Deletion of the C26 Methyl Substituent from the Bryostatin Analogue Merle 23 Has Negligible Impact on Its Biological Profile and Potency

Article abstract of DOI:10.1002/cbic.201700677

Important strides are being made in understanding the effects of structural features of bryostatin 1, a candidate therapeutic agent for cancer and dementia, in conferring its potency toward protein kinase C and the unique spectrum of biological responses that it induces. A critical pharmacophoric element in bryostatin 1 is the secondary hydroxy group at the C26 position, with a corresponding primary hydroxy group playing an analogous role in binding of phorbol esters to protein kinase C. Herein, we describe the synthesis of a bryostatin homologue in which the C26 hydroxy group is primary, as it is in the phorbol esters, and show that its biological activity is almost indistinguishable from that of the corresponding compound with a secondary hydroxy group.

Full text of DOI:10.1002/cbic.201700677

DOI: 10.1002/cbic.201700677
Full Papers
Deletion of the C26 Methyl Substituent from the
Bryostatin Analogue Merle 23 Has Negligible Impact on Its
Biological Profile and Potency
Xiguang Zhao+,[b] Noemi Kedei+,[a] Alexandra Michalowski,[a] Nancy E. Lewin,[a] Gary E. Keck,[b]
and Peter M. Blumberg*[a]
Important strides are being made in understanding the effects
of structural features of bryostatin 1, a candidate therapeutic
agent for cancer and dementia, in conferring its potency
toward protein kinase C and the unique spectrum of biological
responses that it induces. A critical pharmacophoric element in
bryostatin 1 is the secondary hydroxy group at the C26 posi-
tion, with a corresponding primary hydroxy group playing an
analogous role in binding of phorbol esters to protein kinase
C. Herein, we describe the synthesis of a bryostatin homologue
in which the C26 hydroxy group is primary, as it is in the phor-
bol esters, and show that its biological activity is almost indis-
tinguishable from that of the corresponding compound with a
secondary hydroxy group.
Introduction
The bryostatins are a family of complex macrolactone marine
natural products originally isolated through the pioneering
efforts of Pettit and co-workers.[1] The flagship member of the
family, bryostatin 1 (1), has received enormous attention due
to the range of important and medicinally relevant biological
activities it induces. Originally isolated due to the potent anti-
cancer activity shown by extracts of the marine organism
Bugula neritina against a cancer cell line, it has since been
found to be a potent activator of protein kinase C (PKC) and
other proteins that, like PKC, are activated by diacylglycerols
(DAGs).[2,3] As a potent activator of PKC, the biological activities
of bryostatin 1 share similarities with those of other known
high affinity ligands for PKC (such as phorbol esters, exempli-
fied by phorbol 12-myristate 13-acetate (PMA; 2)), but impor-
tantly, unlike the phorbol esters, bryostatin 1 is not tumor-
promoting in a two-stage mouse model for carcinogenesis.[4]
Bryostatin 1 has been utilized in numerous clinical trials for
cancer[3] and has more recently been of interest with respect
to Alzheimer’s disease[5] and HIV infection.[6] Other members of
the bryostatin family mainly differ from bryostatin 1 in having
different substituents at C7 and/or C20, although some, such
as bryostatin 3, are more heavily modified. However, all pos-
sess the same basic macrocyclic lactone core structure, incor-
porating three pyran rings. Pettit named this common core
structural feature a “bryopyran” motif (Scheme 1).
As might be expected, the complex structure and potent
biological activity of bryostatin 1 has led to great interest in
the chemical synthesis of these compounds.[7] The first synthe-
sis of a bryostatin, bryostatin 7, was reported by Masamune
and co-workers in 1990.[8] Despite the intense interest in these
compounds, it was not until 1998 that a second synthesis was
reported, this of bryostatin 2, by Evans and co-workers.[9] This
was followed in 2000 by a synthesis of bryostatin 3 by Yama-
mura and co-workers.[10] It was roughly a decade later before
other syntheses of bryostatins were reported. The first total
synthesis of bryostatin 1 was reported by Keck and co-workers
in 2011.[11] Other elegant total syntheses in this area were also
published, including bryostatin 16 by Trost,[12] bryostatin 7 by
Krische,[13] bryostatin 9 by Wender,[14] and bryostatin 8 by
Song.[15]
However, in 1998, Wender and co-workers reported the syn-
thesis of a simplified analogue of bryostatin 1 (7) in which the
B-ring pyran was replaced by a 1,3-dioxane for ease of synthe-
sis of this acetal subunit.[16] This bryostatin analogue was
shown to have similar affinity for PKCs (by using a mixture of
isozymes isolated from rat brain) to that of bryostatin 1 (3 vs.
