Total synthesis of clavatadine A

Article abstract of DOI:10.1021/np500772u

The first total synthesis of the potent and selective human blood coagulation factor XIa inhibitor clavatadine A (1) is described. Direct, early-stage guanidinylation enabled rapid, convergent access to an immediate clavatadine A precursor. Concomitant lactone hydrolysis and guanidine deprotection with aqueous acid cleanly provided clavatadine A (1) in only four steps (longest linear sequence, 41-43% overall yield).

Full text of DOI:10.1021/np500772u

Article
pubs.acs.org/jnp
Total Synthesis of Clavatadine A
Stephanie J. Conn,†,§,⊥ Shannon M. Vreeland,†,§,∥ Alexandra N. Wexler,†,§,∇ Rebecca N. Pouwer,‡
Ronald J. Quinn,‡ and Stephen Chamberland*,†
†Department of Chemistry, Central Washington University, 400 East University Way, Ellensburg, Washington 98926-7539, United
States
‡Eskitis Institute, Griffith University, Brisbane, Queensland 4111, Australia
S
* Supporting Information
ABSTRACT: The first total synthesis of the potent and selective human
blood coagulation factor XIa inhibitor clavatadine A (1) is described. Direct,
early-stage guanidinylation enabled rapid, convergent access to an immediate
clavatadine A precursor. Concomitant lactone hydrolysis and guanidine
deprotection with aqueous acid cleanly provided clavatadine A (1) in only
four steps (longest linear sequence, 41−43% overall yield).
n 2008, after an extensive screen of over 38 000 biota
extracts for anticoagulant activity, Quinn and co-workers
but still allow coagulation to occur.2 Further, because bleeding
associated with genetic FXIa deficiency (hemophilia C) is rarely
spontaneous and results only from significant trauma, such as
major surgery or a car accident, treatment with a specific FXIa
inhibitor may not present an excessive bleeding risk like current
drugs on the market.2c Accordingly, clavatadine A (1) may
represent a lead compound in the development of the next-
generation anticoagulant drug to treat thrombosis.
I
reported the isolation of two new dibromophenol alkaloids,
clavatadines A (1) and B (2).1 These new alkaloids were
obtained from an extract of the marine sponge Suberea clavata
collected at Swain Reefs, Great Barrier Reef, Queensland,
Australia. Despite a relatively minor difference in the
functionality of these molecules, only clavatadine A (1) was
found to be a potent (IC50 = 1.3 μM) and selective inhibitor of
human blood coagulation factor XIa (FXIa). X-ray crystallo-
graphic studies revealed that precise placement of the linear,
arginine-like chain and the phenylacetic acid moiety at
opposing termini of clavatadine A (1) maximizes two key
noncovalent interactions within the FXIa active site.1
In conjunction with its intriguing biological activity,
clavatadine A (1) was an ideal template to probe the generality
of our early-stage guanidinylation approach for organic
synthesis.3 The retrosynthetic analysis of clavatadine A (1)
was also guided by a desire to construct the central carbamate
moiety in a convergent fashion in the key step. Before uniting
both halves of the molecule, however, it was sought to prepare
the terminal guanidine-containing, linear portion of clavatadine
A (1) by installing the guanidine moiety in the first step. Direct
guanidinylation would obviate the circuitous approach conven-
tionally used in synthesis to incorporate a terminal guanidine.
Namely, two unnecessary steps involving amine protecting
groups were eliminated by allowing a protected guanidinylation
reagent to react with a commercially available or readily
prepared primary amine rather than with an amine that had first
been introduced as a latent amine precursor (azide,
phthalimide, etc.). A recent proof of concept study demon-
strated that this strategy could be used successfully to prepare
the fully functionalized terminal guanidine-containing portion
of the indole alkaloids phidianidines A and B and ultimately led
to the total synthesis of these bioactive natural products.3
Our laboratory was drawn to clavatadine A (1) due to its
relative structural simplicity and noted activity toward a
promising new protein target in anticoagulant drug develop-
ment. Recent studies suggest that targeted FXIa inhibition may
reduce the likelihood of excessive clot formation (thrombosis),
Received: October 2, 2014
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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Adopting this enabling and efficient synthetic strategy to
prepare clavatadine A (1) in a similar fashion became necessary
after an advanced azide-containing intermediate could not be
converted into the primary amine needed to host the guanidine
found in clavatadine A (1).
