Large scale synthesis of the Cdc42 inhibitor secramine A and its inhibition of cell spreading

Article abstract of DOI:10.1039/b609143a

We describe a large scale synthesis of secramine A. Consistent with its ability to inhibit activation of the small GTPase Cdc42, we find that secramine A inhibits cell spreading, a process previously shown to be Cdc42-dependent. The Royal Society of Chemistry 2006.

Full text of DOI:10.1039/b609143a

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Large scale synthesis of the Cdc42 inhibitor secramine A and its inhibition of
cell spreading
Bo Xu,a Henry Pelish,b Tomas Kirchhausenb and Gerald B. Hammond*a
Received 27th June 2006, Accepted 25th September 2006
First published as an Advance Article on the web 5th October 2006
DOI: 10.1039/b609143a
We describe a large scale synthesis of secramine A. Consistent with its ability to inhibit activation of the
small GTPase Cdc42, we find that secramine A inhibits cell spreading, a process previously shown to be
Cdc42-dependent.
Introduction
The synthesis and phenotypic screening of natural product-like
libraries has emerged as a promising approach to reagent and
drug discovery.1 This strategy led to the discovery of secramine2
(later renamed secramine A3) (1), a synthetic analog of galan-
thamine that inhibits some forms of membrane traffic out of the
Golgi apparatus. Secramine A emerged from a library of ∼2500
compounds that were synthesized by mimicking the biosynthesis
of galanthamine on a solid support.2 Recently, Kirchhausen, Shair
and coworkers reported that secramine A inhibits activation of the
Rho GTPase Cdc42, a protein involved in membrane traffic, by
a mechanism dependent upon the guanine dissociation inhibitor
RhoGDI.3 RhoGDI1 shuttles Cdc42 between the cytosol and in-
tracellular membranes.4 Through precise subcellular localization
and the binding and hydrolysis of GTP, Cdc42 contributes to
numerous cellular processes, including polarization, migration and
spreading.5 Many of these events stem from the ability of Cdc42
Scheme 1 Retrosynthesis of secramine A (1).
to regulate actin polymerization and in vitro, secramine A inhibits
Cdc42-dependent actin polymerization.3 As shown in Scheme 1,
synthetic route towards an early stage intermediate. First we
the most salient features in the synthesis of 1 are the bis-cyclization
optimized the synthesis of precursors 2 and 3.
of intermediate B, followed by a Mitsunobu reaction to give A,
Because both precursors are polar compounds, we minimized
chromatography in the preliminary stages. We obtained aldehyde
which then undergoes a conjugate addition and hydroxylamine
formation. Intermediate B was prepared by reductive amination
2 by recrystallization from methanol in 75% overall yield (3 steps)
of the tyrosine derivative 3 with substituted benzaldehyde 2. In
from 4-bromo-3,5-dihydroxybenzoic acid (4) without the use of
view of its distinctive biological activity, we sought a synthesis
chromatography (Scheme 2). Similarly, we prepared amine 3 in five
protocol to generate large amounts of biological active secramine
successive steps before purification by column chromatography in
A by improving the low-yielding, small-scale (<50 mg) synthesis
40% overall yield (Scheme 3). Both compounds 2 and 3 have been
of the original procedure (3.8% yield from 4).
synthesized in ∼20 g scale.
Here we report a scaled-up synthesis of secramine A, including
some attempted variations to our synthetic route and the ability
of secramine A to inhibit cell spreading.
Results and discussion
Our synthesis strategy maintained the biomimetic approach used
in the initial secramine A synthesis, but focused on improved
purification, reduction of chromatographic steps and altering the
Scheme 2 Synthesis of bisallyloxy-4-bromobenzaldehyde (2). Reagents
and conditions: (i) allyl bromide K2CO3, DMF, rt, overnight; (ii) LAH,
aDepartment of Chemistry, University of Louisville, Louisville, Kentucky,
40292, USA
ether, 0 ◦C, 2 h; (iii) PCC, CH2Cl2, rt, 3 h. Overall yield: 75% from 4.
bCBR Institute for Biomedical Research and Department of Cell Biology,
Harvard Medical School, 200 Longwood Ave., Boston, Massachusetts,
02115, USA
With substantial amounts of 2 and 3 in hand, the successive steps
in the synthesis of secramine A were based on small modifications
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Scheme 3 Synthesis of 4-[(S)-2-amino-3-(triisopropylsilanoxyl)propyl]-
phenol (3). Reagents and conditions: (i) allyl chloroformate, i-Pr2NEt,
THF–CH2Cl2 2 : 1, 0 ◦C, 2 h; (ii) allyl bromide, K2CO3, DMF, rt, overnight;
(iii) LiCl, NaBH4, THF–EtOH, rt, overnight; (iv) TIPSOTf, i-Pr2NEt,
CH2Cl2, 0 C, 2 h; (v) Pd(PPh3)4, morpholine, THF, 47 ◦C, overnight.
Scheme 5 Attempted oxidative intramolecular phenolic coupling of
19. Reagents and conditions: (i) NaBH3CN, MEOH, NEt3, 0 ◦C to rt,
overnight, (ii) PhI(OAc)2, HFIPA, 0 ◦C, 3 h.
of the published procedure.2 This new synthesis produced 1
(800 mg) in 9.0% overall yield from 4 as a 1 : 1.6 mixture of
oxime isomers (Scheme 4).
Having accomplished a practical synthesis of 1, we explored
other synthetic approaches that could bypass or reduce the number
of protection–deprotection steps. Accordingly, we first investigated
the use of commercially available D-tyrosinol. The reductive
amination of tyrosinol with aldehyde 2 proceeded efficiently,
but the resulting unprotected vicinal amino alcohol 19 did not
undergo the desired oxidative intramolecular phenolic coupling
and gave a complex mixture of compounds (Scheme 5). The
amine and alcohol moieties of 19 likely act as nucleophiles (intra-
or intermolecular) leading to a mixture of products. Also, these
moieties may subsequently react with the presumed spirodienone
intermediate, leading to undesired heterocyles.
however, the oxidative intramolecular phenolic coupling reaction
of compounds 20, 21 and 22 still furnished intractable mixtures.
In the original hypervalent iodine-mediated intramolecular phe-
nolic coupling reaction, the amino group was protected as its
corresponding amide or carbamate.2,6 We therefore attempted
to protect the vicinal amino alcohol as a cyclic carbamate.
The reaction of diphenyl carbonate with amino alcohol 19
produced cyclic carbamate 23 in high yield (Scheme 7), and the
next successive steps—oxidative intramolecular phenolic coupling
reaction, deprotection, Mitsunobu and condensation reactions—
proceeded efficiently to yield the cyclic carbamate protected
secramine 28 (Scheme 7). However, deprotection of 28 using
Katz’s method (ethylenediamine–THF)5 at rt failed. Increasing
the temperature led to a complex reaction mixture. We then
The vicinal aminoalcohol group of 19 was then protected as
an oxazolidine (compounds 20–22, Scheme 6). In each case,
Scheme 4 Synthesis of secramine A (1). Reagents and conditions: (i) NaBH3CN, MeOH, AcOH, 0 ◦C to rt overnight; (ii) allyl chloroformate, 2,6-lutidine,
CH2Cl2, 0 ◦C to rt, 8 h; (iii) PhI(OAc)2, HFPIA, 0 ◦C, 3 h; (iv) Pd(PPh3)4, morpholine, THF, rt, overnight; (v) cyclopropyl methanol, PPh3, DIAD or
DEAD, THF, 0 ◦C, 2 h; (vi) 2,6-lutidine, THF, 0 ◦C for 15 min, then n-BuLi, 0 ◦C to rt, 1 h; (vii) 2,6-lutidine, CH2Cl2, rt, 1 h; (viii) HF–pyridine, THF,
rt, 2 h.
