Synthesis and biological evaluation of 12-aminoacylphorboids

Article abstract of DOI:10.1021/np9006553

Spurred by the paradoxical anti-inflammatory activity of some aminoacylphorbol derivatives, the naturally occurring and epimeric N,N-dimethylvalinoyl-4α-4-deoxyphorbol derivatives 3b and 3d have been prepared from 4α-4-deoxyphorbol (3e), a byproduct of the isolation of phorbol from Croton oil and a phorboid polyol so far largely overlooked in terms of biological activity. The configuration of the side chain stereocenter was confirmed for both natural products, and to investigate the side chain structure-activity relationships within this class of compounds, their corresponding N,N-dimethylglycinate (3g) and nor (3h) and di-nor derivatives (3i, 3j) were also prepared. By using a PKC-sensitive model of HIV-1 latency (activation of HIV- gene expression in Jurkat-LAT-GFP cells), it was found that both 3b and 3d can activate PKC-dependent responses, while a series of experiments with isoform-specific PKC inhibitors showed that these compounds target PKCα and -δ. Both N,N-dimethylation and the presence of side chain α-substitution were critical for activity. Selective PKC binding, rather than COX inhibition, might explain the paradoxical anti-inflammatory activity of extracts containing aminoacylphorboids in the mouse ear edema assay.

Full text of DOI:10.1021/np9006553

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
J. Nat. Prod. 2010, 73, 447–451
447
Synthesis and Biological Evaluation of 12-Aminoacylphorboids|
Alberto Pagani,† Carmen Navarrete,‡ Bernd L. Fiebich,§ Eduardo Mun˜oz,*,⊥ and Giovanni Appendino*,†
Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche e Farmacologiche, UniVersita` del Piemonte Orientale, Via BoVio 6,
28100, NoVara, Italy, ViVacell Biotechnology Espan˜a S. L., AVenida Conde de Vallellano 15, 14004 Co´rdoba, Spain, Neurochemistry Research
Group, Department of Psychiatry, UniVersity of Freiburg Medical School, Hauptstrasse 5, D-79104 Germany, and Departamento de Biolog´ıa
Celular, Fisiolog´ıa e Inmunolog´ıa, UniVersidad de Co´rdoba, Facultad de Medicina, AVenida de Mene´ndez Pidal s/n, 14004 Co´rdoba, Spain
ReceiVed October 17, 2009
Spurred by the paradoxical anti-inflammatory activity of some aminoacylphorbol derivatives, the naturally occurring
and epimeric N,N-dimethylvalinoyl-4R-4-deoxyphorbol derivatives 3b and 3d have been prepared from 4R-4-deoxyphorbol
(3e), a byproduct of the isolation of phorbol from Croton oil and a phorboid polyol so far largely overlooked in terms
of biological activity. The configuration of the side chain stereocenter was confirmed for both natural products, and to
investigate the side chain structure-activity relationships within this class of compounds, their corresponding N,N-
dimethylglycinate (3g) and nor (3h) and di-nor derivatives (3i, 3j) were also prepared. By using a PKC-sensitive model
of HIV-1 latency (activation of HIV- gene expression in Jurkat-LAT-GFP cells), it was found that both 3b and 3d can
activate PKC-dependent responses, while a series of experiments with isoform-specific PKC inhibitors showed that
these compounds target PKCR and -δ. Both N,N-dimethylation and the presence of side chain R-substitution were
critical for activity. Selective PKC binding, rather than COX inhibition, might explain the paradoxical anti-inflammatory
activity of extracts containing aminoacylphorboids in the mouse ear edema assay.
