Antineoplastic agents. 592. Highly effective cancer cell growth inhibitory structural modifications of dolastatin 10

Article abstract of DOI:10.1021/np1007334

The dolastatin series of unique peptides, origi-nally discovered as constituents of the sea hare Dolabella auricularia, is of increasing importance in providing biological leads, especially to new and useful anticancer drugs. Dolastatin 10 and three analo

Full text of DOI:10.1021/np1007334

ARTICLE
pubs.acs.org/jnp
Antineoplastic Agents. 592. Highly Effective Cancer Cell Growth
Inhibitory Structural Modifications of Dolastatin 10
George R. Pettit,* Fiona Hogan, and Steven Toms
Cancer Research Institute and Department of Chemistry and Biochemistry, Arizona State University, P.O. Box 871604, Tempe,
Arizona 85287-1604, United States
S Supporting Information
b
ABSTRACT: The dolastatin series of unique peptides, origi-
nally discovered as constituents of the sea hare Dolabella auricularia,
is of increasing importance in providing biological leads, especially
to new and useful anticancer drugs. Dolastatin 10 and three ana-
logues, minor structural modifications designated auristatins, are
currently in human cancer clinical trials. The present study was un-
dertaken to explore delivery to the cancer sites by way of phosphate
or quinoline modifications. The initial objectives, auristatin TP as
sodium phosphate 3b (GI50 10-2-10-4 μg/mL), auristatin 2-AQ
(4, GI50 10-2-10-3 μg/mL), and auristatin 6-AQ (5, GI50 10-4 μg/mL), exhibited superior cancer cell growth inhibitory properties.
he remarkable anticancer properties of dolastatin 10 (1), a
unique pentapeptide that we isolated from the sea hare
soluble salts. The synthesis of auristatins suitable for formulation
of such salts is of considerable interest because the use of water-
soluble phosphate derivatives has increased the bioavailability of
a number of anticancer drugs, including combretastatins A-18
and A-4,8b,9 pancratistatin,10 taxol,11 and etoposide.12 The salts
are dephosphorylated by serum phosphatases to yield the active
drug, which is then transported intracellularly. We also report the
syntheses of aminoquinoline (AQ) auristatin modifications 4 and
5. A number of 4- and 8-AQ derivatives have been used his-
torically as antimalarial agents,13 and various biological activities
have been reported for 2-AQ itself14a and for derivatives of the 3,
4-, 5-, 6-, and 8-AQ isomers.14b-f Use of the readily available
isomers 2- and 6-AQ led to new auristatins 4 and 5, respectively.
Each of the new auristatins displayed very strong cancer cell
growth inhibition against a panel of murine and human cancer
cell lines.
T
Dolabella auricularia,2a,b has led to intense interest in closely
related derivatives (auristatins) that are suitable for clinical trial.
Such structural modifications have provided a number of potential
clinical candidates with enhanced efficacy and pharmacological
characteristics.2c,d Replacement of the dolaphenine (Doe) unit with
phenethylamides, to give auristatins PE2c,3,4 (2a), PHE2d,3,5 (2b), and
E,3,6 and with pyridylethylamide (auristatin PYE, 2c)2d,3,7 has led to
active analogues that are undergoing preclinical7 and clinical
development.3 Dolastatin 10 and three of the auristatins are in human
cancer clinical trials, ranging from phase I to phase III.
’ RESULTS AND DISCUSSION
The synthesis of 3 was carried out as shown in Scheme 1.