Table 1. Bromination of Phenol 8
a
yield (%)
RESULTS AND DISCUSSION
■
entry
conditions
(a) Br2, Et2O, rt, 2 h;
9
11
To explore whether a direct guanidinylation approach could be
used to prepare clavatadine A (1), the known4,5 N,N′-di-Boc-
protected guanidine 5 was assembled by chemoselective
guanidinylation of 1,4-butanediamine (4) using Goodman’s
reagent6,7 (3) and Et3N under high dilution conditions
(Scheme 1). A modified isolation protocol combining an
b
1
0
85
0
(b) 11, Br2, Et2O, rt, 6−8 h
8, Br2, Et2O, rt, 25 min
70
0
c
2
90
96
12
11
c
3
8, Br2, Et2O, rt, 6 h
0
d
4
NBS (2 equiv), p-TsOH, CH2Cl2, rt, 12 h
21
c
5
Br2, NaOAc, HOAc, rt, 7.25 h
68
a
b
c
Scheme 1. Synthesis of Clavatadine A (1)
Isolated yields of purified reaction products. Ref 11. This work.
Ref 15.
d
out the second bromination in a separate flask.11 Subsequent
bromination of the resulting attenuated arene 11 to form 9 was
considerably slower (6−8 h) (Table 1, entry 1b).11 Results
from the present study confirm these observations (Table 1,
entries 2 and 3). Even upon prolonged exposure (6 h) of a
solution of phenol 8 to excess Br2, the formation of compound
9 was never observed. In this instance, only monobrominated
phenol 11 was recovered, albeit in good yield (Table 1, entry
3).12 Without a base to neutralize the HBr generated during the
reaction, perhaps the strong Brønsted acid deactivated the
aromatic ring by protonating the hydroxy group to form an
oxonium ion.13 Strongly acidic solutions may also cause
hydrolytic cleavage of the benzofuranone ring, leading to
decomposition.14 A recent attempt by Lebouvier and co-
workers to perform both bromination reactions in one flask
using NBS and p-toluenesulfonic acid (p-TsOH) led to a
mixture of 9 and 11 in low yield (Table 1, entry 4); thus, a
more efficient two-step, one-flask method was desired.15
Accordingly, a slight excess of a weak base, sodium acetate,
was added to a solution of compound 8 and bromine that
neutralized both equivalents of HBr formed during the reaction
and led to the desired lactone 9 (Table 1, entry 5).16 This
improvement to the reported sequential11 and nonspecific15
bromination of homogentisic acid lactone (8) provided 4,6-
dibromohomogentisic acid lactone (9) in good yield.
aqueous NaHCO3 wash with subsequent column chromatog-
raphy on silica gel was necessary to separate compound 5 from
excess diamine 4 and from the principal byproduct of the
reaction, triethylammonium trifluoromethanesulfonamide.
It was envisaged that the aromatic portion of clavatadine A
(1) could be prepared from the known homogentisic acid
lactone8 (8), which was prepared from commercially available
2,5-(dimethoxyphenyl)acetic acid (7).9,10 Ideally suited for the
present purposes, compound 8 cloaks the highly polar and
reactive o-hydroxyphenylacetic acid moiety as a lactone, thereby
facilitating handling of derivatives and enabling purification by
silica gel chromatography. It also provides an activating,
ortho,para-directing hydroxy group that orchestrates regiose-
lective installation of two bromine atoms: the first bromine
adds ortho to the hydroxy at the most sterically accessible
position (C-6), and the second is introduced at a more
hindered location (C-4), but at a site that is also ortho to the
strongest activator, the hydroxy group.
Although dibromination of compound 8 has been explored,
prior efforts have not addressed the need for an efficient two-
step, one-flask method to prepare valuable synthetic
intermediate 9. For example, Krohn observed that regiospecific
monobromination of the electron-rich phenol 8 occurred
within 20−30 min when the reaction was conducted in diethyl
ether with Br2 to provide compound 11 in 85% yield (Table 1,
entry 1a).11 Even in the presence of excess bromine,
decomposition rather than the second bromination ensued if
the reaction mixture was left to stir for 2 days.11 To address this
problem, Krohn isolated bromophenol 11 by washing the
reaction mixture with aqueous sodium sulfite and then carried
Armed with an efficient method to prepare both amine 5 and
lactone 9, the two halves were joined together to build the
complete clavatadine A (1) scaffold. In practice, exposure of
amine 5 to triphosgene afforded the semistable isocyanate 6,
which was isolated in quantitative yield (Scheme 1).