Over the past few years, there has been a growing awareness
that the phorbol core is a privileged structure for bioactivity, which
can be directed to different targets, or even reversed from agonist
to antagonist, by modification of the acyl decoration of the basic
polyol or by isomerization of the A,B-ring junction. Thus, acylation
of the 20-hydroxy group with homovanillic acid redirects the
activity of phorbol 12-phenylacetate-13-acetate (1a) from various
PKC enzyme isoforms to the structurally unrelated ion channel
TRPV1, generating the species-selective vanilloid ligand PPAHV
(1b),1 while epimerization at C-4 turns the ultrapotent PKC activator
phorbol didecanoate (1c) into a TRPV4 ligand (4R-PDD, 1d) devoid
of PKC affinity,2 and iodination of the side chain of resiniferatoxin
(2a) converts this ultrapotent TRPV1 agonist into an ultrapotent
TRPV1 antagonist (I-RTX, 2b).3
In this context, our attention was piqued by the discovery of the
potent COX-inhibiting activity of two aminoacylphorboids isolated
from a medicinal Mexican Croton species, one of the many “sangre
de drago” plants of South American ethnomedicine.4 The discovery
of these compounds came from an unexpected and somewhat
paradoxical observation, namely, the inhibition of the phorbol ester-
induced mouse ear edema formation by extracts from Croton
ciliatoglandulifer Ortega (Euphorbiaceae). Surprisingly, fraction-
ation of the active hexane extract afforded as anti-inflammatory
principles a pair of 4R-phorboids bearing modified valine residues
at the 12-hydroxy position (3a and 3b). While the structure of 3a
is incompatible with its spectroscopic data and requires revision to
its corresponding phorbol (and not 4R-phorbol) derivative (3c),5
that of the R(D)-N,N-dimethylvalinoyl ester 3b seems correct and
shows several intriguing features. Thus, 3b is the first compound
from the 4R-4-deoxyphorbol series to show bioactivity. Remarkably,
this activity is directed to a paradoxical end-point (COX inhibition)
for phorbol esters, the archetypal obnoxious and irritant compounds,
and is critically dependent on the configuration at C-2′, with the
side chain epimer from the S(L)-series (3d) being inactive. Finally,
acylation by an amino acid and not by a fatty acid is also unusual,
although not unprecedented, in phorboids.6 4R-4-Deoxyphorbol (3e)
is available as a byproduct from the isolation of phorbol from
Croton oil (Croton tiglium L.). Armed with a good supply of crude
fractions containing a mixture of 3e and phorbol from previous
studies in this area,7 we have embarked on a study of the biological
profile of the 12-valinyl esters of 13-acetyl-4R-4-deoxyphorbol. To
shed light on the structure-activity relationships and assess the
biological relevance of the side chain nitrogen decoration and of
the R-alkyl substitution, the synthesis of analogues characterized
| Dedicated to the late Dr. John W. Daly of NIDDK, NIH, Bethesda,
Maryland, for his pioneering work on bioactive natural products.
* To whom correspondence should be addressed. Tel: +39 0321 373744
(G.A.); +34 957 218267 (E.M.). Fax: +39 0321 375621 (G.A.); +34 957
218229 (E.M.). E-mail: appendino@pharm.unipmn.it (G.A.); fi1muble@uco.es
(E.M.).
† Universita` del Piemonte Orientale.
‡ Vivacell Biotechnology Espan˜a S. L.
§ University of Freiburg.
⊥ Universidad de Co´rdoba.
10.1021/np9006553  2010 American Chemical Society and American Society of Pharmacognosy
Published on Web 02/02/2010

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
448 Journal of Natural Products, 2010, Vol. 73, No. 3
Notes
Scheme 1. Synthesis of the 12-Acetyl-13-aminoacylphorboids
3b,d,g-j
Figure 1. Effects of nitrogen-containing 4-deoxyphorbol esters in
HIV-1 reactivation from latency. Jurkat LAT-GFP cells were
stimulated with the compounds at the indicated concentrations for
6 h and next analyzed by flow cytometry. Results are represented
as the percentage of GFP positive cells ( SD of four different
experiments.
by the stepwise removal of the N-methyls and of the R-isopropyl
group was planned.
