Total synthesis of bastadins 2, 3, and 6

Article abstract of DOI:10.1021/jo9721204

A chemoenzymatic strategy has been developed for the synthesis of bastadins 2, 3, and 6. The requisite dimeric dityrosine and isodityrosine building blocks were successfully prepared by oxidative C - C and C - O phenolic coupling of mono- and dihalogenated derivatives of tyrosine and tyramine using horseradish and soybean peroxidases. By carefully controlling the experimental parameters, the requisite synthons may now be prepared in synthetically useful yields without the exhaustive protection and deprotection of the sensitive functional groups.

Full text of DOI:10.1021/jo9721204

J . Org. Chem. 1998, 63, 4269-4276
4269
Tota l Syn th esis of Ba sta d in s 2, 3, a n d 6
Zhi-wei Guo, Koji Machiya, Grzegorz M. Salamonczyk, and Charles J . Sih*
School of Pharmacy, University of Wisconsin, 425 N. Charter Street, Madison, Wisconsin 53706-1515
Received November 19, 1997
A chemoenzymatic strategy has been developed for the synthesis of bastadins 2, 3, and 6. The
requisite dimeric dityrosine and isodityrosine building blocks were successfully prepared by oxidative
C-C and C-O phenolic coupling of mono- and dihalogenated derivatives of tyrosine and tyramine
using horseradish and soybean peroxidases. By carefully controlling the experimental parameters,
the requisite synthons may now be prepared in synthetically useful yields without the exhaustive
protection and deprotection of the sensitive functional groups.
In tr od u ction
thallium trinitrate (TTN) oxidation. However, the syn-
thetic routes required extensive protection and depro-
tection of functional groups which resulted in low yields
of bastadins.
The bastadins are a family of modified macrocyclic
tetrapeptides isolated from the marine sponges Ianthella
basta1 and Psammaplysilla purpurea.2 They are bioge-
netically derived from tyrosines via oxidative phenolic
coupling of two tyramine-tyrosine units.3 The first seven
members of this family were described in 1981 by
Kazlauskas et al.1 Since then more than a dozen ad-
ditional members have been isolated.2,3 Bastadins 1-7
exhibited moderate antibacterial activity, whereas other
bastadins have been shown to possess cytotoxic activity
against human tumor cell lines or antiinflammatory
activity.2,3a More recently, bastadin 5 was shown to
increase the ryanodine binding capacity of SR mem-
branes by stabilizing the high affinity conformation of
RyR-1 for ryanodine without shifting the affinity of
the activator site for Ca2+ or altering the response to
caffeine or adenine nucleotides.4 Most binding and Ca2+
transport are antagonized by the immunosuppressant FK
506.
Unlike FK 506, bastadin 5 does not directly promote
the dissociation of FKBP 12 from the RyR-1 membrane
complex, but it markedly enhances the release of FKBP
125 induced by FK 506. Thus, bastadins represent a new
class of chemical probes for studying the functional
significance of immunophilin/ryanodine-sensitive Ca2+
channel interactions in skeletal muscle. In view of their
limited supply from natural sources and their interesting
biological activities,2,3 bastadins serve as an attractive
total synthetic target. To date, Yamamura’s group6 has
reported the syntheses of bastadins 1, 2, 3, and 6 using
a method, in which the aryl ethers were synthesized via
Recently, we reported the successful oxidative C-O
phenolic coupling of dihalogenated tyrosine and tyramine
derivatives to form the isodityrosine framework using
horseradish peroxidase.7 As an extension of these in-
vestigations, we have applied this oxidative coupling
technology to the synthesis of bastadins. Herein we
report a chemoenzymatic strategy for the synthesis of
bastadins 2, 3, and 6.
Retr osyn th etic An a lysis
Retrosynthetic analysis of the bastadin 6 molecule
reveals that the macrocycle may be dissected into two
units constituting the eastern and western segments
joined to each other via amide bonds. The eastern unit
is composed of two brominated tyrosine derivatives (R-
hydroxyimino group) bonded to each other by an aryl
ether linkage whereas the western unit is composed of
two brominated tyramines linked similarly to each other
via a diaryl ether bond. Our synthetic strategy entails
the use of enzymes to catalyze the oxidative coupling of
the respective monomers to form the diaryl ether bond
in the eastern and western units. The amide bonds of
the bastarane carbon skeleton are generated by joining
the dimeric units using conventional chemical methods.
The acyclic bastadin 2 molecule may be synthesized by
the condensation of the eastern segment with 2 equiv of
3-bromotyramine. Bastadin 3 may be constructed in a
similar fashion but, in this case, the requisite dimeric
synthon must be prepared via the oxidative C-C bond
coupling of two brominated tyrosine derivatives using
peroxidases (Scheme 1).
(1) Kazlauskas, R.; Lidgard, R. O.; Murphy, P. T.; Wells, R. J .;
Blount, J . F. Aust. J . Chem. 1981, 34, 765-786.
(2) Carney, J . R.; Scheuer, P. J .; Kelly-Borges, M. J . Nat. Prod. 1993,
56, 153-157.
(3) (a) Pordesimo, E. O.; Schmitz, F. J . J . Org. Chem. 1990, 55,
4704-4709. (b) Miao, S.; Andersen, R. J . J . Nat. Prod. 1990, 53, 1441-
1446. (c) Dexter, A. F.; Garscon, M. J .; Hemling, M. E. Ibid. 1993, 56,
782-786. (d) Butler, M. S.; Lim, T. K.; Capon, R. J . Aust. J . Chem.
1991, 44, 287-296. (e) Gulavita, N. K.; Wright, A. E.; McCarthy, P.
J .; Pomponi, S. A.; Kelly-Borges, M.; Chin, M.; Sills, M. A. J . Nat. Prod.
1993, 56, 1613-1617.
En zym a tic Oxid a tive P h en olic Cou p lin g
The synthesis of bastadin 6 was first investigated with
the attempted oxidative coupling of 3,5-dibromotyramine
(1a ) using horseradish peroxidase (HRP) with a view to
preparing the western hemibastadin segment. When the
enzymatic reaction was conducted in NaOAc-HOAc
buffer at pH 4.0, the major product formed was 4a , which
(4) Mack, M. M.; Molinski, T. F.; Buck, E. D.; Pessah, I. N. J . Biol.
Chem. 1994, 269, 23236-23249.
(5) Schreiber, S. L. Science 1991, 251, 283-287.
(6) (a) Nishiyama, S.; Yamamura, S. Tetrahedron Lett. 1982, 23,
1281-1284. (b) Nishiyama, S.; Suzuki, T.; Yamamura, S. Tetrahedron
Lett. 1982, 23, 3699-3702. (c) Nishiyama, S.; Suzuki, T.; Yamamura,
S. Chem. Lett. (Chem. Soc. J pn.) 1982, 1851-1852.
(7) Guo, Z. W.; Salamonczyk, G. M.; Han, K.; Machiya, K.; Sih, C.
J . J . Org. Chem. 1997, 62, 6700-6701.
S0022-3263(97)02120-8 CCC: $15.00 © 1998 American Chemical Society
Published on Web 06/05/1998

4270 J . Org. Chem., Vol. 63, No. 13, 1998
Guo et al.