Immediately, isocyanate 6 was added to a solution of lactone
9 and a catalytic amount of base to give the carbamate 10.17,18
Subsequent attempts to hydrolyze the lactone with aqueous
base followed by acidification of the reaction mixture led only
to quantitative recovery of carbamate 10.19 It was then
discovered that addition of a solution of carbamate 10 to
dilute aqueous hydrochloric acid promoted hydrolysis of the
lactone and deprotected the di-Boc guanidine to provide
clavatadine A hydrochloride (1·HCl) in 93−96% yield.14
1
Analysis of the H NMR spectrum of unpurified, synthetic
clavatadine A (1) in DMSO-d6 revealed relatively few
impurities, but the spectrum did not precisely match the
reported spectrum of the natural product. There were two
prominent differences. First, the 1H NMR spectrum of
synthetic 1 lacked an apparent 1:1:1 triplet at approximately
δH 7.1 that was visible in the 1H NMR spectrum20 of natural 1,
B
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which was isolated by HPLC as its hydrotrifluoroacetate salt.1
This triplet is often observed in the H NMR spectrum of
tion circumvented the cumbersome protected amine-to-amine-
to-guanidine sequence traditionally employed in the synthesis
of terminal guanidine-containing molecules and should enable
the rapid preparation of analogues for biological evaluation. It is
planned to use this basic approach to prepare clavatadine B (2)
and to synthesize non-natural analogues of clavatadine A (1) as
novel, reversible FXIa inhibitors.
1
natural compounds purified by HPLC when a dilute methanolic
or aqueous solution of trifluoroacetic acid is used as the eluent.
The sharp, spin 1 triplet (J ≈ 48 Hz) observed at δH 7.1 in the
1,21
1
natural sample likely arises from H coupling to 14N.
Sharp
quadrupole coupling such as this is only observed in small,
symmetric molecules, not complex molecules such as
clavatadine A (1).22 Thus, the peak at δH 7.1 in the H NMR
1
EXPERIMENTAL SECTION
■
spectrum of natural 1 is likely attributable to the presence of an
General Experimental Procedures. Infrared (IR) spectra were
obtained on neat solids or liquids using an ATR FT-IR spectrometer.
One- and two-dimensional 1H and 13C NMR spectra were recorded at
ambient temperature at 400 and 100 MHz, respectively, and calibrated
using tetramethylsilane at δ 0.00, unless otherwise stated. All chemical
shifts are reported in ppm on the δ scale, integration, multiplicity (br =
broad, s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet,
or combinations thereof), and coupling constants in Hz. All 2D NMR
spectra were recorded in DMSO-d6. Carbon−hydrogen coupling
constant values in inverse-detection 2D NMR experiments, which
included gradient HSQC (1JCH = 140 Hz) and gradient HMBC (nJCH
= 8.3 Hz), matched those used in the clavatadine A (1) isolation paper
by Quinn and co-workers.1 Accurate mass (MS) measurements were
performed on an LCT premier time-of-flight (TOF) instrument using
electrospray ionization in positive ion mode (ES+) by Dr. John
Greaves and Ms. Shirin Sorooshian. Liquid chromatography was
performed using variable forced air flow (flash chromatography) of the
indicated solvent system or solvent gradient through 60 Å silica gel
(SiO2) (40−63 μm, 230−400 mesh). Analytical thin-layer chromatog-
raphy (TLC) was performed using 0.25 mm silica gel 60 (F254)
plates. Spots were visualized by short-wave (254 nm) UV irradiation
and/or by dipping the plates in phosphomolybdic acid (PMA), cerium
ammonium molybdate (CAM), ferric chloride, or ninhydrin solutions
followed by heating. All reaction mixtures not containing aqueous
reagents were carried out under an atmosphere of dry nitrogen using
standard syringe/septum techniques. Glassware was oven-dried
overnight at 130 °C, flame-dried using a propane torch, and then
allowed to cool to ambient temperature under a stream of dry
nitrogen. Unless otherwise noted, all reagents were used as received
from commercial suppliers. Dichloromethane (CH2Cl2), tetrahydro-
artifact: ammonium trifluoroacetate.21,22 Second, the singlet at
1
δH 6.51 in the H NMR spectrum of natural 1 is a common
impurity that appeared only in highly diluted samples in
DMSO-d6. Its presence was ascribed to the manufacturer of the
DMSO-d6 that was used to dissolve natural clavatadine A (1),
1
because the peak at δH 6.51 was no longer observed in H
NMR spectra after the supplier was changed. Aside from
impurities found only in the natural sample of 1, slight
differences were observed in the chemical shift values reported
for some NH and OH resonances in the synthetic sample:
carboxylic acid (δH 12.41 vs 12.38), phenol (δH 10.45 vs 10.42),
and guanidinium NH (δH 7.52 vs 7.45). Sample concentration
1
seemed to have the biggest impact on the observed H NMR
chemical shifts. Accordingly, the present data most closely
matched the reported values for the natural compound when
dilute solutions (1−2 mg/mL) of synthetic clavatadine A (1)
were prepared in DMSO-d6. Although the 400 MHz NMR
spectrometer used could not completely resolve the peaks at
approximately δH 3.1 (δH 3.11 and 3.08 for natural 1) or those
near δH 1.5 (δH 1.52 and 1.49 for natural 1), data gathered on a
600 MHz NMR instrument exhibited matching δH and J values
to the natural compound.1 All other 1D (13C and DEPT) and
2D NMR spectra of unpurified, synthetic clavatadine A (1)
were identical to those reported for natural 1. In addition, the
1H NMR spectrum of synthetic clavatadine A (1) that had been
purified by HPLC to provide the corresponding hydro-
trifluoroacetate salt (1·CF3CO2H) revealed coincidental
resonances with the natural material. The present preparation
of the title compound was further confirmed by an FXIa
enzyme-inhibition assay, which revealed nearly identical IC50
values for the natural (1.3 μM)1 and synthetic (6.3 μM)
samples of clavatadine A (1) (Figure 1).