Reaction of the γ-amino acid Boc-Dap (6)15 with tyramine in the
presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydro-
chloride (EDCl) and 1-hydroxybenzotriazole (HOBT) gave the
protected amide 7a. Removal of the Boc group with bromotri-
methylsilane (TMSBr) yielded the hydrobromide salt (7b), which
was coupled with Dov-Val-Dil.TFA (8)16 in the presence of EDCI
and HOBT to give the parent auristatin tyramide (9). The doubling
of signals in the 1Hand13C NMR spectra of 9indicated the presence
of two isomers, a pattern similar to that of dolastatin 10 and due to
conformational isomers arising from cis-trans isomerism at the
Dil-Dap bond.16
We now report the synthesis of auristatin TP (3), a tyramide
phosphate modification of dolastatin 10 in the form of water-
Received: October 13, 2010
Published: May 02, 2011
r
Copyright
2011 American Chemical Society and
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American Society of Pharmacognosy
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Scheme 1
Formation of phosphate diester 10a was achieved via in situ
generation of dibenzyl chlorophosphate, from reaction of dibenzyl
phosphite and carbon tetrachloride, and was followed by
removal of the benzyl ester groups by hydrogenolysis to provide
the free phosphoric acid 10b. The pure 10b was quite unstable
but could be stored for short periods as a methanolic solution
(<0.01 M at 4 °C) and was generally used immediately as follows
to provide compounds 3a-h. The passage of acid 10b through a
Dowex cation exchange resin (Naþ form) provided the sodium
salt (3b), and compounds 3a,c,d were similarly produced by ion
exchange of the free acid or of either the sodium or potassium
salts in the appropriate Dowex resin. The remaining salts (3e-h)
were prepared directly from the free acid 10b by treatment with
the appropriate base or amino acid. The solubilities of each salt
and of precursor 9 were measured in distilled water at room
temperature. The most soluble were the sodium (3b) and
potassium (3c) salts (Table 1).
A similar convergent synthesis was planned for the preparation
of the auristatin aminoquinoline modifications (4, 5), that is,
formation of the Dap-aminoquinoline unit, followed by condensa-
tion with tripeptide 8. As shown in Scheme 2, Boc-Dap (6) and
2-aminoquinoline (2-AQ) were condensed to give Boc-Dap-2-AQ
(11), diethylcyanophosphonate (DEPC) being used as coupling
reagent, followed by deprotection to give the amine TFA salt (12).
Coupling of 12 and 8 with use of DEPC gave the desired auristatin
2-AQ (4). The doubling of signals in the 1H NMR spectrum of 4
because of conformational changes was also noted.
5-AQ) was detected after 100 h. The activity of the aminoqui-
nolines varies with the position of the amino group,13a,17 and
they are in general poor nucleophiles. Therefore, we considered a
route involving an activated intermediate preformed from the
amino acid. Pozdnev18a used di-tert-butyl dicarbonate (Boc
anhydride, Boc2O), in the presence of pyridine, to form activated
esters of a number of protected amino acid derivatives, which
were then condensed successfully with 6-aminoquinoline (6-
AQ). Use of this method to couple 5-AQ and compound 6 failed
and was not further pursued, and the condensation of 6-amino-
quinoline (6-AQ) with 6, via mixed anhydride 13 (Scheme 3),
was next attempted.
A mixture of Boc2O and 6 in pyridine and dimethylformamide
(DMF) was allowed to stir for 10 min, and 6-AQ was then
added.18a After isolation of products, the reaction was found to
have given the desired Boc-Dap-6-AQ (14), along with Boc-6-
AQ (at least half of the 6-AQ was used in formation of this
product). When 6 and Boc2O were allowed to stir in base for an
hour so that formation of ester 13 could go to completion (with
evolution of CO2) before addition of 6-AQ ,18b formation of
Boc-6-AQ was avoided, and in isolation of the desired product a
Preparation of auristatins from other aminoquinoline isomers
proved more difficult. First, the coupling of 5-aminoquinoline
(5-AQ) with compound 6 was attempted, but use of DEPC failed
to give the desired product. The coupling agent PyBroP was next
used under standard conditions, but only starting material (6 and
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Table 1. Solubility of Compounds 3a-h and 10a
attempted (Scheme 3). Reaction of cyanuric fluoride (15) with
Boc-Dap (6) to give Boc-Dap-C(O)F (16) was carried out under
mild conditions, and the crude product was used immediately in a
condensation reaction with 6-AQ, in the presence of pyridine.
The reaction did not go to completion (there was no detectable
reduction in the amounts of unreacted compounds from 6 to 20 h
later), and the desired Boc-Dap-6-AQ (14) was isolated in 26%
yield. Compound 14 was then treated with TFA to give the
compound no.
mg/mL
compound no.
mg/mL
3a
3b
3c
3d
3e
>65
>236
>120
>72
3f
3g
3h
9
>51
7
7
<1
<5
Dap-6-AQ TFA salt (17), which was condensed with Dov-
a In distilled water at 23 °C.