1.4 nm). Subsequently, numerous such acetal analogues of
bryostatin 1 were prepared by the Wender group, most nota-
bly, perhaps, acetal 8, which was dubbed “pico” by Wender
et al. to reflect the reported very high PKC binding affinity of
0.25 nm (250 pm).[17] The less potent original acetal analogue,
7, was then named “nano”; these identifiers were subsequently
used in numerous Wender publications and remain in use
today (Scheme 2).
[a] Dr. N. Kedei,+ Dr. A. Michalowski, N. E. Lewin, Dr. P. M. Blumberg
Laboratory of Cancer Biology and Genetics
Center for Cancer Research, National Cancer Institute
Building 37, Room 4048, 37 Convent Drive MSC4255
Bethesda, MD 20892-4255 (USA)
E-mail: blumberp@dc37a.nci.nih
[b] Dr. X. Zhao,+ Dr. G. E. Keck
Department of Chemistry, University of Utah
315 South 1400 East, RM 2020, Salt Lake City, Utah 84112 (USA)
[+] These authors contributed equally to this work.
Supporting information and the ORCID identification numbers for the
authors of this article can be found under https://doi.org/10.1002/
cbic.201700677.
Our own interest in these fascinating compounds originally
focused on the development of methodology that might be of
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Scheme 1. Structures of representative bryostatins and PMA.
biologically active bryostatin analogues that possessed the
core bryopyran carbon skeleton.[20] The lead analogue here,
Merle 23, also possessed the unusual dienoate C20 substituent
found in bryostatin 1 but lacked much of the functionalization
in the A and B rings. This compound was found to have bind-
ing affinity for the PKCa isozyme, very similar to that of bryo-
statin 1 (0.70 vs. 0.4 nm), but surprisingly functioned like the
tumor-promoting ligand PMA in U937 lymphoma cells, rather
than like bryostatin 1. U937 cells are known[21] to provide a
well-characterized biological system that discriminates be-
tween PMA and bryostatin 1 activities: PMA induced attach-
ment and blocked proliferation of these cells, whereas bryo-
statin 1 had little effect in either the attachment or the prolifer-
ation assay. Moreover, bryostatin 1 blocked the effect of PMA
when the U937 cells were treated with the two agents togeth-
er, thus confirming that bryostatin 1 was not simply being de-
graded. At the same time, Wender also reported the synthesis
of some bryopyrans by using our pyran annulation strategy,
but in an intramolecular context, and described their binding
affinity for a mixture of PKC isozymes from rat brain.[22] Sub-
sequent studies from our group, which examined substituent
effects on biological activity, led to a model in which the lower
portion of the bryopyran was largely responsible for its bind-
ing to PKC, whereas the functional response in living cells
depended heavily upon substitution in the upper half of the
structure.[23]
Scheme 2. Structures of the Wender acetal analogues nano (7) and pico (8).
general utility in accessing the bryopyran core structures. The
most powerful of these methodologies, termed “pyran annula-
tion”, described a highly convergent asymmetric methodology
by which a hydroxyallyl silane (11) could be annealed with an
aldehyde (12) under acidic conditions to provide a disubstitut-
ed pyran (13) of the type that occurs repeatedly in the bryo-
pyran core structure. One method by which the hydroxyallyl
silanes (11) were readily available was by nucleophilic addition
of a conjunctive reagent (10) to an aldehyde (9); thus, overall,
two aldehydes plus the conjunctive reagent were annealed to
provide a pyran (Scheme 3). This initial report[18] was followed
by a demonstration of application to the synthesis of the bryo-
pyran core structure of the bryostatins[19] and then to the first
The 2008 paper from the Wender group also made an im-
portant correction, namely, that there really was no “pico”. A
table footnote directs the reader to another table footnote in
the Supplementary Material that indicates that the original
report of binding affinity for “pico” was believed to be an
error, and that the new binding affinity was 3.1 nm, that is,
essentially the same as that for “nano”. However, a report from
the Wender lab published subsequent to this paper still re-
Scheme 3. Pyran annulation. a) BITIP, CH2Cl2; b) R2CHO (12), TMSOTf, Et2O,
À788C.
