Nineteen-step total synthesis of (+)-phorbol

Article abstract of DOI:10.1038/nature17153

Phorbol, the flagship member of the tigliane diterpene family, has been known for over 80 years and has attracted attention from many chemists and biologists owing to its intriguing chemical structure and the medicinal potential of phorbol esters. Access to useful quantities of phorbol and related analogues has relied on isolation from natural sources and semisynthesis. Despite efforts spanning 40 years, chemical synthesis has been unable to compete with these strategies, owing to its complexity and unusual placement of oxygen atoms. Purely synthetic enantiopure phorbol has remained elusive, and biological synthesis has not led to even the simplest members of this terpene family. Recently, the chemical syntheses of eudesmanes, germacrenes, taxanes and ingenanes have all benefited from a strategy inspired by the logic of two-phase terpene biosynthesis in which powerful C-C bond constructions and C-H bond oxidations go hand in hand. Here we implement a two-phase terpene synthesis strategy to achieve enantiospecific total synthesis of (+)-phorbol in only 19 steps from the abundant monoterpene (+)-3-carene. The purpose of this synthesis route is not to displace isolation or semisynthesis as a means of generating the natural product per se, but rather to enable access to analogues containing unique placements of oxygen atoms that are otherwise inaccessible.

Full text of DOI:10.1038/nature17153

LETTER
doi:10.1038/nature17153
Nineteen-step total synthesis of (+)-phorbol
Shuhei Kawamura1, Hang Chu1, Jakob Felding2 & Phil S. Baran1
Phorbol, the flagship member of the tigliane diterpene family, has The route described in refs 17 and 18 is particularly instructive and
been known for over 80 years and has attracted attention from many illustrates the inherent challenge of using (+)-3-carene (6) as a start-
chemists and biologists owing to its intriguing chemical structure ing material to (+)-phorbol (1): 38 steps were required to reach a
and the medicinal potential of phorbol esters1. Access to useful phorbol analogue lacking the C11 methyl and C13 oxygen from 6
quantities of phorbol and related analogues has relied on isolation (refs 17, 18). (Here we define a reaction step as one in which a sub-
from natural sources and semisynthesis. Despite efforts spanning strate is converted to a product in a single reaction flask (irrespective
40 years, chemical synthesis has been unable to compete with of the number of transformations) without intermediate workup or
these strategies, owing to its complexity and unusual placement of purification.) Our studies, outlined below, culminated in a two-phase,
oxygen atoms. Purely synthetic enantiopure phorbol has remained 19-step synthesis of 1 that solves these problems in a concise way.
elusive, and biological synthesis has not led to even the simplest
The synthesis commenced with intermediate 5 (Fig. 2), provided
members of this terpene family. Recently, the chemical syntheses in large quantities using a previously described route6. Installation of
of eudesmanes2, germacrenes3, taxanes4,5 and ingenanes6–8 have all the C4 oxygen was accomplished using a Mukaiyama hydration and
benefited from a strategy inspired by the logic of two-phase terpene in situ silyl group installation to furnish 7 in 70% yield (gram scale).
biosynthesis in which powerful C–C bond constructions and C–H At this juncture, we pursued installation of the C12 oxygen atom,
bond oxidations go hand in hand. Here we implement a two-phase which required the selective oxidation of a methylene position in the
terpene synthesis strategy to achieve enantiospecific total synthesis presence of an enone, six tertiary C–H bonds and two other compet-
of (+)-phorbol in only 19 steps from the abundant monoterpene ing methylene sites. To choose the proper oxidant for this transfor-
(+)-3-carene. The purpose of this synthesis route is not to displace mation, we analysed the structure computationally (Fig. 3a) and by
isolation or semisynthesis as a means of generating the natural
product per se, but rather to enable access to analogues containing
12
18
13
17
unique placements of oxygen atoms that are otherwise inaccessible.