Sch em e 1
Sch em e 2
Sch em e 3
was isolated in 30% yield along with another product
(20%), not characterized, but it afforded a very polar
compound upon reduction with CrCl2. When the reaction
was carried out in Na2HPO4-citrate buffer at pH 4.0 or
6.0, a complex mixture of products were obtained. How-
ever, when N-acetyl-3,5-dibromotyramine (1b) was ex-
posed to HRP at pH 5.0, three products were isolated and
identified to be 2b (10%), 3b (15%), 4b (24%), and
recovered 1b (35%). By carrying out the oxidative coupl-
ing at pH 4.0 in sodium acetate buffer, the quantity of
4b was markedly reduced. Further, isolation of the pro-
ducts could be simplified considerably by in situ treat-
ment of the reaction mixture with CrCl2. Although 3b
was obtained in 65% yield, it did not possess the requisite
regiochemical differentiation of the amino functional
groups necessary for further synthetic elaborations.
When 3b was exposed to the action of several proteases
[R-chymotrypsin (Sigma type II); Subtilisin Carlsberg
(Sigma type VIII); Protease 2A and Prozyme 6 (Amano)],
the hydrolytic reaction could not be efficiently terminated
at the monoamine stage. Hence the regioselectivity of
hydrolysis was not further examined (Scheme 2).
optimum conditions, 2c was obtained in 53% yield
using soybean peroxidase (SPO) as the catalyst (Scheme
3).
As shown in Table 1, several experimental parameters,
which are interrelated, have to be carefully controlled and
defined to obtain optimum yield of the product. These
included the amount of H2O2 and organic solvent, pH,
enzyme-to-substrate ratio, and reaction time.
Like other heme peroxidases, HRP exhibits two cata-
lytically active intermediates, compound I and II. When
HRP reacts with H2O2, compound I (FeV), which is two
oxidizing equivalents above the ferric state, is formed.
Compound I is converted into compound II (FeIV) by one-
electron reduction. Further reduction of compound II to
native enzyme (FeIII) is often rate-limiting in the overall
catalytic cycle.8
In most of our experiments, a molar ratio of 1.2 of H2O2
to substrate (2.4 equiv of H2O2) was used. Higher
concentrations (5 equiv) led to the inhibition of enzyme
activity, which may be due to the accumulation of
compound III (FeIII) during turnover since compound III
is not involved in the peroxidase reaction cycle. More-
over, excess H2O2 causes the oxidative destruction of the
porphyrin ring of HRP.
On the other hand, N-(tert-butoxycarbonyl)-3,5-dibro-
motyramine (1c) was found to be an excellent substrate
for the heme peroxidases. More importantly, the oxygen
atom of the tert-butoxycarbonyl group reacted at the
ipso position of the aromatic ring to form a cyclic
carbamate (2c) with the concomitant generation of
the cyclohexadienone ring system as shown. Under
In general, C-O coupling reactions proceeded ef-
ficiently in the pH range of 4-6. The reaction rate was
(8) Walsh, C. Enzymatic Reaction Mechanisms; W. H. Freeman
Co.: San Francisco, 1979; pp 464-500.

Total Synthesis of Bastadins 2, 3, and 6
J . Org. Chem., Vol. 63, No. 13, 1998 4271
Ta ble 1. Effect of Rea ction Con d ition s on
The enzyme-substrate ratio was also an important
factor and this ratio varied for each substrate and
depended on other factors as well, such as pH and
cosolvent concentration. This phenomenon has been
observed in other peroxidase-catalyzed oxidations of
aromatic substrates and may thus be rationalized simi-
larly. For example, in the oxidation of indole-3-acetate
by peroxidase, a high enzyme-substrate ratio resulted
in the formation of peroxidase compound II, and 3-me-
thyleneoxindole was obtained as the major end product.11
On the other hand, when indole-3-acetate and peroxidase
were mixed in stoichiometric amounts, only ferroperoxi-
dase and compound III were formed, and indole-3-
aldehyde became the major product of oxidation. In
general, the reaction time for C-O coupling should be
completed within a period of 5 to 20 min under optimum
conditions.
P er oxid a se-Ca ta lyzed C-O Cou p lin g of 1ca
yield (%, by HPLC)
acetonitrile enzyme/1c reaction time
pH
(%)
(unit/µmol)
(min)
2c remaining 1c
6
4
4
4
4
8
7
6
5
5
5
5
4
4
35
35
35
35
35
35
35
35
35
25
35
35
40
40
40 HRP
40 HRP
20 HRP
20 HRP
10 HRP
6 SPO
3 SPO
3 SPO
6 SPO
5 SPO
10
20
20
20
20
10
20
20
20
20
20
20
10
10
33
61
62
46b
35
0
0
0
0
18
18
39
7
0
46
42
50
53c
39
35
50
3
5
5 SPO
3 SPO
40 SPO
10 SPO
16
0
6
a
The experiments were carried out at 24 °C, at a scale of 10
When a low enzyme-substrate ratio was used, longer
reaction times were required which resulted in more side
reactions. On the other hand, when too much enzyme
was used, nonhomogeneous polymers were formed as
evidenced by the appearance of broad HPLC peaks.
When in situ reduction was employed, some residual
substrate was always recovered. However, this did not
necessarily mean that the reaction did not go to comple-
tion but rather monomeric quinol derivatives, such as 6
and 7, were reduced back to the starting material.
The requisite eastern segment, 8, was prepared using
3-(3,5-dibromo-4-hydroxyphenyl)-2-(hydroxyimino)propi-
onic acid (5) as the starting material.12 After much
experimentation, we found that the optimum condition
for C-O coupling occurred at pH 4.0 in buffer containing
10% acetonitrile. Under these conditions, 8 was obtained
in 46% yield accompanied by two monomeric side prod-
ucts, 6 and 7. Under mildly acidic conditions, 8 was
found to undergo isomerization to yield 9. The avail-
ability of 2c and 8 provided us with the requisite
synthons for the synthesis of bastadin 6. Reduction of
8 gave the key intermediate 10 for the synthesis of
bastadin 2. Under optimized conditions 10 was secured
in 49% overall yield by the in situ treatment of the
reaction mixture with NaHSO3 prior to workup (Scheme
4).
For the synthesis of bastadin 3, it was necessary to
prepare the dimeric synthon 11b. The dimerization of
the monomer 12a by means of C-C ortho phenolic
coupling, catalyzed by HRP, was achieved at pH 9.0 to
give 11a in 20% yield. It was transformed into 11b by
reaction with dioxane-bromine complex. Alternatively,
11b was obtained in 30% yield by the exposure of 12b to
HRP at pH 9.0 (Scheme 5).
In general the oxidative phenolic C-C coupling of
unhalogenated or monohalognated tyrosine derivatives
was best achieved in the pH range of 8 to 10. Although
the reaction rate was faster at lower pH, more polymeric
products were formed. As a rule, less enzyme was needed
for C-C than for C-O coupling reactions. The avail-
ability of the requisite synthons, successfully prepared
by enzymatic oxidative C-C and C-O couplings, allowed
us to proceed with the synthesis of the proposed targets
via amide bond formation using conventional methods.
µmol of 1c, and 5 mM of substrate concentration. The reaction
mixtures were analyzed by RP-HPLC (8 × 100 mm cartridge, 1.5
mL/min, solvent B from 20% to 80% in 12 min). The retention
times of 1c and 2c were 11.6 and 11.9 min, respectively. HRP and
SOP were abbreviations of horseradish peroxidase and soybean
b
peroxidase, respectively. Isolated yield at a scale of 5 mmol of
substrate 1c. c Isolated yield at a scale of 3 mmol of substrate 1c.
slower at lower pH, but more byproducts were formed at
neutral to alkaline pH. For example, there was a marked
difference in the product profiles between pH 6 and 7.