4R-4-Deoxyphorbol (3e) was purified from the mother liquor of
phorbol after conversion to a triacetate (3f). While phorbol and its
4R-4-deoxy derivative are difficult to separate, their 12,13,20-
triacetyl derivatives could be satisfactorily separated by gravity
column chromatography on normal silica gel. After rehydrolysis,
further acylative elaboration (Scheme 1) was done according to an
established protocol for phorbol derivatives, namely, (a) selective
acetylation of the 13- and 20-hydroxy groups, (b) esterification with
a N-BOC-protected amino acid (BOC ) tert-butoxycarbonyl), and
(c) final stepwise deprotection of the amino group (TFA in CH2Cl2)
and of the 20-hydroxy group (HClO4 in methanol).7 The failure to
remove both acid-sensitive protecting groups (N-BOC and 20-
acetyl) in a single step was not surprising, since different mecha-
nisms are involved (protonolysis for the removal of the N-BOC-
group and transesterification for the removal of the 20-acetyl). D-
and L-N-BOC-valines are commercially available, as is the N-methyl
derivative of L-N-BOC-valine, and esterification of 4R-4-deoxy-
phorbol-13,20-diacetate with these amino acids was uneventful. The
N,N-dimethyl decoration had to be introduced at the stage of 12,13-
phorboid diesters, because of the failure of all our attempts to
prepare enantiopure N,N-dimethylvalines from the reaction of
dimethylamine and their corresponding and commercially available
enantiopure bromoisovalerates. Despite the risk of having to run a
somewhat capricious reaction like the reductive N,N-dialkylation
at the level of multifunctional compounds, we were, however,
delighted to discover that the ring A enone system of the terpenoid
core of valinoylphorboids was stable under the conditions of
reductive dimethylation (aqueous formaldehyde and sodium triac-
etoxyborohydride). Overall, this semisynthetic route to aminoa-
cylphorboids seems robust and amenable to the preparation of
further analogues. Semisynthetic 3b and 3d had NMR spectra
identical to those reported for the natural products, thus confirming
their structural assignment based on molecular mechanic calcula-
tions and NMR experiments.4
response (Figure 2). Remarkably, the structure-activity relation-
ships seem very strict, since both the N.N-dimethyl groups (cf. 3b,d
vs 3e) and the side chain R-substitution (cf. 3b,d vs 3g) are critical
for activity.
To investigate the PKC activity at a molecular level, the ability
of compounds 3d and 3g, as representative of a positive and a
negative probe, respectively, to activate PKCs (R and δ isotypes)
in vitro10 was used. A substrate peptide that can be efficiently
phosphorylated by both PKCR and PKCδ was employed.12 Only
3d could activate both PKC isoenzymes in a concentration-
dependent manner (Figure 3A). Since PKCs are also regulated by
phosphorylation, and activated PKCδ exhibits a critical phorpho-
rylation of Thr-505 in the activation loop,10 we used a specific
antibody antiphospho PKCδ (Thr505) to confirm the results of the
biochemical assay. In full accordance with the previous observa-
tions, 3d, as well as 12-O-tetradecanoylphorbol 13-acetate (TPA),
could activate PKCδ (Figure 3B), while PKCδ phosphorylation was
undetectable with 3g. Compound 3b has been reported to inhibit
COX-1 and COX-2 in a recombinant assay.4 Using this approach,
we indeed detected a marginal COX-2-inhibiting activity12 in all
compounds assayed. However, these compounds did not inhibit the
release of PGE2 in activated primary monocytes,12 strongly
suggesting that the activity observed in the recombinant assay is
probably due to an artifact.
Long-chain phorbol esters require a 4ꢀ-hydroxy group to bind
PKC,8 but, surprisingly, this hydroxy is redundant for PKC binding
in short-chain phorbol esters from the 4ꢀ-deoxy series.8 Intrigued
by these observations, we investigated the activity of our 4RH-
deoxyphorbol esters in a PKC-sensitive model of HIV-1 latency,
namely, the activation of HIV gene expression in Jurkat-LAT-GFP
cells.9 Surprisingly, it was found that the naturally occurring N,N-
dimethylvalinates 3b and 3d, but not their semisynthetic analogues
3g-j, could induce HIV-1 reactivation from latency (Figure 1).
Pretreatment with the inhibitors Go¨6976 (classical PKCs inhibitor)
and Go¨6983 (pan-PKC inhibitor) strongly inhibited GFP expression
induced by 3b and 3d, confirming the PKC dependence of this
Taken together, our results suggest that the natural N,N-
dimethylvalinoylphorboids 3b and 3d are potent and isoform-
specific activators of PKC, whose binding to the C1 pocket of this
enzyme could explain the paradoxical inhibitory activity of extracts
containing these compounds in the TPA mouse ear edema assay.

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
Notes
Journal of Natural Products, 2010, Vol. 73, No. 3 449
Figure 2. Compounds 3b and 3d antagonize HIV-1 latency through a PKC-dependent pathway. Jurkat LAT-GFP cells were pretreated with
the indicated inhibitors (1 µM) for 30 min and then stimulated with 3b and 3b (10 µM) for 6 h. The percentage of GFP+ cells was
measured by flow cytometry. Histograms represent one out three independent experiments, and the percentage of GFP positive cells is
indicated.