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ferred to the 0.25 nm value originally claimed for pico.[24] This
confusing state of affairs, and the lack of other biological
characterization for pico, prompted us to examine the same
structural change on the bryopyran platform, particularly as
Merle 23 had been extensively characterized in terms of the
biological effects it induced.[25] Here, we report the synthesis
and biological activity of the new bryopyran analogue,
Merle 41, in which the C26 methyl substituent was deleted
from structure Merle 23 (Scheme 4).
stereocenter at C25 was then established by using a catalytic
asymmetric allylation (CAA) reaction,[26] which afforded the
desired homoallylic alcohol (19) in excellent yield (93%) and
essentially as a single enantiomer (99% ee). The new hydroxy
group was protected as a PMB ether, with an eye toward con-
ducting a diastereoselective nucleophilic addition to aldehyde
20, available from the olefin by oxidative cleavage. Unfortu-
nately, neither chelation-controlled nor non-chelation-con-
trolled reaction conditions provided a satisfying level of stereo-
selectivity upon addition to aldehyde 20. Finally, a second CAA
reaction was applied to afford alcohol 21 as essentially a single
diastereomer (minor isomer not detected).
After conversion of the free hydroxy group to a TBS ether,
the alkene was regioselectively hydroformylated to give alde-
hyde 22 in 87% yield.[27] Next, we carried out a prenylation re-
action to add a reverse prenyl group to aldehyde 22. Although
we had done such reactions previously using indium metal
and an allylic halide, here we found that the use of inexpensive
zinc dust in an aqueous medium gave excellent results; subse-
quent Swern oxidation of the resulting secondary alcohol af-
forded ketone 23. Ozonolysis of the vinyl group, followed by a
phosphonate Wittig reaction on the resulting aldehyde, afford-
ed a,b-unsaturated thiol ester 24 in 86% yield over the two
steps.[28] After removal of the TBS group by reaction with aque-
ous HF buffered with pyridine, acid-promoted cyclization of
the hydroxy ketone with concomitant dehydration was accom-
plished by using CSA in toluene at reflux to afford glycal 25.
Low temperature reduction with DIBAL in dichloromethane
then gave the desired enal (14; Scheme 6).
The hydroxyallylstannane required for the pyran annulation
had been previously prepared by CAA reaction in our labs.[18,19]
However, several examples of pyran annulation reactions, con-
ducted with glycals such as 14 as substrates, had been exam-
ined previously and had generally proven to be problematic,
in that yields were significantly lower than for most other
cases examined. Similar complications were observed in at-
tempted pyran annulations with 14. Thinking that the problem
could potentially be decomposition due to the presence of
acid-sensitive regions of the substrate, we examined the use of
various additives in the pyran annulation by using substrate
14. It was found that the use of substoichiometric amounts of
pyridine greatly improved the yields and reproducibility of this
reaction, with the yield improvement being in the 20–30%
range. Under optimal conditions, the desired pyran product 26
was obtained much more cleanly, and in 93% isolated yield
(Scheme 7).
Scheme 4. Structures of Merle 23 and Merle 41.
Results
Synthesis of bryopyran Merle 41
The synthetic plan for Merle 41 followed the scheme demon-
strated in 2005, utilizing two consecutive pyran annulations to
prepare the B and C rings. However, the absence of the C26
stereogenic center required that the very beginning steps of
the synthesis (which occur in our C ring fragment) be re-
worked. Previously, we had used a commercially available start-
ing material containing the C26 stereogenic center, and this
stereochemistry was then utilized to construct additional ste-
reogenic centers through a series of diastereoselective reac-
tions. With this center absent, an asymmetric approach to gen-
erating the C25 stereogenic center was required (Scheme 5).
The synthesis of the C ring segment commenced from allyl
alcohol (17; Scheme 6). The hydroxy group was protected,
after which ozonolysis afforded aldehyde 18. The requisite
To avoid the impediment of the labile glycal in the remain-
ing synthetic steps, the C ring was fully functionalized at this
point. After chemoselective epoxidation and methanolysis in
situ, the resulting ketal was subjected to a catalytic amount of
PPTS to enrich the mixture of diastereomers (at C19) in favor
of the desired and thermodynamically more stable isomer.