furan (THF), and Hunig’s base (i-Pr NEt) were dried over anhydrous
̈
2
CaH2 and distilled under N2. Triethylamine (Et3N) was dried over
KOH and distilled under N2. When anhydrous reaction conditions
were required, solvents were first distilled under nitrogen onto
activated 3 Å molecular sieves and then were transferred under
nitrogen by cannula into amber-colored glass bottles for storage. These
bottles were capped with an Aldrich (St. Louis, MO, USA) Sure-Seal
PTFE disk and a metal crown cap using an Aldrich crown-cap crimper.
Factor XIa was purchased from Haematologic Technologies, Inc.
(Essex Junction, VT, USA), and substrate s2366 from Chromogenix
(manufactured by Instrumentation Laboratory, Bedford, MA, USA).
Clear 96-well Nunc microplates were purchased from Thermo
Scientific (Waltham, MA, USA). Absorbance was measured on a
PerkinElmer (Waltham, MA, USA) Envision 2104 multilabel reader.
N,N′-Di-Boc Guanidine 5. The known N,N′-di-Boc guanidine 5 was
prepared using an adaptation of the procedures of Botta and co-
CONCLUSIONS
■
In conclusion, the first total synthesis of clavatadine A (1) has
been completed in four steps (longest linear sequence). The
overall yield of the convergent approach used was 41−43%
from commercially available compound 7. Direct guanidinyla-
workers and Munoz and co-workers.4,5 Spectroscopic data and copies
̃
of spectra are also provided for compound 5 because complete data
have not yet been published. To a solution of 1,4-butanediamine (4)
(2.32 mL, 23.1 mmol, 3.0 equiv) in 320 mL of CH2Cl2 was added
Et3N (1.07 mL, 7.69 mmol, 1.0 equiv). Then, a solution of N,N′-di-
Boc-N″-triflylguanidine7 (3) (3.01 g, 7.69 mmol, 1.0 equiv) in 25 mL
of CH2Cl2 was added dropwise by addition funnel over approximately
1−2 h (the reaction mixture grew cloudy), and the reaction mixture
was stirred at ambient temperature for 12 h. After 12 h, the reaction
mixture was washed with saturated aqueous NaHCO3 (2 × 50 mL),
H2O (2 × 50 mL), and brine (1 × 50 mL), dried over anhydrous
Na2SO4, filtered, and concentrated in vacuo. Purification by flash
column chromatography on silica gel (5:3:2 EtOAc−MeOH−Et3N)
afforded the product as a pale yellow oil (2.49 g, 98%): IR (neat) νmax
Figure 1. Results of FXIa inhibition assay (IC50 6.3 μM) using
synthetic clavatadine A (1).
C
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3333, 2977, 2932, 1719, 1636, 1613, 1572, 1414, 1365, 1328, 1131,
and was concentrated in vacuo to provide a foamy, dark-brown-
colored solid (2.20 g, 110% mass balance, presuming i-Pr2NEt was
removed in vacuo). This solid was purified by flash column
chromatography on silica gel (1:0 to 1:1 CH2Cl2−Et2O) to provide
carbamate 10 as a maroon-colored, foamy solid (1.36 g, 68% from
amine 5): IR (neat) νmax 3335, 2927, 1822, 1755, 1720, 1639, 1616,
1
1053 cm−1; H NMR (CDCl3, 400 MHz) δ 11.50 (1H, br s), 8.35
(1H, br s), 3.43 (2H, td, J = 7.2, 4.8 Hz), 2.74 (2H, t, J = 7.0 Hz), 1.63
(2H, m), 1.53 (2H, m), 1.51 (9H, s), 1.49 (9H, s); 13C NMR (CDCl3,
100 MHz) δ 163.6 (C), 156.1 (C), 153.3 (C), 83.1 (C), 79.3 (C), 41.8
(CH2), 40.7 (CH2), 30.8 (CH2), 28.3 (CH3), 28.1 (CH3), 26.4
(CH2); (+)-HRTOFESIMS m/z 353.2168 (calcd for C15H30N4O4Na
[M + Na]+, 353.2165); Rf = 0.39, 5:3:2 EtOAc−MeOH−Et3N.
