Antineoplastic agents. 400. Synthesis of the Indian ocean marine sponge cyclic heptapeptide phakellistatin 2

Article abstract of DOI:10.1021/np980168m

Solution-phase synthesis of the marine sponge constituent phakellistatin 2 (1), cyclo(Tyr-Pro-Phe-Pro-Ile-Ile-Pro), was completed using a combination of stepwise coupling and (4 + 3) segment condensation. Use of diethyl phosphorocyanidate for the peptide bond formations gave the linear heptapeptide in 54% yield. Cyclization was achieved in high yields utilizing TBTU (2), BOP-C1 (3), PyBroP (4), and HOAt (5), resulting in 50-65% yields of phakellistatin 2 (1) depending on the method employed. The synthetic cyclic peptide was chemically but not biologically identical with the natural product.

Full text of DOI:10.1021/np980168m

J . Nat. Prod. 1999, 62, 409-414
409
An tin eop la stic Agen ts. 400. Syn th esis of th e In d ia n Ocea n Ma r in e Sp on ge Cyclic
Hep ta p ep tid e P h a k ellista tin 2†,1a
George R. Pettit,* Monte R. Rhodes, and Rui Tan
Cancer Research Institute and Department of Chemistry, Arizona State University, Tempe, Arizona 85287-2404
Received April 27, 1998
Solution-phase synthesis of the marine sponge constituent phakellistatin 2 (1), cyclo(Tyr-Pro-Phe-Pro-
Ile-Ile-Pro), was completed using a combination of stepwise coupling and (4 + 3) segment condensation.
Use of diethyl phosphorocyanidate for the peptide bond formations gave the linear heptapeptide in 54%
yield. Cyclization was achieved in high yields utilizing TBTU (2), BOP-C1 (3), PyBroP (4), and HOAt (5),
resulting in 50-65% yields of phakellistatin 2 (1) depending on the method employed. The synthetic
cyclic peptide was chemically but not biologically identical with the natural product.
The isolation of new cyclic peptides from marine sponges
has been increasingly productive. Illustrative are the
Pohnpei Cribrochalina olemda kapakahines2a (related to
phakellistatin 32b), the Philippine Aciculites orientalis
aciculitins,2c the Federated State of Micronesia Phakellia
sp. phakellistatins,2d and the Indonesia Ircinia dendroides
waiakeamide,2e of which the first three groups contain
cancer cell growth inhibitory members. An early advance
with such potentiallyusefulPorifera cyclicpeptideconstituents2b,3
was our isolation [9.6 × 10-5% yield and structural
elucidation (by high-field 2D-NMR and HRFABMS tech-
niques)] of phakellistatin 2 (1) from the Republic of
Comoros Phakellia carteri.4 This cyclic heptapeptide (1)
exhibited cell growth inhibitory properties against the
murine P388 lymphocytic leukemia (ED50 0.34 µg/mL) and
a selection of human cancer cell lines. A total synthesis of
phakellistatin 2 (1) was undertaken to increase the supply
for further biological studies and to ascertain whether the
natural product might contain an exceptionally potent
antineoplastic substance5 in a trace amount too small to
detect6 by the chemical and physical techniques employed
for isolation and structure determination.
For the synthesis of the cyclic heptapeptide 1, a solution-
phase synthetic strategy7 was employed involving Fmoc
N-terminal protection8,9 and tert-butyl ester C-terminal
protection, similar to that which we recently used to obtain
axinastatins 2 and 36a and stylopeptide 1.6b The tert-butyl
ester for C-terminal protection was utilized owing to its
ability to resist nucleophilic attack and diketopiperazine
formation.10 Peptide bond formation was accomplished with
DEPC,1b,11 starting from tert-butyl proline (7).6a,12 When
DEA9a was used as the base for Fmoc cleavage, a large
amount of dibenzofulvene (DBF) was formed, accompanied
by significant (5-10%) epimerization and overall lowered
yields. Therefore, TAEA1b,13 with a phosphate buffer (pH
units were then coupled to form the linear heptapeptide
5.5) was used to assist in removing DBF, reduce epimer-
(Scheme 3). Since cyclization would be based on a proline
ization, and result in 70% or higher yields on the average.
unit, which is turn inducing16a and minimizes racemiza-
Peptide bond formation was conducted with DIEA as the
tion,16b,c the formation of phakellistatin 2 was expected to
be favored by this approach.
base in dichloromethane (DCM shown to give less epimer-
ization14 than DMF15). The use of this base was further
supported by previous studies6,8 and the stability of the
Fmoc group toward DIEA.10c These improvements were
employed to synthesize the requisite tri- and tetrapeptide
segments shown in Schemes 1 and 2, respectively. The two
Cyclization of heptapeptide 13 to provide phakellistatin
2 was readily accomplished utilizing the following tech-
niques: TBTU17 (2)/DIEA in DCM, BOP-C118 (3)/DIEA in
DCM, PyBroP19 (4)/DIEA in DCM, and TBTU with HOAt
(5),20 the pyridyl variant of the well-known21 HOBt (6). All
of these methods afforded yields of 45% or better. The use
of TBTU (2) gave the highest consistent yields (55%). The
synthetic phakellistatin 2 (1) was found to be chemically
† Dedicated to the memory of Dr. C. Gordon Zubrod (1914-1999), a great
pioneer of cancer chemotherapy and friend.
* To whom correspondence should be addressed. Phone: (602) 965-0186.
Fax: (602) 965-8558. E-mail: bpettit@asu.edu.
10.1021/np980168m CCC: $18.00
© 1999 American Chemical Society and American Society of Pharmacognosy
Published on Web 03/04/1999

410 J ournal of Natural Products, 1999, Vol. 62, No. 3
Pettit et al.
Sch em e 1a
Sch em e 3a
a
Key: (i) TAEA, DCM; (ii) Fmoc-L-Ile, DEPC, DIEA, DCM, 72%; (iii)
Fmoc-L-Ile, DEPC, DIEA, DCM, 84%.
Sch em e 2a
a
a
Key: (i) TFA, DCM; (ii) TAEA, DCM; (iii) DEPC, DIEA, DCM, 54%;
(iv) method A, B, C, or D.
