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Tigliane Diterpenoids 93
rather than a lactone. This next loses carbon dioxide in a vinylogous fashion, eventu-
ally generating the norphorbol furan derivative (523) (Scheme
16)[44]. The config-
urational retention at C-4 is associated with the presence of a 20-trityl group, which
possibly for steric reasons, slows down the retro-aldol epimerization, also giving
an equilibrium mixture containing both C-4 epimers [
49]. Remarkably, the 20-trityl
group additionally protects the ring B Δ
6
-double bond by oxygen attack, and phorbol
20-trityl ether is indeed much more stable than phorbol (1) and the present authors
have stored this derivative for several years in a well-closed flask at a refrigerator
temperature of 4°C.
Epimerization at C-4 as well as oxidative opening of ring A limit the hydrolysis/
transesterification of phorbol esters under basic conditions. Apart from a careful
control of pH and temperature, the reaction would therefore benefit from being
carried out under an inert gas atmosphere to minimize degradation. The formation
of the furan derivative 523 during the hydrolysis of croton oil is easily shown by the
strong purple color this compound exhibits on TLC plates as revealed with sulfuric
acid, which contrast vividly with the brown-orange color of the phorbol (1) and
4α-phorbol (14) zones [
268].
The tertiary C-4 hydroxy group of phorbol (1) can be acetylated only under forced
conditions (i.e. heating of phorbol 12,13,20-triacetate with acetic anhydride in the
presence of p-toluenesulfonic acid) under which also the tertiary C-9 hydroxy group
is acetylated [
261]. The methylation of the 4-hydroxy group of phorbol myristate
acetate (2) is an important reaction, since it has a detrimental effect on the interac-
tion with protein kinase C, and the 4-O-methyl derivative of PMA 524 is used as
negative control in biological experiments when the involvement of protein kinase
C is suspected [
269]. The methylation reaction is carried out in DMF, when treating
phorbol esters with excess methyl iodide in the presence of Ag
2
O. The reaction
cannot be carried out on phorbol (1) itself, due to the oxidative lability of the 13-
hydroxy group (see Sect.
4.3)[269]. Remarkably, under these conditions, vinylogous
retro-aldol epimerization at C-4 does not take place.
O
OAc
HO
O
OH
HO
2
4
O
C
13
H
27
O
OAc
HO
O
OH
O
12
13
O
C
13
H
27
4
524
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94 G. Appendino and S. Gaeta
4.2 The Ring B Allyl Alcohol Hotspot of Reactivity
The reactivity associated with the ring B Δ
6(7)
double bond also affects its three allylic
carbons (C-5, C-7, and C-20). The hydroxylation of C-5 induces a reactivity profile
which, just like that of the C-9 hydroxy group, can bridge the tigliane molecular
regions of reactivity, and will be treated separately.
The Δ
6(7)
-Double Bond
The ring B endocyclic Δ
6(7)
-double bond is a key element of the tigliane struc-
tural motif. It has never been found in a reduced form in any naturally occur-
ring compounds, and its main natural oxidative modification (epoxide) is rare, and
always associated with β-hydroxylation at C-5, as in the tigilanoids (5β-hydroxy-6α-
epoxyphorbol derivatives). Remarkably, the 5β-hydroxy-6α-epoxy functionality is
instead standard in daphnane phorboids from thymelaeaceous plants [
270]. Aerobic
oxidation, a major oxidative degradation pathway for phorbol esters, is associated
with the Δ
6(7)
-double bond and has been investigated extensively in connection with
studies on the stability of phorbol myristate acetate (2)[
271]. It was discovered that
an ene reaction with single oxygen occurred spontaneously when 2 was stored in
the presence of air and light, with oxygen activation being presumably triggered by
traces of impurities. The mixture of degraded products included, in addition to the
hydroperoxide 525, its further transformation products 526 and 527,aswellasthe
seco-tigliane 528, derived from the dioxetane 529 via the dicarbonyl intermediate
530 (Scheme
17). The isomeric hydroperoxide of the 6-hydroxy-Δ
7,8
-series was not
detected in these studies, but compounds of this type are known to occur in their
reduced hydroxy version in thymelaeaceous plants [
270]. Alternative aerobic degra-
dation pathways of phorbol myristate acetate (2) afforded the β-epoxide 531 and the
unsaturated aldehyde 532 [
271].