6-Bromohomogentisic Acid Lactone (11). To a solution of
homogentisic acid lactone 8 (0.150 g, 1.00 mmol, 1.00 equiv)9,10 in
10 mL of Et2O was added bromine (0.154 mL, 3.00 mmol, 3.00 equiv)
dropwise by syringe. The reaction mixture was stirred at ambient
temperature for 25 min and then was poured into a separatory funnel
containing 10 mL of saturated aqueous Na2SO3. The layers were
separated, and the organic phase was washed with an additional 2 × 10
mL of saturated aqueous Na2SO3 and 1 × 10 mL of brine, dried over
anhydrous Na2SO4, filtered, and concentrated in vacuo to provide the
product as a brown-colored solid (0.207 g, 90%): Rf of 11 = 0.34, 1:1
EtOAc−hexanes. Krohn first reported this method to prepare
compound 11, but did not provide experimental details.11 Spectro-
scopic data for 11 match previously reported data for compound 6b
prepared by Lebouvier.15 If, instead, the above reaction mixture was
stirred at ambient temperature for 6 h, compound 11 was formed in
96% yield.
4,6-Dibromohomogentisic Acid Lactone (9). To a suspension of
homogentisic acid lactone 8 (0.601 g, 4.00 mmol, 1.00 equiv)9,10 and
sodium acetate (0.673 g, 8.20 mmol, 2.05 equiv) in 30 mL of acetic
acid was added a solution of bromine (0.62 mL, 12.0 mmol, 3.00
equiv) in 1.25 mL of acetic acid dropwise by syringe. The reaction
mixture was stirred at ambient temperature for 7 h, 15 min and then
was concentrated in vacuo to provide a brown solid (1.146 g). This
solid was dissolved in EtOAc, adsorbed onto 5 g of silica gel, and then
purified by flash column chromatography on silica gel (1:4 EtOAc−
hexanes, to recover the desired product, compound 9, and then 1:1
EtOAc−hexanes to elute 6-bromohomogentisic acid lactone (11)).
This purification provided the desired product, compound 9, as a
brownish-purple-colored powder (0.834 g, 68%, based on a theoretical
yield of 1.232 g, 4.00 mmol), and compound 11, as a brown-colored
powder (0.102 g, 11%, based upon a theoretical yield of 0.916 g, 4.00
mmol): Rf of 9 = 0.66, 1:1 EtOAc−hexanes. The spectroscopic data for
9 matched previously reported data for compound 6a prepared by
Lebouvier.15
N,N′-Di-Boc Guanidine-Containing Carbamate 10. To a cooled
(0 °C) solution of N,N′-di-Boc guanidine 5 (1.00 g, 3.03 mmol, 1.00
equiv) in 25 mL of CH2Cl2 was added triphosgene (0.297 g, 1.01
mmol, 0.33 equiv; CAUTION: highly toxic; open, weigh, and handle
only in a fume hood and while wearing two pairs of nitrile gloves). Then, a
solution of saturated aqueous NaHCO3 (25 mL) was added, and the
biphasic reaction mixture was stirred vigorously at 0 °C for 30 min.
After 30 min, the reaction mixture was partitioned between 100 mL of
CH2Cl2 and 100 mL of H2O, the layers were separated, and the
aqueous layer was extracted with CH2Cl2 (3 × 15 mL). The combined
organic extracts were dried over anhydrous Na2SO4, filtered, and
concentrated in vacuo to provide semistable, crude isocyanate 6 as a
light-brown-colored oil (1.11 g, 103% of theoretical). This oil was used
immediately in the next step without purification. Diagnostic data: IR
(neat) νmax 3331, 2978, 2934, 2265, 1719, 1635, 1613, 1574, 1327,
1129, 731 cm−1; 1H NMR (CDCl3, 400 MHz) δ 11.50 (1H, br s), 8.36
(1H, br s), 3.46 (2H, m), 3.37 (2H, m), 1.67 (4H, m), 1.51 (9H, s),
1.50 (9H, s); 13C NMR (CDCl3, 100 MHz) δ 163.5 (C), 156.2 (C),
153.4 (C), 122.0 (NCO), 83.3 (C), 79.4 (C), 42.6 (CH2), 40.1 (CH2),
28.5 (CH2), 28.3 (CH3), 28.1 (CH3), 26.2 (CH2).