Dess–Martin periodinane (DMP) oxidation then provided
ketone 27.[29] This sequence of three steps was accomplished
in excellent (89%) yield. To complete the elaboration of the
C ring, an aldol reaction with freshly distilled methyl glyoxalate
was used to give enoate 28 in 76% isolated yield. Luche
Scheme 5. Retrosynthetic plan for synthesis of Merle 41.
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Scheme 6. Synthesis of the C ring fragment. a) NaH, BOMCl, THF, 08C–RT; b) O3, CH2Cl2, À788C, then DMS (84% over 2 steps); c) (R)-BITIP, TFA, 4 ꢁ MS, CH2Cl2,
À788C to À308C (93%, 99% ee); d) KH, PMBBr, THF, 08C (96%); e) O3, CH2Cl2, À788C, then PPh3 (83%); f) (S)-BITIP, TFA, 4 ꢁ MS, CH2Cl2, À78 to À308C (89%,
>99% dr); g) TBSCl, Im, DMF, RT (99%); h) BIPHEPHOS, CO/H2 (1:1), Rh(CO)2(acac), THF, 608C (87%); i) Zn, NH4Cl (aq.), THF, RT; j) (COCl)2, DMSO, NEt3, CH2Cl2,
À788C (91% over 2 steps); k) O3, PPh3, CH2Cl2, À788C; l) NaH, THF, 08C (86% over 2 steps); m) HF (aq.), Py, MeCN, RT; n) CSA, PhMe, reflux (79% over 2
steps); o) DIBAL, CH2Cl2, À788C (80%).
reduction of the ketone[30] gave an unstable alcohol, which
was immediately esterified with the requisite octadienoic acid
under Yamaguchi conditions.[31] The desired C20 ester 29 was
obtained in an excellent 93% yield over this two-step se-
quence.
over two steps. The aldehyde was then oxidized to the corre-
sponding carboxylic acid under Pinnick conditions.[33] Next, the
removal of the C25 TBS group furnished a seco-acid, which
then underwent macrolactonization according to Yamaguchi’s
protocol under high dilution techniques (slow addition by sy-
ringe pump). These three steps transpired in amazingly high
overall yield, affording the macrolactone 39 in 95% isolated
yield. With the bryopyran skeleton now complete, all that re-
mained was removal of the final three protecting groups. This
was accomplished by initial DDQ-mediated PMB removal,
followed by a global deprotection with LiBF4,[34] to afford the
des-methyl analogue Merle 41 in 69% yield.
The stage was now set for incorporation of the A ring. Re-
moval of the BPS group by using ammonium fluoride in meth-
anol at reflux gave the corresponding alcohol (92% yield),
which was oxidized by using Dess–Martin reagent to the corre-
sponding aldehyde. At this point, it was necessary to perform
a protecting group swap of the PMB group in aldehyde 30,
due to the presence of another PMB group on the hydroxyallyl
silane partner for the pyran annulation. This was accomplished
by removal of the PMB group by reaction with DDQ, followed
by silylation of the alcohol with TBS triflate at À788C, to give
aldehyde 31.
An A ring hydroxyallyl silane was easily prepared from ho-
moallylic alcohol 34, itself available through a CAA reaction as
shown (Scheme 8). The alcohol was protected as a PMB ether
to allow for a high level of diastereoselectivity in the subse-
quent allylation of aldehyde 35 (Scheme 8).
Thus, after introduction of the PMB group, the vinyl group
was cleaved by ozonolysis to afford aldehyde 35. Chelation-
controlled addition of silyl stannane reagent 10, promoted by
magnesium bromide etherate in dichloromethane at À788C,
afforded essentially exclusively (>99:1) the desired diastereo-
mer of hydroxyallyl silane 36, as a consequence of chelation
control in addition to a preformed magnesium chelate.[32]
With both aldehyde 31 and b-hydroxyallylsilane 36 in hand,
the second pyran annulation was conducted to afford tris
pyran 37 in excellent yield (90%) under the modified (pyridine
additive) conditions (Scheme 9). The BPS silyl ether at C1 was
selectively deprotected by NH4F, and the resulting alcohol was
subject to DMP oxidation to afford aldehyde 38 in 57% yield
Biological characterization of Merle 41
Merle 41 bound to PKCa with a Ki value of (0.73Æ0.05) nm
(n=3 experiments). This value closely matches the Ki of (0.70Æ
0.06) nm that we reported previously for Merle 23.[20] We previ-
ously described that bryostatin 1 and bryostatin 7 showed little
PKC isoform selectivity,[23b] with no more than a threefold
difference in Ki values for human PKCbII, PKCd, and PKCe com-
pared to that for human PKCa. In preliminary experiments, we
found that this was likewise the case for Merle 41. Ki values of
Merle 41 for human PKCbII, PKCd, and PKCe assayed in vitro
were (2.1Æ0.6), (0.8Æ0.2), and (0.9Æ0.2) nm, respectively.