Key: (i) TAEA, DCM; (ii) Fmoc-L-Phe, DEPC, DIEA, DCM, 71%; (iii)
Fmoc-L-Pro, DEPC, DIEA, DCM, 81%; (iv) Fmoc-L-Tyr-OtBu, DEPC, DIEA,
DCM, 75%.
assumption that Pro would induce a â-turn in the i + 1
position. That strategy might explain their observation that
the product did not coincide exactly with the NMR data
we reported for natural phakellistatin 2. Furthermore,
their product was not compared with an authentic speci-
men of natural phakellistatin 2. To remove any conceivable
doubt about the correctness of the structure we originally
assigned to natural phakellistatin 2 and confirmed in the
present study, we again meticulously purified specimens
of the natural and synthetic phakellistatin 2 and repeated
the comparison investigation. Both specimens were purified
by the same HPLC techniques and individually restudied
by high-field NMR (500 MHz). In addition, the high-field
1H NMR and 13C NMR spectra were repeated using an
equiweight mixture (cf. Table 1) of the natural and
synthetic phakellistatin 2 (1). All of the chromatographic
and spectral comparison procedures using the natural and
synthetic specimens gave identical results.
identical with the natural product by comparison of high-
1
field (500 MHz) H and 13C NMR data (see Tables 1 and
2), IR, TLC, mixture mp, and [R]D, but not biologically
identical as noted below.
Biological evaluation of the synthetic phakellistatin 2
and the natural sample against the P388 lymphocytic
leukemia cell line showed cell growth inhibitory activity
of ED50 24 µg/mL as compared with the natural peptide’s
activity of ED50, 0.34 µg/mL. Comparison of the synthetic
cyclic peptide with a minipanel of six cell lines from the
NCI’s primary screening panel of human cancer cell lines22
showed no activity compared with the natural peptide. The
results of these biological experiments again suggest that
cyclic peptides of this nature may be capable of capturing/
complexing strongly active antineoplastic agents in amounts
undetectable by current high-field NMR, high-resolution
MS, and chromatographic techniques, as we have recently
discovered.23,6 Alternatively, a reviewer has thoughtfully
suggested that these differences might result from confor-
mational changes arising from the proline units.
Exp er im en ta l Section
Gen er a l P r oced u r e. All solvents except for acetonitrile
(HPLC grade, EM Science) and DMF (anhyd, Aldrich) were
redistilled. Dichloromethane was distilled from calcium hy-
dride and stored over 4-Å molecular sieves. The Fmoc L-amino
acids (NovaBiochem and Sigma-Aldrich) were used as re-
After this study was completed, Kessler24 summarized
in preliminary form a solid-phase peptide synthesis based
on the structure assigned phakellistatin 2. In the final step,
they chose cyclization between Ile and Ile under the

Antineoplastic Agents
J ournal of Natural Products, 1999, Vol. 62, No. 3 411
Ta ble 1. Phakellistatin 2a High-Field (500 MHz) NMR
Ta ble 2. 13C NMR Spectral Data Comparison of Natural and
Assignments Recorded in CD3OD
Synthetic Phakellistatin 2
carbon
13C NMR
1H NMR
carbon
natural
synthetic
assignment
phakellistatin 2
phakellistatin 2
Pro*
CO
a-CH
b-CH2
172.74p
59.89n
31.78p
Ile-CO
Pro-CO
Pro-CO/Ile-CO
Phe-CO
Pro-CO
174.5
173.22
172.99
172.78
172.74
170.46
157.77
136.87
131.98
130.64
130.06
128.68
127.35
116.13
63.25
62.34
59.93
59.89
55.80
55.62
54.33
48.24
47.68
38.41
38.35
37.63
35.71
32.77
31.78
31.69
27.12
26.08
23.01
22.85
22.56
16.28
15.16
11.03
10.65
174.45
173.17
172.94
172.76
172.70
170.42
157.74
136.85
131.96
130.60
130.03
128.64
127.33
116.11
63.22
62.31
59.91
59.86
55.77
55.60
54.30
48.22
47.67
38.39
38.33
37.63
35.69
32.77
31.77
31.68
27.10
26.06
23.00
22.84
22.54
16.27
15.17
11.03
10.65
4.44 (1H, d, 8.0)
1.96 (1H)
2.12 (1H)
1.89 (1H)
2.12 (1H)
c-CH2
d-CH2
22.56p
48.24p
Tyr-CO
3.41 (1H, m)
3.59 (1H, m)
Tyr-Ar-C4
Phe-Ar-C1
Tyr-Ar-C2,6
Phe-Ar-C2,6
Phe-Ar-C3.5
Phe-Ar-C4
Tyr-Ar-C1
Tyr-Ar-C3,5
Pro-RCH
Pro-RCH
Ile-RCH
Pro**
CO
a-CH
b-CH2
172.99p
63.26n
32.78p
4.54 (1H, brd, 8.5)
2.16 (1H)
2.27 (1H, m)
1.67 (1H)
1.91 (1H)
3.45 (1H, m)
3.60 (1H, m)
c-CH2
d-CH2
23.01p
47.67p
Pro-RCH
Ile-RCH
Pro***
CO
a-CH
b-CH2
Phe-RCH
Tyr-RCH
Pro-RCH2
(Pro-δCH2) × 2
Phe-â-CH2
Ile-âCH2
173.23p
62.34n
31.70p
3.21 (1H, d, 8.0)
0.96 (1H, m)
1.91 (1H)
1.40 (1H, m)
1.69 (1H)
c-CH2
d-CH2
22.85p
47.67p
Tyr-âCH2
Ile-âCH2
3.34 (1H)
3.36 (1H)
Pro-âCH2
Pro-âCH2
Pro-âCH2
Ile-γCH2
Ile*
CO
a-CH
b-CH
b′-CH3
c-CH2
172.99p
59.94n
35.71n
16.28n
27.12p
3.77 (1H, d, 10.5)
1.90 (1H, m)
0.73 (3H, d, 7.0)
1.13 (1H, m)
1.36 (1H, m)
0.79 (3H, t, 7.0)
Ile-γCH2
Pro-γCH2
Pro-γCH2
Pro-γCH2
Ile-â′-CH3
Ile-â′-CH3
Ile-δCH3
d-CH3
10.64
Ile**
CO
a-CH
b-CH
b′-CH3
c-CH2
174.51p
55.80n
38.35n
15.16n
26.08p
Ile-δCH3
4.33 (1H, d, 9.5)
1.58 (1H, m)
0.89 (3H, d, 7.2)
1.18 (1H, m)
1.67 (1H)
and trifluoroacetic acid were used as received from Sigma-
Aldrich Co. The HOAt synthesis was performed by nitration
of commercially available 3-hydroxypyridine,25 methylation of
the resulting 2-nitro-3-pyridinol,26 and treatment of the result-
ing methyl ether with excess hydrazine.27 Solvent extracts of
aqueous solutions were dried over anhydrous sodium sulfate
unless noted otherwise. Thin-layer chromatography was per-
formed using silica gel GHLF Uniplates (Analtech), and the
plates were visualized by UV light (254 nm) and/or 2%
Ninhydrin-ethanol (by heating for 2-3 min). Chromato-
graphic purification of products was accomplished by flash
chromatography28 using Merck silica gel 60 (230-400 mesh).