Tigliane diterpenes have been viewed as promising leads in many
different medicinal applications including immunomodulatory9,
anti-viral10 and anti-cancer11 applications. The most progressed
compound in this class of natural products is phorbol 12-myristate
13-acetate, which is currently in phase II clinical trials for treatment
of acute myeloid leukaemia. Subtle perturbations of their structures
can have a marked effect on their biological profiles, perhaps as a
result of differing protein kinase C subtype selectivity9,10. As such,
phorbol esters and related terpenes have been a focus of natural
products research1. Our interest in this family stemmed from the
anti-cancer effects of certain phorbol analogues whose access was
limited. Numerous eudesmane family members have previously been
accessed in a concise way via a strategy for the chemical synthesis
of complex terpenes that follows the underlying logic of biosyn-
thesis2. This strategy has subsequently been successfully applied to
germacrenes3, complex taxanes4,5 and ingenanes6–8. In particular, the
two-phase synthesis of (–)-ingenol (2, Fig. 1) is only 14 steps from
(+)-3-carene (6) and has enabled access to a variety of analogues that
were not accessible by any other means6–8. Because ingenanes (4)
and tiglianes (3) are closely related in nature, we hypothesized that
intermediate 5, which is available in quantities of more than 100g,
might serve as an ideal starting point for a short route to 1. The exe-
cution of this plan depended on the invention of a simple solution
to the challenge of incorporating the C12 and C13 oxygen atoms
of 1; the difficulty in installing this functionality is well known in the
field of organic chemistry. So far, only two total syntheses12–14 and
two formal syntheses15,16 of 1 have been reported in 40–52 steps (see
Supplementary Information for a summary)12–16. In these studies, an
α-oxygenated enone was prepared from a ketone and subsequently
cyclopropanated to build in the C13 oxidation via a six-step sequence.
Many other efforts towards 1 have also been reported17,18 (for a full
11
16
15
14
19
1
9
8
10
5
2
7
4
3
6
20
3
4
Tigliane skeleton
Ingenane skeleton
Biosynthetic oxidase phase
Key challenge
HO
H
Me
Me
H
13 OH
Me
HO
Me
12
O
Me
Me
Me
Me
H
H
H
Phorbol (1)
O
OH
HO
HO
HO
OH
OH
• Previously 40–52 steps
• Not enantioselective
Ingenol (2)
13 steps,
this work
8 steps,
ref. 6
Synthetic oxidase phase
H
13 H
Me
Me
Me
12
TMSO
O
H
OTBS
Me
5
6 steps,
>100-g scale
Ref. 6
Me
Me
Me
(+)-3-carene (6)
Figure 1 | A two-phase approach to ingenanes and tiglianes enables a
listing of the 36 papers in this area, see Supplementary Information). concise approach to the phorbol structure.
1Department of Chemistry, The Scripps Research Institute, La Jolla, California 92037, USA. 2Front End Innovation, LEO Pharma A/S Industriparken 55, 2750 Ballerup, Denmark.
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© 2016 Macmillan Publishers Limited. All rights reserved

RESEARCH LETTER
HO
Me
Me
Me
Me
Me
Me
Me
(7)[Mn], O2
PhSiH3
Me
Me
TMSO
TMSO
6 steps
(8) TFDO
TMSO
Me
Me
O
O
H
H
OTBS
O
H
then
TMSOTf
(70%)
OTBS
1-g scale
Me
Ref. 6
OTBS
TMSO
TMSO
Me
Me
>100-g scale
Me
(+)-3-carene (6)
5
7
8
1-g scale
then MgI2
ZnI2
O
OH
O
OH
H
CF3
(9) [Mn], O2
PhSiH3,
PPh3
Me
Me
O
Me
Me
(10) RuCl3
NaBrO3
TMSO
TMSO
O
Me
OH
(11) TFAA
then Zn
TMSO
TMSO
Me
OH
O
O
H
H
O
O
Me
Me
Me
H
Me
(96%)
2 steps
(34% 10 +
62% 7)
