1104, 1063, 1025, 1006, 980, 917, 885, 859, 834, 806, 760, 734, 703,
665 cmꢁ1. 1H NMR (500 MHz, CDCl3): 8.06e8.02 (m, 1H, aryl-H);
7.99e7.97 (m, 1H, aryl-H); 7.58e7.55 (m, 1H, aryl-H); 7.51e7.47
(m, 1H, aryl-H); 6.25 (d, J¼16.0 Hz, 1H, H-13); 6.01 (d, J¼2.0 Hz, 1H,
H-23); 5.45 (dd, J¼7.5, 16.0 Hz, 1H, H-12); 5.45e5.39 (m, 1H, H-
21); 5.17 (s, 1H, H-16 or C15eOH); 5.12 (d, J¼7.5 Hz, 1H, H-11); 5.11
(s, 1H, H-16 or C15eOH); 4.45 (dd, J¼2.4, 12.0 Hz, 1H, H-6); 4.39 (d,
J¼12.0 Hz, 1H, C3eOH); 4.34e4.26 (m, 1H, H-3); 4.14e4.02 (m, 3H,
H-8/H-10/H-19); 3.92 (dt, J¼1.8, 12.0 Hz, 1H, H-100); 3.88 (dd,
J¼3.2, 12.0 Hz, 1H, H-22); 3.68 (s, 3H, OMe); 3.73 (dd, J¼1.9,
13.5 Hz, 1H, H-18); 3.70e3.65 (m, 1H, H-220); 3.45e3.40 (m, 1H, H-
5); 3.36 (mc, 1H, H-50); 2.59 (dd, J¼11.0, 12.5 Hz, 1H, H-2); 2.53 (dd,
J¼2.9, 12.5 Hz, 1H, H-20); 2.30 (dt, J¼3.3, 7.5 Hz, 2H, H-24);
2.27e2.22 (m, 1H, H-4); 2.15 (mc, 1H, H-7); 2.10e1.98 (mc, 2H, H-
20/H-180); 1.82 (ddd, J¼2.8, 12.0, 13.5 Hz, 1H, H-200); 1.76 (dq,
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