Tigliane esters oxygenated at C-7 and with a Δ
5
-double bonds, analogs to 525–
527, occasionally have been reported from natural sources, but, given the presence
in crude plant extracts of chlorophyll, an oxygen sensitizer, they are most likely
extraction and/or isolation artefacts. Reaction with singlet oxygen and epoxidation
are substrate-controlled processes, and proceed from the more accessible β-face of
the phorbol myristate acetate B ring, notwithstanding the potential directing effect on
the α-face that the 9-hydroxy group could exert by engaging activated oxygen with
dipolar or hydrogen bonding interactions. The composition of the auto-oxidation
mixture of phorbol myristate acetate (2) was strongly dependent on its physical
state (solid/oil or solution), the nature of the solvent, and the matrix of adsorp-
tion. In solvent media, and especially acetone, the β-epoxide 531 was formed as the
major product, along with the aldehyde 532 [
271]. Conversely, formation of products
derived from an ene-reaction (525–527) prevailed in the solid state, with the semi-
acetal 533 becoming the major product when 2 was adsorbed on silica gel [
271].
Auto-oxidation was relatively fast at room temperature, requiring 2–3 months, but
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Tigliane Diterpenoids 95
OTetradec
OAc
HO
O
OH
525
HO
OOH
OTetradec
OAc
HO
O
OH
526
HO
OH
OTetradec
OAc
HO
O
OH
527
HO
O
OTetradec
OAc
HO
O
528
OTetradec
OAc
HO
O
OH
529
HO
O
O
OTetradec
OAc
HO
O
OH
530
HO
O
O
OTetradec
OAc
HO
O
OH
2 (phorbol myristate acetate)
HO
+
hυ, air
O
O
OH
7
5
6
20
5
6
20
6
7
Scheme 17 Air degradation of phorbol myristate acetate (2) in the presence of light
was slowed significantly by cooling to −20°C. Storage in well-stoppered amber-
colored vials is therefore important, possibly also under an inert gas blanket, and it
has been estimated that stock solutions of phorbol esters can be stored for at least
three months if maintained refrigerated (0°C or lower temperatures) and in the dark
[
271].
Epoxidation of the 6,7-double bond is also a major degradation pathway of phorbol
esters under γ-ray radiolytic conditions [
272]. The reaction was investigated in the
context of studies aimed at the detoxification of waste products associated with the
production of biodiesel from phorbol-ester rich oleaginous euphorbiaceous plants
like Jatropha curcas, but the nature of the final degradation products could not be
clarified [
272].
For preparative purposes, the hydroperoxide 525 can be obtained by dye-
sensitized photooxygenation of phorbol myristate acetate (2), and the epoxide 531
by epoxidation with peracids [
271]. Remarkably, peracid epoxidation affords unnat-
ural phorboid β-epoxides. The orientation of the oxirane oxygen is important for
bioactivity, since pyramidalization of the olefin C-6 carbon affects the orientation
of the primary 20-hydroxy, a group critically involved, in its dual role of hydrogen
bonding-donor and acceptor, in the interaction of phorbol esters with protein kinase
C[
273]. In accordance with the stereochemical course of the ene-reaction, treatment
of phorbol (1)[
274], phorbol 12,13-diesters [275], and phorbol 12,13,20-triesters
[
275] with peracids afforded exclusively the corresponding unnatural β-epoxides.
The reaction was first reported by Kauffmann in 1966 in the course of studies on the
structure of 1 [
274], and the reaction product was named phorboloxide (Phorboloxyd
in German), shortened into TPA-oxide (531).