For a summary of the instrumental methods and equipment
refer to ref 6.
d-CH3
Phe
CO
a-CH
b-CH2
11.01n
0.89 (1H, t, 7.2)
172.79p
55.63n
38.41p
4.41 (1H, dd, 12/5.0)
2.93 (1H, t, 12.5)
3.18 (1H)
Ar-C1
136.88p
130.63n
130.05n
128.68n
Ar-C2, 6
Ar-C3, 5
Ar-C4
7.22 (2H, d, 7.5)
7.32 (2H, t, 7.5)
7.29 (1H, t, 7.3)
Tyr
CO
a-CH
b-CH2
170.48p
54.34n
37.66p
Gen er a l Dep r otection Meth od .13 A solution of the N-
Fmoc tert-butyl ester (5.00 g) in dichloromethane (50 mL) was
stirred (under argon) at room temperature, and TAEA (20
equiv) was added. After addition of the yellow-green base, the
solution became yellow and cloudy. Stirring was continued for
1 h, and deprotection was observed (by TLC) to be complete.
The mixture was washed with brine (3 × 25 mL), followed by
a phosphate buffer solution (25.3 g K2HPO4/12.3 g KH2PO4 in
250 mL of water, 2 × 25 mL). The total aqueous phase was
extracted with dichloromethane (25 mL), and the combined
organic solution was dried (MgSO4), filtered, and reduced (∼20
mL) in volume. The amino acid tert-butyl ester was used
directly without further purification.
4.66 (1H, dd, 7.0/3.5)
3.08 (1H)
3.14 (1H)
Ar-C1
127.37p
131.98n
116.14n
157.77p
Ar-C2, 6
Ar-C3, 5
Ar-C4
6.87 (2H, d, 8.3)
6.68 (2H, d, 8.5)
*,**,***Denotes individual amino acid units of the same type
a
without any implied sequence information. Data resulting from
an equiweight (2.5 mg each) mixture of natural and synthetic.b The
coupling constants refer to Hz. The n and p notations correspond
to APT results in which n indicates one or three protons and p
refers to none or two protons attached.
Gen er a l P ep tid e Bon d -F or m in g Meth od . The N-Fmoc-
amino acid (1 equiv) was added to a flame-dried flask under
argon and dissolved in anhydrous dichloromethane (∼0.3 M)
containing several drops of DMF to assist dissolution (if
ceived. BOP-Cl was supplied by TCI America, and PyBroP was
supplied by Advanced ChemTech. Diethyl phosphorocyanidate
(93%), diisopropylethylamine, diethylamine, TBTU, TAEA,

412 J ournal of Natural Products, 1999, Vol. 62, No. 3
Pettit et al.
necessary). The solution was cooled (-10 °C) and stirred, and
DEPC (calculated volume of 93% pure DEPC was added to
give 1.1 equiv) was added via syringe. After being stirred for
15 min, a solution of the amino acid tert-butyl ester in
dichloromethane was added. DIEA (1.1 equiv) was added via
syringe and the solution allowed to warm to rt over the course
of 2 h. The reaction mixture was stirred at rt for 6 h,
whereupon TLC monitoring indicated the desired N-Fmoc-
peptide-OBut and a minor amount of dibenzofulvene (DBF).
The reaction mixture was washed successively with 10%
aqueous citric acid, saturated aqueous NaHCO3, water, and
brine. Drying, filtration, and solvent removal gave the crude
product.
N-F m oc-4Tyr (OBu t)-3P r o-2P h e-1P r o-OBu t (12). N-Fmoc-
Pro-Phe-Pro-OBut (11, 5.1 g, 8 mmol) was N-deprotected and
coupled (cf. the general methods) with N-Fmoc-OBut-Tyr. The
oily product was subjected to chromatographic separation
using 3:1 EtOAc-hexane to provide the tetrapeptide as a
colorless foam that precipitated from ether-hexane (5.14 g,
75%) to give a colorless solid: mp 90-92 °C; Rf 0.45 (3:1
EtOAc-hexane); [R]24 -47° (c 0.32, CHCl3); λmax (CHCl3/nm
D
(log ꢀ) 228 (4.1), 267 (4.3), 289 (3.8), 300 (3.8); IR νmax (Nujol)/
cm-1 3408, 3292, 3057, 2978, 2879, 1722, 1641, 1506, 1477,
1367, 1255, 1161, 1037, 898, 738; 1H NMR δ 1.26 (d, J ) 10.8
Hz, 2H, Pro γ-CH2), 1.27 (s, 9H, Pro C(CH3)3), 1.33 (dd, J )
10.8, 1.2 Hz, 2H, Pro γ-CH2), 1.37 (d, J ) 2.1 Hz, 1H, Pro
γ-CH2), 1.47 (s, 9H, Tyr C(CH3)3), 1.65 (m, 1H, Pro CH2), 1.80
(m, 1H, Pro CH2), 1.95 (m, 2H, Pro â-CH2), 2.14 (m, 2H, Pro
â-CH2), 2.86 (d, J ) 6.6 Hz, 2H, Tyr â-CH2), 2.97 (dd, J ) 15,
6.6 Hz, 2H, Phe â-CH2), 3.10 (m, 2H), 3.20 (dd, Pro δ-CH2),
3.31 (dd, Pro δ-CH2), 3.37 (m, 1H, Pro δ-CH2), 3.65 (m, 1H,
Pro δ-CH2), 4.11 (dd, Fmoc â-CH2), 4.25 (m, 2H, Fmoc â-CH2),
4.38 (m, 1H, Fmoc R-CH), 4.43 (m, 1H, Pro R-CH), 4.49 (m,
1H, Pro R-CH), 4.63 (m, 1H, Phe R-CH), 4.91 (dd, J ) 13.5,
6.6 Hz, 1H, Tyr R-CH), 5.69 (d, J ) 9 Hz, 1H, Phe NH), 5.75
(d, J ) 8.7 Hz, 1H, Tyr NH), 6.88 (d, J ) 8.1 Hz), 7.06 (d, J )
8.7 Hz), 7.18 (t, J ) 8.7 Hz), 7.30 (t, J ) 7.2 Hz), 7.39 (t, J )
7.2 Hz), 7.57 (d, J ) 7.2 Hz, 2H, Fmoc Ar-CH), 7.75 (d, J )
7.2 Hz, 2H, Fmoc Ar-CH); 13C NMR (75 MHz) δ 21.85 (3Pro
γ-CH2), 24.56 (1Pro γ-CH2), 27.53 (3Pro â-CH2), 27.75 (Tyr
C(CH3)3), 28.52 (Pro C(CH3)3), 28.74 (1Pro â-CH2), 38.04 (Phe
â-CH2), 38.44 (Tyr â-CH2), 46.65 (1Pro δ-CH2), 46.86 (3Pro
δ-CH2), 52.04 (Tyr R-CH), 53.81 (Phe R-CH), 59.54 (1Pro R-CH),
59.77 (3Pro-R-CH), 65.33 (Fmoc R-CH), 66.80 (Fmoc â-CH2),
77.99 (Tyr C(CH3)3), 80.96 (Pro C(CH3)3), 119.73 (Fmoc Ar-
CH), 123.95 (Tyr Ar-CH), 125.03 (Fmoc Ar-CH), 126.54 (Tyr
Ar-CH), 126.87 (Phe Ar-CH), 127.48 (Fmoc Ar-CH), 128.12
(Phe Ar-CH), 129.74 (Fmoc Ar-CH), 129.86 (Phe Ar-pCH),
131.11 (Tyr γ-Cq), 136.25 (Phe γ-Cq), 141.06 (Fmoc Ar-Cq),
143.72 (Fmoc Ar-Cq), 154.02 (Tyr Ar-Cq-OBut), 155.64 (ure-
thane CO), 169.60 (Phe CO), 170.42 (Tyr CO), 171.01 (3Pr CO),
171.13 (1Pro CO); anal. C 70.67%, H 7.28%, N 6.36%, calcd
for C51H60N4O8‚1/2H2O, C 70.73%, H 6.93%, N 6.47%.