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Scheme 6 Protection of 19 as its oxazolidine. Reagents and condi-
˚
tions: for 20–21: (i) RCHO, MS 4 A, CH2Cl2, rt, overnight; for 22:
(ii) dimethoxypropane, reflux, 2 d.
Scheme 8 Synthesis of 3 by a protection and deprotection sequence.
Reagents and conditions: (i) TIPSOTf (1 eq.), Et3N, CH2Cl2, 0 ◦C to rt,
24 h; (ii) TIPSOTf (2 eq.), Et3N, CH2Cl2, 0 C to rt, 24 h; (iii) TIPSOTf
(3 eq.), Et3N, CH2Cl2, 0 ◦C to rt, 24 h; (iv) TBAF (1 eq.), −78 ◦C to rt,
THF, 1 h.
opted for the deprotection of the cyclic carbamate at an earlier
stage (using compounds 25, 26 and 27).7 Katz’s method8 or more
vigorous conditions (KOH–EtOH–reflux)9 led to either recovery
of starting material or complex reaction products and therefore
we abandoned this strategy.
Our synthesis of TIPS-protected tyrosinol 3 (Scheme 3) needed
five steps because of the number of protection–deprotection
steps employed. If this number of steps could be reduced, then
the synthesis of 1 would be greatly simplified. Collington and
coworkers10 have reported the selective deprotection of alcoholic
and phenolic silyl ethers. Following their protocol, we protected
both the alcohol and phenol using an excess of TIPSOTf in 55%
yield (Scheme 8).
Selective deprotection conditions (1 eq. TBAF–THF) cleaved
the TIPS group on the phenol group and compound 3 was the
only product isolated. Its 1H and 13C data matched those recorded
for the same compound using the sequence shown in Scheme 3.
Further proof of the success of this approach was an NMR
experiment in which NaOH was added to a solution of 3 in CDCl3.
A significant upfield shift of the aromatic proton took place,
indicating that the deprotection had taken place on the phenol
TIPS group. Hence, using this strategy, the synthesis of 3 was
carried out in one (Scheme 8, top) or two simple steps (Scheme 8,
bottom). Following our synthesis of secramine A, we tested its
biological activity. As expected, secramine A inhibited the Golgi
apparatus to plasma membrane transport of VSVGts-EGFP in
monkey kidney epithelial (BS–C-1) cells as previously described3
(data not shown). We subsequently tested whether secramine A
inhibited cell spreading. In culture, at a low density of plating, BS–
C-1 cells spread out to a flattened, fried-egg shaped morphology
within 35 min of plating (Fig. 1a, vehicle panels). This spreading
is thought to be mediated by integrins, a family of transmem-
brane proteins that form contacts with the extracellular matrix
Scheme 7 The synthesis of 28, a carbamate protected derivative of secramine A (1). Reagents and conditions: (i) (PhO)2CO, CH3CN, 60 ◦C, 18 h;
(ii) PhI(OAc)2, morpholine, THF, rt, overnight; (iv) cyclopropyl methanol, PPh3, DIAD or DEAD, THF, 0 ◦C, 2 h; (v) 2,6-lutidine, THF, 0 ◦C for 15 min,
then n-BuLi, 0 ◦C to rt, 1 h; (vi) 2,6-lutidine, CH2Cl2, rt, 1 h.
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Fig. 1 Effect of secramine A on cell spreading. 1 h after trypsinization, BS–C-1 cells were mixed with vehicle (1% DMSO) or secramine A (1) and
allowed to adhere and spread on plastic. The cells were visualized live by phase contrast microscopy (20× magnification) at regular intervals and scored
as fully spread, partially spread, or round. (A) Representative images of the same cells at 13 and 35 min after deposition on plastic. Bar is 20 lm. The
shapes of the cells were designated as previously described:11 round indicates that they did no spread; partially spread indicates some thinning of the
outer boundaries of the cells and fully spread corresponds to cells with a clear fried-egg shaped morphology. White arrowhead indicates a cell scored as
round and the black arrowhead indicates the same cell scored as fully spread. The gray arrowhead indicates another cell that was scored as a partially
spread cell. (B) Quantification of cell morphology at 35 min. Values are means SD from two independent experiments in which >20 cells were scored
per condition. (C) Representative time course for cell spreading. (D) Representative time course for cell spreading upon washout of secramine A (1) at
30 min.
to regulate adhesion and subsequent morphological changes.
Integrins were shown to mediate cell spreading through activation
of the Rho GTPases Rac and Cdc42.11 Rac and Cdc42 regulate
the formation of distinct actin-dependent structures through the
binding and hydrolysis of GTP. Introduction of an activated
form of Cdc42 (a mutant unable to hydrolyze GTP) into cells
stimulates the extension of long, thin membrane projections called
filopodia.12 In contrast, introduction of an activated form of
Rac (a mutant unable to hydrolyze GTP) into cells stimulates
the extension of thin, broad membrane sheets called lamelipodia
and ruffles.12 Overexpression of dominant negative mutants of
either Cdc42 or Rac (mutants unable to bind to GTP) caused
a marked inhibition of cell spreading.11 Indeed, incubation with
secramine A inhibited cell spreading. BS–C-1 cells were dispensed
onto plastic surfaces, adhered and spread; secramine A inhibited
this cell spreading process (see the Experimental section) (Fig. 1).
The cells were imaged periodically and designated as round, par-
tially spread and fully spread as previously described11 (Fig. 1a).
As indicated in Fig. 1b, vehicle-treated cells fully spread to greater
than 70% in 35 min, whereas greater than 50% remained round
in the presence of secramine A. This cell spreading process was
reversible upon washout of secramine A at 30 min (Fig. 1c, d).
A, which can also be applied to other analogs. Furthermore,
selective deprotection of the TIPS group further shortens the
synthesis. Secramine A made with the improved synthesis is
bioactive and was used to provide a complementary approach
to corroborate the importance of Rho GTPase activation in cell
spreading.