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96 G. Appendino and S. Gaeta
There has been contrasting information in the literature on the effect of epoxidation
on the activity of phorbol esters. Thus, 531 was reported to be more potent than 2 in
promoting radiation-induced transformation in C3H10T1/2 cells, and contamination
with 531 was even suggested as a reason for the differences in potency observed
between different batches of 2 [
276]. However, this is in contrast with the general
observation of the decreased activity of a solution of 2 on storage, and cellular and
biochemically experiments of more direct mechanistic interpretation gave opposite
results. Thus, complete loss of cancer-related activity was observed when phorbol
12,13-dibenzoate (533) was converted to its β-epoxide [
275], and similar results
were observed with the synthetic daphnanes des-epoxy-yuanhuapin (534) and its
β-epoxide (535)[
277]. The lack of cytotoxicity and protein kinase C affinity of the
β-epoxide 535 stands in sharp contrast with the potency of the natural α-epoxide
yuanhuapin (536)[
277].
OBz
OBz
HO
O
OH
533
HO
OAc
O
O
OH
534
HO
O
O
HO
OAc
O
O
OH
HO
O
O
HO
535
OAc
O
O
OH
HO
O
O
HO
536
O
O
OTetradec
OAc
HO
O
OH
531
HO
O
OTetradec
OAc
HO
O
O
HO
532
Since the α-orientation of the epoxide oxygen is critical for bioactivity of phor-
boids [
275–277], a detailed study to reverse the intrinsic bias for generating unnatural
and inactive β-epoxides was carried out by Wender, using phorbol 12,13-dibutyrate
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Tigliane Diterpenoids 97
(503) as a substrate (Scheme 18)[278]. Treatment with mCPBA afforded the expected
β-epoxide 537 (Scheme
18). To overcome the steric bias, an alternative two-step
formation of the α-epoxide 538 via the bromohydrin 540 was first attempted. Owing
to the steric bulk of bromine, formation of the bromonium ion 539 is expected to
take place from the convex β-face, while, for stereoelectronic reasons, attack of water
should take place, at least under neutral conditions, from the α-side and on the less
substituted C-7 carbon of the bromonium ion. This strategy was, however, thwarted
by the propensity of bromine to oxidize the 20-hydroxy group, even when protected
by esterification or by silylation, with formation of a 20-dehydro derivative 541,de
facto quenching the nucleophilicity of the adjacent double bond. A possible mecha-
nism involves an alternative regiochemistry of electrophilic attack of Br
+
, that, rather
than binding to the double bond, attacks the 20-oxygen and forms a hypobromite
ester, next evolving into the aldehyde 541 by β-elimination of HBr [
278].
Alternative strategies based on reagent control were therefore pursued (Shi- and
Sharpless asymmetric epoxidations). Epoxidation with oxone in the presence of a d-
fructose-derived Shi catalyst afforded only the β-epoxide of phorbol 12,13-dibutyrate
(538), while the l-configured enantiomer of the catalyst was unreactive, due to a
mismatch between the configuration of the substrate and that of the catalyst. Better
results were obtained with Sharpless epoxidation, which afforded a 1.5:1 mixture
of β- and α-epoxides, validating the possibility of overcoming the β-facial selec-
tivity under conditions of reagent control. Good results were obtained eventually
with the Yamamoto epoxidation, a protocol that uses a combination of VO(acac)
2
,
t-butylhydroperoxide (TBHP) and a C
2
-symmetric diamide as catalyst. Under opti-
mized conditions of catalyst, solvent and temperature, a 1:9 mixture of β- and α-
epoxides (538) eventually could be obtained is an overall 90% yield [
278]. Taken
together, these studies show that the double bond epoxidation of phorbol and its esters
has a strong bias for the generation of unnatural and bioactivity-depleted β-epoxides.
Conversely, the presence of an α-epoxide positively modulates the interaction with
protein kinase C and bioactivity, but its introduction by direct epoxidation is not
trivial.