N-F m oc-Ile-P r o-OBu t (8). Using the above general pro-
cedures, N-Fmoc-Pro-OBut (7, 10.0 g; 25.4 mmol) was N-
deprotected and coupled with N-Fmoc-Ile. The resulting crude
product was purified chromatographically (3:1 hexane-EtOAc
as eluent) to give 9.3 g (72%) of a foamy solid that crystallized
slowly from dichloromethane: mp 47-49 °C; Rf 0.24 (3:1
hexanes-EtOAc); [R]24 -37° (c 0.31, CHCl3); EIMS m/z 506
D
(15), 433 (12), 394 (5), 336 (7), 308 (35), 284 (75), 264 (13), 211
(100), 178 (100); anal. C 70.82%, H 7.42%, N 5.81%, calcd for
C
30H38N2O5, C 71.12%, H 7.56%, N 5.53%.
N-F m oc-3Ile-2Ile-1P r o-OBu t (9). As summarized above,
N-Fmoc-Ile-Pro-OBut (8, 8.00 g; 15.8 mmol) was N-deprotected
and condensed with N-Fmoc-Ile. The clear oily product was
chromatographed (2:1 hexanes-EtOAc) to give 8.65 g (88.3%)
of a colorless foam: mp 96-98 °C; Rf 0.23 (2:1 hexane-EtOAc);
[R]24 -58° (c 0.20, CHCl3); λmax (CHCl3)/nm (log ꢀ) 267 (4.35),
D
289 (3.87), 301 (3.91); IR νmax (Nujol)/cm-1 3290, 3067, 2966,
2877, 1724, 1641, 1537, 1448, 1367, 1242, 1155, 1035, 910, 758;
EIMS m/z 619 (10), 546 (5), 449 (10), 367 (2), 311 (4), 255 (14),
225 (12), 178 (100); 1H NMR δ 0.86 (t, J ) 7.5 Hz, 3H, 2Ile
3
γ-CH3), 0.90 (t, J ) 7.5 Hz, 3H, Ile γ-CH3), 1.01 (d, J ) 7.0
Hz, 6H, 2,3Ile δ-CH3), 1.12 (m, 4H, 2,3Ile γ-CH2), 1.45 (s, 9H,
C(CH3)3), 1.57-1.85 (m, 2H, 2,3Ile â-CH), 1.94 (m, 2H, Pro
γ-CH2), 2.04 (t, J ) 8.5 Hz, 1H, Pro â-CH2), 2.17 (m, 1H, Pro
â-CH2), 3.65 (dd, J ) 13, 7 Hz, 1H, Pro δ-CH2), 3.81 (dd, J )
13, 7 Hz, 1H, Pro δ-CH2), 4.06 (t, J ) 7 Hz, 1H, 2Ile R-CH),
4.22 (t, J ) 7 Hz, 1H, 3Ile R-CH), 4.38 (m, 3H, Pro R-CH, Fmoc
â-CH2), 4.63 (t, J ) 8 Hz, 1H, Ile R-CH), 5.38 (d, J ) 8.5 Hz,
1H, 2Ile-NH), 6.50 (d, J ) 9.0 Hz, 1H, 3Ile-NH), 7.31 (t, J )
7.5 Hz, 2H, Fmoc Ar-H), 7.40 (t, J ) 7.5 Hz, 2H, Fmoc Ar-H),
7.59 (t, J ) 7.5 Hz, 2H, Fmoc Ar-H), 7.76 (t, J ) 7.5 Hz, 2H,
Fmoc Ar-H); 13C NMR (100 MHz) δ 10.82 (2Ile δ-CH3), 11.18
(3Ile δ-CH3), 15.05 (2Ile γ-CH3), 15.33 (3Ile γ-CH3), 24.28 (Pro
γ-CH2), 24.60 (2Ile γ-CH2), 24.65 (3Ile γ-CH2), 27.74 (Pro
C(CH3)3), 28.96 (Pro â-CH2), 37.19 (2Ile â-CH), 37.65 (3Ile
â-CH), 46.97 (Fmoc R-CH), 47.25 (Pro δ-CH2), 54.74 (2Ile
R-CH), 59.38 (3Ile R-CH), 59.54 (Pro R-CH), 66.80 (Fmoc
-CH2), 81.00 (Pro C(CH3)3), 119.68, 124.93, 124.99, 126.83,
127.40 (Fmoc Ar-CH), 141.02 (Fmoc Ar-Cq), 143.67 (Fmoc Ar-
Cq), 156.02 (urethane CO), 170.23 (2Ile CO), 170.88 (3Ile CO),
171.33 (Pro CO); anal. C 69.78%, H 8.16%, N 6.72%, calcd for
N -F m o c -7I le -6I le -5P r o -4T y r (O B u t )-3P r o -2P h e -1P r o -
OBu t (13). N-Fmoc-Tyr(OBut)-Pro-Phe-Pro-OBut (12, 2.50 g,
2.92 mmol) was N-deprotected (see above). Simultaneously,
N-Fmoc-Ile-Ile-Pro-OBut (9, 1.81 g; 2.92 mmol) was treated
with TFA (10 mL) for 1 h, and the solvents were removed
(azeotropically) under vacuum. Peptide bond formation led to
a faint yellow oil that was purified by chromatography in
EtOAc to give the product as a foam that crystallized from
EtOAc-hexane (2.50 g, 71%) as a colorless powder: mp 123-
125 °C; Rf 0.24 (EtOAc); [R]24 -80.9° (c 0.22, CHCl3); λmax
D
(CHCl3)/nm (log ꢀ) 267 (4.29), 289 (3.66), 301 (3.62); IR νmax
(KBr)/cm-1 3414, 3306, 2972, 2933, 2877, 1728, 1631, 1508,
1448, 1365, 1236, 1159, 1033, 898, 742; 1H NMR δ 0.81 (t, J )
6
6
7.0 Hz, 3H, Ile δ-CH3), 0.85 (d, J ) 5.5 Hz, 3H, Ile γ-CH3),
7
C
36H49N3O6, C 69.76%, H 7.97%, N 6.78%.