Although these measurements were not carried out in parallel
with the above, and thus do not provide a precise comparison,
they give no indication of substantial in vitro binding selectivi-
ty among PKC isoforms for Merle 41.
The Toledo cell line is derived from a non-Hodgkin’s B cell
lymphoma. It is among the most sensitive cell lines for growth
inhibition by phorbol ester and, unlike leukemia cell lines such
as U937, K562, or MV-4–11,[35] is similarly growth inhibited by
bryostatin 1.[36,37] Merle 41 and Merle 23, like PMA and bryosta-
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Scheme 7. Pyran annulation and C ring elaboration. a) TMSOTf, Py, Et2O, À788C (93%); b) m-CPBA, MeOH, CH2Cl2, À108C; c) PPTS, MeOH, RT; d) DMP, tBuOH,
Py, CH2Cl2, RT (89% over 3 steps); e) MeO2CCHO, K2CO3, MeOH, THF, RT (76%, E/Z>95:5); f) NaBH4, CeCl3·7H2O, MeOH, À788C; g) 2,4,6-(Cl3C6H2)COCl, DMAP,
NEt3, PhMe, RT (93% over 2 steps); h) NH4F, MeOH, reflux (92%); i) DMP, tBuOH, Py, CH2Cl2, RT (97%); j) DDQ, pH 6 buffer, CH2Cl2, 08C (98%); k) TBSOTf, 2,6-
lutidine, CH2Cl2, À788C (90%).
nism of the PMA response at high concentrations of Merle 23
or Merle 41 was the same for each.
TNFa was released onto the medium of the U937 cells in re-
sponse to PMA and contributed to growth inhibition. Merle 41
and Merle 23 displayed identical dose–response curves for
release of TNFa, with absolute maximal levels of release of
(65.2Æ2.6)% and (66.6Æ2.6)%, respectively, of that for PMA
(Figure 1D). Once again, in combination with PMA, both com-
pounds reduced the level of TNFa release in response to PMA
to the level induced by the compound alone. By all of these
measures, Merle 41 was very similar to Merle 23.
Scheme 8. Preparation of A ring allyl silane. a) NaH, BPSCl, THF, RT (96%);
PMA, bryostatin 1, and Merle 23 caused distinct patterns of
b) (COCl)2, DMSO, NEt3, CH2Cl2, À788C (97%); c) TFA, (R)-BITIP, 4 ꢁ MS,
downregulation of PKC isozymes in the human prostate cancer
CH2Cl2, À308C (98%, 98% ee); d) PMB-imidate, PhMe, Sc(OTf)3, 08C (76%);
cell line LNCaP as a function of time.[25] As previously observed,
PKCd was downregulated by PMA, bryostatin 1 caused bi-
phasic downregulation of PKCd with protection at higher bryo-
statin 1 doses, and Merle 23 caused more extensive downregu-
lation of PKCd than did either PMA or bryostatin 1 (Figure 2A).
Merle 41 acted similarly to Merle 23. PKCa was downregulated
by PMA, bryostatin 1 was more potent at PKCa downregula-
tion, and Merle 23 was less potent than either and indeed, at
low doses, caused a modest increase in PKCa (Figure 2B).
Merle 41 again acted similarly to Merle 23. PKCe showed
modest downregulation in response to PMA, bryostatin 1
caused even less downregulation, and Merle 23 resembled
PMA in inducing weak PKCe downregulation (Figure 2C).
Merle 41 showed a pattern of response identical to that of
Merle 23. Finally, PMA caused strong downregulation of PKD1.