1
1583, 1413, 1158, 733 cm−1; H NMR (CDCl3, 400 MHz) δ 11.48
(1H, br s), 8.40 (1H, apparent br t, J value indecipherable), 7.32 (1H,
s), 5.78 (1H, br t, J = 6.0 Hz), 3.69 (2H, s), 3.45 (2H, dt, J = 6.6, 6.0
Hz), 3.38 (2H, dt, J = 6.6, 6.0 Hz), 1.75−1.64 (4H, m), 1.50 (18H, s);
13C NMR (CDCl3, 100 MHz) δ 171.3 (C), 163.5 (C), 156.3 (C),
153.3 (C), 152.3 (C), 151.7 (C), 142.7 (C), 125.5 (C), 118.3 (C),
115.0 (C), 114.4 (CH), 83.2 (C), 79.4 (C), 40.7 (CH2), 40.0 (CH2),
1
34.7 (CH2), 28.3 (CH3), 28.1 (CH3), 26.5 (CH2), 26.1 (CH2); H
NMR (DMSO-d6, 400 MHz) δ 11.51 (1H, br s), 8.32 (1H, apparent
br t, J value indecipherable), 8.13 (1H, t, J = 5.6 Hz), 7.66 (1H, s), 3.91
(2H, s), 3.28 (2H, m), 3.10 (2H, m), 1.58−1.50 (2H, m), 1.48 (9H, s,
and 2H, m), 1.40 (9H, s); 13C NMR (DMSO-d6, 100 MHz) δ 172.2
(C), 163.1 (C), 155.1 (C), 152.1 (C), 152.0 (C), 151.2 (C), 141.8
(C), 127.0 (C), 116.6 (C), 114.3 (C), 113.5 (CH), 82.8 (C), 78.0 (C),
40.1 (CH2), 39.4 (CH2), 34.7 (CH2), 27.9 (CH3), 27.5 (CH3), 26.5
(CH2), 25.8 (CH2); (+)-HRTOFESIMS m/z 685.0468 (calcd for
C24H3279Br2N4O8Na [M + Na]+, 685.0485); Rf = 0.36, 2:3 EtOAc−
hexanes, visualized with short-wave UV light, I2, and CAM/heat.
Clavatadine A (1). To a round-bottomed flask charged with
carbamate 10 (1.205 g, 1.810 mmol) was successively added 12 mL of
THF and 48 mL of 1 M HCl(aq). The flask was gently covered with a
glass stopper to prevent evaporation and to avoid plasticizer leaching
into the mixture, the latter of which occurred when a standard rubber
septum or plastic yellow cap was used to cover the aperture. The
resulting solution was heated at 30 °C (water bath) and stirred
vigorously for 20 h. After 20 h, the resulting suspension was vacuum
filtered to remove 9 mg of a black-colored solid. The yellow-orange-
colored filtrate was then concentrated in vacuo to afford clavatadine A
hydrochloride (1·HCl) as a peach-colored, amorphous solid (0.866 g,
1
93%). The H NMR chemical shifts for dilute solutions of synthetic
clavatadine A (1−2 mg of 1·HCl per milliliter of DMSO-d61) differed
very slightly from those found in the previously reported H NMR
spectrum of natural clavatadine A hydrotrifluoroacetate (1·CF3CO2H)
(1.5 mg in DMSO-d6).1 All other NMR data, including 13C, DEPT-
135, and all correlations within gradient COSY, gradient HSQC, and
gradient HMBC spectra of unpurified synthetic clavatadine A (1),
matched previously reported spectra for natural clavatadine A (1):1 IR
1
(neat) νmax 3166, 2942, 1714, 1648, 1398, 1200, 953, 754 cm−1; H
NMR (DMSO-d6, 400 MHz, referenced to residual solvent peak at δ
2.50) δ 12.41 (1H, v br s), 10.45 (1H, s), 8.02 (1H, t, J = 5.6 Hz), 7.52
(1H, t, J = 5.6 Hz), 7.40−7.10 (2H, v br s), 7.10 (1H, s), 6.95−6.65
(2H, v br s), 3.66 (2H, s), 3.12 (2H, dt, J = 5.6, 5.6 Hz), 3.07 (2H, dt, J
= 5.6, 5.6 Hz), 1.50 (4H, m); 13C NMR (DMSO-d6, 100 MHz,
referenced to residual solvent peak at δ 39.50) δ 170.8 (C), 156.8 (C),
154.2 (C), 152.7 (C), 138.0 (C), 123.2 (C), 121.9 (C), 117.1 (DEPT-
135, CH), 115.7 (C), 40.4 (DEPT-135, CH2), 40.0 (DEPT-135, CH2),
35.4 (DEPT-135, CH2), 26.5 (DEPT-135, CH2), 25.8 (DEPT-135,
CH2); (+)-HRTOFESIMS m/z 480.9716 (calcd for C14H1979Br2N4O5
[M + H]+, 480.9722).