3
Val-Dil TFA (8) to give auristatin-6-AQ (5).
3
In a repeat of the synthesis of acid fluoride 16, diisopropy-
lethylamine (DIEA) was used as base, and purification of 16 was
carried out on silica gel before condensation with 6-AQ, in the
presence of DIEA, to give Boc-Dap-6-AQ (14). Reaction was
slow, and at 44 h no change was apparent compared to the
mixture at 32 h. The colorless oil that was isolated contained both
product 14 and unreacted 16 (by TLC). According to the
literature, the reaction of Fmoc amino acid fluorides with amines
is often very slow and is not dependent on base20b,c (the presence
of base can increase the reaction rate, but lack of it can result in a
cleaner reaction). Of the two methods to synthesize Boc-Dap-6-
AQ (5), use of Boc2O to form active intermediate 13 led
consistently to a yield of about 24%, whereas the yield from
the Boc-Dap-C(O)F (16) method varied from 26% (using
pyridine) to 6.6% (using DIEA and purifying the intermediate).
Compounds 3b, 3c, 4, 5, and 9 were evaluated against the
murine P388 lymphocytic leukemia cell line and showed excep-
tional activity; auristatins 3b, 4, and 5 were also tested against a
minipanel of human cancer cell lines in our laboratories, with
similarly strong activity evident (Table 2), especially from
compounds 3b and 5. These in vitro data are quite comparable
to those of dolastatin 10 (1) and auristatin PE (2a), each of which
had GI50 values of 10-5-10-6 μg/mL (10-2-10-3 nM) against
a similar minipanel of human cell lines.2b,c,3 Further biological
testing of the new auristatins is under way.
Scheme 2
Scheme 3
’ EXPERIMENTAL SECTION
General Experimental Procedures. N-Boc-Dolaproine and
Dov-Val-Dil TFA were synthesized as described earlier.15,16 Reagents
3
and anhydrous solvents were purchased from Acros Organics (Fisher
Scientific), Sigma-Aldrich Chemical Co., and Lancaster Synthesis and were
used as received. Diisopropylethylamine (DIEA) was redistilled over
potassium hydroxide. Dibenzylphosphite was redistilled before use (bp
160 °C at 0.1 mmHg). For thin-layer chromatography, Analtech silica gel
GHLF Uniplates were used and visualized with short-wave UV irradiation
and use of a permanganate dip followed by heating. Solvent extracts of
aqueous solutions were dried over magnesium sulfate. For column chro-
matography, silica gel (230-400 mesh ASTM) from E. Merck (Darmstadt,
Germany) was used. For ion-exchange chromatography, Dowex 50Wꢀ8-
400 hydrogen form resin (Sigma-Aldrich) was washed with MeOH,
hydrochloric acid (1 M), and deionized H2O before use. The cation forms
of the resin were prepared by elution of an aqueous solution (1 M) of the
corresponding base followed by deionized H2O.
Melting points are uncorrected and were determined with a Fischer-
Johns melting point apparatus. Optical rotations were measured by use
of a Perkin-Elmer 241 polarimeter, and the [R]D values are given in 10-1
deg cm2 g-1. The 1H, 13C, and 31P NMR spectra were recorded using
Varian Gemini 300 and Unity 400 and 500 instruments with deuterated
solvents. The 31P spectra were referenced to 80% phosphoric acid or to
the corresponding 1H spectra. High-resolution mass spectra were
obtained with a Jeol JMS-LCmate mass spectrometer. Elemental
analyses were determined by Galbraith Laboratories, Inc.
citric acid wash was found useful for removal of unreacted
aminoquinoline. However, the yield of product was still quite
low, at 25%, and another method was sought.
Among the most reactive of the common activated intermedi-
ates are the amino acid fluorides,19 which have been shown to be
very efficient reagents for peptide bond formation.20 With a
sample of Boc-Dap-6-AQ (14) in hand for comparison, the
condensation of the acid fluoride of 6 with 6-AQ was next
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Table 2. Murine and Human Cancer Cell Line Results [ED50 and GI50, μg/mL (nM)]a
cell lineb
compound no.
