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Tigliane Diterpenoids 23
The hydrogen resonances span the chemical shift interval between the downfield
signal of H-1 (δ = 7.55 ppm, s) and the upfield one of the cyclopropane C-14 proton
(δ = 0.54 ppm, d, J = 5.5 Hz). The signal of H-1 resonates in a spectroscopic area
scarcely populated in lipophilic extracts and can be used to quantify the presence
of tigliane esters in crude extracts and in euphorbiaceous seed oils using the H-8
signal of caffeine (δ = 7.56 ppm in CDCl
3
) as internal standard [36]. The signal of
the methines H-8 and H-10 resonate rather downfield, at, respectively, around δ =
3.80 ppm (d, J = 5.4 Hz), and δ = 4.35 ppm (br s), but no other significant chemical
shift peculiarities are present in the
1
H NMR spectrum. Conversely, the
13
CNMR
spectrum shows some unexpected features. The resonances are spread between those
of the cyclopentenone carbonyl C-4 (δ = 209.2 ppm, s) and the allylic methyl C-19
(δ = 10.4 ppm, q). Of note, the upfield chemical shift of the tertiary cyclopropanol
carbon C-13 (δ = 61.7 ppm, s) is in line with the effect of hybridization on the parent
system, and contrasts with the relatively downfield chemical shift of the two other
cyclopropane carbons (δ = 36.4 ppm, d for C-14 and δ = 25.0 ppm, s for C-15). Also
surprising is the observation that the secondary oxymethine C-12 resonates downfield
(δ = 80.1 ppm, d) of the tertiary hydroxylated carbons C-4 (δ = 79.4 ppm, s) and
C-9 (δ = 74.1 ppm, s). The remarkable difference between the chemical shifts of
the cyclopropane gem-dimethyls is due to γ -shielding experienced by the β-methyl
group (C-17, δ = 18.1 ppm, q) from the syn-oriented C-12 and C-8 carbon atoms. This
shielding is not experienced by the C-16 methyl, which resonates at δ = 24.4 ppm
(q). These general features are also observed in the modified tigliane polyols 4- and
12-deoxyphorbol, 16-hydroxyphorbol, and 5-hydroxy-6,7-epoxyphorbol.
Two empirical correlations have emerged from the analysis of the
1
H NMR spectra
of tigliane esters:
1. The position of H-1 in CDCl
3
can differentiate esters of phorbol (1) from those of
4α-phorbol (14). In compounds from the native series, H-1 resonates downfield
close to the CDCl
3
residual proton at δ = 7.27 ppm, around δ = 7.30–7.40 ppm.
Conversely, in compounds from the 4α-series, the signal of H-1 appears upfield
of the CDCl
3
residual proton, around δ = 6.90–7.10 ppm.
2. The diastereotopic H-20 protons are quasi-isochronous when the 20-hydroxy
group is free, but form a well-defined A,B-system (J = ca. 12 Hz) in the 20-esters.
The nature of the acyl decoration of the parent tigliane polyol can be established
by combining information from NMR and mass spectra, and of great help is, in this
context, the availability of tabulated
1
H and
13
C NMR values for the most common
ester groups bound to isoprenoid alcohols [
55]. The location of ester groups at C-12
and C-20 can be secured by HMBC measurements, since in both cases proton(s)
are present on the carbon bearing the acylated hydroxy group, while for the tertiary
13-ester groups NOE-measurements with the α-oriented cyclopropane methyl H
3
-16
and with H-8 are highly diagnostic. The two other tertiary hydroxy groups, located
at C-4 and C-9 are never found acylated in natural tiglianes (see Sect.
3.3).
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24 G. Appendino and S. Gaeta
Fig. 5
1
H NMR spectrum of tigilanol tiglate (3) (CDCl
3
, 400 MHz, residual CDCl
3
proton at δ =
7.27 ppm as reference)
Acylation shifts aside, ester decoration with acids bearing aromatic ring(s) does
not change the basic
1
H NMR spectroscopic features of the parent polyol, unlike
what is observed, for instance, in certain taxane pseudoalkaloids, where esterifica-
tion with acids bearing aryl groups can dramatically affect ring protons, even bringing
them to negative δ-values [
56]. Figure 5 shows the
1
H NMR spectrum of the anti-
cancer epoxytigliane diester tigilanol tiglate (3), where resonances of the core tigliane
skeleton are easily identified [
8].