Experimental
General
NMR spectra were recorded on Varian Inova 500 (500 MHz) in-
struments. 1H and 13C spectra were recorded at 500 and 126 MHz,
respectively, using CDCl3 as a solvent. The chemical shifts are re-
ported in d (ppm) values relative to CHCl3 (7.26 ppm for 1H NMR
and 77.0 ppm for 13C NMR). Coupling constants are reported in
hertz (Hz). All air and/or moisture sensitive reactions were carried
out under an argon atmosphere. Dry solvents (tetrahydrofuran,
ether, CH2Cl2 and DMF) were purchased from Aldrich and were
additionally purified on PureSolv PS-400-4 purification system
(Innovative Technology, Inc.). All other reagents and solvents
were employed without further purification. The products were
purified by Biotage FLASH+ system or Chromatotron (thin layer
chromatograph system). TLC was developed on Merck silica gel
60 F254 aluminum sheets.
Conclusions
In summary, we have developed a synthetic sequence using
reaction conditions amenable to a scaled-up synthesis of secramine
3,5-Bisallyloxy-4-bromobenzoic acid allyl ester (5). To a solu-
tion of 4-bromo-3,5-dihydroxybenzoic acid 4 (20.5 g, 87.5 mmol)
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in DMF 120 mL at rt was added solid K2CO3 (60.5 g, 438 mmol)
during stirring. To the suspension was added allylbromide (30 mL,
358 mmol) dropwise over 40 min, then the reaction mixture was
stirred for another 18 h at rt, then the reaction mixture was poured
into water (500 mL), after stirring for 30 min, the water layer was
decanted and the solid was filtered and washed with water, then the
residue solid was dissolved in ether (200 mL), dried over Na2SO4,
condensation of the solvent gave the crude ester 5 (30 g). The crude
ester was used without further purification. 1H NMR (500 MHz,
CDCl3): d 7.24 (s, 2H), 6.12–5.98 (m, 3H), 5.51 (dd, J = 17.6,
1.6 Hz, 2H), 5.40 (dd, J = 17.2, 1.2 Hz, 2H), 5.32 (dd, J = 10.2,
1.2 Hz, 2H), 5.30 (dd, J = 17.6, 1.6 Hz, 2H), 4.81 (d, J = 6.0 Hz,
2H), 4.66 (d, J = 3.4 Hz, 4H).13
4H), 4.65–4.60 (m, 1H), 4.53 (d, J = 5.2 Hz, 2H), 3.71 (s, 3H),
3.04–3.00 (m, 2H).13
(S)-2-Allyoxycarbonylamino-3-(4-allyloxyphenyl)propionic acid
methyl ester (9). To a solution of crude 8 (30.0 g, ca. 107 mmol)
in DMF (170 mL) at rt was added solid K2CO3 (29.6 g, 214 mmol)
then allylbromide (10.2 mL, 116 mmol) was added dropwise. The
suspension was stirred overnight at rt; the reaction mixture was
poured into water (500 mL) under stirring and then extracted with
a mixture of EtOAc (300 mL) and hexane (100 mL). The organic
layer was combined and washed first by water and then by brine,
and then dried with Na2SO4. Condensation of the solvent gave the
crude product 30 g. The crude product was used without further
purification. 1H NMR (500 MHz, CDCl3): d 7.00 ((d, J = 8.4 Hz,
2H), 6.79 (d, J = 8.8 Hz, 2H), 5.99–5.86 (m, 1H), 5.85–5.80 (m,
1H), 5.44 (d, J = 8.0 Hz, 1H), 5.36 (dd, J = 17.4, 1.2 Hz, 1H),
4.56 (dd, J = 14.2, 6.0 Hz, 1H), 4.51 (d, J = 5.6, 2H), 4.45 (d, J =
5.2 Hz, 2H), 3.66 (s, 3H), 3.03 (dd, J = 14.4, 5.2 Hz, 1H), 2.96 (dd,
J = 14.0, 6.4 Hz, 1H).13
[(S)-2-(4-Allyloxyphenyl)-1-hydroxymethylethyl]carbamic acid
allyl ester (10). To a solution of crude 9 (30.0 g, ca. 94 mmol) in
THF (170 mL) at rt was added solid LiCl (7.95 g, 94 mmol), sodium
borohydride (7.18 g, 188 mmol) and then ethanol (200 mL).
The suspension was stirred overnight at rt and then saturated
ammonium chloride (100 mL) was added. Most of the solvent
was removed in reduced pressure, the residue was extracted EtOAc
(2 × 150 mL), the organic layer was combined and washed first by
water and then by brine, and then dried by Na2SO4. Condensation
of solvent gave the crude product (30 g). The crude product was
used without further purification. 1H NMR (500 MHz, CDCl3) d
7.11 (d, J = 8.8 Hz, 2H), 6.85 (d, J = 8.8 Hz, 2H), 6.09–6.00 (m,
1H), 5.92–5.84 (m, 1H), 5.40 (dd, J = 15.6, 2.0 Hz, 1H), 5.28 (dd,
J = 10.4, 1.2 Hz, 1H), 5.27 (dd, J = 17.2, 1.6 Hz, 1H), 5.20 (dd,
J = 10.0, 1.6, 1H), 4.97 (d, J = 7.2 Hz, 1H), 4.54–4.50 (m, 4H),
3.89–3.86 (m, 1H), 3.68–3.65 (b, 1H), 3.57 (b, 1H), 2.80 (d, J =
7.2 Hz, 2H), 2.29 (br-s, 1H).13
(3,5-Bisallyloxy-4-bromophenyl)methanol (6). Lithium alu-
minium hydride (7 g, 175 mmol) was added to anhydrous ether
◦
(150 mL) slowly, the mixture was cooled to 0 C, under stirring
the solution of the above crude ester 5 (30 g, ca. 87 mmol) which
was dissolved in anhydrous THF (150 mL) was added slowly,
after completion of addition, the reaction mixture was allowed
to warm to rt, then the reaction mixture was stirred for 4 h at
rt, then saturated aqueous potassium sodium tartrate was added
dropwise until bubbling ceased (25 ml, over 30 min), then the
mixture was stirred overnight, filtered, the solid was washed with
ether (150 mL), the combined organic phase was washed with
water and brine, dried over Na2SO4, distillation of the solvent in
1
vacuum gave the crude alcohol 6 (26 g). H NMR (500 MHz,
CDCl3): d 6.65 (s, 2H), 6.10–6.00 (m, 2H), 5.48 (dd, J = 17.4,
1.6 Hz, 2H), 5.29 (dd, J = 10.6, 1.6 Hz, 2H), 4.62 (s, 2H), 4.60 (d,
J = 5.2 Hz, 4H).13
Bisallyloxy-4-bromobenzaldehyde (2). To a suspension of pyri-
dinium chlorochromate (34.7 g, 161 mmol) and celite (34.7 g) and
sodium acetate hydrate (5.44 g, 40 mmol) in CH2Cl2 (300 mL)
at 0 ◦C, the solution of crude alcohol 6 (26 g, ca. 87 mmol) in
CH2Cl2 (120 mL) was added slowly. After completion, the reaction
mixture was stirred for another 2 h at 0 ◦C. Then hexane (300 mL)
was added, stirred for 30 min at rt, filtered, the solid was washed
by CH2Cl2, the combined organic solution was distilled off, the
residue was recrystallized from hot methanol (50 mL) to give 19.5 g
[(S)-2-(4-Allyloxybenzyl)-2-(triisopropylsilanyl)ethyl]carbamic
acid allyl ester (11). To a solution ◦of crude 10 (7.07 g, ca.