Bryostatin 1 and Merle 23 caused little PKD1 downregulation
(Figure 2D). Merle 41, again, closely paralleled Merle 23.
e) O3, DMS, CH2Cl2, À788C (92%); f) CH2Cl2, MgBr2·OEt2, À788C (85%, >99%
dr).
tin 1, inhibited Toledo cell growth (Figure 1A). Merle 41 closely
resembled Merle 23, both in potency and in the extent of
growth inhibition. Unlike the Toledo cells, U937 promyelocytic
leukemia cells respond to phorbol ester with growth inhibition
and cell attachment, whereas bryostatin 1 causes little growth
inhibition and almost no cell attachment. Merle 41 elicited a
response virtually identical to that to Merle 23 (Figure 1B and
C). For both compounds, U937 cell growth was inhibited
almost to the level caused by PMA but with a somewhat
biphasic dose response, with reduced inhibition at 100 or
1000 nm. Attachment was induced to the same level as for
PMA, but again, the dose–response was biphasic, with reduced
attachment at 300 nm or above. The combination of PMA plus
Merle 41 or Merle 23 gave a response similar to that of
Merle 23 or Merle 41 alone, and the pattern of partial antago-
Although the data clearly showed that Merle 41 resembled
Merle 23 in its downregulation of the PKC isoforms examined,
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Scheme 9. Completion of the synthesis of Merle 41. a) TMSOTf, Py, Et2O, À788C (90%); b) NH4F, MeOH, reflux; c) DMP, tBuOH, Py, CH2Cl2, RT (57% over 2
steps); d) NaClO2, NaHPO4, H2O, isoamylene, tBuOH, MeCN, 08C; e) HF·Py (20 w%), THF, RT; f) 2,4,6-Cl3BzCl, DIPEA, DMAP, PhCH3, 308C (95% over 3 steps);
g) DDQ, pH 6 buffer, CH2Cl2, 08C; h) LiBF4, H2O, MeCN, reflux (69% over 2 steps).
in support of the lack of a role for the C26 methyl group in
biological activity, it should be noted that the relative dose–
response curves for downregulation of the PKC isoforms by
Merle 41 do not parallel its in vitro binding potencies, as we
described above. Likewise, the marked differences between it
and bryostatin 1 in downregulation of the PKC isoforms are
not reflected in appreciable differences in their selectivity for
in vitro binding. Although the basis for these differences re-
mains unresolved, downregulation represents the interplay of
numerous factors, both reflecting the initial ligand–PKC inter-
action and feedback from downstream consequences, as well
as multiple pathways. The profound differences in biological
response between bryostatin 1 and Merle 23, as previously re-
ported, illustrated that binding alone was only one element,
and we and others have speculated that the different surfaces
provided by Merle 23/Merle 41 versus bryostatin 1 might be an
important contributor.[23c]
subsequent localization to internal membranes, whereas bryos-
tatin 1 caused less initial plasma membrane translocation, with
more staining of nuclear and internal membranes.[38] These
differences were likewise found in the current experiments
(Figure 4). As previously reported,[25] Merle 23 behaved more
like bryostatin 1 than like PMA (Figure 4). Merle 41 behaved
similarly to Merle 23 (Figure 4).
PKCe in LNCaP cells was translocated to the plasma mem-
brane in response to PMA and more weakly in response to
bryostatin 1 (Figure 5), as had been reported previously.[23b]
Merle 23 and Merle 41 resembled one another in their behav-
ior, which was intermediate between that of PMA and bryosta-
tin 1 (Figure 5). Because of the extent of variability of response
among cells, it should be noted that the comparisons of trans-
location for PKCd and PKCe would not be able to define minor
differences in behavior.