An alternative multi-step α-epoxidation strategy was developed by Wender as
part of the conversion of phorbol (1) into the anticancer agent tigilanol tiglate (3)
by hydroxylation of the C-5 methylene and epoxidation of the adjacent double bond
(Scheme
19)[45]. The double oxygenation was associated with the isomerization of
the Δ
6(7)
double bond into its Δ
5
- and Δ
6(20)
isomeric arrangements, and capitalizes
on three oxidative steps and one reduction. Photo-oxygenation of phorbol 12,13-
diacetate (542) regio-and stereoselectively afforded the Δ
5
-7β-hydroperoxide 543,
and was then reduced to its corresponding alcohol 544 with thiourea [
279]. Peracid
epoxidation of the latter was stereoselective, and afforded the expected β-epoxide
545, as a result of the convergence of steric (attack from the less encumbered convex
β-face) and directing effects (hydrogen bonding of the 7β-hydroxy group with a
peroxyacid oxygen). Tosylation of the 20-hydroxy group to generate 546 and treat-
ment of the tosylate with NaI next triggered the transpositive deoxygenation of the
glycidyl system, with the formation of the bis-allylic Δ
6(20)
alcohol 548. The reaction
is presumably a retro-type I Ferrier reaction [280], with the fleeting 20-iodide 547
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98 G. Appendino and S. Gaeta
OBut
OBut
HO
O
OH
503
HO
OBut
OBut
HO
O
OH
537
HO
mCPBA
OBut
OBut
HO
O
OH
HO
O
OBut
OBut
HO
O
OH
538
HO
O
Br
X
Br
2
, H
2
O
OBut
OBut
HO
O
O
541
HO
539
7
20
OBut
OBut
HO
O
OH
HO
OH
Br
540
7
a)
Scheme 18 Epoxidation studies on phorbol-12,13-dibutyrate (503). a) 10% VO(Oi-Prop)
3
, 20%
ligand ((R,S )-N,N
'
-dihydroxy-N,N’-3,3-diphenylpyhenylacetyl-trans-1,2-cyclohexanediamine), 2
equiv. TBHP, C
6
H
6
, 4°C; yield 90% ((1:9 mixture of β-epoxide and α-epoxide (538))
as a substrate. The reductive opening of the epoxide was then triggered by the attack
of an iodide anion on the 20-iodine atom. This generated molecular iodine and a
C-5 alcoholate (Scheme
19), formally reversing the electron movements involved
in the type I Ferrier reaction, a Lewis acid-mediated cationic process producing an
intermediate oxonium ion [
280].
The bis-allylic diol 548 was then isomerized regioselectivity with rhenium (VII)
oxide [
281] to the endocyclic Δ
6(7)
-β-hydroxy derivative 549, with the driving force
of the reaction being the higher stability of a trisubstituted endocyclic bond compared
to a disubstituted exocyclic one. The peracid epoxidation of 549 gave the expected
β-epoxide, but the formation of the six-membered C-5,20-acetonide 550 remarkably
reversed the regiochemistry of the epoxidation, affording in excellent yield the α-
epoxide 551 when mCPBA was replaced by dimethyldioxirane, a less encumbered
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Tigliane Diterpenoids 99
O
OH
HO
OAc
AcO
HO
542 (phorbol
12,13-diacetate)
O
OH
HO
OAc
AcO
HO
OR
R
543 OH
544 H
thiourea
O
OR
HO
OAc
AcO
HO
OH
O
1
O
2
mCPBA
(66% from 542)
5
7
R
545 H
546 Ts
TsCl, NMI
O
I
HO
OAc
AcO
HO
OH
O
I
547
O
HO
OAc
AcO
HO
OH
OH
20
20
7
5
O
HO
OAc
AcO
HO
OH
5
OH
548
549
O
HO
OAc
AcO
HO
O
5
O
550
O
HO
OAc
AcO
HO
O
5
O
O
O
OH
HO
O
O
HO
OH
O
O
3 (tigilanol tiglate)
551
NaI
(88% from 545)
Re
2
O
7
76%
92%
DMP, PPTS
DMDO
(63%)
12
13
Scheme 19 Conversion of phorbol diacetate (542) into tigilanol tiglate (3). TsCl = p-tosyl chloride;
NMI = N-methylimidazole; DMP = dimethoxypropane; PPTS = pyridinium p-toluene sulfonate;
DMDO = dimethyldioxirane
and more reactive reagent. Completion of the semi-synthesis of tigilanol tiglate (3)
from phorbol then involved the uneventful remodulation of the ring C acylating
decoration and final deprotection [
279].