Purification. An aliquot (4.0 mg) of synthetic clavatadine A
hydrochloride salt (1·HCl) was subjected to purification by mass-
directed semipreparative HPLC to yield 3.4 mg of clavatadine A
hydrotrifluoroacetate (1·CF3CO2H) as a colorless oil (Agilent
Technologies (Santa Clara, CA, USA) 1200 system, G1311A pump,
G1315D diode array detector, Agilent 6120 quadrupole MS). HPLC
conditions: Zorbax C18 column (9.4 × 50 mm), flow rate 5 mL/min,
solvent solvent A = 0.1% TFA in H2O, B = 0.1% TFA in CH3CN,
linear gradient from 95% solvent A, 5% solvent B to 5% solvent A, 95%
solvent B over 6 min, then to 95% solvent A, 5% solvent B in 2 min.
The oil was dissolved in DMSO to give a 10 mM stock solution.
Factor XIa Assay. Factor XIa enzyme (received as a 3.9 mg/mL
solution in glycerol−H2O, 1:1) was serially diluted to a working
concentration of 0.54 nM in assay buffer (pH 7.4 at rt) consisting of
50 mM Tris, 100 mM NaCl, 5 mM CaCl2, and 0.1 mg/mL BSA. The
To a round-bottomed flask containing a solution of dibromophenol
9 (0.933 g, 3.03 mmol, 1.00 equiv) and N,N-diisopropylethylamine
(0.103 mL, 0.606 mmol, 0.200 equiv) in 30 mL of CH2Cl2 was added
a solution of freshly prepared isocyanate 6 (1.11 g, 3.03 mmol, 1.00
equiv) in 30 mL of CH2Cl2 dropwise by cannula over 1 h. The flask
containing isocyanate 6 was rinsed with CH2Cl2 (2 × 5 mL), and these
washings were added to the reaction flask containing dibromophenol
9. The resulting solution was stirred at ambient temperature for 3 h
D
dx.doi.org/10.1021/np500772u | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
assay was performed in triplicate in 96-well microplates and consisted
of the following: 1 μL of compound in DMSO, 31 μL of factor XIa
buffer, 20 μL of factor XIa, followed by incubation at rt for 15 min.
Then, 20 μL of substrate S2366 (0.9 mM in FXIa assay buffer) was
added, followed by incubation for 2 h at rt. Absorbance was then read
at 405 nM. In-plate controls consisted of 0% inhibition (1 μL of
DMSO, 31 μL of buffer, 20 μL of factor XIa, and 20 μL of substrate
S2366) and 100% inhibition (1 μL of DMSO, 51 μL of buffer, 20 μL
of substrate S2366).
(3) Buchanan, J. C.; Petersen, B. P.; Chamberland, S. Tetrahedron
Lett. 2013, 54, 6002−6004.
(4) Castagnolo, D.; Raffi, F.; Giorgi, G.; Botta, M. Eur. J. Org. Chem.
2009, 334−337.
́ ́ ́
(5) Exposito, A.; Fernandez-Suarez, M.; Iglesias, T.; Munoz, L.;
̃
Riguera, R. J. Org. Chem. 2001, 66, 4206−4213.
(6) Feichtinger, K.; Zapf, C.; Sings, H. L.; Goodman, M. J. Org. Chem.
1998, 63, 3804−3805.
(7) Baker, T. J.; Tomioka, M.; Goodman, M. Organic Syntheses;
Wiley: New York, 2004; Coll. Vol. 10, pp 266−270.
(8) This compound, also known as 5-hydroxy-2(3H)-benzofuranone,
is commercially available but prohibitively expensive ($67.70/g, Sigma-
Aldrich).
Data Analysis. Percent inhibition was calculated as follows:
%inhibition = 100 − {[(Abscompound − Abs100%inhibition
)
/(Abs0%inhibition − Abs100%inhibition)] × 100}
(9) Wolkowitz, H.; Dunn, M. S. Biochem. Prep. 1955, 4, 6−11.
(10) Abbott, L. D., Jr.; Smith, J. D. J. Biol. Chem. 1949, 179, 359−364.
(11) Krohn, K. Tetrahedron Lett. 1975, 52, 4667−4668.
(12) The spectroscopic data matched previously reported data for
this compound.
(13) The equilibrium constant for protonation of the hydroxy group
by HBr can be estimated by extrapolating pKa data of similar species.