This observation is consistent with the published report
that the reactivity of compound II was pH dependent due
to changes in the bond length of the sixth coordinated
ligand of HRP compound II with pH.10 At pH 3.0, SPO
was more active and stable than HRP but the reaction
rate was too slow to be useful in most cases. At pH 5-6,
the reactivities of the two enzymes were virtually identi-
cal. The concentrations of substrates were in the range
of 5 to 20 mM, which depended on their solubilities.
Higher concentrations of substrates were used for
intermolecular coupling reactions, but the tendencies to
form polymers were also enhanced. The amount of
organic solvent used depended on the solubility of
substrate in buffer of defined pH. Under acidic condi-
tions, most of the substrates, except 3,5-dibromotyramine,
cannot be dissolved in aqueous buffer without the addi-
tion of a cosolvent. It is known that addition of cosolvents
may prevent the inactivation of heme peroxidases during
catalytic turnover. For example, tert-butyl alcohol is
known to protect the deactivation of chloroperoxidase
during its oxidation of indole.9 Of the organic solvents
(dioxane, acetone, methanol, DMSO, DMF, and acetoni-
trile) tested, acetonitrile was found to be the best cosol-
vent for it gave less byproducts. At a 10% acetonitrile
concentration the peroxidases were inhibited only slightly,
but at higher concentrations the inhibition became very
pronounced and more enzyme must be added to compen-
sate the reaction rate. In most cases, a clear solution of
5 to 10 mM substrate could be obtained in aqueous buffer
containing 10% acetonitrile. At pH 3, more dilute
substrate solution was used or more acetonitrile was
added to ensure a clear reaction mixture. Acetonitrile
concentrations as high as 35% could be used as in the
case of N-(tert-butoxycarbonyl)-3,5-dibromotyramine to
solubilize a 5 mM substrate solution.
(11) Ricard, J .; J ob, D. Eur. J . Biochem. 1974, 44, 359-374.
(12) Compound 5 was prepared from 4-hydroxybenzaldehyde. (a)
Forrester, A. R.; Thomson, R. H.; Woo, S. O. J . Chem. Soc., Perkin
Trans. 1 1975, 2340-2348. (b) Shiba, T.; Cahnman, H. J . J . Org. Chem.
1964, 29, 3061-3063.
(9) Van Deurzen, M. P. J .; Rantwijk, F. V.; Sheldon, R. A. Tetrahe-
dron 1997, 53, 13183-13220.
(10) Chang, C. S.; Yamazaki, I.; Sinclair, R.; Khalid, S.; Powers, L.
Biochemistry 1993, 32, 923-928.

4272 J . Org. Chem., Vol. 63, No. 13, 1998
Guo et al.
Sch em e 4
Sch em e 5
Con clu sion
In summary, we have achieved the synthesis of the
proposed bastadin targets using a chemoenzymatic ap-
proach, which avoids the extensive protection and depro-
tection of functional groups thereby demonstrating the
usefulness of peroxidases for the synthesis of the diaryl
ether bonds. To our knowledge, this approach represents
a markedly improved method for the preparation of
macrocyclic bastadins. In this connection it is also
noteworthy that this mode of oxidative coupling may
represent the biogenetic route for bastadin formation.1
That is, the isodityrosine and isodityramine frameworks
are formed via the coupling of dihalogenated tyrosine and
tyramine derivatives respectively rather than oxidative
coupling of tyrosine and tyramine followed by bromina-
tion. Although one cytochrome P450 enzyme of plant
origin was reported to catalyze diaryl ether formation of
phenolic compounds,14 it is, unfortunately, not readily
accessible for synthetic use. Until now, the yields of C-O
coupling of tyrosine derivatives assisted by peroxidases
were extremely low. We have shown that by carefully
controlling the experimental parameters, isodityrosine
derivatives can now be prepared in synthetically useful
yields without the exhaustive protection and deprotection
of the sensitive functional groups. Further application
of this oxidative enzyme technology to the synthesis of
complex natural products are currently in progress.
Com p letion of th e Syn th esis of Ba sta d in s
To complete the synthesis of bastadin 6, the cyclic
carbamate 2c was reduced with CrCl2 to give 13 in 98%
yield. Surprisingly, treatment of 2c with NaHSO3 af-
forded a complex mixture of products. Reaction of 13
with 8 in the presence of DCC and l-hydroxybenzotriazole
(HOBt) gave 14 in 72% yield, which upon reduction of
the cyclohexadienone with NaHSO3/NaOH at pH 8 fol-
lowed by the removal of the t-BOC group with TFA gave
16 (95%). The formation of the macrocycle was ac-
complished by the treatment of 16 with DCC/HOBt
giving bastadin 6 in 64% yield, whose physical properties
(1H NMR) were identical to reported values.1 The
structure of the synthetic bastadin 6 was further con-
firmed by converting it to the tetramethyl derivative, 17,
using methyl iodide. No tetramethyl isobastadine 61,13
was detected by 1H NMR analysis (Scheme 6). Bastadin
2 was prepared by the condensation of 10 with an excess
of 3-bromotyramine in the presence of DCC/HOBt to
afford bastadin 2 in 68% yield (Scheme 4). It is worthy
of note that when extra base such as diisopropylethy-
lamine or triethylamine were added to the reaction
mixture, lower yield of the product was obtained. Instead
a large amount of a less polar byproduct appeared, whose
structure was not identified.
Exp er im en ta l Section
General experimental techniques and analytical measure-
ments were applied as previously described.15 Horseradish
peroxidase (HRP, type 1) was purchased from Sigma Chemical
Co. Soybean peroxidase (SPO) was a product of Enzymol
International, Inc. The following aqueous buffers were used
in this work unless otherwise stated: 0.1 M sodium borate-
boric acid at pH 9 to 10; 0.2 M Na2HPO4-NaH2PO4 at pH 6 to
8; 0.2 M Na2HPO4-0.1 M citric acid at pH 3 to 5. All NMR
spectra were recorded on a spectrometer operating at 300 MHz
for 1H and 75 MHz for 13C, in a solvent as indicated. Reverse
phase high performance liquid chromatography (RP-HPLC)
was carried out on a Waters Nova-Pak C18 cartridge (8 × 100
mm) or a Prep Nova-Pak HR C18 column (19 × 300 mm,
preparative column) using an elution gradient from solvent A
Two methods were used for the synthesis of bastadin
3. The first of these entailed the condensation of 11b
with 3-bromotyramine to give the bastadin 3 derivative
18 in 57% yield. After debenzylation with BBr3, bastadin
3 was obtained in 80% yield. Alternatively, 11b was first
hydrogenated to remove the benzyl protecting group
(74%), and the resulting intermediate 19 was then trans-
formed into bastadin 3 by reaction with excess 3-bromo-
tyramine, which proceeded in 73% yield (Scheme 7).
(14) (a) Zenk, M. H.; Gerardy, R.; Stadler, R. J . Chem. Soc., Chem.
Commun. 1989, 1725-1727. (b) Stadler, R.; Zenk, M. H. J . Biol. Chem.
1993, 268, 823-831.
(15) Salamonczyk, G. M.; Han, K.; Guo, Z. W.; Sih, C. J . J . Org.
Chem. 1996, 61, 6893-6900.