As a powerful approach to detect more subtle differences in
the responses to Merle 41 and Merle 23, we examined the time
and dose dependence of the induction of gene expression by
Merle 41 and Merle 23, and we further compared those re-
sponses to those induced by PMA and bryostatin 1. Responses
were characterized in both LNCaP cells (Figure 6) and U937
cells (Figure 7), two systems that we have studied extensively
for their responses to bryostatin derivatives. The genes we
examined had been previously observed to illustrate different
patterns of response to PMA and bryostatin 1. As we had de-
scribed before,[39] bryostatin 1 and Merle 23 caused responses
in LNCaP cells very similar to those caused by PMA at 2 h (Fig-
ure 6A). By 6 h, the response to bryostatin 1 had decreased to
variable extents for different genes relative to the PMA re-
sponse, and the response to Merle 23 began to separate from
that of PMA (Figure 6B). By 24 h, the response to Merle 23 had
Comparison of the patterns of downregulation at 100 nm
levels of the various ligands emphasizes a very important con-
cept (Figure 3). Whereas “PMA-like” or “bryostatin-like” may
afford a convenient summary of the pattern of behavior in a
system like the U937 cells, detailed characterization shows that
different ligands have different patterns of interaction with var-
ious PKC isoforms or other C1 domain-containing targets like
PKD1. The patterns of downregulation of PKC isoforms by
Merle 41 or Merle 23 were unique and distinct from those of
either PMA or bryostatin 1. A powerful prediction, therefore, is
that such derivatives should have a unique pattern of biology,
as we indeed observed in our detailed characterization of the
action of Merle 23 in LNCaP cells.[25]
We previously described that PMA initially induced translo-
cation of PKCd to the plasma membrane of LNCaP cells, with
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Figure 1. Comparison of the biological effects of Merle 23 and Merle 41. A) Dose–response curves for inhibition of growth of Toledo cells after 72 h of treat-
ment. B) Inhibition of growth of U937 cells after 60 h of treatment. C) Induction of attachment of U937 cells after 60 h of treatment. D) Secretion of TNFa
from U937 cells after 60 h of treatment. All points represent the meanÆSEM of triplicate experiments. Methods were as previously described.[23b,39]
further separated and more approached that of bryostatin 1
(Figure 6C). In each case, the response to Merle 41 was almost
identical to that of Merle 23. A similar pattern was observed in
the U937 cells, except that the response to bryostatin 1
diverged at later times for some genes, and the response to
Merle 23 remained closer to the PMA response (Figure 7). In
each case, the response to Merle 41 remained very similar to
that of Merle 23.
gave maximal induction, whereas maximal induction by
Merle 23/41 occurred at 10 nm. Because of the biphasic dose–
response curves, response to bryostatin 1 at 10 nm was some-
what lower, and response to PMA was lower still. Because the
dose–response to bryostatin 1 was substantially less biphasic
than that of PMA or Merle 23/41, at 100 nm, the response to
bryostatin 1 was greater than that of PMA or Merle 23/41. De-
spite such complexities, Merle 41 behaved similarly to Merle 23
under virtually all assay conditions. Of the 15 genes examined
in LNCaP cells and the 12 genes examined in U937 cells, a
modest, statistically significant difference in the responses to
Merle 23 and Merle 41 was observed in only one case, for in-
duction of TNFa expression in U937 cells (Figure S1 in the Sup-
porting Information).
The above summaries reflect behavior at 100 nm concentra-
tions of each ligand. The complete dose–response curves pro-
vide further insight. For example, at 2 h in the U937 cells, the
response of TNFAIP3 was greater for bryostatin 1 than it was
for PMA or for Merle 23/41 (Figure 7A). The complete dose–re-
sponse curves revealed that bryostatin 1 was not actually more
effective at induction of TNFAIP3 gene expression (Figure 8);
rather, the dose–response curves for PMA and Merle 23/41
were markedly biphasic, although that for bryostatin 1 was less
so. Equal levels of TNFAIP3 were induced at optimal levels of
each ligand, but a 1 nm concentration of PMA and bryostatin 1
Conclusion
Our extensive analysis failed to reveal any substantial influence
of the C26 methyl group on the pattern of biological response
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Figure 2. Dose–response curves for downregulation of PKC isoforms and PKD1 in LNCaP cells. LNCaP cells were treated as indicated for 24 h. All points repre-
sent the meanÆSEM of quadruplicate experiments. Methods are described in the Supporting Information.
Figure 3. Comparison of the effects of compounds on the levels of PKC iso-
forms and of PKD1 in LNCaP cells. Levels following treatment for 24 h with
the indicated compounds (100 nm) are expressed relative to those for the
vehicle control (DMSO; data from Figure 6).
Figure 4. Comparison of the effects of compounds on translocation of
mouse GFP-PKCd. LNCaP cells were imaged at the indicated times after
treatment with 1000 nm concentrations of the compounds. Results are rep-
of this pair of bryostatin derivatives, Merle 23 and Merle 41.