0.85 (t, J ) 6.0 Hz, 3H, Ile δ-CH3), 0.95 (d, J ) 6.0 Hz, 3H,
N-F m oc-P h e-P r o-OBu t (10). For preparation of this dipep-
7Ile δ-CH3), 1.14-1.46 (2m, 4H, 7,6Ile γ-CH2), 1.25 (s, 9H, Tyr
C(CH3)3), 1.38 (s, 9H, Pro C(CH3)3), 1.59, 1.87 (2m, 2H, 7,6Ile
â-CH), 1.87-1.95 (m, 3H, 5,3,1Pro â-CH2), 1.93-2.17 (m, 6H,
5,3,1Pro γ-CH2), 2.06-2.17 (m, 3H, 5,3,1Pro â-CH2), 2.83 (dd, J
) 13.0, 6.0 Hz, 2H, Tyr â-CH2), 2.96 (dd, J ) 14.0, 6.6 Hz, 2H,
tide N-Fmoc-Pro-OBut (7, 12.0 g; 30.5 mmol) was N-depro-
tected and combined with N-Fmoc-Phe. The pale yellow oily
product was purified by chromatography in 5:2 hexane-EtOAc
to afford a solid that crystallized from EtOAc-hexane to give
colorless prisms (13.1 g; 80%): mp 145.1-145.4 °C; Rf 0.48
1
Phe â-CH2), 3.15 (d, J ) 6.0 Hz, 2H, Pro δ-CH2), 3.22 (d, J )
(2:1 hexanes-EtOAc); [R]25 -78° (c 0.54, CHCl3); EIMS m/z
6.0 Hz, 2H, Pro δ-CH2), 3.42-3.55 (m, 1H, Pro δ-CH2), 3.70,
3.90 (2m, 1H, 5Pro δ-CH2), 4.11 (t, J ) 7.0 Hz, 1H, 7,6Ile R-CH),
4.20 (m, 2H, Fmoc â-CH2), 4.26 (t, J ) 8.0 Hz, 1H, 6Ile R-CH),
3
5
D
540 (20), 467 (10), 393 (20), 342 (15), 227 (10), 178 (100); anal.
C 73.6%, H 6.89%, N 5.09%, calcd for C33H46N2O5, C 73.3%, H
6.71%, N 5.18%.
3
4.35 (m, 1H, Fmoc R-CH), 4.37 (m, 1H, Pro R-CH), 4.48 (bd,
N-F m oc-3P r o-2P h e-1P r o-OBu t (11). The preceding N-
Fmoc-Phe-Pro-OBut (10, 10.0 g; 18.5 mmol) was N-deprotected
and coupled with N-Fmoc-Pro. The resulting clear oil was
separated chromatographically employing 3:1 EtOAc-hexane
as eluent to give a solid that crystallized from ether-hexane
(9.44 g, 80%) as a colorless solid: mp 110-111 °C; Rf 0.27 (3:1
EtOAc-hexane); [R]25D -86° (c 1.0, CHCl3); EIMS m/z 637 (1),
564 (1), 467 (1), 415 (4), 346 (6), 303 (20), 247 (50), 178 (100);
anal. C 71.66%, H 7.02%, N 6.49%, calcd for C38H43N3O6, C
71.6%, H 6.8%, N 6.58%.
J ) 8.0 Hz, 2H, 5,1Pro R-CH), 4.76 (t, J ) 8.0 Hz, 1H, Phe
R-CH), 4.98 (dd, J ) 14.0, 6.0 Hz, 1H, Tyr R-CH), 5.53 (d, J )
9.0 Hz, 1H, Phe NH), 6.80 (d, J ) 8.0 Hz, 1H, Tyr NH), 6.85
(d, J ) 8.5, 1H, 7Ile NH), 7.07 (d, J ) 8.0 Hz, 1H, 6Ile NH),
7.17 (t, J ) 8.0 Hz, 2H, Fmoc Ar-H), 7.21-7.40 (m, 9H, Phe
Ar-H and Tyr Ar-H), 7.60 (t, J ) 6.0 Hz, 2H, Fmoc Ar-H), 7.72
(t, J ) 5.5 Hz, 2H, Fmoc Ar-H), 8.21 (bd, J ) 6.0 Hz, 2H, Fmoc
Ar-H); 13C NMR δ 10.92 (Ile δ-CH3), 11.36 (Ile δ-CH3), 15.07
(Ile γ-CH3), 15.91 (Ile γ-CH3), 24.50 (Pro δ-CH2), 24.53 (Pro
δ-CH2), 24.59 (Pro δ-CH2), 24.65 (Ile CH2), 24.77 (Ile CH2),

Antineoplastic Agents
J ournal of Natural Products, 1999, Vol. 62, No. 3 413
27.69 (Pro â-CH2), 27.84 (Tyr C(CH3)3), 28.66 (Pro C(CH3)3),
28.91 (Pro â-CH2), 30.54 (Pro â-CH2), 37.89 (Ile â-CH), 38.37
(Ile â-CH), 39.00 (Tyr/Phe â-CH2), 46.72 (Pro γ-CH2), 47.08
(Fmoc R-CH), 47.23 (Pro γ-CH2), 47.86 (Pro γ-CH2), 52.18 (Tyr
R-CH), 52.34 (Ile R-CH), 54.43 (Phe R-CH), 54.70 (Ile R-CH),
58.80 (Pro R-CH), 59.24 (Pro R-CH), 59.69 (Pro R-CH), 65.09
(Fmoc â-CH2), 77.96 (Tyr C(CH3)3), 80.71 (Pro C(CH3)3), 119.75
(d, J ) 6.0 Hz, Fmoc Ar-CH), 124.09 (Tyr Ar-CH), 125.10 (d,
J ) 12.0 Hz, Fmoc Ar-CH), 126.39 (Phe Ar-CH), 126.89 (Phe
Ar-CH), 127.46 (Fmoc Ar-CH), 128.13 (Tyr Ar-CH), 129.72
(Fmoc Ar-CH), 130.04 (Phe Ar-pCH), 131.26 (Tyr Ar-γCq),
137.00 (Phe γ-Cq), 141.16 (Fmoc Ar-Cq), 143.85 (d, J ) 26.0
Hz, Fmoc Ar-Cq), 153.88 (Tyr Ar-Cq-OBut), 156.19 (urethane
CO), 169.47 (Phe CO), 169.94 (Tyr CO), 170.39 (Ile CO), 170.92
(Ile CO), 171.09 (Pro CO), 171.35 (Pro CO), 171.75 (Pro CO);
EIMS m/z calcd 1180.5, found 1180.7; anal. C 68.88%, H 7.56%,
N 8.40%, calcd for C68H89N7O11, C 69.19%, H 7.60%, N 8.306%.