Experimental Section
General Experimental Procedures. IR spectra were obtained on a
1
Shimadzu DR 8001 spectrophotometer. H NMR (300 MHz) and 13C
NMR (75 MHz) spectra were obtained at room temperature with a JEOL
Eclipse spectrometer. The spectra were recorded in CDCl3, and the
solvent signals (7.26 and 77.0 ppm, respectively) were used as reference.
The chemical shifts (δ) are given in ppm, and the coupling constants
(J) in Hz. CIMS were taken on a VG Prospec (FISONS) mass
spectrometer. Silica gel 60 (70-230 mesh) was used for gravity column
chromatography. Reactions were monitored by TLC on Merck 60 F254
(0.25 mm) plates and were visualized by UV inspection and/or staining
with 5% H2SO4 in ethanol and heating. Organic phases were dried with
Na2SO4 before evaporation.
12,13,20-Triacetyl-4r-4-deoxyphorbol (3f). Phorbol was obtained
from Croton oil as previously reported.7 Crude semisolid phorbol (ca.
0.1% from the oil) obtained from the gravity column chromatography
purification of the defattened oil hydrolysate was triturated with EtOAc
and filtered. The filtrate (ca. 44% of the crude phorbol) was a ca. 3:1
mixtureofphorboland4R-4-deoxyphorbol(Rf 0.17in9:1EtOAc-MeOH
for both compounds). To 5 g of this crude mixture were added Ac2O
(110 mL) and cat. DMAP (ca. 100 mg). After stirring at room
temperature for 1 h, the reaction was worked up by the addition of
MeOH (ca. 75 mL) to destroy the excess acetic anhydride and next
washed with 2 N H2SO4 (5 × ca. 50 mL) and brine. The organic phase
was dried, filtered, and evaporated to afford a semisolid residue, which
Figure 3. Activation of PKCR and PKCδ by 3d and 3 g. (A)
was fractionated by gravity column chromatography on silica gel (ca.
PKCR and PKCδ activities were assayed in the presence of
35 g) to afford 12,13,20-triacetylphorbol (petroleum ether-EtOAc, 8:2,
as eluant, 2 g) and 12,13,20-triacetyl-4R-4deoxyphorbol (3f, petroleum
ether-EtOAc, 7:3, as eluant, 600 mg): white powder; IR (liquid film)
phospholipid vesicles (20% phosphatidylserine, 80% phosphati-
dylcholine) and increasing concentration of 3d and 3g. Results
are expressed as percentage of the activation observed with 1
µM TPA (100%) and represent the mean ( SE of triplicate
determination in a single experiment. Two additional experiments
gave similar results. (B) TPA and 3d phosphorylate PKCδ. Jurkat
LAT-GFP cells were incubated with either TPA (1 µM) or
increasing concentrations of 3d and 3g during 15 min. Total
PKCs and phosphorylated PKCδ (Thr505) were analyzed using
specific antibodies by western blot.
1
νmax 3416, 2950, 1738, 1432, 1375, 1320, 1022, 978 cm-1; H NMR
(300 MHz, CDCl3) δ 6.96 (1H, brs, H-1), 5.43 (1H, d, J ) 10.7 Hz,
H-12), 5.15 (1H, brs, OH-10), 5.08 (1H, brs, H-7), 4.45 (1H, brs, J )
12.6 Hz H-20a), 4.32 (1H, brd, J ) 12.6 Hz, H-20b), 3.46 (1H, m,
H-10), 3.44 (1H, brdd, J ) 11.0 Hz, H-5a), 2,.72 (1H, m, H-4), 2.45
(1H, brdd, J ) 11.0 Hz, H-5b), 2.06 (9H, s, OAc), 1.90 (1H, m, H-8),
1.75 (3H, brs, H-19), 1.70 (1H, m, H-11), 1.24 (3H, s, H-17), 1.17
(3H, d, J ) 7.0 Hz, H-18), 1.07 (3H, s, H-16), 0.78 (1H, d, J ) 5.2
Hz, H-14); CIMS m/z 475 [M + 1]+ [C26H34O8 + H]+.