24.3 mmol) in CH2Cl2 (100 mL) at 0 C was added diisopropy-
lethylamine 9.43 g (29.2 mmol) and triisopropylsilyltriflate 8.94 g
(29.2 mmol) dropwise with stirring. The reaction mixture was
stirred at 0 ◦C for 2 h and then warmed to rt, then saturated
ammonium chloride (80 mL) was added, the aqueous suspension
was extracted by EtOAc (2 × 200 mL), the organic layer was
combined and washed first by water and then by brine, and
then dried by Na2SO4. Condensation gave crude 11 (11 g). The
1
pure aldehyde 2 (75%, 3 steps). H NMR (500 MHz, CDCl3): d
9.88 (s, 1H), 7.02 (s, 2H), 6.18–6.02 (m, 2H), 5.50 (dd, J = 17.4,
2.0 Hz, 2H), 5.33 (dd, J = 10.6, 1.2 Hz, 2H), 4.70 (ddd, J = 5.2,
1.6, 1.6 Hz, 4H).13
(S)-2-Allyoxycarbonylamino-3-(4-hydroxyphenyl)propionic acid
methyl ester (8). To a solution of L-tyrosine methyl ester 7 (23.0 g,
99.3 mmol) in THF (100 mL) and CH2Cl2 (100 mL) at rt was added
diisopropylethylamine (50 mL) during stirring. The solution was
cooled to 0 ◦C (some solid formed when the solution was cooled
1
crude product was used without further purification. H NMR
(500 MHz, CDCl3): d 7.11 (d, J = 8.0 Hz, 2H), 6.82 (d, J =
8.0 Hz, 2H), 6.08–6.00 (m, 1H), 5.92–5.82 (m, 1H), 5.39 (dd, J =
17.2, 1.6 Hz, 1H), 5.27–5.22 (m, 2H), 5.17 (dd, J = 10.0, 0.8 Hz,
1H), 4.95 (d, J = 8.4 Hz, 1H), 4.52–4.48 (m, 4H), 3.80 (br s, 1H),
3.60 (d, J = 3.6 Hz, 2H), 2.81 (d, J = 7.2 Hz, 2H), 1.10–1.00 (m,
21H).13
◦
to 0 C) and allylchoroformate (11.97 g, 99.3 mmol), was added
dropwise. The reaction mixture was stirred at 0 ◦C for 2 h, after that
saturated ammonium chloride solution (100 mL) was added, then
most solvent was removed under reduced pressure. The aqueous
suspension was extracted by EtOAc (2 × 200 mL), the organic layer
was combined and was washed first by water and then by brine,
and then dried by Na2SO4, condensation of the solvent gave the
crude product 30 g. The crude product was used without further
purification. 1H NMR (500 MHz, CDCl3): d 6.95 ( (d, J = 7.9 Hz,
2H), 6.71 (d, J = 8.0 Hz, 2H), 5.92–5.83 (m, 1H), 5.30–5.18 (m,
4-[(S)-2-Amino-3-(triisopropylsilanoxyl)propyl]phenol (3). To
a solution of crude 11 (10.8 g, 24.3 mmol) in THF 100 mL
at rt was added morpholine 20 mL, then tetrakis(triphenyl-
phosphine)palladium(0) (280 mg, 0.24 mmol) in the dark. The
solution was warmed to 47 ◦C and then stirred overnight.
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Condensation of the solvent gave the crude product. Purification
by flash chromatography (30% EtOAc–hexane to 100% EtOAc)
afforded 3 (3.05 g, 40% overall yield from 7). 1H NMR (500 MHz,
CDCl3): d 7.01 (d, J = 8.0 Hz, 2H), 6.71 (d, J = 8.0 Hz, 2H), 4.50
(br s, 2H), 3.75 (dd, J = 10.0, 4.0 Hz, 1H), 3.61 (dd, J = 10.0,
7.0 Hz, 1H), 3.19 (br s, 1H), 2.79 (dd, J = 13.3, 6.0 Hz, 1H), 2.64
(dd, J = 13.8, 7.5 Hz, 1H), 1.14–1.01 (m, 21H).13
(m, 2H), 4.3–4.27 (m, 1H), 4.22–4.16 (m, 1H), 4.14–4.06 (m, 2H),
3.92 (dd, J = 4, 10 Hz, 1H) 3.83 (dd, J = 4.75, 10 Hz, 1H), 2.99
(dd, J = 15, 14 Hz, 1H), 2.86 (dd, J = 13, 15 Hz, 1H), 1.75 (dd,
J = 4.5, 15 Hz), 1.70 (dd, J = 4, 15 Hz, 1H), 1.10–1.03 (m, 42H).13
6H-Benzofuro[3a,3,2-ef ][2]benzazepin-6-one, 3-bromo-4a,5,9,
10,11,12-hexahydro-2-hydroxy-10-(triisopropylsilanyloxymethyl)-
[(4aR,8aR,10S)-15]. To a solution of 14 (2.30 g, 3.35 mmol)
in THF (50 mL) was added morpholine (2.92 g, 33.5 mmol)
followed by solid Pd(PPh3)4 (116 mg, 0.1 mmol) and PPh3 (26.4 mg,
0.01 mmol) at rt in the dark. After stirring overnight, the solution
was concentrated under reduced pressure and purified by flash
chromatography (10–100% EtOAc–hexane) to obtain 15 as a solid
(1.723 g, 98%). 1H NMR (500 MHz, CDCl3) d 6.82 (d, J = 15 Hz,
1H), 6.33 (s, 1H), 6.0 (d, J = 15 Hz, 1H), 4.85 (br s, 1H), 4.04 (d,
J = 15 Hz, 1H), 3.96 (d, J = 15 Hz, 1H), 3.70 (dd, J = 10, 5 Hz,
1H), 3.62 (dd, J = 10, 10 Hz, 1H), 3.3–3.2 (m, 1H), 3.15 (dd, J =
17.5, 2.5 Hz, 1H), 2.75 (dd, J = 17.5, 2.5 Hz, 1H), 1.95–1.9 (m,
2H), 1.20–1.00 (m, 21H).13
4-[(S)-2-(3,5-Bisallyloxy-4-bromobenzylamino)-3-(triisopropyl-
silanyloxy)propyl]phenol (12).