(13) Franklin, M. A.; Penn, S. G.; Lebrilla, C. B.; Lam, T. H.; Pessah,
I. N.; Molinski, T. F. J . Nat. Prod. 1996, 59, 1121-1127.

Total Synthesis of Bastadins 2, 3, and 6
J . Org. Chem., Vol. 63, No. 13, 1998 4273
Sch em e 6
3.42 (m, 1H), 3.38-3.29 (m, 1H), 3.33 (t, 7.0, 2H), 2.80 (t, 7.0,
2H), 2.19-2.02 (m, 2H), 1.44 (s, 9H) ppm; δ 13C 172.0, 156.0,
151.5, 146.3, 146.1, 145.5, 140.8, 133.6 (2C), 124.3 (2C), 118.4,
116.7, 78.94, 76.91, 41.07, 37.15, 34.99, 30.99, 28.41 (3C) ppm;
MS FAB m/z (relative intensity) 649/651/653/655 [52/98/100/
54, (M + H)]. Similarly, 2c was obtained in 46% yield at a
larger scale (5 mmol of 1c) with HRP at pH 4.0.
Sch em e 7
3-[2-(Acetyla m in o)eth yl]-5-[4-(2-a cetyla m in oeth yl)-2,6-
d ib r om op h en oxy]-1-b r om o-3-h yd r oxy-6-oxo-1,4-cyclo-
h exa d ien e (2b); N-Aectyl-3-[4-[2-(a cetyla m in o)eth yl]-2,6-
d ibr om op h en oxy]-5-br om otyr a m in e (3b); a n d N-Acetyl-
2-(3,5-d ib r o m o -4-h y d r o x y p h e n y l)-2-(h y d r o x y e t h y l)-
a m in e (4b). To a clear solution of N-acetyl-3,5-dibromo-
tyramine (1b, 169 mg, 0.5 mmol) in 80 mL of pH 5.0 buffer
and 20 mL of acetonitrile was added 0.5 mL of HRP (1000 unit/
ml) followed by 0.6 mL of 1 M H2O2. The resulting reaction
mixture was stirred at 24 °C for 15 min, adjusted to pH 3 with
0.5 mL of 1 M NaHSO3 and 2 mL of 1 M HCl, and then
concentrated to dryness under reduced pressure. The residue
was extracted with 20 mL of dichloromethane-acetone (1:3),
and the solids were removed by filtration. The organic solution
was concentrated to dryness under reduced pressure. The
residue was then dissolved in 10 mL of 30% aqueous aceto-
nitrile. After centrifugation, the supernatant was analyzed,
and the products were isolated by RP-HPLC (8 × 100 mm
cartridge, 1.5 mL/min, solvent B from 20% to 60% in 12 min).
The retention times and yields were as follows: 4b (3.8 min,
23%), recovered 1b (6.4 min, 30%), 2b (7.6 min, 10%), 3b (9.8
min, 15%). Compound 2b, NMR (acetone-d6-DMSO-d6 9:1)
δ 1H: 7.62 (s, 2H), 7.50 (d, 1.8, 1H), 5.65 (d, 1.8, 1H), 3.44 (m,
1H), 3.32 (m, 1H), 2.82 (t, 7.2, 1H), 1.94 (m, 1H), 1.88 (s, 3H),
1.86 (s, 3H) ppm; δ 13C: 173.4, 171.4, 171.1, 154.2, 146.8, 146.0,
141.9, 134.4 (2C), 124.6, 121.8, 117.6 (2C), 71.89, 41.21, 40.64,
35.05 (2C), 22.83, 22.71 ppm; MS FAB m/z (relative intensity)
607/609/611/613 [39/98/100/34, (M + H)]. 3b, NMR (acetone-
d6-DMSO-d6 9:1) δ 1H: 7.66 (s, 2H), 7.10 (d, 1.9, 1H), 6.23 (d,
1.9, 1H), 3.45 (t, 6.9, 1H), 3.23 (t, 7.2, 1H), 2.85 (t, 6.9, 1H),
2.60 (t, 7.2, 1H), 1.89 (s, 3H), 1.83 (s, 3H) ppm; δ 13C: 171.6,
171.5, 147.6, 145.7, 142.9, 141.6, 134.4 (2C), 132.5, 127.3, 118.3
(2C), 113.8, 110.8, 41.15, 40.84, 35.02, 34.75, 22.78 (2C) ppm;
MS FAB m/z (relative intensity) 591/593/595/597 [38/100/92/
(0.1% TFA in water) to solvent B (0.1% TFA in 90% aqueous
acetonitrile). Baker silica gel (40 µm) was used for flash
chromatography.
N-(ter t-Bu toxyca r bon yl)-3,5-d ibr om otyr a m in e (1c). 1c
was prepared from tyramine. NMR (CDCl3): δ 1H 7.22 (s, 2H),
6.01 (brs, 1H), 4.60 (brs, 1H), 3.29 (brq, 2H), 2.67 (t, 6.9, 2H),
1.42 (s, 9H) ppm; δ 13C 155.9, 148.1, 133.7, 132.3 (2C), 109.9
(2C), 79.54, 41.61, 34.73, 28.39 (3C) ppm; MS FAB m/z (relative
intensity) 394/396/398 [80/100/48, (M + H)].
10-Br om o-8-[4-[2-(ter t-b u t oxyca r b on yla m in o)et h yl]-
2,6-d ib r om op h e n oxy]-2,9-d ioxo-3-a za -1-oxa sp ir o[5.5]-
u n d eca -7,10-d ien e (2c). To a clear solution of 1c (1185 mg,
3 mmol) in 340 mL of pH 5.0 buffer and 210 mL of acetonitrile
was added 50 mL of SPO (300 unit/mL) followed by 3.6 mL of
1 M H2O2. The resulting mixture was stirred at 24 °C for 20
min, quenched with 10 mL of 1 M KHSO4, and extracted with
ethyl acetate (3 × 300 mL). The combined organic extracts
were washed with brine (150 mL), dried over MgSO4, and
concentrated to dryness under reduced pressure. The residue
was subjected to flash chromatography (hexanes-ethyl acetate
2:1 to 0:1) to give 2c (520 mg, 53%). NMR (CDCl3-DMSO-d6
4:1): δ 1H 7.52 (d, 2.6, 1H), 7.48 (s, 2H), 5.63 (d, 2.6, 1H), 3.51-
1
34, (M + H)]. 4b, NMR (acetone-d6) δ H: 7.52 (s, 2H), 4.70
(dd, 7.6, 4.4, 1H), 3.45 (m, 1H), 3.28 (m, 1H), 1.90 (s, 3H).
Red u ction of 2b. Compound 2b (12 mg, 0.02 mmol) was
dissolved in 2 mL of methanol and 2 mL of pH 3 buffer. To
this mixture was added CrCl2 (20 mg, 0.16 mmol), and the
contents were stirred at 24 °C for 10 min and analyzed, and
3b (10 mg, 83%) was isolated by RP-HPLC, whose physical

4274 J . Org. Chem., Vol. 63, No. 13, 1998
Guo et al.
data were identical to a sample isolated directly from the
enzymatic reaction mixture.
7-Br om o-3-ca r boxy-8-oxo-9-[2,6-d ibr om o-4-[2-ca r boxy-
2-(h yd r oxyim in o)eth yl]p h en oxy]-2-a za -1-oxa -sp ir o[4.5]-
d eca -2,6,9-tr ien e (9). A clear solution of 50 mg of 8 in 9 mL
of dichloromethane and 1 mL of TFA was stirred at 24 °C.