Me
OTBS
OTBS
TMSO
TMSO
TMSO
OTBS
TMSO
OTBS
Me
10
Me
Me
12 [X-ray]
Me
1-g scale
(62%)
11
9
HO
O
O
OH
H
HO
OR
Me
TMSO
Me
Me
Me
Me
Me
Me
Sc(OTf)3
TMSO
TMSO
(R = H)
Me
O
O
O
OTFA
H
H
Me
Undesired
OTBS
OTBS
TMSO
OTBS
TMSO
TMSO
Me
Me
Me
A
B
13
then Ac2O
Desired
(R = Ac) 1-g scale
Me
O
HO
O
OAc
O
O
OAc
OAc
O
Me
Me
Me
Me
Me (13) CrO3
3,5-DMP
Me
Me
(12) TsNHNH2
O
CF3
TMSO
TMSO
TMSO
NaBH3CN
Et3N
Me
Me
OTBS
Me
O
O
TMSO
H
H
OTBS
H
OTBS
H
H
(40%)
(46%)
O
H
Me
Me
TMSO
TMSO
O
OTMS
Me
15 (64%)
Me
16
Me
17
(14) TMSN3 (15) Me4Sn
I2
[Pd]
TMSO
OTBS
Me
14
O
HO
O
O
OAc
OH
OAc
OAc
Me
HO
Me
HO
Me
HO
Me
TMSO
(18) NaBH4
Me
Me
Me
Me
(19) NaBH(OAc)3
then TBAF
Me
Me
then Ac2O
(16) HF-Py
(17) Martin
Me
Me
Me
Me
Me
Me
H
H
H
H
OTBS
then
Ba(OH)2
H
H
H
H
(93%)
Sulfurane
O
O
O
O
then SeO2
OTMS
OH
OTMS
20
OTMS
18
(72%)
CHO
19 [X-ray]
Me
OH
OAc
2 steps (51%)
(+)-phorbol (1)
19 steps from (+)-3-carene
(64% 18 + 33%17)
Figure 2 | 19-step total synthesis of 1. Reagents and conditions are as
follows. (7) Mn(acac)2, PhSiH3, O2, EtOH, then TMSOTf, Et3N, CH2Cl2,
0 °C, 70%. (8) TFDO, CH2Cl2, 0 °C, then ZnI2, MgI2, Et2O. (9) Mn(acac)2,
PhSiH3, O2, PPh3, EtOH, 2 steps (34% 10 + 62% 7). (10) RuCl3, NaBrO3,
NaHCO3, EtOAc, CH3CN, H2O, 96%. (11) TFAA, DMAP, CH2Cl2, 0 °C,
then Zn, AcOH, CH2Cl2, then Ac2O, DMAP, CH2Cl2, 0 °C, then Et3N,
DMF, 60 °C, 64%. (12) TsNHNH2, MeOH, reflux, then NaBH3CN, AcOH,
reflux, 40%. (13) CrO3, 3,5-DMP, CH2Cl2, 0 °C to room temperature,
46%. (14) TMSN3, DCE, 70 °C, then I2, DCE, pyridine, 70 °C. (15) Me4Sn,
PdCl2(PhCN)2, AsPh3, CuI, NMP, 80 °C, 2 steps (64% 18 + 33% 17).
(16) HF ⋅ pyridine, THF, 0 °C. (17) Martin Sulfurane, DCE, 60 °C, then
SeO2, benzene, 80 °C, 2 steps 51%. (18) NaBH4, MeOH, −40 °C, then
Ac2O, DMAP, CH2Cl2, 93%. (19) NaBH(OAc)3, benzene, reflux, then
TBAF, THF, 0 °C, then Ba(OH)2, MeOH, 72%.
inference of innate reactivity using NMR (Fig. 3b). Thus, we predicted same flask with MgI2/ZnI2 to elicit a dehydrative ring opening22 to
the pseudo-equatorial C–H bond at C12 to be the most reactive on furnish diene 9 along with unreacted enone 7. We did not observe the
the basis of the following considerations19: (1) steric shielding of the anticipated tertiary iodide product, presumably owing to rapid elim-
C6, C7, C8 and C11 positions would decrease their rate of oxidation; ination to form a diene system. The mixture of 7 and 9 was directly
(2) the higher s-character of the tertiary cyclopropane C–H bonds subjected to another Mukaiyama hydration to furnish the tertiary
(C13/14) makes them very difficult to oxidize; (3) of the remaining carbinol 10 in 34% isolated yield from 7 along with 62% recovered 7.
carbon centres, 13C NMR indicated that C12 is the most ‘nucleop- The addition of PPh3 to this type of reaction has not previously been
hilic’; (4) hyperconjugation from the π-like cyclopropane system reported and was found to be essential to prevent the reactive inter-
should facilitate oxidation of the pseudo-equatorial C–H bond on mediate peroxide adduct from generating an unwanted by-product
C12; and (5) strain-release might, to a small extent, accelerate such an (see Supplementary Information for details).
oxidation20. Therefore, we chose the small, reactive, electrophilic
With the cyclopropane ruptured, the next step was to install the
oxidant methyl(trifluoromethyl)dioxirane (TFDO), owing to its C12/13 oxygenation pattern along with reclosure of the cyclopropane.