P388
NCI-H460
KM20L2
DU-145
BXPC-3
MCF-7
SF-268
3b
<0.001
(<1.2)
0.0076
(8.7)
0.00088
(1.05)
0.00061
(0.72)
0.00054
(0.64)
0.046
0.00068
(0.81)
0.00125
(1.48)
(54.6)
3c
4
0.031
0.016
0.0077
(10.6)
0.00025
(0.35)
0.023
0.029
0.0046
(6.35)
0.00014
(0.19)
0.029
(42.8)
0.0026
(3.59)
0.0036
(5.0)
(22.1)
0.00036
(0.50)
(31.8)
0.00030
(0.41)
(40.1)
0.00031
(0.43)
(40.1)
0.00016
(0.22)
5
9
a Cytotoxicity concentrations as nanomolar values are given in parentheses. b Cancer cell lines in order: murine lymphocytic leukemia (P388); lung
(NCI-H460); colon (KM20L2); prostate (DU-145); pancreas (BXPC-3); breast (MCF-7); CNS (SF-268).
N-Boc-Dap-4-hydroxyphenethylamide (7a). To a solution
of Boc-Dap15 (6, 0.49 g, 1.71 mmol) in dry DMF (3 mL) that was stirring at
20 °C was added 1-hydroxybenzotriazole (HOBT, 0.37 g, 2.74 mmol).
Diisopropylethylamine (DIEA, 0.95 mL, 5.48 mmol) was added, followed by
N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide hydrochloride (EDCI),
and the reaction mixture was stirred for 10 min before the addition of
tyramine (0.28 g, 2.05 mmol). The mixture was stirred at 20 °C for 16 h
before termination of reaction by addition of saturated NaHCO3 solution
(5 mL) and extraction with EtOAc (4 ꢀ 5 mL). The combined organic
extract was washed with brine (20 mL) and dried. Removal of solvent yielded
a yellow oil (0.89 g), which was fractionated by column chromatography
(eluent: 2.5-6.0% CH3OH in CH2Cl2) to provide 7a as a colorless oil
(0.57 g, 82%) that crystallized from 1:1 CH2Cl2-hexane: mp 163-164 °C;
[R]23D -30.4 (c 1.9, CHCl3); IR (neat) νmax 3305, 2975, 2935, 1650, 1515,
1168, 755 cm-1; 1H NMR (CD3OD, 300 MHz) δ 6.92 (d, J = 8.4 Hz, 2H),
6.57 (d, J = 8.4 Hz, 2H), 3.55 (br m, 1H), 3.49- 3.33 (m, 3H), 3.29 (s, 3H),
3.26-3.19 (m, 2H), 3.19-3.03 (m, 2H), 2.70-2.53 (m, 2H), 2.11 (m, 1H),
1.81-1.70 (m, 2H), 1.61-1.50 (m, 2H), 1.40 (br s, 9H), 1.05 (d, J = 6.3 Hz,
3H); 13C NMR (CDCl3, 100.5 MHz) (two conformers observed) δ 174.7,
174.1, 155.5, 155.3, 154.8, 154.4, 129.5, 129.3, 115.5, 115.4, 83.7, 82.0, 80.1,
79.5, 60.6, 59.1, 58.6, 46.9, 46.5, 44.2, 43.8, 40.9, 40.7, 34.3, 28.5, 28.4, 25.7,
25.1, 24.4, 24.0, 14.3, 14.0; HRMS (FAB) m/z 407.2565 [M þ H]þ (calcd
for C22H35N2O5, 407.2546).