The allylic reactivity of the Δ
6(7)
double bond is associated with the f unction-
alization of C-7 and its Δ
5
relocation, as shown in Scheme 18. Attempts to func-
tionalize the allylic carbons with retention of the Δ
6(7)
position have so far failed
[
44, 282]. Thus, allylic oxidation of phorbol 12,13,20-triacetate (552) with selenium
(IV)-reagents (selenium dioxide, selenium dioxide/t-butylhydroperoxide, phenylse-
leninic anhydride) gave the 20-aldehyde 553 as the only reaction product, echoing
the Wender results on bromine addition to the Δ
6(7)−
double bond discussed previ-
ously. Removal of the protecting group of the 20-hydroxy group and its oxidation
were also observed when a trityl group replaced the 20-acetyl.
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100 G. Appendino and S. Gaeta
OAc
OAc
HO
O
OAc
552 (phorbol 12,13,20-triacetate)
(phorbol 20-aldehyde)
HO
OAc
OAc
HO
O
O
553
HO
A notable rearrangement involving the Δ
6(7)
double bond was observed instead
when the acetonide of 3αH-dihydrophorbol 12,13,20-triacetate (554) was treated
with pyridinium chlorochromate and sodium acetate (Scheme
20). Initial forma-
tion of the cyclic chromium (IV) diester 555 was followed by displacement of
the chromium ester bond at C-6 by the 9-hydroxy group, with generation of the
chromium(IV) ester 556, eventually evolving into the C-8 ketone 557 [
282]. The
intramolecular photochemistry of the Δ
6(7)
double bond has been discussed in
Sect.
4.1.
The Δ
6(7)
double bond of phorbol 12,13,20-triacetate (552) has been dihydroxy-
lated chemoselectively with OsO
4
in pyridine [283]. In accordance with the stereos-
electivity observed in the epoxidation, attack occurred exclusively from the β-face,
with formation of the 6β,7β-glycol 558. Treatment of this compound with lead
O
HO
OAc
AcO
O
554
OAc
Cr
O
O
O
O
OH
OAc
AcO
O
555
AcO
Cr
O
O
O
O
O
OAc
AcO
O
556
AcO
Cr
O
O
OH
H
PCC
H
O
O
OAc
AcO
O
557
OAc
O
46%
3
9
Scheme 20 Reaction of 3αH-dihydrophorbol triacetate 3,4-acetonide (554) with Cr(VI) reagents.
In the mechanistic scheme, pyridinium chlorochromate (PCC) is simplified into chromium(VI)
anhydride
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Tigliane Diterpenoids 101
tetraacetate or sodium periodate cleaved ring B, with formation of the 6,7-seco-
tigliane 559, for which the C-7 aldehyde carbonyl engaged the 4-hydroxy group into
a δ-lactone ring. Historically, this reaction was critical for the localization of the
single unfunctionalized methylene of phorbol (1), which evidently was adjacent to
the non-conjugated double bond [
283], contributing to the rejection of the formula
where this methylene had been located on the five-membered ring (see Sect.
2).
OAc
OAc
HO
O
OAc
HO
558
OH
OH
OAc
OAc
HO
O
O
559
OH
O
AcO
20
6
6
4
Due to the presence of an allylic hydroxy group, hydrogenation of the ring B
double bond is generally accompanied by deoxygenation of C-20, even when working
at hydrogen pressure [
284]. Hydrogenation shows, however, selectivity for the Δ
6(7)
double bond compared to the ring A enone system.
The C-20 Hydroxymethylene Group
The 20-hydroxy group is not able to be directly acetylated in a selective way, since
acetyl chloride and acetic anhydride cannot discriminate the primary allylic C-20
hydroxy and the tertiary cyclopropyl C-13 hydroxy groups [
261]. Due to its enol-
like nature, the latter is highly reactive, and even can be acetylated chemoselectively
in the presence of the 20-hydroxy group if strictly stoichiometric amounts of the
acylating reagents are used and the reaction is carried out at low temperature [
261].