A solution of 3 (3.49 g,
10.79 mmol) and aldehyde 2 (3.20 g, 10.79 mmol) was cooled
◦
to 0 C, then acetic acid (7.5 mL) was added, then a solution of
NaBH3CN 0.745 g (11.8 mmol, 1.1 eq.) in methanol (50 mL) was
added dropwise. After addition, the reaction mixture warmed to
rt and stirred overnight. Then Et3N (10 mL) was added, most
of the solvent was removed under reduced pressure and brine
(100 mL) and saturated NaHCO3 (30 mL) were added. The
mixture was extracted by EtOAc (150 mL × 2). The organic
layers were combined and dried over Na2SO4. Purification by flash
chromatography (10–40% EtOAc–hexane) afforded 12 (5.29 g,
6H-Benzofuro[3a,3,2-ef ][2]benzazepin-6-one, 3-bromo-2-(cyclo-
propylmethoxy)-4a,5,9,10,11,12-hexahydro-10-(triisopropysilanyl-
oxymethyl)- [(4aR,8aR,10S)-16]. To a solution of 15 (1.72 g,
3.30 mmol) in THF (30 mL) was added PPh3 (1.041 g, 3.96 mmol)
and cyclopropylmethylalcohol (0.356 g, 4.95 mmol). The solution
1
82%). H NMR (500 MHz, CDCl3): d 6.98 (d, J = 8.0 Hz, 2H),
6.69 (d, J = 8.0 Hz, 2H), 6.45 (s, 2H), 6.09–5.99 (m, 2H), 5.46 (dd,
J = 17.3, 1.5 Hz, 2H), 5.27 (dd, J = 11.0, 1.5 Hz, 2H), 4.49 (d,
J = 5.0 Hz, 4H), 3.80 (d, J = 13.0 Hz, 1H), 3.75 (d, J = 13.5,
1H), 3.68–3.61 (m, 2H), 2.93–2.88 (m, 1H), 2.76–2.67 (m, 2H),
1.05–1.00 (m, 21H).13
◦
was cooled to 0 C and DIAD (0.866 g, 4.29 mmol) was added
with stirring. After 2 h, the solution was concentrated under
reduced pressure and purified by flash chromatography (0–50%
EtOAc–hexane) to obtain 16 (1.85 g, 97%). Rf = 0.29 (50%
(3,5-Bisallyloxy-4-bromobenzyl)-[(S)-2-(4-hydroxyphenyl)-1-
(triisopropylsilanyloxymethyl)ethyl]carbamic acid allyl ester (13).
To a solution of 12 (5.20 g, 8.62 mmol) in THF 150 mL at 0 ◦C was
added 2,6-lutidine (1.38 g, 8.62 mmol), then allylchloroformate
(1.25 g, 10.34 mmol) and warmed to rt. After 8 h, saturated
aqueous NH4Cl (100 mL) was added, THF was removed under
reduced pressure, and the aqueous suspension was extracted with
EtOAc. The organics were combined, dried over Na2SO4, filtered,
and concentrated. Purification by flash chromatography (10–30%
EtOAc in hexane) afforded 13 as a white solid (3.46 g, 58%); The rt
1H NMR was complex due to presence of amide group, 1H NMR
(400 MHz, (CD3)2SO, 80 ◦C) d 8.90 (s, 1H), 6.88 (d, J = 8, 2H),
6.639 (d, J = 8.4, 2H) 6.55 (s, 2H), 6.05–5.97 (m, 2H), 5.96–5.83
(m, 1H), 5.41 (dd, J = 19.2, 1.4 Hz, 2H), 5.24 (dd, J = 11.2, 1.4 Hz
2H), 5.22–5.13 (m, 2H), 4.52 (br s, 6H), 4.37 (d, J = 16 Hz, 1H),
4.23 (d, J = 16.4 Hz, 1H), 4.12 (br s, 1H), 3.70 (m, 2H), 2.77 (dd,
J = 18.4, 13.6 Hz, 2H), 1.30–0.81 (m, 21H).13
Spiro-[5H-2-benzazepine-5,1ꢀ-[2,5]cyclohexadiene]-2(1H) carb-
oxylic acid, 7-bromo-3,4-dihydro-3-(triisopropylsilanyloxymethyl)-
4ꢀ-oxo-6,8-bis(2-propenyloxy)-, 2-propenyl ester [(3S)-14]. Com-
pound 13 (3.46 g, 5.03 mmol) was dissolved in the HFIPA (12 mL)
and cooled to 0 ◦C. Then PhI(OAc)2 (1.95 g, 6.04 mmol) dissolved
in CH2Cl2 (12 mL) was added and the reaction mixture was stirred
for 3 h. Then the reaction was quenched by NaHCO3 solution,
extracted by EtOAc, and the organic layer was dried over Na2SO4.
Flash chromatography (10–30% EtOAc in hexane) afforded 14
(2.32 g, 59%). 1H NMR (500 MHz, CDCl3) At rt, 2 rotamers are
present in a ratio of 1 : 1.1, d 7.08–7.04 (m, 2H), 6.96–6.92 (m, 2H),
6.60 (s, 1H), 6.51 (s, 1H), 6.35–6.31 (m, 2H), 6.22–6.19 (m, 2H),
6.09–6.03 (m, 2H), 5.96–5.78 (m, 4H), 5.50 (dd, J = 1.25, 17.8 Hz,
2H), 5.35–5.08 (m, 10H), 4.90 (d, J = 16.5 Hz, 1H), 4.80 (d, J =
22 Hz, 1H), 4.74 (d, J = 22 Hz, 1H), 4.68–4.56 (m, 9H), 4.52–4.42
1
EtOAc–hexane). H NMR (500 MHz, CDCl3) d 6.84 (d, J =
10.4 Hz, 1H), 6.24 (s, 1H), 6.0 (d, 10.4 Hz, 1H), 4.74 (br s, 1H),
4.04 (d, J = 15.6 Hz, 1H), 3.98 (d, J = 16.6 Hz, 1H), 3.84 (dd,
J = 6.4, 10 Hz, 1H), 3.78 (dd, J = 6.8, 6.6 Hz), 3.67–3.57 (m,
2H), 3.22–3.16 (m, 1H), 3.14 (d, J = 16 Hz, 1H), 2.72 (dd, J =
3.4, 17.8 Hz, 1H), 2.01 (d, J = 12.8 Hz, 1H), 1.69 (dd, J = 12.4,
12.4 Hz, 1H), 1.2–1.0 (m, 22H), 0.62–0.57 (m, 2H), 0.36–0.33 (m,
1H).13
6H-Benzofuro[3a,3,2-ef ][2]benzazepin-6-one, 3-bromo-2-(cyclo-
propylmethoxy)-4a,5,7,8,9,10,11,12-octahydro-10-(triisopropylsil-
anyloxymethyl)-8-[[(4-methoxyphenyl)methyl]thio]-
[(4aR,8aR,
10S)-17]. To a solution of 16 (1.85 g, 3.21 mmol) in THF
(30 mL) at 0 ◦C was added 4-methoxybenzyl mercaptan (0.643 g,
4.17 mmol) and 2,6-lutidine (0.446 g, 4.17 mmol). After 15 min,
n-BuLi (0.05 mL of 2.5 M solution in hexane, 0.125 mmol) was
added and the solution was warmed to rt. After 1 h, anhydrous
acetic acid (0.05 mL, 0.87 mmol) was added, the solution was
concentrated under reduced pressure, and purified by flash
chromatography (0–40% EtOAc–hexane) to obtain compound
17 (1.20 g, 52%). Rf = 0.5 (50% EtOAc–hexane). 1H NMR
(500 MHz, CDCl3) d 7.19 (d, J = 8.5 Hz, 2H), 6.84 (d, J = 8.5 Hz,
2H), 6.18 (s, 1H), 4.71 (dd, J = 2.75, 2.75 Hz, 1H), 3.84 (dd, J =
6.5, 9.5 Hz, 1H), 3.80 (s, 3H), 3.79 (dd, J = 6.5, 9.5 Hz, 1H), 3.74
(d, J = 13.7 Hz, 1H), 3.73–3.70 (m, 1H), 3.60 (d, J = 13.7 Hz,
1H), 3.52 (dd, J = 8.5, 8.5 Hz, 1H), 3.37 (d, J = 15 Hz, 1H),
3.14–3.08 (m, 2H), 3.05 (dd, J = 2.75, 18.3 Hz, 1H), 2.97 (dd, J =
2.75, 18.3 Hz, 1H), 2.61 (dd, J = 4, 17 Hz, 1H), 2.40 (dd, J = 3,
17), 2.08 (d, J = 13 Hz, 1H), 1.46 (dd, J = 17, 17 Hz), 1.30–1.26
(m, 1H), 1.16–1.07 (m, 21H), 0.64–0.61 (m, 2H), 0.38–0.34 (m,
2H).13
4154 | Org. Biomol. Chem., 2006, 4, 4149–4157
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6H-Benzofuro[3a,3,2-ef ][2]benzazepin-6-one, 3-bromo-2-(cyclo-
propylmethoxy)-4a,5,7,8,9,10,11,12-octahydro-10-(triisopropysil-
1H), 2.79 (dd, J = 13.3, 6.0 Hz, 1H), 2.64 (dd, J = 13.8, 7.5 Hz,
1H), 1.05 (m, 21H).