Dap-4-hydroxyphenethylamide Hydrobromide (7b). Bro-
motrimethylsilane (0.46 mL, 3.5 mmol) was added to a stirred solution of
7a (0.57 g, 1.4 mmol) in dry CH2Cl2 at 20 °C, and stirring was continued for
18 h. Water (5 mL) was added, and the mixture was stirred vigorously for
30 min. The aqueous layer was removed, and the organic phase was extracted
again with H2O (5 mL). Freeze-drying of the combined aqueous phase
provided the hydrobromide salt 7b as a colorless solid (0.54 g, 99%), which
was used without further purification. A sample was crystallized from
CH2Cl2-hexane: mp 79-81 °C; IR (neat) νmax 3275, 2980, 1640, 1515,
1235, 830 cm-1; 1H NMR (CD3OD, 300 MHz) δ 6.95 (d, J = 8.4 Hz, 2H),
6.60 (d, J = 8.4 Hz, 2H), 3.47 (m, 1H), 3.41 (s, 3H), 3.25-3.02 (m, 5H),
2.72-2.62 (m, 2H), 2.33 (m, 1H), 1.88-1.76 (m, 2H), 1.72-1.64 (m, 2H),
1.13 (d, J = 7.2 Hz, 3H); 13C NMR (CD3OD, 125.5 MHz) δ 175.6, 157.0,
131.0, 130.8, 116.2, 81.6, 62.9, 61.8, 46.5, 45.5, 41.3, 35.2, 24.1, 23.9, 15.5;
HRMS (APCI) m/z 307.2026 [(M - HBr) þ H]þ (calcd for C17H27-
N2O3, 307.2022).
20 °C for 6 h, and then reaction was terminated by addition of saturated
NaHCO3 solution (10 mL), followed by extraction of the mixture with
EtOAc (4 ꢀ 10 mL). The combined organic extract was washed with
brine (50 mL) and dried. Removal of solvent yielded a viscous, brown oil
(0.83 g), which was fractionated by column chromatography (eluent:
5-10% MeOH in CH2Cl2) to provide 9 as a viscous, colorless oil (0.61
g, 65%): [R]23D -44.0 (c 2.2, CHCl3); IR (neat) νmax 3295, 2965, 1620,
1515, 1100, 755 cm-1; 1H NMR (CD3OD, 400 MHz) δ 7.25 (m, 1H),
7.05-7.01 (m, 4H), 6.68 (t, J = 8.5 Hz, 4H), 4.74 (d, J = 8.4 Hz, 1H),
4.71 (d, J = 8.4 Hz, 1H), 4.63 (d, J = 8.8 Hz, 1H), 4.15 (m, 1H), 4.07 (m,
1H), 3.90-3.83 (m, 2H), 3.78 (m, 1H), 3.68 (m, 1H), 3.57 (m, 6H),
3.40-3.32 (m, 2H), 3.38 (s, 3H), 3.36 (s, 3H), 3.29 (s, 6H), 3.26 (s,
3H), 3.13 (s, 3H), 2.81-2.68 (m, 4H), 2.65-2.62 (m, 2H), 2.48 (d, J =
6.6 Hz, 2H), 2.31 (s, 6H), 2.29 (s, 6H), 2.27-2.19 (m, 2H), 2.08-1.86
(m, 10H), 1.78-1.63 (m, 4H), 1.44-1.35 (m, 2H), 1.16 (t, J = 7.1 Hz,
6H), 1.05-0.95 (m, 28H), 0.90-0.84 (m, 12H); 13C NMR (CD3OD,
100.5 MHz) δ 176.5, 176.4, 175.3, 173.3, 173.2, 171.9, 157.0, 156.9,
136.5, 131.2, 130.9, 130.8, 130.7, 116.3, 116.2, 87.2, 83.6, 79.8, 76.0, 75.8,
62.1, 61.4, 61.0, 60.8, 58.6, 58.3, 57.8, 56.2, 56.0, 45.9, 45.7, 42.5, 42.4,
41.8, 41.4, 38.2, 35.3, 33.8, 33.1, 31.8, 31.7, 28.8, 27.0, 26.8, 25.8, 24.5,
20.2, 20.2, 19.9, 19.5, 19.3, 16.3, 16.0, 15.8, 15.1, 10.9, 10.8; HRMS
(FAB) m/z 718.5084 [M þ H]þ (calcd for C39H68N5O7, 718.5119).