Mass Spectrometry and Mass Spectrometry-Based Hyphenated
Techniques
Mass spectrometry is a powerful technique for the identification of the basic structure
of tigliane esters, providing, under widely available ionization techniques (chemical-
or electrospray ionization), information on the ester decoration and the nature of a
tigliane polyol. In early studies based on electron-impact ionization, it was noticed
that in phorbol 12,13-diesters, a long-chain ester group at C-12 is lost as an acyloxy
radical (RCOO
.
), but as a neutral molecule (RCOOH) from C-13. Based on this
observation, diesters could be sorted out into the A- and B-type, depending on
the location of the long-chain acyl moiety at C-12 (A-group) or C-13 (B-group)
[
57
]. The intensity of the molecular ion is in the range of 1% or even lower in the
electron-impact ionization spectra, but chemical-ionization can provide more intense
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Tigliane Diterpenoids 25
M+1/M−1 peaks, facilitating the assignment of molecular weight. Mass spectrom-
etry can complement NMR spectroscopy to locate functionalization in acyl moieties
having functional groups (double bond(s), hydroxy, carbonyl) separated by several
chain methylenes. Under electrospray ionization (ESI), double bond location can be
assessed by ozone-induced dissociation (OzID), a method based on in situ ozonolysis
[
58], or, alternatively, by the previous photochemical Paternó-Büchi reaction with
2-acetylpyridine [
59].
The coupling of LC-HRMS and MS/MS is a powerful technique to analyze
complex mixtures of tigliane esters, identifying the polyol(s) present in the mixture
and the molecular weight of its acyl moieties. The polyol parent peak is gener-
ally detectable, with an intensity highly variable between the various ester types,
depending on the number and location of the acyl moieties. When interfaced with
bioactivity data, tandem MS-based molecular networking can identify putative active
molecules in bioactive extracts, accelerating dereplication and focusing the purifi-
cation steps on the most active constituents of an extract [
60]. For this purpose, a
crude extract is fractionated, and the fractions are then evaluated by bioassay and
next by MS/MS. By calculating the relative abundance of a specific compound in
a fraction and comparing it to the activity level of the fraction, a bioactivity score
can be calculated for a single constituent, providing a quick alternative to classical
bioassay-guided fractionation for the identification of active constituents in complex
extracts. Structural identification is assisted by the Global Natural Product Social
(GNPS) molecular networking web platform (at
http://gnps.ucsd.edu), which makes
possible for automatic spectral mining in a database of MS/MS spectra of thousands
of compounds. This strategy has been applied successfully to the identification of
antiviral (chikungunya virus) tigliane esters in extracts of Euphorbia dendroides L.
[
60]. Remarkably, these compounds are minor constituents of the plant extract, which
is made up mostly by macrocyclic jatrophane polyesters, and inactive in this assay.
Crystallographic Studies
With some notable exceptions, including phorbol myristate acetate (2), native
tiglianes are generally amorphous solids or gums, and are not amenable to crystallo-
graphic studies. These have been reported on the unstable ethanol solvate of phorbol
(1)[
61], on its derivatives (e.g. neophorbol 13,20-diacetate-3-p-bromobenzoate (8)
[
62]), semi-synthetic esters (20-(5-bromofuroate) 7 [63]) or more modified deriva-
tives, like a 20-deoxy-20-chlorotigilanol tiglate adduct with triphenylphosphine
oxide (15)[
8]. 4α-4-Deoxyphorbol-12-tigliate-13-isobutyrate (16), possibly an arte-
fact from its corresponding 4β-configured native isomer, is one of the few tiglianes
directly obtained from isolation solved by X-ray analysis [
64].
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26 G. Appendino and S. Gaeta
HO
O
Cl
O
O
O
O
O
O
O
15 (20-deoxy-20-chlorotigilanol tiglate
triphenylphosphine adduct)
HO
O
OH
O
O
O
O
16 (4α -4-deoxyphorbol-
12-tigliate-13-isobutyrate)
P
Ph
Ph
Ph
Optical Rotation and Electronic Circular Dichroism Studies
While the optical rotation of ingenol has caused considerable confusion in the liter-
ature [
65], phorbol (1) has always been reported as a dextrorotatory compound, with
optical rotations ranging between +100° and +120° depending on the solvent and
the concentration used. Conversely, the electronic circular dichroism (ECD) of 1
originally was wrongly correlated to that of other chiral cyclopentenones, leading
to the assignment of an incorrect absolute configuration of the natural product [
66].