The reaction progress was monitored by RP-HPLC (8 × 100
mm cartridge, 1.5 mL/min, solvent B from 20% to 80% in 12
min). The retention times of 8 and 9 were 10.5 and 10.2 min,
respectively. The conversion was 35% at 6 h, and the isomer-
ization was complete after 24 h. The mixture was concen-
trated to dryness under reduced pressure, and the residue was
recrystallized from hexanes-ethyl acetate to give 9 (40 mg,
80%), NMR (DMSO-d6): δ 1H 12.55 (s, 1H, NOH), 7.82 (d, 2.4,
1H), 7.56 (s, 2H), 6.10 (d, 2.4, 1H), 3.85 (s, 2H), 3.51 (d, 17.6,
1H), 3.32 (d, 17.6, 1H) ppm; δ 13C 172.2, 165.0, 160.6, 153.5,
149.0, 147.2, 145.2, 145.1, 138.0, 133.2 (2C), 122.3, 118.7, 116.5
(2C), 85.09, 43.19, 28.80 ppm.
3-[3-Br om o-5-[2,6-d ib r om o-4-[2-ca r b oxy-2-(h yd r oxy-
im in o)eth yl]p h en oxy]-4-h yd r oxyp h en yl]-2-(h yd r oxyim i-
n o)p r op ion ic Acid (10). To a clear solution of 5 (353 mg, 1
mmol) in 90 mL of pH 5.0 buffer and 10 mL of acetonitrile
was added 5 mL of HRP (1000 unit/mL) followed by 1.2 mL of
1 M H2O2. The resulting reaction mixture was stirred at 24
°C for 10 min, quenched with 1 M NaHSO3 (3.0 mL), adjusted
to pH 7.5 with 1 M NaOH, stirred for 10 min, acidified to pH
3.0 with KHSO4 (3 g), and then extracted with ethyl acetate
(2 × 120 mL). The combined organic extracts were washed
with brine (2 × 50 mL), dried over MgSO4, and concentrated
to dryness under reduced pressure. The residue was subjected
to flash chromatography (dichloromethane-acetone-acetic
acid 4:1:0.1 to 0:5:0.1) to give recovered 5 (28 mg, 8%) and
product 10 (153 mg, 49%), NMR (acetone-d6): δ 1H 7.65 (s,
2H), 7.16 (d, 1.8, 1H). 6.42 (d, 1.8, 1H), 3.99 (s, 2H), 3.70 (s,
2H) ppm; δ 13C 164.9, 164.7, 150.8, 150.3, 147.8, 145.3, 143.2,
138.4, 134.4 (2C), 129.5, 127.6, 118.3 (2C), 114.5, 110.3, 29.52,
29.39 ppm; MS FAB m/z (relative intensity) 623/625/627/629
[42/100/100/40, (M + H)].
En zym a tic Oxid a tive C-O Cou p lin g of 1b F ollow ed
by in situ Red u ction . To a clear solution of 1b (337 mg, 1
mmol) in 160 mL of pH 4.0, 0.2 M NaOAc-HOAc buffer and
40 mL of acetonitrile was added 5 mL of HRP (1000 unit/mL)
followed by 1.2 mL of 1 M H2O2. The resulting reaction
mixture was stirred at 24 °C for 5 min and quenched with 1
mL of 1 M NaHSO3, and CrCl2 (984 mg, 8 mmol) was then
added. The pH of the mixture was adjusted to 3 with 1 M
HCl, stirred for additional 20 min, and then extracted with
ethyl acetate (3 × 200 mL). The extracts were concentrated
to dryness under reduced pressure. The residue was extracted
with acetonitrile (2 × 50 mL), and the solid salt was removed
by filtration. The acetonitrile solution was concentrated to
dryness to give 310 mg of solids, which were stirred in 2 mL
of water for 20 min. The remaining solids were collected by
filtration, washed with another 1 mL of water, and dried under
1
reduced pressure to give 3b (192 mg, 65% overall), whose H
NMR spectrum was identical to a sample isolated by RP-HPLC
in the previous experiment.
2-Ac e t oxy-2-(3,5-d ib r o m o-4-h y d r ox y p h e n yl)e t h y l-
a m in e (4a ). To a clear solution of 3,5-dibromotyramine (1a ,
30 mg, 0.1 mmol) in 5 mL of pH 4.0, 0.2 M NaOAc-HOAc
buffer was added 0.1 mL of HRP (1000 unit/mL) followed by
0.12 mL of 1 M H2O2. The resulting reaction mixture was
stirred at 24 °C for 10 min and quenched with 0.2 mL of 1 M
NaHSO3 and 1 mL of acetonitrile. The pH of the mixture was
then adjusted to 3 with 1 M HCl and was then analyzed by
RP-HPLC (8 × 100 mm cartridge, 1.0 mL/min, from solvent A
to B in 20 min). The elution profile showed three major
peaks: peak 1 (25%, 11.8 min, the same retention time as 1a ),
peak 2 (30%, 12.3 min), peak 3 (20%, 13.9 min). After the
addition of CrCl2 (98 mg, 0.8 mmol), the mixture was stirred
for 10 min and analyzed, and the products were isolated by
RP-HPLC. Peaks 1 and 2 remained unchanged, whereas peak
3 was transformed into a new peak with a retention time of
3.2 min, whose chemical identity was not determined. 1H
NMR analysis confirmed peak 1 was recovered 1a . The
2-(Ben zyloxyim in o)-3-[3-[5-[2-(ben zyloxyim in o)-2-ca r -
b oxyet h yl]-2-h yd r oxyp h en yl]-4-h yd r oxyp h en yl]p r op i-
on ic Acid (11a ). To a clear solution of 2-(benyloxyimino)-3-
(4-hydroxyphenyl)propionic acid (12a , 286 mg) in 52 mL of pH
9.0 buffer and 6 mL of dioxane was added 1430 units of HRP
followed by 136 µL of 30% H2O2. The reaction mixture was
stirred at 24 °C for 10 min, quenched and adjusted to pH 2
with 5 M HCl, and extracted with 50 mL of ethyl acetate. The
organic extracts were washed with 30 mL of water and
concentrated to dryness under reduced pressure. The residue
was subjected to flash chromatography (dichloromethane-
acetone 100:0 to 7:1) to afford 11a (58 mg, 20%), NMR
(CDCl3): δ 1H 7.20-7.16 (m, 12H), 7.09 (d, 8.1, 2H), 6.81 (dd,
8.1, 2.0, 2H), 5.16 (s, 4H), 3.76 (s, 4H) ppm; δ 13C 164.3 (2C),
151.7 (2C), 150.3 (2C), 135.9 (2C), 132.3 (2C), 130.3 (2C), 128.6
(4C), 128.5 (2C), 128.3 (4C), 128.0 (2C), 124.9 (2C), 117.0 (2C),
78.25 (2C), 29.75 (2C) ppm; MS FAB m/z 569 (M + H).
2-(Ben zyloxyim in o)-3-[3-br om o-5-[5-[2-(ben zyloxyim i-
n o)-2-ca r b oxye t h yl]-3-b r om o-2-h yd r oxyp h e n yl]-4-h y-
d r oxyp h en yl]p r op ion ic Acid (11b). To a clear solution of
2-(benzyloxyimino)-3-(3-bromo-4-hydroxyphenyl)propionic acid
(12b, 510 mg) in 92 mL of pH 9.0 buffer and 10 mL of dioxane
was added 1275 units of HRP followed by 190 µL of 30% H2O2.