anyloxymethyl)-8-[[(4-methoxyphenyl)methyl]thio]-,
O-(phenyl-
(S)-1-(Triisopropylsilyloxy)-3-(4-(triisopropylsilyloxy)phenyl)-
propan-2-amine (30). To a solution of tyrosinol hydrochloride
(206 mg, 1.0 mmol) in CH2Cl2 (5 mL) at 0 ◦C was added TIPSOTf
(918 mg, 3.0 mmol), Et3N (505 mg, 5.0 mmol), the solution was
warmed to rt and then stirred for 24 h. The reaction mixture
was quenched by sat. NH4Cl solution and extracted by ethyl
acetate, the organic layer was dried over Na2SO4, condensation
of the solvent gave the crude product. Purification by flash
chromatography (10–50% EtOAc in hexane) afforded 30 (311 mg,
65%). 1H NMR (500 MHz, CDCl3): d 7.05 ( (d, J = 8.0 Hz, 2H),
6.81 (d, J = 8.0 Hz, 2H), 3.66–3.64 (m, 2H), 3.52–3.49 (m, 2H),
3.08–3.05 (m, 1H), 2.74–2.70 (m, 1H), 2.52–2.48 (m, 1H), 1.54
(br s, 2H), 1.28–1.22 (m, 6H), 1.11–1.02 (m, 36H).
methyl)oxime [(4aR,8aR,10S)-18]. To a solution of compound
17 (1.20 g, 1.64 mmol) in CH2Cl2 (2 mL) was added O-
benzylhydroxylamine hydrochloride (392 mg, 2.46 mmol) and
2,6-lutidine (350 mg, 3.28 mmol) in CH2Cl2 (10 mL) at rt. After
10 min, anhydrous acetic acid (0.12 mL, 2.1 mmol) was added.
After 1 h, the solution was concentrated under reduced pressure
and immediately purified by flash chromatography (5–30%
EtOAc–hexane) to obtain 18 as a 1.6 : 1 mixture of isomers
(1.32 g, 96%) as a white solid. (0–2% MeOH–CH2Cl2). Rf = 0.5
(66% EtOAc–hexane). The isomer mixture can be isolated by
careful chromatograph separation if a single isomer of secramine
A is needed, this separation should be done before TIPS removal.
1H NMR (500 MHz, CDCl3) d 7.41 (d, J = 7.5 Hz, 2H), 7.36 (dd,
J = 7.7, 7.7 Hz, 2H), 7.30 (d, J = 6.5 Hz), 7.17 (d, J = 8.5 Hz,
2H), 6.81 (d, J = 9 Hz, 2H), 6.16 (s, 1H), 5.20 (d, J = 13 Hz,
1H), 5.15 (d, J = 12.5 Hz, 1H), 4.57 (dd, J = 2.5, 2.5 Hz, 1H),
3.83 (dd, J = 6.75, 9.75 Hz, 1H), 3.79 (s, 3H), 3.78 (dd, J = 6.75,
9.75 Hz, 1H), 3.71 (br s, 1H), 3.63 (d, J = 13 Hz, 1H), 3.53 (d,
J = 14 Hz, 1H), 3.51 (dd, J = 2.5, 18.2 Hz, 1H), 3.44 (d, J =
15 Hz, 1H), 3.23 (br s, 1H), 3.02 (br s, 1H), 2.80 (dd, J = 2.5, 18
Hz), 2.55 (dd, J = 3.75, 11.25 Hz, 1H), 2.34 (d, J = 10 Hz, 1H),
1.97 (d, J = 13 Hz, 1H), 1.29–1.25 (m, 2H), 1.16–1.07 (m, 21H),
0.63–0.61 (m, 2H), 0.37–0.34 (m, 2H).13
4-[(S)-2-Amino-3-(triisopropylsilanoxyl)propyl]phenol (3) (through
selective deprotection of 30)
To a solution of 30 (239 mg, 0.5 mmol) in THF (3 mL) at −78 ◦C
was added TBAF (1 M solution in THF, 0.5 mL, 0.5 mmol) the
◦
solution was warmed to 0 C slowly, then reaction mixture was
quenched by sat. NH4Cl solution and extracted by ethyl actate, the
organic layer was dried over Na2SO4. Condensation of the solvent
gave the crude product. Purification by flash chromatography (30%
1
EtOAc–hexane to 100% EtOAc) afforded 3 (93 mg, 58%). H
NMR (500 MHz, CDCl3): d 7.01 (d, J = 8.0 Hz, 2H), 6.71 (d,
J = 8.5 Hz, 2H), 4.50 (br s, 2H), 3.75 (dd, J = 10.0, 3.5 Hz, 1H),
3.61 (dd, J = 9.5, 6.5 Hz, 1H), 3.19 (br s, 1H), 2.79 (dd, J = 13.3,
6.0 Hz, 1H), 2.64 (dd, J = 13.8, 7.5 Hz, 1H), 1.05 (m, 21H).
6H-Benzofuro[3a,3,2-ef ][2]benzazepin-6-one, 3-bromo-2-(cyclo-
propylmethoxy)-4a,5,7,8,9,10,11,12-octahydro-10-(hydroxymeth-
yl)-8-[[(4-methoxyphenyl)methyl]thio],
O-(phenylmethyl)oxime,
(4aR,8aR,10S)-secramine A (1). To a solution of 18 (1.32 g,
1.58 mmol, 1 eq.) in THF (10 mL) in a high density polyethylene
vial was added HF–pyridine (3 mL) slowly with stirring. After
2 h, the solution was concentrated under reduced pressure and
purified by flash chromatography (0–5% MeOH–CH2Cl2) to
obtain 1 0.79 g (yield 73%, 1.6 : 1 mixture of isomers) white solid.