The ECD spectrum of phorbol in dioxane shows negative bands at 346 (Δε =−
1.11 M
–1
cm
–1
) and 270 (Δε =−1.51 M
–1
cm
–1
), and a larger positive band at 250
(Δε =+2.8 M
–1
cm
–1
). Calculations with ester derivatives fit well with the observed
spectra, but in protic solvents the intensity of the negative bands is decreased, while
the positive band is unaffected in intensity but shifted at slightly lower wavelength
values. In esters of 4α-phorbol (14) and its 4-deoxy derivative, the sign of the Cotton
effect is reversed for both the π–π* and the n–π* transitions [
66].
3.3 Distribution and Structural Diversity
Tigliane derivatives have a point-like distribution in Nature, being typical of plants
from the Euphorbiaceae and the Thymelaeaceae families. Both families are, however,
large, encompassing, respectively, 228 and 52 genera, with over 8,000 and 900
species, resulting in a large number of potential plant sources for these compounds
[
67]. Tigliane derivatives have a broader distribution compared to the two other
classes of phorboids (ingenanes and daphnanes), in having been isolated from over
thirty genera. Conversely, ingenanes only occur in three genera, and daphnanes in
less than fifteen genera, with uncertainties in species calculation related to issues of
botanical taxonomy [
67]. The largest sources of tigliane derivatives are plants from
the genera Euphorbia and Croton. While the presence of tiglianes and ingenanes is,
with only a few exception(s), mutually exclusive (see Sect.
3.4), the co-occurrence
of tiglianes and daphnanes is, conversely, not uncommon.
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Tigliane Diterpenoids 27
Plate 2 Fontainea picrosperma C.T. White. Water color painting by Ellis Rowan (1916)
Tigliane esters are lipophilic compounds, found especially in latices and in fatty
seeds, where their concentration is, however, much lower than those of the dominant
triterpenoids, triglycerides and fatty acids. A significant accumulation has so far
been reported only in the seed kernels of Croton tiglium (Plate
1; see Sect. 2) and
Fontainea picrosperma (Plate
2), where the concentration of tigliane derivatives can
reach 0.3% [
32] and >2% [36], with higher concentrations (ca. 1% and up to 6%)
being found in the kernel seed oils. Important commercial sources of tigliane esters
are the seed oils from C. tiglium (croton oil), Jatropha curcas (jatropha oil) (Plate
3),
and Sapium fordii Hemsl. (Vernicia fordii (Hemsl.) Airy Shaw) (Plate
4) (tung oil).
These oils are used for the production of biodiesel (croton and jatropha oils) or for
the preparation of paints (tung oil). From croton oil, making up ca. 30% of C. tiglium
seeds, the parent tigliane polyol phorbol (1) can be obtained in a ca. 1% yield [
32].
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28 G. Appendino and S. Gaeta
OH
OH
HO
O
OH
HO
5
2
3
4
12
16
20
6
7
9
1 (phorbol)
deoxygenative modifications
oxygenative modifications
Fig. 6 Main modifications of the phorbol oxygenation profile occurring in natural tigliane esters
Unfortunately, oxygenation at C-16 makes the major polyols from jatropha- and tung
oils unstable in the basic conditions required for their hydrolysis [
38] (see Sect. 4.4).
In addition, the concentration of tiglianes in tung oil is at least one order of magnitude
lower than in croton oil [
38].
Plate 3 Jatropha curcas L. Photograph courtesy R. K. Henning at www.jatropha.org; Creative
Commons 2.5
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Tigliane Diterpenoids 29
Plate 4 Vernicia fordii (Hemsl.) Airy Shaw. Photograph courtesy KENPAI; Creative Commons
3.0
Starting from the pioneering work of Hecker [32] and Evans [33] in the 1960s
and 1970s of the past century, the taxonomy of naturally occurring tigliane diter-
penoids has expanded significantly, reaching an inventory of over 300 different
compounds. Compared to other classes of diterpenoids, the structural diversity of
tiglianes is, however, relatively limited, and focused on a combination of oxygena-
tive/deoxygenative modifications of phorbol (1) with changes in the acyl decoration
of the various hydroxy groups. In addition to 1, major polyols have been described,
differing from the parent compound in terms of deoxygenation at C-4, C-12, or C-20,
epoxidation of the Δ
6,7
-double bond, and hydroxylation of C-5 and C-16 (Fig. 6).