P h a k ellista tin 2 (1). Meth od A. TBTU. N-Fmoc-Ile-Ile-
Pro-Tyr(OBut)-Pro-Phe-Pro-OBut (13, 0.30 g, 0.254 mmol) was
N-deprotected. After removal of solvent, TFA (10 mL) was
added to the crude Ile-Ile-Pro-Tyr(OBut)-Pro-Phe-Pro-OBut at
room temperature, and the mixture was stirred for 2.5 h.
Excess TFA was removed in vacuo, followed by addition and
evaporation of two 5-mL portions of toluene and dichlo-
romethane. To the TFA salt in dichloromethane (170 mL) was
added TBTU (0.42 g; 1.7 mmol; 5 equiv) in acetonitrile (5 mL
at 0 °C under argon). DIEA (1.7 mL, 1% v/v) was added slowly
(∼15 min) at room temperature with stirring. Dilution of the
heptapeptide corresponded to 1.5 mM. The reaction mixture
was stirred for 2 h at ice-bath temperature and at room
temperature for 14 days. After removal of solvent in vacuo, a
solution of the crude product in CH2Cl2 (100 mL) was washed
with 10% aqueous citric acid (3 × 10 mL), saturated aqueous
NaHCO3 (3 × 10 mL), and water (10 mL). The combined
aqueous phase was extracted with EtOAc (15 mL). The organic
solvents were combined, dried, and filtered, and the solvent
was removed in vacuo to yield a brown oil (0.59 g). Chromato-
graphic separation with a 25-mm × 20-cm (silica depth)
column, elution with hexane-EtOAc-MeOH (4:2:1) under
positive pressure, and collection of 5-mL fractions gave phakel-
listatin 2 (1, 117 mg, 56%) as a colorless solid (72% overall).
Comparison of the natural and synthetic specimens of
and washed with 1 N hydrochloric acid (5 × 20 mL), saturated
aqueous sodium bicarbonate (2 × 25 mL), and brine (25 mL).
The synthetic phakellistatin 2 was isolated (see method A) as
a colorless solid (34.0 mg, 51% yield) that was identical (refer
to method A) to the natural specimen (1).
Meth od D. TBTU/HOAt.30 The linear heptapeptide (13,
300 mg; 0.254 mmol) was N-deprotected as described above.
After removal of solvent, TFA (10 mL) was added to the crude
residue at room temperature, and the mixture was stirred for
2.5 h. Excess TFA was removed in vacuo, followed by evapora-
tion with two 5-mL portions of toluene and dichloromethane.
To the TFA salt in dichloromethane (170 mL) was added TBTU
(246 mg; 0.762 mmol; 3 equiv) in acetonitrile (5 mL at 0 °C
under argon) followed by HOAt (0.108 g, 0.762 mmol, 3 equiv).
DIEA (1.7 mL, 1% v/v) was added slowly (∼15 min) at room
temperature with stirring. Dilution of the heptapeptide cor-
responded to 1.5 mM. The reaction mixture was stirred for 2
h at ice-bath temperature and at room temperature for 14
days. After removal of solvent in vacuo, a solution of the crude
product in dichloromethane (100 mL) was washed with 10%
aqueous citric acid (3 × 10 mL), saturated aqueous NaHCO3
(3 × 10 mL), and water (10 mL). The combined aqueous phase
was extracted with EtOAc (15 mL). The organic solvents were
combined, dried, and filtered, and the solvent was removed in
vacuo to yield a brown oil (0.78 g). Chromatographic separation
and elution with hexane-EtOAc-MeOH (4:2:1) under positive
pressure gave phakellistatin 2 (1, 136 mg, 65%) as a colorless
solid identical (see method A) with the natural specimen.
Com p a r ison of Na tu r a l a n d Syn th etic P h a k ellista tin
2 (1). Specimens of both the natural and synthetic phakel-
listatin 2 were repurified using reversed phase HPLC on a
C8 column in methanol-acetonitrile-water (50:50:55) at λ 235
nm. Both separately and in a mixture, the natural and
synthetic phakellistatin 2 exhibited the same retention time.
When the synthetic specimen was repurified by HPLC to
remove two trace impurities, a mixture of the natural and
synthetic phakellistatin 2 again showed only one peak in the
HPLC recording. Direct comparison of the pure natural and
synthetic phakellistatin 2 specimens employing high-field (500
MHz) NMR techniques showed the specimens to be identical;
a comparison made by combination of 2.5-mg specimens of
each in the same NMR sample tube again showed no struc-
tural differences, and the result has been recorded in Table 1.
1
phakellistatin 2 (1) by mixture mp, TLC, [R]D, IR, and H and
13C NMR (400 MHz, CD3OD) gave identical results.