4r-4-Deoxyphorbol (3e). 12,13,20-Triacetyl-4R-4-deoxyphorbol (3f,
100 mg; 0,23 mmol) was dissolved in 0.01 N NaOMe (2.0 mL), and
the solution was stirred at room temperature, maintaining the pH below
12 with the addition of further amounts of 0.01 N NaOMe. After 1 h,
the reaction was worked up by neutralization with acetic acid, filtration,
and evaporation. The residue was purified by gravity column chroma-
tography on silica gel (2 g; petroleum ether-EtOAc gradient, from
PKC functional activity was found for the first time in 4R-4-
deoxyphorbol esters, qualifying these compounds as interesting and
so far overlooked noninflammatory probes to study PKC functions
and to design small molecules capable of modulating this enzyme
in a therapeutically useful way.

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
450 Journal of Natural Products, 2010, Vol. 73, No. 3
Notes
7:3 to pure EtOAc) to afford 4R-4-deoxyphorbol as a white powder
(80 mg): IR (liquid film) νmax 3345, 2933, 1704, 1454, 1378, 1331,
(161 mg, 0.76 mmol, 3.8 equiv) and glacial acetic acid (38 µL) were
added, and stirring was continued for 1 h. The reaction was next worked
up by dilution with saturated NaHCO3, and the organic layer was
washed with brine. After drying and evaporation, the residue was pu-
rified by gravity column chromatography on silica gel (10 g, EtOAc-
MeOH gradient, from 6:4 to 2:8, as eluant) to afford the N,N-dimethyl
derivatives 3b, 3d, and 3g (ca. 80% yield).
1
1067, 1018 cm-1; H NMR (300 MHz, MeOH-d4) δ 7.29 (1H, brs,
H-1), 5.13 (1H, brs, H-7), 4.02 (1H, d, J ) 10.0 Hz, H-12), 3.84 (2H,
brs, H-20a,b), 3.49 (1H, brs, H-10), 3.18 (1H, brd, J ) 15.0 Hz, H-5a),
2.69 (1H, m, H-4), 2.26 (1H, brd, J ) 15.0 Hz, H-5b), 1.84 (1H, m,
H-8), 1.71 (3H, brs, H-19), 1.59 (1H, m, H-11), 1.23 (3H, s, H-17),
1.21 (3H, d, J ) 7.0 Hz, H-18), 1.13 (3H, s, H-16), 0.52 (1H, d, J )
5.3 Hz, H-14); 13C NMR (75 MHz, CDCl3) δ 212.8 (s, C-3), 155.8 (d,
C-1), 130.4 (s, C-2), 137.3 (s, C-6), 126.2 (d, C-7), 78.2 (s, C-9), 74.8
(d, C-12), 69.2 (t, C-20), 65.1 (s, C-13), 49.6 (d, C-4), 47.4 (d, C-10),
43.0 (d, C-11), 40.5 (d, C-8), 37.2 (d, C-14), 25.9 (t, C-5), 25.1 (s,
C-15), 16.3 (t, C-16), 11.9 (t, C-18), 10.4 (t, C-19); CIMS m/z 349 [M
+ 1]+ [C20H28O5 + H]+.
N,N-Dimethyl-12-(R-valinoyl)-13-acetyl-4r-4-deoxyphorbol (3b).
1H NMR (300 MHz, CDCl3) δ 7.06 (1H, brs, H-1), 5.50 (1H, d, J )
10.5 Hz, H-12), 5.16 (1H, brs, H-7), 5.12 (1H, s, OH-9), 4.00 (1H,
brd, J ) 10.8 Hz H-20a), 3.90 (1H, brd, J ) 10.8 Hz, H-20b), 3.58
(1H, m, H-10), 3.44 (1H, brdd, J ) 16.8 Hz, H-5a), 2.79 (1H, brs,
H-4), 2.45 (1H, brdd, J ) 15.6 Hz, H-5b), 2.29 (6H, s, N-Me2), 2.06
(3H, s, OAc), 2.02 (1H, m, H-3′), 1.97 (1H, brs, H-8), 1.79 (3H, brs,
H-19), 1.71 (1H, m, H-11), 1.21 (3H, s, H-16), 1.20 (3H, d, J ) 3.9
Hz, H-18), 1.13 (3H, s, H-17), 1.04 (1H, d, J ) 6.6 Hz, H-4′), 0.98
(3H, d, J ) 6.6 Hz, H-5′), 0.80 (1H, d, J ) 5.1 Hz, H-14); 13C NMR
(75 MHz, CDCl3) δ 212.8 (s, C-3), 155.9 (d, C-1), 173.4 (s, OAc),
172.1 (s, C-1′), 155.9 (d, C-1), 143.4 (s, C-2), 137.4 (s, C-6), 126.3 (d,
C-7), 78.2 (s, C-9), 74.8 (d, C-12), 74.5 (d, C-2′), 69.3 (t, C-20), 65.3
(s, C-13), 49.6 (d, C-4), 47.4 (d, C-10), 42.6 (d, C-11), 41.4 (q, N-Me2),
40.6 (d, C-8), 36.8 (d, C-14), 28.2 (d, C-3′), 25.0 (t, C-5), 25.2 (s,
C-15), 24.0 (q, C-17), 21.0 (q, OAc), 19.6 (t, C-5′), 19.3 (t, C-4′), 16.3
(q, C-16), 12.0 (q, C-18), 10.4 (q, C-19); CIMS m/z 518 [M + 1]+
[C29H43NO7 + H]+.