straightforward preparation and success in other challenging methyl- This manoeuvre has no precedent, and needed to be accomplished
ene oxidations21. As predicted, TFDO cleanly achieved C–H oxidation rapidly and in a scalable fashion. To this end, chemoselective oxida-
at C12 to deliver the intermediate cyclopropyl carbinol intermediate 8 tion of the C12/13 olefin to diketone 11 took place in near quantitative
(single diastereomer), which could be isolated and fully characterized yield using Ru/NaBrO3. To reform the gem-dimethylcyclopropane
on a gram scale. In practice, however, it was directly treated in the moiety and access the correct oxidation state, a two-electron reduction
2
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LETTER RESEARCH
a
desired cyclopropane is concerted because no elimination products
were observed (see above). This reaction cascade consists of thirteen
discreet events occurring in the same flask, forming two new C–C
bonds, three new ring systems and three C–O bonds, along with the
cleavage of one C–C bond, two ring systems and two C–O bonds. It
unravels a complex polycyclic network to a kinetic endpoint using
simple reagents thereby overcoming the greatest challenge posed by 1.
The final eight steps of the synthesis served to install two addi-
tional oxygen atoms, one methyl group and the proper C12 and C10
stereochemistry. Attempts to install the key C10 stereocentre by way
of conjugate reduction were met with failure despite extensive exper-
imentation (see Supplementary Information for details). Thus, to
achieve allylic transposition of 15, the weakest point of the synthesis
from an efficiency standpoint, we used TsNHNH2 under reductive
conditions, which led to 16 in 40% yield. Allylic oxidation with CrO3
(ref. 26) delivered enone 17 in 46% yield. A two-step methyl group
introduction was accomplished by formation of an α-iodoenone
(TMSN3, I2)27 followed by a Stille coupling28, which afforded 18 in
64% overall yield along with 33% recovered 17. Selective removal of
the C7 and C9 silyl groups with the HF ⋅ pyridine complex led to a
diol that could be selectively dehydrated using the Martin Sulfurane
reagent to an unstable olefin that was directly oxidized using SeO2 to
aldehyde 19 in 51% overall yield. The selective cleavage of the C7 and
C9 silyl groups could stem from the inductive effect of the C3 carbonyl
group, which could make the C4 silyl group less reactive to acids. X-ray
crystallographic analysis of 19 confirmed the structural assignments
made thus far. To complete the synthesis, 19 was reduced to the allylic
alcohol and shielded as the acetate ester. Attempts to directly reduce
the C20 aldehyde and the C12 ketone in 19 were unsuccessful, owing
to the rapid rate of the C3 enone carbonyl reduction in the presence of
12
13
11
8
14
3
7
6
5
b
More nucleophilic
C5
C12
25
C3
55
50
45
40
35
30
20
15
G (p.p.m.)
Figure 3 | Predicting the site of C–H functionalization on intermediate
7. a, Structural analysis of intermediate 7 calculated using MacroModel
10.8 (Schrödinger LLC; http://www.schrodinger.com/citations/41/11/1/).
Grey, carbon; white, hydrogen; red, oxygen; orange, silicon. b, 13C NMR
can be used to predict the most nucleophilic methylene C–H bond after
taking into account the role of other factors (see text).
of the diketone to an enediol would need to take place followed by the C20 free primary alcohol (the C20 hydroxy group is actually prox-
intramolecular cyclization. Whereas attempts at acetylation of the imate to C3 as judged by molecular models). Stereoselective ketone
hindered tertiary alcohol were fruitless and mesylation led to rapid reduction using NaBH(OAc)3 followed by successive treatment with
elimination (as with the putative iodide intermediate in 8 → 9), treat- fluoride and hydroxide sources (TBAF and Ba(OH)2, respectively)
ment with trifluoroacetic anhydride smoothly led to an intermediate led to optically pure (+)-1 (synthetic, [α]26D 105.37 (c 0.23, CH3OH);
trifluoroacetate. This ester could be treated in the same flask with natural, [α]27D 104.25 (c 0.20, CH3OH)) in 72% isolated yield (15mg
Zn to deliver an intermediate (12) whose structure was confirmed prepared by this route).