Within the tigliane derivatives, some point-like mutations only occur in combi-
nation, as shown by the epoxidation of the ring B double bond, always observed
as being associated with hydroxylation of C-5. Oxygenation is stereoselective, with
epoxidation of the ring B double bond always occurring from the concave α-face,
with hydroxylation of the C-5 methylene almost exclusively seen from the convex
β-face.
We have summarized the structural diversity of tiglianes in a series of tables.
Table 1 reports the ester groups found in tigliane derivatives. Some acyl groups
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30 G. Appendino and S. Gaeta
are unique to tigliane esters, like the cyclobutane- and bicyclo[3.1.0]-dicarboxylates
found in Jatropha factors C1–C6 (17–21)[
68] and pycnocomolides (22–24)[69].
The other tables (Tables 2–26) report all known tigliane esters, along with their
plant sources. Authority abbreviations for the binomials were assigned using the
International Plant Name Index [
67] and could differ from those used in the original
reports. In the primary literature, different systematic names have been used for the
same species, and their pooling in the tables under the same name has been carried
out according to reference [
70].
O
3
O
HO
O
OH
HO
17 (C1)
12
13
16
O
O
O
3
O
HO
O
OH
HO
18 (C2)
O
O
O
HO
O
OH
HO
19 (C3)
O
O
O
3
O
HO
O
OH
HO
20 (C4 + C5; epimeric míxture)
12
13
16
O
O
O
4
O
HO
O
OH
HO
21 (C6)
O
O
O
3
2
8
Jatropha factors C1–C6
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Tigliane Diterpenoids 31
Tabl e 1 Ester groups found in phorboids
Abbreviation Name of corresponding
acid
Formula of corresponding acid
Ac Acetic acid
HO
O
Pr Propionic acid
HO
O
Bu Butanoic acid
HO
O
iBu Isobutanoic acid
HO
O
2MeBu 2-Methylbutanoic acid
HO
O
2,3MeBu 2,3-Dimethylbutanoic acid
HO
O
(S)-2MeBu (+)-(S)-2-Methylbutanoic
acid
HO
O
3MeBu 3-Methylbutanoic acid
(isovaleric acid)
HO
O
Ang (2E)-2-Methyl-2-butenoic
acid (angelic acid)
HO
O
Tigl (2Z)-2-Methyl-2-butenoic
acid (tiglic acid)
HO
O
Sen 3-Methylbut-2-enoic acid
(senecioic acid)
HO
O
(2S,3S)-3-OH-
2MeBu
(2S,3S)-3-Hydroxy-2-
methylbutanoic acid
HO
O
OH
(R)-2-Me
2
NH
2
-
3MeBu
(R)-2-(N,N-
Dimethylamino)-3-methyl
butanoic acid
HO
O
N
(continued)
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32 G. Appendino and S. Gaeta
Tabl e 1 (continued)
Abbreviation Name of corresponding
acid
Formula of corresponding acid
(S)-2-Me
2
NH
2
-
3MeBu
(S)-2-(N,N-
Dimethylamino)-3-methyl
butanoic acid
HO
O
N
Hex Hexanoic acid
(caproic acid)
HO
O
(2E,4E)-6-Oxo-
hex
(2E,4E)-6-Oxohexa-2,4-
dienoic acid
HO
O
O
Hept Heptanoic acid
(enanthic acid)
HO
O
Oct Octanoic acid
(caprylic acid)
HO
O
Oct:1 Octenoic acid (double bond
location unspecified)
HO
O
Δ
2
-Oct 2-Octenoic acid
HO
O
Δ
2,4
-Oct 2,4-Octadienoic acid
HO
O
(2Z,4E)-Oct (2Z,4E)-Octa-2,4-
dienoic acid
HO
O
(2E,4E,6E)-8-
Oxo-oct
(2E,4E,6E)-8-Oxoocta-
2,4,6-trienoic acid
HO
O
O
Non Nonanoic acid
(pelargonic acid)
HO
O
(2Z,4E,6E)-Non (2Z,4E,6E )-Nona-2,4,6-
trienoic acid
HO
O
Dec Decanoic acid (capric acid)
HO
O
Dec:2 Decadienoic acid (double
bond location unspecified)
HO
O
(continued)
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