Meth od B. BOP -Cl. The preceding Fmoc and tert-butyl
ester deprotection reactions (method A) were repeated using
heptapeptide 13 (77 mg; 0.065 mmol). Dichloromethane (400
mL) and BOP-Cl (0.37 g; 1.43 mmol; 22 equiv) were added
under argon to a dry flask (ice bath). The solution was
maintained at ice-bath temperature, and the TFA salt and
DIEA (1.82 mL; 10.4 mmol; 160 equiv) in dichloromethane (35
mL) were added slowly (over 1 h) via cannula to the dropping
funnel. After addition, the solution was stirred at room
temperature under argon for 7 days. The solvent was removed
(in vacuo), and the residue was dissolved in EtOAc (50 mL).
The EtOAc solution was washed with water (3 × 10 mL), and
the aqueous phase was extracted with EtOAc (3 × 5 mL). The
combined EtOAc extract was washed with saturated aqueous
sodium bicarbonate (3 × 5 mL) and brine (10 mL) and dried.
Filtration and solvent removal afforded a clear oil (0.17 g). The
product was isolated by chromatography (4:2:1 hexane-
EtOAc-MeOH) as described in method A to afford the cyclic
heptapeptide as a colorless amorphous solid (25.4 mg, 50%),
identical (cf. method A) with authentic natural phakellistatin
2.
Ack n ow led gm en t. For the very necessary financial as-
sistance we thank with appreciation the following: Outstand-
ing Investigator Grant CA44344-05-09 awarded by the Divi-
sion of Cancer Treatment, National Cancer Institute, DHHS;
the Arizona Disease Control Research Commission; the Fannie
E. Rippel Foundation; the Robert B. Dalton Endowment Fund;
Eleanor W. Libby; Diane Cummings Halle, the Nathan Cum-
mings Foundation, Inc.; Gary L. and Diane Tooker; Dr. J ohn
C. Budzinski; Polly J . Trautman; J ohn and Edith Reyno; Lottie
Fugal; the Eagles Art Ehrmann Cancer Fund; and the Ladies
Auxiliary to the Veterans of Foreign Wars. For other helpful
assistance we thank Dr. Fiona Hogan.
Refer en ces a n d Notes
(1) (a) For part 399 see: Kalemkerian, G. P.; Ou, X.; Adil, M. R.; Rosati,
R.; Khoulani, M. M.; Madan, S. K.; Pettit, G. R. J . Natl. Cancer Inst.,
submitted. (b) Abbreviations for amino acids and peptides used in
this paper are those recommended by the IUPAC-IUPAB Commis-
sion of Biochemical Nomenclature (Eur. J . Biochem. 1984, 138, 9-37)
and: Pettit, G. R. Synthetic Peptides; Elsevier Scientific Pub. Co.:
Amsterdam, The Netherlands, 1982; Vol. 6 and preceding volumes.
Additional abbreviations are as follows: Fmoc, fluoren-9-ylmethoxy-
carbonyl; DMF, N,N-dimethylformamide; DEA, N,N-diethylamine;
DIEA, N,N-diisopropylethylamine; TAEA, tris(2-aminoethyl)amine;
TFA, trifluoracetic acid; HOBt, 1-hydroxybenzotriazole; DEPC, di-
ethyl phosphorocyanidate; BOP-Cl, N,N-bis(2-oxo-3-oxazolidinyl)-
phosphinine chloride; HOAt, 1-hydroxy-7-azabenzotriazole; TBTU,
O-(1H-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluo-
roborate; PyBroP, bromotrispyrrolidinophosphonium hexafluorophos-
phate; NMP, N-methyl 2-pyrrolidinone.
Meth od C. P yBr oP .29 The Fmoc and tert-butyl ester
deprotection reactions (method A) were repeated using the
heptapeptide (13, 0.10 g; 0.085 mmol). The TFA salt and
PyBroP (0.20 g; 0.43 mmol; 5 equiv) were dissolved in dry
dichloromethane (100 mL), and the solution was cooled (ice
bath) and stirred. DIEA (0.059 mL, 0.34 mmol; 4 equiv) was
added (syringe) slowly, and the mixture was allowed to warm
to room temperature and then stirred under argon at room
temperature for 4 days. The solvent was removed under
reduced pressure. The residue was dissolved in EtOAc (50 mL)
(2) (a) Yeung, B. K. S.; Nakao, Y.; Kinnel, R. B.; Carney, J . R.; Yoshida,
W. Y.; Scheuer, P. J .; Kelly-Borges, M. J . Org. Chem. 1996, 61, 7168-
7173. (b) Pettit, G. R.; Tan, R.; Herald, D. L.; Cerny, R. L.; Williams,
M. D. J . Org. Chem. 1994, 59, 1593-1595. (c) Bewley, C. A.; He, H.;

414 J ournal of Natural Products, 1999, Vol. 62, No. 3
Pettit et al.
Williams, D. H.; Faulkner, D. J . J . Am. Chem. Soc. 1996, 118, 4314-
4321. (d) Pettit, G. R.; Tan, R.; Ichihara, Y.; Williams, M. D.; Doubek,
D. L.; Tackett, L. P.; Schmidt, J . M. J . Nat. Prod. 1995, 58, 961-
965. (e) Mau, C. M. S.; Nakao, Y.; Yoshida, W. Y.; Scheuer, P. J .;
Kelly-Borges, M. J . Org. Chem. 1996, 61, 6302-6304.
(13) Carpino, L. A.; Sadat-Aalaee, D.; Beyermann, M. J . Org. Chem. 1990,
55, 1673-1675.
(14) Benoiton, N. L. Int. J . Pept. Protein Res. 1994, 44, 399-400.
(15) Haver, A. C.; Smith, D. D. Tetrahedron Lett. 1993, 34, 2239-2242.
Benoiton, N. L.; Kuroda, K. Int. J . Pept. Protein Res. 1981, 17, 197-
204.
(16) (a) Bodanszky, M. Principles of Peptide Synthesis, 2nd ed; Springer-
Verlag: New York, 1993; p 232. (b) J ones, J . The Chemical Synthesis
of Peptides; Clarendon Press: Oxford, 1991; p 93. (c) Goodman, M.;
Steuben, K. C. J . Org. Chem. 1962, 27, 3409-3416.
(17) (a) Zimmer, S.; Hoffmann, E.; J ung, G.; Kessler, H. Leibigs Ann.
Chem. 1993, 497-501. (b) Ehrlich, A.; Rothemund, S.; Brudel, M.;
Beyermann, M.; Carpino, L. A.; Bienert, M. Tetrahedron Lett. 1993,
34, 4781-4784. (c) Trzeciak, A.; Bannwarth, W. Tetrahedron Lett.
1992, 33, 4557-4560. (d) Knorr, R.; Trzeciak, A.; Bannwarth, W.;
Gillessen, G. Tetrahedron Lett. 1989, 30, 1927-1930.