Until shown otherwise, it therefore seems appropriate to
assume that differences in bryostatin structure–function rela-
tionships reflect structural differences, exclusive of the pres-
ence or absence of a C26 methyl group.
resentative of five experiments for PMA and bryostatin 1, 12 experiments for
Merle 23, and ten experiments for Merle 41. Scale bars: 10 mm. Methods
were as previously described.[23b]
As we learn more about bryostatin structure–function rela-
tionships, the importance of detailed functional characteriza-
tion has become ever clearer. PKC binding potency represents
an initial determinant of potential activity and is predominantly
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Figure 5. Comparison of the effects of compounds on translocation of
human YFP-PKCe. LNCaP cells were imaged at the indicated times after
treatment with 1000 nm concentrations of the compounds. Results are rep-
resentative of 4 experiments for PMA, bryostatin 1, and Merle 23, and six
experiments for Merle 41. Scale bars: 10 mm. Methods were as previously
described.[23b]
conferred by pharmacophoric elements in the lower half of the
molecule.[16] In contrast, the failure of bryostatin 1 to induce
PMA-like responses, despite PKC activation, is largely conferred
by the top (A, B rings) portion of the molecule,[20,40] provided
some domain capable of high affinity PKC binding is present.
Bryostatin-like activity is not an all-or-none phenomenon, how-
ever. A series of bryostatin derivatives with progressive modifi-
cations in the patterns of substitution in the A and/or B ring
portion showed variable extents to which they resembled PMA
versus bryostatin 1 in their activity in a U937 cell system.[39]
The extents of bryostatin-like activity correlated with the
extent to which they could induce a more bryostatin-like pat-
tern of transient gene expression. Comparison of the behavior
of Merle 23 in U937 cells with its activity in LNCaP cells further
emphasized that the cellular pattern of response depended
not only on the ligand but also on the cell system.[25] Thus, in
LNCaP cells, Merle 23 was like bryostatin 1, not like PMA, in its
inability to inhibit cell proliferation. A plausible rationale is that
biological responses in different cells show different depend-
ence on the levels of expression of different PKC isoforms and
on the variable sensitivities of these isoforms to the com-
pounds. The biological outcome depends on the extent of
downregulation of the various isoforms, as well as on their cel-
lular localization.[25,39,41]
Figure 6. Induction of gene expression in LNCaP cells as a function of time
*
following treatment with 100 nm concentrations of PMA ( ), bryostatin 1
* * *
( ), Merle 23 ( ), and Merle 41 ( ). Levels of mRNA expression of the indi-
cated genes were measured by qPCR as described previously[39] after treat-
ment for A) 2 h, B) 6 h, and C) 24 h. Values are expressed relative to those for
the vehicle control (DMSO) and represent the meanÆSEM of triplicate ex-
periments.
tural modification might yield important drug leads beyond
the initial goal of structurally simplified, synthetically accessible
bryostatin 1 mimetics.
An important implication for therapeutic development is
that different ligands that cause different patterns of PKC iso-
form downregulation and localization should have different
impacts, depending on the specific cell type and the specific
set of responses of importance for that cell. As illustrated here, Acknowledgements
Merle 23/41 have their own patterns of PKC regulation. These
compounds could thus be of particular utility for the appropri-
ate targets. By extension, the current efforts in bryostatin struc-
This research was supported in part by the intramural research
program of the National Institutes of Health (NIH), Center for
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Figure 8. Dose–response for induction of gene expression of TNFAIP3 in
U937 cells after treatment for 2 or 6 h. Values are expressed relative to those
for the vehicle control (DMSO) and represent the meanÆSEM of triplicate
experiments.
Conflict of Interest
The authors declare no conflict of interest.
Keywords: drug design · natural products · protein kinase C ·
signal transduction · structure–activity relationships
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Manuscript received: December 19, 2017
Accepted manuscript online: March 8, 2018
Version of record online: && &&, 0000
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X. Zhao, N. Kedei, A. Michalowski,
Missing but not missed: Work is under-
way to understand how bryostatin 1, a
potential therapeutic agent for cancer
and dementia, confers potency toward
protein kinase C and induces unique
biological responses. Although the sec-
ondary C26 hydroxy group was consid-
ered a critical pharmacophoric element,
the presence or absence of C26 methyl
substitution of bryostatin analogues did
not affect their induced biological re-
sponses.
N. E. Lewin, G. E. Keck, P. M. Blumberg*
&& – &&
Deletion of the C26 Methyl
Substituent from the Bryostatin
Analogue Merle 23 Has Negligible
Impact on Its Biological Profile and
Potency
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