Dov-Val-Dil-Dap-4-(dibenzylphosphoryloxy)phenethyl-
amide (10a). To a solution of 9 (0.51 g, 0.70 mmol) in dry CH3CN
(4 mL) at -15 °C (ice/salt) was added carbon tetrachloride (0.34 mL, 1.02
mmol), followed by DIEA (0.26 mL, 1.50 mmol) and 4-dimethylaminopyr-
idine (9 mg, 0.07 mmol). Dibenzylphosphite (0.23 mL, 1.02 mmol) was next
added over a 20 min period to the mixture, the temperature being maintained
between -15 and -18 °C. After addition, the mixture was cooled to -20 °C
and then allowed to warm to 5 °C over 90 min, and reaction was terminated
by addition of saturated NaHCO3 solution (10 mL). The mixture was
extracted with EtOAc (3 ꢀ 10 mL), and the combined organic extract was
washed with brine (50 mL) and dried. Removal of solvent yielded a viscous,
pale yellow oil (0.60 g), which was fractionated by column chromatography
(eluent: 5-10% MeOH in CH2Cl2) to provide 10a as a colorless oil (0.34 g,
49%): IR (neat) νmax 3305, 2965, 1620, 1455, 1215, 1015, 955 cm-1; 1H
NMR (CD3OD, 500 MHz) δ7.36-7.31 (m, 20H), 7.21 (d, J= 6.6 Hz, 2H),
7.20 (d, J = 6.6 Hz, 2H), 7.07 (d, J = 6.6 Hz, 2H), 7.03 (d, J = 6.6 Hz, 2H),
5.13-5.10 (m, 8H), 4.81-4.71 (m, 2H), 4.71 (d, J= 8.0 Hz, 1H), 4.62 (d, J=
8.0 Hz, 1H), 4.14 (m, 1H), 4.06 (m, 1H), 3.91-3.86 (m, 2H), 3.80 (m, 1H),
3.69 (m, 1H), 3.56-3.47 (m, 4H), 3.43-3.32 (m, 2H), 3.38 (s, 3H), 3.36
(s, 3H), 3.28 (s, 6H), 3.26 (s, 3H), 3.11 (s, 3H), 2.86-2.77 (m, 4H), 2.65-
2.61 (m, 3H), 2.51 (m, 1H), 2.46 (d, J = 6.5 Hz, 2H), 2.30 (s, 6H), 2.29
(s, 6H), 2.28-2.18 (m, 2H), 2.08-1.86 (m, 9H), 1.76-1.64 (m, 5H),
1.43-1.36 (m, 2H), 1.16 (d, J = 7.5 Hz, 3H), 1.15 (d, J = 7.5 Hz, 3H),
Dov-Val-Dil-Dap-4-hydroxyphenethylamide (9). To a solu-
tion of 816 (0.78 g, 1.43 mmol) that was stirring in dry DMF (2 mL) at
20 °C was added HOBT (0.31 g, 2.29 mmol). Next was added DIEA
(0.96 mL, 5.50 mmol), followed by EDCI (0.44 g, 2.29 mmol), and the
reaction mixture was stirred for 15 min before the addition of a solution
of 7b (0.50 g, 1.30 mmol) in DMF (4 mL). The mixture was stirred at
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1.02-0.92 (m, 28H), 0.87-0.81 (m, 12H); 13C NMR (CD3OD, 125.5
(t, J = 6.2 Hz, 6H), 1.03-0.95 (m, 28H), 0.84 (q, J = 6.9 Hz, 12H); 31
NMR (CD3OD, 162.0 MHz) δ -0.42.
P
MHz) δ175.2, 175.1, 171.9, 170.6, 149.0 (d, JC-P = 7.0 Hz), 148.9 (d, JC-P
=
Morpholine Auristatin TP (3d). Ion-exchange chromatography
of potassium salt 3c with aqueous morpholine led to 3d as a colorless
solid: mp 148-150 °C; IR (neat) νmax 3295, 2965, 1620, 1455, 1105,
880 cm-1; 1H NMR (CD3OD, 500 MHz) δ 7.18-7.11 (m, 8H), 4.82-
4.74 (m, 2H), 4.71 (d, J = 8.5 Hz, 1H), 4.65 (d, J = 8.5 Hz, 1H), 4.13 (m,
1H), 4.07 (m, 1H), 3.97 (m, 1H), 3.91 (dd, J = 9.3, 2.3 Hz, 1H), 3.80 (br
s, 16H), 3.71 (m, 1H), 3.60 (m, 1H), 3.52 (d, J = 8.5 Hz, 1H), 3.49-3.43
(m, 3H), 3.40 (s, 3H), 3.39 (s, 3H), 3.37-3.34 (m, 2H), 3.31 (s, 6H),
3.28 (s, 3H), 3.15 (s, 3H), 3.06 (br s, 16H), 2.82 (m, 1H), 2.77 (q, J = 7.2
Hz, 4H), 2.69 (d, J = 8.5 Hz, 1H), 2.67 (m, 1H), 2.54 (d, J = 9.0 Hz, 1H),
2.50 (d, J = 6.0 Hz, 2H), 2.42 (s, 6H), 2.34 (s, 6H), 2.32-2.14 (m, 2H),
2.13-1.88 (m, 9H), 1.81-1.71 (m, 5H), 1.46-1.38 (m, 2H), 1.20 (d,
J = 6.5 Hz, 3H), 1.18 (d, J = 7.5 Hz, 3H), 1.04-0.95 (m, 28H), 0.91-
0.87 (m, 12H); 31P NMR (CD3OD, 162.0 MHz) δ -3.43.