For example, HBr, with a pKa of −9.0 (H2O), would likely protonate
the hydroxy group of homogentisic acid lactone with an approximate
equilibrium constant between 102.5 (methylphenyl ether (PhO+(H)
Me), pKa −6.5 (H2O)) and 106.8 (protonated methanol (MeO+H2,
pKa −2.2 (H2O)). Acidity constant data taken from the pKa table of
Prof. Dave Evans, which can be found at the following Web address:
http://evans.harvard.edu/pdf/evans_pka_table.pdf.
(14) Hillery, P. S.; Cohen, L. A. J. Org. Chem. 1983, 48, 3465−3471.
(15) Lebouvier, N.; Jullian, V.; Desvignes, I.; Maurel, S.; Parenty, A.;
Dorin-Semblat, D.; Doerig, C.; Sauvain, M.; Laurent, D. Mar. Drugs
2009, 7, 640−653.
(16) Cheng, J. F.; Nguyen, B. N.; Liu, X.; Lopaschuk, G. D.; Dyck, J.
R. U.S. Patent 7,696,365 B2, 2010.
ASSOCIATED CONTENT
■
S
* Supporting Information
Comparison of the NMR data of natural and synthetic
1
clavatadine A and copies of H and 13C NMR spectra for
new compounds and compounds prepared using modified
literature procedures, including 2D NMR data for synthetic
clavatadine A (1). This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel: (509) 963-1126. Fax: (509) 963-1050. E-mail:
chambers@cwu.edu.
■
Present Addresses
⊥Department of Chemistry, Washington State University,
Pullman, WA 99164, United States.
∥Moses Lake Industries, Inc., Moses Lake, WA 98837, United
States.
(17) Wu, X.; Starnes, S. D. Org. Lett. 2012, 14, 3652−3655.
(18) Hlavac
́ ̌ ̌ ́ ̌ ́ ̌
ek, J.; Pícha, J.; Vanek, V.; Jiracek, J.; Slaninova, J.; Fucík,
V.; Budesínsky, M.; Gilner, D.; Holz, R. C. Amino Acids 2010, 38,
̌
̌
́
∇Midwestern University College of Dental Medicine, Glendale,
AZ 85308, United States.
1155−1164.
(19) The consumption of carbamate 10 under basic (0.1 M
NaOH(aq)) and reducing (0.21 M Na2SO3(aq)) conditions was
confirmed qualitatively by TLC. In one experiment, in lieu of
acidification, the reaction mixture was concentrated to provide a beige-
colored solid. Analysis of the 1H NMR spectrum of the resulting
unstable compound, which decomposed upon exposure to air and
within hours in DMSO-d6 solution, revealed a highly shielded singlet
at δH 6.74 (1H, aromatic C−H) and a deshielded singlet at δH 3.99
(2H, benzylic CH2). In comparison, the aromatic C−H and benzylic
methylene resonances of synthetic 4,6-dibromohomogentisic acid,
which is similar to the hydrolysis product of compound 10, are δH 6.99
and 3.63, respectively, in DMSO-d6. These results suggest that the
lactone was hydrolyzed under basic reaction conditions to provide a
carboxylate/phenolate dianion intermediate that cyclized upon
acidification.
Notes
The authors declare no competing financial interest.
§Undergraduate research participant.
ACKNOWLEDGMENTS
■
We dedicate this paper to University of California, Irvine
(UCI), Distinguished Professor Larry E. Overman on the
occasion of his 70th birthday this year (2013, when this paper
was first submitted to J. Nat. Prod.). We thank J. Greaves and S.
Sorooshian, Department of Chemistry, UCI Mass Spectrometry
Facility, for mass spectrometric analyses, and J. C. Buchanan
and C. E. Malmberg, Department of Chemistry, Central
Washington University (CWU), for helpful discussions. Partial
funding was provided by the CWU Science Honors Research
Program (S.J.C.), the CWU Seed Grant Program, and the
CWU Faculty Research Program. We acknowledge funding
from the National Health and Medical Research Council
(NHMRC) (R.J.Q; APP1024982).
(20) See ref 1, Supporting Information, p S4.
(21) (a) Hoffman, R.; Ozery, Y. Nitrogen NMR. http://chem.ch.huji.
ac.il/nmr/techniques/1d/row2/n.html#n (accessed Nov 20, 2014).
(b) Silverstein, R. M.; Webster, F. X.; Kiemle, D. J. Spectrometric
Identification of Organic Compounds, 7th ed.; John Wiley & Sons:
Hoboken, NJ, 2005; pp 153−155.
(22) We acknowledge a reviewer for bringing this information to our
attention.
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dx.doi.org/10.1021/np500772u | J. Nat. Prod. XXXX, XXX, XXX−XXX