Rf = 0.24 (5% MeOH–CH2Cl2). 1H NMR (500 MHz, (CD3)2SO)
major isomer: d 7.39–7.29 (m, 5H), 7.19 (d, J = 8.5 Hz, 2H) 6.84
(d, J = 8.5 Hz, 2H), 6.38 (s, 1H), 5.11 (d, J = 12.5 Hz, 1H), 5.08
(d, J = 12.5 Hz, 1H), 4.60 (dd, J = 3, 3 Hz, 1H), 4.54 (br s, 1H),
3.85 (dd, J = 7, 6.5 Hz, 1H), 3.79 (dd, J = 7.5, 6.5 Hz, 1H),
3.72 (s, 3H), 3.89 (d, J = 12.5 Hz, 1H), 3.68 (d, J = 16 Hz, 1H),
3.57 (d, J = 13 Hz, 1H), 3.37–3.24 (m, 3H), 3.14–3.10 (m, 1H),
3.07 (br s, 1H), 2.99–2.93 (m, 1H), 2.63 (d, J = 19 Hz, 1H), 2.07
(d, J = 15.5 Hz, 1H), 1.90 (d, J = 13.5 Hz), 1.27–1.16 (m, 2H),
0.56–0.52 (m, 2H), 0.32–0.28 (m, 2H).13
(S) - 4 - (2 - (3,5 - bis(allyloxy)-4-bromobenzylamino)-3-hydroxy-
propyl)phenol (19).
A solution of tyrosinol hydrochloride
(814 mg, 4.0 mmol) and aldehyde 2 (1.32 g, 4.45 mmol) in methanol
(20 mL) was cooled to 0 ◦C, then a solution of NaBH3CN
(315 mg, 5.0 mmol) in methanol (20 mL) was added dropwise.
After addition, the reaction mixture warmed to rt and stirred
overnight. Then Et3N (5 ml) was added, most of the solvent was
removed under reduced pressure and brine (100 mL) and saturated
NaHCO3 (30 mL) were added. The mixture was extracted by
EtOAc (50 mL × 2). The organic layers were combined and dried
over Na2SO4. Purification by flash chromatography (50%–100%
EtOAc in hexane) afforded 19 (1.40 g, 78%). 1H NMR (300 MHz,
CDCl3): d 6.98 (d, J = 8.4 Hz, 2H), 6.74 (d, J = 8.4 Hz, 2H), 6.41
(s, 2H), 6.11–5.99 (m, 2H), 5.52–5.44 (m, 2H), 5.31–5.27 (m, 2H),
4.54 (td, J = 4.8, 1.6 Hz, 4H), 3.71 (s, 2H), 3.41–3.36 (m, 1H),
2.91–2.89 (m, 2H), 2.73–2.68 (m, 2H).
Synthesis of 4-[(S)-2-amino-3-(triisopropylsilanoxyl)propyl]-
phenol (3) through direct protection. To a solution of tyrosinol
hydrochloride (206 mg, 1.0 mmol) in CH2Cl2 (5 mL) at 0 ◦C was
added TIPSOTf (612 mg, 2.0 mmol), Et3N (253 mg, 3.5 mmol),
then the solution was warmed to rt and then stirred for 24 h.
The reaction mixture was quenched by sat. NH4Cl solution and
extracted by ethyl acetate, the organic layer was dried over Na2SO4.
Condensation of the solvent gave the crude product. Purification
by flash chromatography (30% EtOAc–hexane to 100% EtOAc)
afforded 3 (103 mg, 32%). 1H NMR (500 MHz, CDCl3): d 7.01 (d,
J = 8.0 Hz, 2H), 6.71 (d, J = 8.5 Hz, 2H), 4.50 (br s, 2H), 3.75 (dd,
J = 10.0, 3.5 Hz, 1H), 3.61 (dd, J = 9.5, 6.5 Hz, 1H), 3.19 (br s,
(S) - 4 - ((3 - (3,5 - bis(allyloxy)-4-bromobenzyl)-2,2-dimethyloxa-
zolidin-4-yl)methyl)phenol (22). A solution of compound 19
(700 mg, 1.56 mmol), TsOH monohydrate (12 mg, 0.06 mmol)
and 2,2-dimethoxypropane (2.5 mL) was refluxed for 2 d, then
saturated NaHCO3 (30 mL) were added to quench the reaction.
The mixture was extracted by EtOAc (50 mL × 2). The organic
layers were combined, washed by brine and dried over Na2SO4.
Purification by flash chromatography (10–50% EtOAc in hexane)
afforded 22 (192 mg, 25%). 1H NMR (300 MHz, CDCl3): d 6.97 (d,
J = 8.4 Hz, 2H), 6.69 (d, J = 8.4 Hz, 2H), 6.43 (s, 2H), 6.10–5.97
(m, 2H), 527 (dq, J = 10.4, 1.5 Hz, 4H), 4.52–4.49 (m, 4H), 3.77
(s, 2H), 3.42–3.36 (m, 2H), 3.16 (s, 2H), 2.94–2.91 (m, 1H), 2.70
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(d, J = 6.8 Hz, 2H), 1.32 (s, 6H); 13C NMR (300 MHz, CDCl3):
d 24.4, 37.3, 48.5, 51.4, 57.9, 62.4, 69.7, 100.1, 106.0, 115.4, 117.5,
130.3, 132.7, 140.7, 154.6, 156.1.
27: To a solution of 26 (60 mg, 0.134 mmol, 1 eq.) in THF
(2 mL) at 0 ◦C was added 4-methoxybenzyl mercaptan (27 mg,
0.174 mmol) and 2,6-lutidine (18 mg, 0.174 mmol). After 15 min,
n-BuLi (0.005 mL of 2.5M solution in hexane, 0.008 mmol) was
added and the solution was warmed to rt. After 1 h, anhydrous
acetic acid (0.01 mL) was added, the solution was concentrated
under reduced pressure, and purified by flash chromatography
(10–40% EtOAc–hexane) to obtain compound 27 (30 mg, 52%) as
a white solid. 1H NMR (500 MHz, CDCl3) d 7.17 (d, J = 8.6 Hz,
2H), 6.89 (d, J = 8.6 Hz, 2H), 6.30 (s, 1H), 4.72 (t, J = 2.7 Hz,
1H), 4.46 (d, J = 15.8 Hz, 1H), 4.34 (t, J = 7.8 Hz, 1H), 3.85–3.75
(m, H), 3.55 (d, J = 13.8 Hz, 1H), 3.00 (d, J = 2.8 Hz, 2H), 2.65
(dd, J = 17.2, 3.5 Hz, 1H), 2.53 (s, 1H), 2.38 (dd, J = 17.2, 2.9 Hz,
1H), 2.24 (dd, J = 1.8, 13.3 Hz, 1H), 1.75–1.85 (m, 1H), 0.67–0.60
(m, 2H), 0.39–0.34 (m, 2H).