Conversely, and in blatant defiance of the principle of microscopic reversibility,
a 20-ester group can be removed selectively in the presence of 12- and 13-esters
by acidic transesterification with perchloric acid in methanol, making it possible to
synthesize 12,13-phorbol diesters from their corresponding 12,13,20-triesters [
261].
Alternatively, the 20-hydroxy group can be protected by tritylation or silylation, the
12- and 13-hydroxy groups are esterified, and eventually the protecting group (trityl
or silyl) of the 20-hydroxy group is removed [
263]. A vis-à-vis comparison of the two
strategies was reported for the synthesis of phorbol 12,13-diacetate (542) by Wender,
and the three-step protocol (protection-acetylation-deprotection) gave a better yield
than the two-step protocol (acetylation-selective deacetylation) (82% vs. 65%) [
279].
Quaternization of C-7 as in lumiphorbol (500) quenches the selective reactivity
of the 20-ester [
263], which is, however, retained in tigilanoids, where the inductive
effect of the electronegative oxirane oxygen seemingly compensates for the increased
steric congestion associated with the quaternization of C-6 [
8]. A surprising and
disturbing loss of the 20-protecting group followed by oxidation of the 20-hydroxy
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102 G. Appendino and S. Gaeta
OAcyl
OAcyl
HO
O
OH
HO
560a Acyl = COR, COR'
560b Acyl = COR'', COR'''
5
HO
OAcyl
OAcyl
HO
O
HO
5
O
O
561
O
O
PPTS
DMF
1. KOH
2. R''COCl
2. H
+
Scheme 21 Reversible protection of the 5β,20-diol system of tigilanoids as exemplified by 561
group was observed during the addition of bromine to the 6,7-double bond and during
its allylic oxidation, as discussed in the previous section [
279].
Alkylation of the 20-hydroxy group is complicated by the instability of the
vinylogous aldol system at the A/B ring junction under basic conditions, leading
to the formation of 20-alkyl 4α-phorbol derivatives. Acetalization with a 5β-
hydroxy group is, however, easy under acidic conditions with both acetone and 2,2-
dimethoxypropane, and the mild acid pyridinium p-toluenesulfonate proved suffi-
cient for the reaction [
8, 279]. Deprotection with diluted strong acids (perchloric,
p-toluenesulfonic) is then possible, although care should be taken not to trigger
cationic rearrangements associated with the cyclopropylic C-13 ester group [
8] (see
also Sect.
4.4). Capitalizing on the reversibility of acetonide formation, the ring C
acylation profile ca be modulated, converting diesters of the 560a type into diesters
of the 560b type via the acetonide 561 (Scheme
21) (see Sect. 4.4).
The 20-hydroxy group could be replaced by a chlorine by reacting phorbol 12,13-
diacetate (542) with mesyl chloride in pyridine. The resulting 20-deoxy-20-chloro
derivative (562) next could be dehalogenated reductively with Raney nickel, which
occurs more easily compared to the deoxygenation of the 20-acetate [
32]. Surpris-
ingly, the corresponding 12,13-didecanoate was unreactive under these conditions
[
32].
An unexpected reaction occurred when tigilanol tiglate (3) was subjected to the
Appel reaction with CCl
4
and triphenylphosphine (TPP) [8]. Rather than the expected
20-deoxy-20-chloro derivative, its 4,5-acetal with triphenylphosphine oxide 15 was
obtained in 19% yield. The X-ray analysis of 15 was useful in confirming the structure
of tigilanol tiglate (3) and in securing the configuration of its α-methylbutyric ester
group. The unusual course of the Appel reaction can be rationalized in terms of
intramolecular attack of the 5-hydroxy group on the oxyphosphonium intermediate
563, produced from the interaction of the 20-hydroxy with the phosphonium salt
generated by the interaction of triphenylphosphine and CCl
4
. This could originate
from the interaction of the 20-hydroxy with the phosphonium salt generated from
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