N,N-Dimethyl-12-(S-valinoyl)-13-acetyl-4r-4-deoxyphorbol (3d).
1H NMR (300 MHz, CDCl3) δ 7.04 (1H, brs, H-1), 5.67 (1H, d, J )
10.5 Hz, H-12), 5.09 (1H, s, OH-9), 5.11 (1H, brs, H-7), 4.01 (1H,
brd, J ) 11.7 Hz, H-20a), 3.89 (1H, brd, J ) 11.7 Hz, H-20b), 3.48
(1H, m, H-10) 3.40 (1H, brdd, J ) 16.8 Hz, H-5a), 2.80 (1H, brs,
H-4), 2.48 (1H, brdd, J ) 20.0 Hz, H-5b), 2.35 (6H, s, N-Me2), 2.10
(3H, s, OAc), 2.77 (1H, m, Hz, H-2′), 1.98 (1H, brs, H-8), 2.02 (1H,
m, H-3′), 1.78 (3H, brs, H-19), 1.71 (1H, m, H-11), 1.13 (3H, s, H-17),
1.20 (3H, d, J ) 13.5 Hz, H-18), 1.20 (3H, s, H-16), 1.00 (3H, d, J )
6.9 Hz, H-4′), 0.92 (3H, d, J ) 6.6 Hz, H-5′), 0.82 (1H, d, J ) 5.1 Hz,
H-14); 13C NMR (75 MHz, CDCl3) δ 212.8 (s, C-3), 155.7 (d, C-1),
173.4 (s, OAc), 171.3 (s, C-1′), 155.7 (d, C-1), 143.4 (s, C-2), 137.2
(s, C-6), 126.3 (d, C-7), 78.2 (s, C-9), 74.8 (d, C-12), 74.7 (d, C-2′),
69.3 (t, C-20), 65.3 (s, C-13), 49.6 (d, C-4), 47.3 (d, C-10), 42.7 (d,
C-11), 41.3 (q, N-Me2), 40.6 (d, C-8), 36.8 (d, C-14), 27.3 (d, C-3′),
24.8 (t, C-5), 25.2 (s, C-15), 24.0 (q, C-17), 21.1 (q, OAc), 19.7 (t,
C-5′), 19.2 (t, C-4′), 16.3 (q, C-16), 11.9 (q, C-18), 10.4 (q, C-19);
CIMS m/z 518 [M + 1]+ [C29H43NO7 + H]+.