(S)-3-(3,5-bis(allyloxy)-4-bromobenzyl)-4-(4-hydroxybenzyl)-
oxazolidin-2-one (23). A solution of compound 19 (1.0 g,
2.24 mmol), Diphenyl carbonate (1.2 g, 5.6 mmol) and acetonitrile
(10 mL) was refluxed for 18 h, then saturated NaHCO3 (30 mL)
was added to quench the reaction. The mixture was extracted
with EtOAc (50 mL × 2). The organic layers were combined was
washed by brine and dried over Na2SO4. Purification by flash
chromatography (10–50% EtOAc in hexane) afforded 23 (0.91 g,
86%). 1H NMR (300 MHz, CDCl3): d 6.90 ((d, J = 8.5 Hz, 2H),
6.78 (d, J = 8.4 Hz, 2H), 6.39 (s, 2H), 6.09–5.99 (m, 2H), 5.47
(ddd, J = 17.3, 1.7, 1.5 Hz, 2H), 5.29 (ddd, J = 10.5, 2.4, 1.5 Hz,
1H), 4.70 (d, J = 15.2 Hz, 1H), 4.59–4.56 (m, 4H), 4.19–4.09 (m,
2H), 4.03–3.97 (m, 2H), 3.81–3.75 (m, 1H), 2.96 (dd, J = 13.8,
5.2 Hz, 1H), 2.60 (dd, J = 13.8, 8.1 Hz, 1H).
28: To a solution of 27 (10 mg, 0.0167 mmol) in CH2Cl2
(1 mL) was added O-benzylhydroxylamine hydrochloride (5.0 mg,
0.03 mmol) and 2,6-lutidine (3.2 mg, 0.03 mmol) in CH2Cl2
(10 mL) at rt. After 10 min, anhydrous acetic acid (0.12 mL,
2.1 mmol, 5 eq.) was added. After 1 h, the solution was concen-
trated under reduced pressure and immediately purified by flash
chromatography (5–30% EtOAc–hexane) to obtain compound 28
as a 1.6 : 1 mixture of isomers (5 mg, 42%) as a white solid. (0–2%
Synthesis of intermediates 24 to 28
Compound 23 (0.620 mg, 1.31 mmol) was dissolved in the HFIPA
(4 mL) and cooled to 0 ◦C. Then a solution of PhI(OAc)2 (505 mg,
1.57 mmol), dissolved in CH2Cl2 (12 mL) was added at 0 ◦C and the
reaction mixture was stirred for 3 h at same temperature. Then the
reaction was quenched by NaHCO3 solution, extracted by EtOAc,
and organic layer was dried over Na2SO4. Flash chromatography
(10–50% EtOAc in hexane) afforded compound 24 as colorless oil
(400 mg, 59%). 1H NMR (500 MHz, CDCl3): d 7.42–7.36 (m, 2H),
6.73 (s, 1H), 6.41–6.37 (m, 1H), 6.17–6.13 (m, 1H), 6.10–6.04 (m,
1H), 6.01–5.85 (m, 1H), 5.53–5.47 (m, 1H), 5.36–5.32 (m, 1H),
5.29–5.21 (m, 3H), 4.82 (d, J = 16.0 Hz, 1H), 4.66–4.62 (m, 2H),
4.57–4.42 (m, 2H), 4.26–4.23 (m, 1H), 4.13–4.05 (m, 2H), 3.91–
3.84 (m, 2H); 13C NMR (300 MHz, CDCl3): d 185.0, 157.0, 156.1,
155.4, 147.7, 136.7, 131.8, 131.7, 128.8, 122.1, 119.0, 118.2, 112.6,
109.0, 74.5, 69.8, 67.3, 55.2, 48.5, 47.3, 46.6
25: To a solution of 24 (127 mg, 0.27 mmol) in THF (3 mL)
was added morpholine (234 mg, 2.7 mmol), followed by solid
Pd(PPh3)4 (2.3 mg, 0.018 mmol) and PPh3 (26.4mg, 0.01 mmol)
at rt in the dark. After stirring overnight, the solution was
concentrated under reduced pressure and purified by flash chro-
matography (10–50% EtOAc–hexane) to obtain compound 25 as
a solid (90 mg, 70%). 1H NMR (500 MHz, CDCl3) d 6.80 (dd, J =
10.3, 1.9 Hz, 1H), 6.11 (d, J = 10.3 Hz, 1H), 4.88 (d, J = 16.4 Hz,
1H), 4.84–4.82 (m, 1H), 4.58–4.52 (m, 1H), 4.33–4.28 (m, 2H),
4.00–3.96 (m, 1H), 3.75–3.68 (m, 1H), 3.22 (d, J = 17.8 Hz, 1 H),
2.76 (dd, J = 3.7 Hz, 17.9 Hz, 1H), 2.19–2.16 (m, 2H).
1
MeOH–CH2Cl2). H NMR (500 MHz, CDCl3) d 7.39–7.33 (m,
5H), 7.15 (d, J = 8.6 Hz, 2H), 6.85 (d, J = 8.6 Hz, 2H), 6.27 (s,
1H), 5.20–5.12 (m, 1H), 4.57–4.49 (m, 1H), 4.46 (d, J = 15.8 Hz,
1H), 4.37 (t, J = 8.4 Hz, 1H), 4.40–4.34 (m, 1H), 3.80 (s, 3H),
3.72–3.50 (m, 1H).
Procedure for cell spreading assay
Secramine A was stored as 20 mM aliquots at −20 ◦C. Confluent
BS-C-1 cells were trypsinized, washed, and resuspended in culture
◦
media containing 10% heat-inactivated FBS at 37 C. After 1 h,
the cells were pelleted and resuspended in DMEMa with 2% Nu-
serum (BD Biosciences) at 37 ◦C. The cells were mixed 1 : 1 in the
same media containing 2× vehicle (2% DMSO) or 2× secramine
A (50 lM or 25 lM) an plated in 6-well plates with transparent
bottom at low density (∼100 000 cells per well). For washout
experiments, theimagingmediawasdiluted ∼3-foldwithDMEMa
containing 10% FBS and exchanged with DMEMa containing
10% FBS. Live cell imaging was done using phase contrast
microscopy at 20× magnification starting 5 min after plating.
>20 cells per condition were imaged for each subsequent time
point using a computer-control inverted Zeiss 200M microscope
under control of Slidebook (Intelligent Imaging Innovations, Inc.).
Experiments were performed in duplicate and scoring was not
carried out in a blinded fashion.
26: To a solution of 25 (70 mg, 0.179 mmol) in THF (2.0 mL) was
added PPh3 (56 mg, 0.215 mmol) and cyclopropylmethylalcohol
(17 mg, 0.233 mmol). The solution was cooled to 0 ◦C and DIAD
(38.5 mg, 0.179 mmol) was added with stirring. After 2 h, the
solution was concentrated under reduced pressure and purified
by flash chromatography (10–50% EtOAc–hexane) to obtain 26
(68.5 mg, 86%). 1H NMR (400 MHz, CDCl3) d 6.81 (dd, J = 10.4,
2.0 Hz, 1H), 6.37 (s, 1H), 6.04 (d, J = 10.4 Hz, 1H), 4.85 (d, J =
16.4 Hz, 1H), 4.51 (t, J = 8.5 Hz, 1H), 4.36–4.29 (m, 2H), 3.96–
3.92 (m, 1H), 3.85 (dd, J = 6.8, 1.3 Hz, 2H), 3.18 (d, J = 18.0 Hz,
1H), 2.76 (dd, J = 18.0 Hz, 1H), 2.06–2.22 (m, 1H), 0.66–0.60 (m,
2H), 0.38–0.34 (m, 2H).
Acknowledgements
We are grateful to the National Institutes of Health (Grant NIH-
GM 62566) for financial support. The contribution of Olusegun
Dele Olubanwo and Satoru Arimitsu towards the synthesis of 2 is
gratefully recognized.
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