The reaction mixture was stirred at 24 °C for 5 min, quenched
with 60 mL of 5% citric acid aqueous solution, and extracted
with 100 mL of ethyl acetate. The organic extracts were
washed with 40 mL of water and concentrated to dryness
under reduced pressure. The residue was subjected to flash
chromatography (dichloromethane-acetone 100:0 to 5:1) to
1
structure of peak 2 was established as 4a , NMR (D2O): δ H
7.60 (s, 2H), 5.83 (dd, 8.8, 4.1, 1H), 3.43 (dd, 14.2, 8.8, 1H),
3.36 (dd, 14.2, 4.1, 1H), 2.20 (s, 3H) ppm; δ 13C 176.8, 154.0,
134.1 (2C), 133.9, 114.8 (2C), 75.0, 47.2, 24.2 ppm; MS FAB
m/z (relative intensity) 352/354/356 [60/100/55, (M + H)].
7,9-Dib r om o-2,8-d ioxo-3-(h yd r oxyim in o)-1-oxa sp ir o-
[4.5]d eca -6,9-d ien e (6); 7,9-Dibr om o-3-ca r boxy-8-oxo-2-
a za -1-oxa sp ir o[4.5]d eca -2,6,9-tr ien e (7); a n d 7-Br om o-2,8-
d ioxo-3-(h yd r oxyim in o)-9-[2,6-d ib r om o-4-[2-ca r b oxy-2-
(h yd r oxyim in o)eth yl]p h en oxy]-1-oxa sp ir o[4.5]d eca -6,9-
d ien e (8). To a clear solution of 3-(3,5-dibromo-4-hydroxy-
phenyl)-2-(hydroxyimino)propionic acid (5, 2120 mg, 6 mmol)
in 420 mL of pH 4.0 buffer and 60 mL of acetonitrile was added
120 mL of SPO (250 unit/mL) followed by 7.2 mL of 1 M H2O2.
The resulting reaction mixture was stirred at 24 °C for 10 min
and extracted with ethyl acetate (3 × 500 mL). The combined
organic extracts were washed with brine (300 mL), dried over
MgSO4, and concentrated to dryness under reduced pressure.
The residue was subjected to flash chromatography (hexanes-
ethyl acetate-acetone 1:1:0 to 0:4:1) to give 6 (42 mg, 4%),
NMR (CDCl3-acetone-d6 4:1): δ 1H 12.18 (brs, 1H, NOH), 7.60
(s, 2H), 3.39 (s, 2H) ppm; δ 13C 171.9, 164.1, 147.2 (2C), 144.8,
123.6 (2C), 79.33, 33.59 ppm, 7 (28 mg, 3%), NMR (CDCl3-
acetone-d6 1:1): δ 1H 7.64 (s, 2H), 3.65 (s, 2H) ppm; δ 13C 171.5,
160.0, 152.7, 145.7 (2C), 122.8 (2C), 86.45, 42.96 ppm, and 8
(860 mg, 46%), NMR (DMSO-d6) δ 1H:12.83 (s, 1H, NOH),
12.55 (s, 1H, NOH), 7.94 (d, 2.2, 1H), 7.56 (s, 2H), 6.33 (d, 2.2,
1H), 3.84 (s, 2H), 3.23 (d, 19.1, 1H), 2.93 (d, 19.1, 1H) ppm;
NMR (acetone-d6): δ 1H 7.80 (d, 2.6, 1H), 7.64 (s, 2H), 6.23 (d,
2.6, 1H), 3.93 (s, 2H), 3.44 (d, 19.5, 1H), 3.21 (d, 19.5, 1H); δ
13C 172.6, 165.3, 164.7, 149.7, 148.3, 146.5 (2C), 145.5, 139.0,
134.5 (2C), 123.9, 120.3, 117.4 (2C), 78.99, 34.21, 29.46 ppm;
MS FAB m/z (relative intensity) 621/623/625/627 [35/92/100/
44, (M + H)]. Compound 8 was also obtained in 47% yield
under the same reaction conditions except HRP was used
instead of SPO on a smaller scale (1 mmol of 5).
1
afford 11b (155 mg, 30%). NMR: δ H (acetone-d6-D2O 9:1)
7.44 (s, 2H), 7.33-7.26 (m, 10H), 7.04 (s, 2H), 5.29 (s, 4H),
3.84 (s, 4H) ppm; δ 13C (CDCl3-acetone-d6 1:2) 164.8 (2C),
151.3 (2C), 150.4 (2C), 137.4 (2C), 133.7 (2C), 132.4 (2C), 129.8
(2C), 129.2 (4C), 129.1 (4C), 128.9 (2C), 127.4 (2C), 111.7 (2C),
78.25 (2C), 30.21 (2C) ppm; MS FAB m/z (relative intensity)
725/727/729 [50/100/56, (M + H)].
3-Br om o-5-[2,6-d ibr om o-4-[2-(ter t-bu toxyca r bon yla m i-
n o)eth yl]p h en oxy]tyr a m in e (13). To a clear solution of 2c
(326 mg, 0.5 mmol) in methanol-dichloromethane (1:1, 50 mL)

Total Synthesis of Bastadins 2, 3, and 6
J . Org. Chem., Vol. 63, No. 13, 1998 4275
at 24 °C was added CrCl2 (492 mg, 4 mmol). The resulting
reaction mixture was stirred at the same temperature for 30
min. Water (50 mL) was added to the mixture after the
solvent was partially removed under reduced pressure. The
mixture was extracted with ethyl acetate (3 × 80 mL). The
combined organic extracts were washed with brine (150 mL),
dried over MgSO4, and concentrated to dryness under reduced
pressure. The residue was mixed with 10 mL of water and
stored at 0 °C overnight after the pH was adjusted to 7.0 with
1 M NaOH. The precipitate was filtered and washed with 2
mL of water and dried under reduced pressure to give 13 (298
mg, 98%). NMR (DMSO-d6-acetone-d6-D2O 8:1:1): δ 1H 7.55
(s, 2H), 7.12 (d, 1.9, 1H), 6.18 (d, 1.9, 1H), 3.20 (t, 6.6, 2H),
2.91 (t, 8.1, 2H), 2.71 (t, 6.6, 2H), 2.64 (t, 8.1, 2H), 1.29 (s, 9H)
ppm; δ 13C 157.2, 146.7, 145.5, 142.8, 141.3, 134.1 (2C), 129.6,
126.9, 117.7 (2C), 113.6, 111.2, 79.49, 41.10, 40.59, 34.89,
32.22, 28.60 (3C) ppm.