(18) Kim, M. H., Patel, D. V. Tetrahedron Lett. 1994, 35, 5603-5606.
(19) Castro, B.; Dormoy, J . R. Tetrahedron Lett. 1973, 3243-3246. Coste,
J .; Frerot, E.; J ouin, P. Tetrahedron Lett. 1991, 32, 1967-1970.
(20) Carpino, L. A.; Elfaham, A.; Albericio, F. J . Org. Chem. 1995, 60,
3561-3564. Carpino, L. A. J . Am. Chem. Soc. 1993, 115, 4397-4398.
(21) Konig, W.; Geiger, R. Chem. Ber. 1970, 103, 788-798. Pettit, G. R.
Synthetic Peptides; Elsevier Publishing Co.: Amsterdam, The Neth-
erlands, 1976; Vol. 4.
(22) Monks, A.; Scudiero, P.; Skehan, P.; Shoemaker, R.; Paull, K.; Vistica,
D.; Hose, C.; Langley, J .; Cronise, P.; Vaigro-Wolff, A.; Gray-Goodrich,
M.; Campbell, H.; Boyd, M. R. J . Natl. Cancer Inst. 1991, 83, 757-
766.
(23) Pettit, G. R.; Srirangam, J . K.; Herald, D. L.; Xu, J .-P.; Boyd, M. R.;
Cichacz, Z.; Kamano, Y.; Schmidt, J . M.; Erickson, K. L. J . Org. Chem.
1995, 60, 8257-8261.
(24) Mechnich, O.; Kessler, H. Lett. Peptide Sci. 1997, 4, 21-28.
(25) De Selms, R. C. J . Org. Chem. 1968, 33, 478-480.
(3) (a) Pettit, G. R.; Cichacz, Z.; Barkoczy, J .; Dorsaz, A.-C.; Herald, D.
L.; Williams, M. D.; Doubek, D. L.; Schmidt, J . M.; Tackett, L. P.;
Brune, D. C. J . Nat. Prod. 1993, 56, 260-267. (b) Pettit, G. R.; Herald,
C. L.; Boyd, M. R.; Leet, J . E.; Dufresne, C.; Doubek, D. L.; Schmidt,
J . M.; Cerny, R. L.; Hooper, J . N. A.; Ru¨tzler, K. C. J . Med. Chem.
1991, 34, 3339-3340.
(4) (a) Pettit, G. R.; Gao, F.; Cerny, R. L.; Doubek, D. L.; Tackett, L. P.;
Schmidt, J . M.; Chapuis, J .-C. J . Med. Chem. 1994, 37, 1165-1168.
(b) Pettit, G. R.; Tan, R.; Williams, M. D.; Tackett, L.; Schmidt, J . M.
Bioorg. Med. Chem. Lett. 1993, 3(12), 2869-2874.
(5) (a) Pettit, G. R.; Tan, R.; Gao, F.; Williams, M. D.; Doubek, D. L.;
Boyd, M. R.; Schmidt, J . M.; Chapuis, J .-C.; Hamel, E.; Bai, R.;
Hooper, J . N. A.; Tackett, L. P. J . Org. Chem. 1993, 58, 2538-2543.
(b) Pettit, G. R.; Gao, F.; Doubek, D. L.; Boyd, M. R.; Hamel, E.; Bai,
R.; Schmidt, J . M.; Tacketet, L. P.; Ru¨tzler, K. Gazz. Chim. Ital. 1993,
123, 371-377.
(6) (a) Pettit, G. R.; Holman, J . W.; Boland, G. M. J . Chem. Soc., Perkin
Trans. 1 1996, 2411-2416. (b) Pettit, G. R.; Taylor, S. R. J . Org.
Chem. 1996, 61, 2322-2325.
(7) (a) J iang, J .; Li, W.-R.; J oullie, M. Synth. Commun. 1994, 24, 187-
195. (b) Trzeciak, A.; Bannwarth, W. Tetrahedron Lett. 1992, 33,
4557-4560.
(8) For a review, see: Fields, G. B.; Noble, R. I. Int. J . Pept. Protein Res.
1990, 35, 161-214.
(9) (a) Tung, R. D.; Dhaon, M. K.; Rich, D. H. J . Org. Chem. 1986, 51,
3350-3354. (b) For an exception, see: Fischer, P. M.; Solbakken, M.;
Undheim, K. Tetrahedron 1994, 50, 2277-2288.
(10) (a) Bodanszky, M. Principles of Peptide Synthesis, 2nd ed.; Springer-
Verlag: New York, 1993; pp 186, 106-107. (b) Hoeg-J ensen, T.;
J akobsen, M. H.; Holm, A. Tetrahedron Lett. 1991, 32, 6387-6390.
(c) Atherton, E.; Sheppard, R. C. In The Peptides: Analysis, Synthesis,
Biology; Udenfriend, S., Meienhofer, J ., Eds.; Academic Press: San
Diego, 1987; Part C, Vol. 9, pp 1-38.
(26) Williamson, D. A.; Bowler, B. E. Tetrahedron 1996, 52, 12357-12372.
(27) Azev, Y.; Mokrushina, G. A.; Postovskii, I. Ya.; Sheinker, Yu. N.;
Anisimova, O. S. Chem. Heterocycl. Compds. 1976, 1172-1176.
(28) Still, W. C.; Kahn, M.; Mitra, A. J . Org. Chem. 1978, 43, 2923-2925.
(29) (a) Sefler, A.; Kozlowski, M. C.; Guo, T.; Bartleett, P. A. J . Org. Chem.
1997, 62, 93-102. (b) Kurome, T.; Inami, K.; Inoue, T.; Ikai, K.;
Takesako, K.; Kato, I.; Shiba, T. Tetrahedron 1996, 52, 4327-4346.
(30) Ehrlich, A.; Heyne, H.-U.; Winter, R.; Beyermann, M.; Haber, H.;
Carpino, L. A.; Bienert, M. J . Org. Chem. 1996, 61, 8831-8838.
(11) (a) Pettit, G. R.; Srirangam, J . K.; Singh, S. B.; Williams, M. D.;
Herald, D. L.; Barkoczy, J .; Kantoci, D.; Hogan, F. J . Chem. Soc.,
Perkin Trans. 1 1996, 853-858. (b) Hamada, Y.; Rishi, S.; Shioiri,
T.; Yamada, S.-I. Chem. Pharm. Bull. 1977, 25, 224-230.
(12) Valerio, R. M.; Alewood, P. F.; J ohns, R. B. Synthesis 1988, 10, 786-
789.
NP980168M