13,20-Diacetyl-4r-4-deoxyphorbol (4). To a solution of 100 mg
of 4R-4deoxyphorbol (3e, 0,23 mM) in 1:1 (v/v) CH2Cl2-THF (5 mL)
were added an excess of TEA (1.64 mL, 18 mM, 8 molar equiv) and
Ac2O (2.1 mL, 16 mM, 8 molar equiv). After stirring at room
temperature for 1.5 h, the reaction was worked up by the addition of
methanol (ca. 1.5 mL) to quench the excess Ac2O and washed (×5)
with 2 N H2SO4 and with brine. The organic layer was dried, filtered,
and evaporated. The residue was purified by gravity column chroma-
tography on silica gel (5 g; petroleum ether-EtOAc eluant, from 6:4
1
to 4:6) to afford 94 mg (87%) of 4 as a white solid: H NMR (300
MHz, CDCl3) δ 6.96 (1H, brs, H-1), 5.43 (1H, d, J ) 10.7 Hz, H-12),
5.15 (1H, brs, OH-10), 5.08 (1H, brs, H-7), 4.39 (2H, brs, J ) 27.2 Hz
H-20a,b), 3.46 (1H, m, H-10), 3.44 (1H, brdd, J ) 11.0 Hz, H-5a),
2.72 (1H, m, H-4), 2.45 (1H, brdd, J ) 11.0 Hz, H-5b), 2.06 (6H, s,
OAc), 1.90 (1H, m, H-8), 1.75 (3H, brs, H-19), 1.70 (1H, m, H-11),
1.24 (3H, s, H-17), 1.17 (3H, d, J ) 7.0 Hz, H-18), 1.07 (3H, s, H-16),
0.78 (1H, d, J ) 5.2 Hz, H-14); CIMS m/z 433 [M + 1]+ [C24H32O7
+ H]+.
General Protocol for the Esterification of 13,20-Diacetyl-4r-4-
deoxyphorbol (4) with BOC-Protected Amino Acids. To a solution
of 13,20-diacetyl-4R-4-deoxyphorbol (4, 100 mg, 0.23 mmol) in 3:1
toluene-CH2Cl2 (20 mL) was added a solution of an N-BOC-amino
acid (for the synthesis of 3g and 3j) or an N-methyl-N-BOC-amino
acid (for the synthesis of 3h) (1.5 molar equiv) and DCC (1.5 molar
equiv) in 3:1 toluene-CH2Cl2 (20 mL). After stirring at room
temperature for 1 h, the reaction was worked up by filtration with
toluene. The organic phase was sequentially washed with 2 N H2SO4,
saturated NaHCO3, and brine. After drying, the organic phase was
filtered and evaporated. The residue was purified by gravity column
chromatography on silica gel (10 g, petroleum ether-EtOAc, 8:2 to
7:3, as eluant) to afford 13,20-diacetyl-12-(N-BOC-aminoacyl)-4R-4-
deoxyphorbols as white powders (yield 80-90%).
General Protocol for the N-Deprotection of 12-(N-BOC-
aminoacyl)-13,20-diacetyl-4r-4-deoxyphorbols. To a solution of 100
mg of a 12-(N-BOC-aminoacyl)-13,20-diacetyl-4R-4-deoxyphorbol in
CH2Cl2 (5 mL) was added trifluoroacetic acid (TFA) (0.25 mL, 5% of
the substrate solution). After stirring at room temperature for 8 h, the
reaction was worked up by filtration and washing with toluene. The
filtrate was washed with saturated NaHCO3 and next with brine. After
drying, filtration, and evaporation, the residue was purified by gravity
column chromatography on silica gel (10 g, petroleum ether-EtOAc
gradient, from 6:4 to pure EtOAc, as eluant) to afford the corresponding
13,20-diacetyl-12-aminoacyl-4R-4-deoxyphorbol (ca. 60% yield).
General Protocol for the Chemoselective 20-Deacetylation of
12-Aminoacyl-13,20-diacetyl-4r-4-deoxyphorbols. A solution of 100
mg of a 13,20-diacetyl-12-aminoacyl-4R-4-deoxyphorbol in MeOH (ca.
5 mL) was brought to pH ca. 0.5 by the dropwise addition of HClO4.
After stirring at room temperature for 24 h, the reaction was worked
up by neutralization with solid sodium acetate, filtration, and removal
of methanol. The residue was dissolved in dichloromethane, and the
organic phase was washed with 5% NaHCO3 and brine. After drying
and evaporation, the residue was purified by gravity column chroma-
tography on silica gel (10 g, EtOAc-MeOH gradient, from 5:% to
2:8, as eluant) to afford the corresponding 13-acetyl-12-aminoacyl-
4R-4-deoxyphorbol in 50-60% yield.