7-Br om o-2,8-d ioxo-3-(h yd r oxyim in o)-9-[2,6-d ibr om o-4-
[2-(h yd r oxyim in o)-2-[2-[3-br om o-4-h yd r oxy-5-[2,6-d ibr o-
m o-4-[2-(t er t -b u t oxyca r b on yla m in o)e t h yl]p h e n oxy]-
p h e n y l]e t h y la m in o c a r b o n y l]e t h y l]p h e n o x y ]-1-o x a -
sp ir o[4.5]d eca -6,9-d ien e (14). To a solution of 8 (62 mg, 0.1
mmol), 13 (61 mg, 0.1 mmol), and 1-hydroxybenzotriazole
(HOBt, 14 mg, 0.1 mmol) in dioxane (8 mL) and DMF (1 mL)
at 0 °C was added a solution of DCC (31 mg, 0.15 mmol) in 1
mL of dichloromethane. The resulting reaction mixture was
stirred at 24 °C for 24 h. Ethyl acetate (40 mL) was added to
the mixture after the solvent was partially removed under
reduced pressure. The organic solution was washed with 1
M KHSO4 (20 mL) and brine (2 × 20 mL), dried over MgSO4,
and concentrated to dryness under reduced pressure. The
residue was subjected to flash chromatography (dichlo-
romethane-acetone 19:1 to 4:1) to give 14 (86 mg, 72%). NMR
(CDCl3-acetone-d6 1:1): δ 1H 12.17 (s, 1H), 11.23 (s, 1H), 8.34
(s, 1H), 7.61 (d, 2.6, 1H), 7.59 (s, 2H), 7.50 (s, 2H), 7.29 (t, 5.8,
1H), 7.06 (d, 1.8, 1H), 6.16 (d, 1.8, 1H), 5.90 (d, 2.6, 1H), 3.87
(s, 2H), 3.46-3.32 (m, 5 H), 3.17 (d, 19.7, 1H), 2.85 (brt, 2H),
2.64 (t, 6.8, 2H), 1.41 (s, 9H) ppm; δ 13C 172.5, 164.4, 163.5,
156.9, 151.3, 147.8, 147.5, 146.7, 146.4, 145.5, 145.3, 142.9,
141.2, 138.9, 134.7 (2C), 134.3 (2C), 131.9, 127.3, 124.4, 119.6,
118.3 (2C), 117.4 (2C), 113.7, 110.7, 79.29, 78.92, 42.09, 41.17,
35.73, 35.15, 34.61, 28.90 (3C), 28.83 ppm.
The mixture was adjusted to pH 7.0 with 1 M NaOH and
stirred for 5 min. The solids were collected by filtration,
washed with water (3 × 1 mL), and dried under reduced
pressure to give 16 (52 mg, 95%), which was used directly for
bastadin 6 synthesis.
Ba sta d in 6. To a solution of 16 (50 mg, 0.045 mmol) and
HOBt (7 mg, 0.054 mmol) in dioxane (16 mL) and DMF (2 mL)
at 0 °C was added a solution of DCC (19 mg, 0.09 mmol) in 2
mL of dichloromethane. The resulting mixture was stirred
at 24 °C for 8 h. Ethyl acetate (50 mL) was added to the
mixture after the solvent was partially removed under reduced
pressure. The organic solution was washed with 1 M KHSO4
(20 mL) and brine (2 × 20 mL), dried over MgSO4, and
concentrated to dryness under reduced pressure. The residue
was subjected to flash chromatography (dichloromethane-
acetone 19:1) to give white solid bastadin 6 (32 mg, 64%), NMR
1H 1D (DMSO-d6) and COSY (DMSO-d6-D2O-acetone-d6
8:1:1) spectra were in accord with the literature data1; NMR
(DMSO-d6):
δ
13C 163.3, 163.1, 156.6, 151.4, 150.5, 146.1,
144.8, 144.7, 141.9, 141.6, 140.2, 137.7, 133.7 (2C), 133.3 (2C),
130.8, 128.2, 127.0, 126.3, 117.6 (2C), 117.2 (2C), 112.7, 111.7,
110.2, 109.8, 40.48, 38.33, 33.95, 32.75, 28.76, 27.36 ppm; MS
FAB m/z (peak maximum mass) 1099 (M + H).
Tetr a m eth yl Ba sta d in 6 (17). The structure of the
synthetic bastadin 6 was further confirmed by converting it
to the tetramethyl derivative 17 with methyl iodide. Bastadin
6 (20 mg) was stirred in DMF (5 mL) with K2CO3 (0.3 g) and
methyl iodide (0.3 mL) at 24 °C for 20 h. The reaction mixture
was diluted with dichloromethane (30 mL) and filtered. The
filtrate was washed with 1 M HCl (15 mL), 5% NaHCO3 (15
mL), 1 M HCl (15 mL), brine (15 mL), dried over MgSO4, and
concentrated to dryness under reduced pressure. The residue
was subjected to flash chromatography (dichloromethane-
acetone 19:1) to give a colorless gum 17 (10 mg). The
structural analysis of 17 was in accord with the literature
1
data;1 NMR (CDCl3): δ H 7.53 (s, 2H), 7.46 (s, 2H), 7.19 (d,
1.8, 1H), 7.08 (d, 1.8, 1H), 6.8-6.7 (m, 2H), 6.25, (d, 1.8, 1H),
6.23 (d, 1.8, 1H), 4.06 (s, 3H), 4.03 (s, 3H), 4.02 (s, 3H), 3.85
(s, 2H), 3.72 (s, 2H), 3.61 (s, 3H), 3.6-3.4 (m, 4H), 3.0-2.6 (m,
4H) ppm; no methoxy singlet at δ 3.92 of tetramethylisobas-
tadin 6 was detected;1,10 MS FAB m/z (relative intensity) 1149/
1151/1153/1155/1157/1159/1161 [22/42/80/100/82/52/20, (M +
H)].
3-[3-Br om o-4-h yd r oxy-5-[2,6-d ib r om o-4-[2-(h yd r oxy-
im in o)-2-[2-[3-br om o-4-h yd r oxy-5-[2,6-d ibr om o-4-[2-(ter t-
b u t oxyca r b on yla m in o)e t h yl]p h e n oxy]p h e n yl]e t h yl-
a m in oca r b on yl]et h yl]p h en oxy]p h en yl]-2-(h yd r oxyim i-
n o)p r op ion ic Acid (15). To a clear solution of 14 (81 mg,
0.068 mmol) in acetonitrile (10 mL) and water (5 mL) was
added 0.27 mL of 1 M NaHSO3 followed by 1 mL of pH 8.0
buffer. The mixture was stirred at 24 °C for 10 min after the
pH was adjusted to 8.0 with a few drops of 1 M NaOH. It
was then acidified to pH 3.5 with 1 M KHSO4, extracted with
ethyl acetate (3 × 30 mL), washed with brine (2 × 30 mL),
dried over MgSO4, and concentrated to dryness under reduced
pressure. The solid residue was washed with 2 mL of hexane
and dried again to give 15 (80 mg, 99%), NMR (acetone-d6): δ
1H 7.64 (s, 2H), 7.60 (s, 2H), 7.48 (t, 6.0, 1H), 7.15 (d, 1.8, 1H),
7.11 (d, 1.9, 1H), 6.39 (d, 1.8, 1H), 6.25 (d, 1.9, 1H), 3.94 (s,
2H), 3.70 (s, 2H), 3.46-3.34 (m, 4H), 2.86 (t, 6.8, 2H), 2.66 (t,
7.3, 2H), 1.39 (s, 9H) ppm; δ 13C 164.8, 164.5, 156.6, 151.9,
150.9, 147.6, 147.4, 145.5, 145.4, 143.2, 143.1, 141.7, 138.8,
134.5 (2C), 134.4 (2C), 132.3, 129.5, 127.6, 127.3, 118.2 (4C),
114.5, 113.7, 110.6, 110.3, 78.68, 41.85, 41.19, 35.48, 34.96,
29.41, 28.82, 28.53 (3C) ppm; MS FAB m/z (relative intensity)
1211/1213/1215/1217/1219/1221/1223 [30/42/74/100/72/40/28,
(M + H)].