with the aid of X-ray crystallography. Although the desired reduction
The concise route to 1 is enabled by a fundamentally different ret-
took place, it was accompanied by the formation of an additional, rosynthetic approach to terpene synthesis in the laboratory, rather
unwanted and bizarre C–C bond between C13 and the carbonyl than by focusing on the invention of a new synthetic method; the
group of the trifluoroester. Although aldol additions of enediols to newest method used in this synthesis is a Stille coupling, invented in
aldehydes23 and ketones24 are known, there is no precedent for such the 1980’s28. Although the development of new synthetic methodolo-
reactivity with esters. We reasoned that this product originated from gies are necessary to push the field of organic chemistry forward, this
a highly reactive enediol intermediate (such as A; R=H) that might work emphasizes the equal importance of strategy design in the total
be regenerated under equilibrating conditions. In support of this synthesis of complex natural products29. By holistically mimicking
hypothesis was the empirical observation that conversion of 11 to 12 the biosynthesis of ingenol (2) in the laboratory6,7, we exploited the
initially resulted in the formation of an approximately 1:1 mixture interrelationship between ingenanes and tiglianes by using the same
of 12 along with an isomeric species tentatively assigned as the dias- building block for both synthetic routes. Because two-phase terpene
tereomeric aldol adduct. Over 24 h this mixture equilibrated to 12 synthesis builds the carbon skeleton with a strategically planned redox
exclusively, perhaps owing to stabilizing intramolecular hydrogen state, the majority of steps can focus on installing key oxidations
bonding interactions. We evaluated various sets of conditions with rather than repositioning functional groups and C–C bonds. Access
the aim of reintroducing the enediol (retro aldol) in the hope that to phorbol (1) requires only five additional steps compared to ingenol
concomitant irreversible cyclopropane formation (via attack at C15) (2), owing to the presence of two additional oxygen atoms (C12/13)
would take place. However, treatment of 12 with catalytic Sc(OTf)3 placed at particularly challenging locations on the carbon skeleton.
in DMF led instead to the cyclobutanone 13. Although clearly unde- Successful installation of these oxygenations and two others directly
sired, this was encouraging because 13 must have been derived from or indirectly relied on the application of C–H functionalization logic
the desired hydroxy-cyclopropane B via a 1,2-shift, which in turn to pinpoint both the location and sequence of reactions19,30. This arti-
was produced via the enediol intermediate A (R =H). Indeed, the ficial oxidase phase will enable the synthesis of analogues containing
formation of cyclobutanones such as 13 from hydroxycyclopropanes deep-seated modifications in the same fashion that has recently been
has a precedent25. To circumvent this undesired pathway, interme- reported for ingenol (2)8. Although the route, in its current concise
diate 12 was converted to hemiorthoester 14 by simple treatment form, cannot compete with isolation for the procurement of large
with Ac2O followed by exposure to Et3N and gentle heating to quantities of phorbol (1), we argue that it could be adapted to become
60 °C to deliver 15. The single flask conversion of 11 to 15 can be truly scalable if the need arose. Rather, the purpose of this synthesis is
conducted on a gram scale, and presumably succeeds owing to the to enable rapid access to new tigliane family members with exciting
in situ formation of intermediate A (R=Ac), which prevents 1,2-shift bioactivity that are either difficult or impossible to access through
after cyclopropanation. We believe that the conversion of A to the isolation or semisynthesis.
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RESEARCH LETTER
Received 4 December 2015; accepted 20 January 2016.
Published online 23 March 2016.
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Acknowledgements This work was supported by LEO Pharma, the Uehara
Memorial Foundation (postdoctoral fellowship to S.K.) and the National Institute
of General Medical Sciences Grant GM-097444. We are especially grateful
to S. Natarajan of KemXtree and his team for providing ample quantities of
compound 5. We thank D.-H. Huang and L. Pasternack for assistance with NMR
spectroscopy, and A. L. Rheingold and C. E. Moore for X-ray crystallographic
analysis.
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Author Contributions S.K. and P.S.B. conceived this work; J.F. provided
compound 5; S.K., H.C. and P.S.B. designed the experiments and analysed that
data; S.K. and H.C. conducted the experiments; S.K. performed the molecular
mechanics calculations; and S.K. and P.S.B. wrote the manuscript.
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Author Information Metrical parameters for the structures of 12 and 19 are
available free of charge from the Cambridge Crystallographic Data Centre
(CCDC) under reference numbers 1434376 and 1434377. Reprints and
permissions information is available at www.nature.com/reprints. The authors
declare no competing financial interests. Readers are welcome to comment
on the online version of the paper. Correspondence and requests for materials
should be addressed to P.S.B. (pbaran@scripps.edu).
19. Newhouse, T. & Baran, P. S. If C–H bonds could talk: selective C–H bond
oxidation. Angew. Chem. Int. Ed. 50, 3362–3374 (2011).
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