N,N-Dimethyl-12-glycinoyl-13-acetyl-4r-4-deoxyphorbol (3g). 1H
NMR (300 MHz, CDCl3) δ 7.01 (1H, brs, H-1), 5.52 (1H, d, J ) 10.5
Hz, H-12), 5.09 (1H, s, OH-9), 5.11 (1H, brs, H-7), 4.00 (1H, brd, J )
12.6 Hz H-20a), 3.88 (1H, brd, J ) 12.6 Hz, H-20b), 3.49 (1H, m,
H-10), 3.42 (2H, brdd, J ) 18.0 Hz, H-5a,b), 3.27 (1H, d, J ) 16.2
Hz, H-2′a), 3.16 (1H, d, J ) 15.9 Hz, H-2′b), 2.78 (1H, brs, H-4), 2.38
(6H, s, N-Me2), 2.06 (3H, s, OAc), 1.96 (1H, brs, H-8), 1.77 (3H, brs,
H-19), 1.71 (1H, m, H-11), 1.16 (3H, s, H-17), 1.19 (3H, s, H-16),
0.82 (1H, d, J ) 5.1 Hz, H-14); 13C NMR (75 MHz, CDCl3) δ 212.8
(s, C-3), 173.3 (s, OAc), 170.4 (s, C-1′), 155.8 (d, C-1), 143.4 (s, C-2),
137.3 (s, C-6), 126.2 (d, C-7), 78.2 (s, C-9), 74.8 (d, C-12), 69.3 (t,
C-20), 65.1 (s, C-13), 60.6 (t, C-2′), 49.6 (d, C-4), 47.4 (d, C-10), 45.2
(q, N-Me2), 43.0 (d, C-11), 40.6 (d, C-8), 37.1 (d, C-14), 25.9 (t, C-5),
25.1 (s, C-15), 24.1 (q, C-17), 21.0 (q, OAc), 16.3 (q, C-16), 11.9 (q,
C-18), 10.4 (q, C-19); CIMS m/z 476 [M + 1]+ [C26H37NO7 + H]+.
1
N-Methyl-12-(S-valinoyl)-13-acetyl-4r-4-deoxyphorbol (3h). H
NMR (300 MHz, CDCl3) δ 7.05 (1H, brs, H-1), 5.52 (1H, d, J ) 10.2
Hz, H-12), 5.14 (1H, s, OH-9), 5.11 (1H, brs, H-7), 4.00 (1H, brd, J )
10.8 Hz, H-20a), 3.90 (1H, brd, J ) 10.8 Hz, H-20b), 3.50 (1H, m,
H-10), 3.45 (1H, brdd, J ) 17.7 Hz, H-5a), 2.80 (1H, m, H-4), 2.48
(1H, brdd, J ) 21.0 Hz, H-5b), 2.43 (3H, s, N-Me), 2.06 (3H, s, OAc),
2.89 (1H, d, J ) 6.9 Hz, H-2′), 1.96 (1H, brs, H-8), 1.89 (1H, m, H-3′),
1.78 (3H, brs, H-19), 1.73 (1H, m, H-11), 1.14 (3H, s, H-17), 1.12
(3H, d, J ) 6.0 Hz, H-18), 1.20 (3H, s, H-16), 1.00 (3H, d, J ) 6.9
Hz, H-4′), 1.00 (3H, d, J ) 6.6 Hz, H-5′), 0.82 (1H, d, J ) 5.4 Hz,
H-14); 13C NMR (75 MHz, CDCl3) δ 212.8 (s, C-3), 174.8 (s, OAc),
173.4 (s, C-1′), 155.8 (d, C-1), 143.5 (s, C-2), 137.3 (s, C-6), 126.1 (d,
C-7), 78.2 (s, C-9), 75.9 (d, C-12), 70.4 (d, C-2′), 69.2 (t, C-20), 65.3
(s, C-13), 49.6 (d, C-4), 47.3 (d, C-10), 42.8 (d, C-11), 40.6 (d, C-8),
37.1 (d, C-14), 35.0 (q, N-Me), 31.4 (d, C-3′), 25.0 (t, C-5), 25.2 (s,
C-15), 24.0 (q, C-17), 21.0 (q, OAc), 19.3 (t, C-5′), 19.0 (t, C-4′), 16.4
General Protocol for the N,N-Dimethylation of 12-Aminoacyl-
13,20-diacetyl-4r-4-deoxyphorbols. To a solution of a 12-aminoacyl-
13-acetyl-4R-4-deoxyphorbol (0-20 mM, ca. 100 mg) in acetonitrile
(570 µL) was added 36.5% aqueous formaldehyde (164 µL). After
stirring at room temperature for 5 min, sodium triacetoxyborohydride