Ba sta d in 2. To a solution of 10 (31 mg, 0.05 mmol),
3-bromotyramine (26 mg, 0.12 mmol), and HOBt (14 mg, 0.1
mmol) in dioxane (8 mL) and DMF (1 mL) at 0 °C was added
a solution of DCC (31 mg, 0.15 mmol) in 1 mL of dichlo-
romethane. The resulting reaction mixture was stirred at 24
°C for 24 h. Ethyl acetate (40 mL) was added to the mixture
after the solvent was partially removed under reduced pres-
sure. The organic solution was washed successively with 1 M
KHSO4 (2 × 10 mL) and brine (2 × 10 mL), dried over MgSO4,
and concentrated to dryness under reduced pressure. The
residue was subjected to flash chromatography (dichlo-
romethane-acetone 19:1 to 4:1) to give a colorless gum, which
was triturated in 2 mL of hexane to afford bastadin 2 (35 mg,
68%). NMR (DMSO-d6) 1H 1D and COSY spectra were in
accord with the literature data1; δ 13C 162.8, 162.7, 152.2 (2C),
151.3, 150.8, 146.2, 144.5, 141.9, 137.6, 133.2 (2C), 132.6 (2C),
131.4, 131.3, 128.7 (2C), 128.3, 126.1, 117.2 (2C), 116.1 (2C),
113.3, 110.0, 109.0 (2C), 40.35 (2C), 33.50, 33.26, 28.01, 27.70
ppm; it should be noted that the signal at δ 117.2 was not
reported in the literature, which corresponds to the carbons
28 and 30 according to the nomenclature proposed by Ka-
zlauskas1; MS FAB m/z (relative intensity) 1018/1020/1022/
1024/1026/1028 [26/64/80/100/64/26, (M + H)].
3-[3-Br om o-4-h yd r oxy-5-[2,6-d ib r om o-4-[2-(h yd r oxy-
im in o)-2-[2-[3-br om o-4-h yd r oxy-5-[2,6-d ibr om o-4-(2-a m i-
n oet h yl)p h en oxy]p h en yl]et h yla m in oca r b on yl]et h yl]-
p h en oxy]p h en yl]-2-(h yd r oxyim in o)p r op ion ic Acid (16).
To a mixture of 15 (60 mg, 0.05 mmol) in dichloromethane (2
mL) at 0 °C was added TFA (2 mL). The reaction mixture
was stirred at 0 °C for 5 min and then at 24 °C for 30 min and
concentrated to dryness under reduced pressure. The residue
was triturated in 2 mL of water and 0.5 mL of pH 7.0 buffer.
Diben zylba sta d in 3 (18). To a solution of 11b (100 mg)
in dichloromethane were added HOBt (41 mg) and 1-[3-
(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride
(EDCI HCl, 63 mg) at 0 °C, followed by 3-bromotyramine
hydrochloride (77 mg), diisopropylethylamine (39 mg), and 2
mL of DMF. The reaction mixture was stirred at the same
temperature for 2 h, quenched with 15 mL of water, and then
extracted with 20 mL of dichloromethane. The organic
extracts were washed with 20 mL of 5% NaHCO3, 20 mL of

4276 J . Org. Chem., Vol. 63, No. 13, 1998
Guo et al.
5% HCl, and 20 mL of water twice, dried over MgSO4, and
concentrated to dryness under reduced pressure. The residue
was subjected to flash chromatography (dichloromethane-
acetone 100:0 to 5:1) to afford 18 (88 mg, 57%). NMR
was subjected to flash chromatography (dichloromethane-
acetone 100:0 to 5:1) to afford bastadin 3 (25.4 mg, 80%), NMR
data (1H and 13C) were in accord with the literature data1; MS
FAB m/z (relative intensity) 939/941/943/945/947 [32/78/100/
78/35, (M + H)].
1
(CDCl3): δ H 7.39 (d, 1.8, 2H), 7.20-7.18 (m, 12H), 7.01 (d,
1.8, 2H), 6.82-6.76 (m, 4H), 6.65 (t, 6.6, 2H), 5.08 (s, 4H), 3.78
(s, 4H), 3.38 (brq, 4H), 2.62, (t, 6.8, 4H) ppm; δ 13C 162.4 (2C),
151.9 (2C), 151.3 (2C), 148.1 (2C), 136.4 (2C), 133.1 (2C), 132.2
(2C), 132.1 (2C), 132.0 (2C), 129.7 (2C), 129.4 (2C), 128.6 (4C),
128.3 (2C), 128.2 (4C), 125.2(2C), 116.2 (2C), 111.2 (2C), 110.1
(2C), 77.5 (2C), 40.72 (2C), 34.36 (2C), 28.80 (2C) ppm; MS
FAB m/z (relative intensity) 1119/1121/1123/1125/1127 [53/
67/100/82/44, (M + H)].
3-[3-Br om o-4-h yd r oxy-5-[3-br om o-2-h yd r oxy-5-[2-ca r -
boxy-2-(h yd r oxyim in o)eth yl]p h en yl]p h en yl]-2-(h yd r oxy-
im in o)p r op ion ic Acid (19). To a clear solution of 11b (10
mg) in 4 mL of ethanol was added 10 mg of 10% Pd/C. The
reaction mixture was stirred under an atmospheric pressure
of hydrogen gas at 24 °C for 30 min. The catalyst was removed
by filtration and washed with methanol. The combined filtrate
was concentrated to dryness under reduced pressure to afford
19 (5.6 mg, 74%), which was used directly for bastadin 3
synthesis.
Ba sta d in 3 fr om 18. To a solution of 18 (38 mg) in 3.8
mL of thioanisole was added 812 µL of 1 M boron tribromide
solution in dichloromethane. The reaction mixture was stirred
at 24 °C for 30 min, quenched with 10 mL of 0.5 M HCl, and
extracted with 20 mL of ethyl acetate. The organic extracts
were washed with 10 mL of water twice, dried over MgSO4,
and concentrated to dryness under reduced pressure. The
residue was triturated in hexane to afford a precipitate, which
Ba sta d in 3 fr om 19. To a solution of 19 (5.4 mg) in 2 mL
of dichloromethane were added HOBt (3.5 mg) and EDCI HCl
(4.5 mg) at 0 °C, followed by 3-bromotyramine hydrochloride
(5.5 mg), diisopropyl ethylamine (2.8 mg), and 0.1 mL of DMF.
The reaction mixture was stirred at the same temperature for
4 h, quenched with 2 mL of water, and extracted with 5 mL of
ethyl acetate. The organic extracts were successively washed
with 2 mL of 5% NaHCO3, 2 mL of 5% HCl, and 2 mL of water
twice, dried over MgSO4, and concentrated to dryness under
reduced pressure. The residue was subjected to flash chro-
matography (dichloromethane-acetone 100:0 to 5:1) to afford
bastadin 3 (6.8 mg, 73%); the analytical data were identical
to the sample prepared from 18.
Ack n ow led gm en t . This investigation was sup-
ported in part by a grant from NIH.
Su p p or tin g In for m a tion Ava ila ble: NMR (1H, 1H-1H
COSY, 13C, DEPT) and MS spectra of compounds 1b, 2c, 2b,
3b, 4b, 4a , 6, 7, 8, 9, 10, 11a , 11b, 13, 14, 15, and 18 (78
pages). This material is contained in libraries on microfiche,
immediately follows this article in the microfilm version of the
journal, and can be ordered from the ACS; see any current
masthead page for ordering information.
J O9721204