7.0 Hz), 136.6, 136.4, 129.9, 129.8, 128.5, 128.3, 127.9 (d, JC-P = 2.6 Hz),
119.8 (d, JC-P = 4.4 Hz), 119.6 (d, JC-P = 4.4 Hz), 85.7, 82.2, 78.5, 78.4, 78.4,
74.5, 74.4, 70.1, 70.1, 60.7, 60.0, 59.6, 57.3, 56.9, 54.8, 54.6, 46.7, 44.5, 44.3, 41.1,
41.1, 40.0, 39.7, 36.8, 35.6, 34.1, 32.3, 32.2, 31.7, 30.4, 30.3, 27.4, 25.7, 25.5, 24.4
(d, JC-P = 3.6 Hz), 23.1, 18.8 (d, JC-P = 4.4 Hz), 18.5, 18.2, 18.0, 14.9, 14.6,
14.5, 13.7, 9.5; 31P NMR (CD3OD, 202.5 MHz) δ -6.51, -6.54; HRMS
(FAB) m/z 978.5811 [M þ H]þ (calcd for C53H81N5O10P 978.5721).
Dov-Val-Dil-Dap-4-(dihydrophosphoryloxy)phenethyl-
amide (10b). To a solution of dibenzyl phosphate 10a (38 mg, 0.04 mmol)
in MeOH (5 mL) was added palladium on activated carbon (10 wt % Pd,
10 mg), and hydrogen gas (balloon) was bubbled through the suspension for
1 h. The mixture was filtered through a plug of Celite, and the filter was washed
with MeOH (2 ꢀ 5 mL). Removal of solvent from the filtrate yielded the free
phosphoric acid 10b as a glassy solid (32 mg, quantitative): mp 168-170 °C;
IR (neat) νmax 3400, 2970, 1635, 1460, 1095, 910 cm-1; 1H NMR (CD3OD,
500 MHz) δ 7.20-7.12 (m, 8H), 4.77-4.71 (m, 2H), 4.67 (d, J = 8.5 Hz,
1H), 4.62 (d, J = 9.0 Hz, 1H), 4.11 (m, 1H), 4.05 (m, 1H), 3.93-3.89 (m,
2H), 3.73-3.68 (m, 2H), 3.61-3.48 (m, 4H), 3.44-3.33 (m, 2H), 3.41 (s,
3H), 3.37 (s, 3H), 3.29 (s, 6H), 3.28 (s, 3H), 3.15 (s, 3H), 2.90 (s, 6H),
2.79-2.73 (m, 4H), 2.66-2.49 (m, 4H), 2.42-2.26 (m, 4H), 2.08-1.64 (m,
14H), 1.46-1.38 (m, 2H), 1.23 (d, J = 7.0 Hz, 3H), 1.17 (d, J = 7.0 Hz, 3H),
1.07-1.00 (m, 20H), 0.97-0.84 (m, 20H); 31P NMR (CD3OD, 202.5
MHz) δ -4.11.
Sodium Auristatin TP (3b). Ion-exchange chromatography of
free acid 10b (32 mg) with aqueous NaOH led to 3b as a colorless solid
(24 mg, 71%): mp 170-171 °C; IR (neat) νmax 3305, 2965, 1625, 1510,
1105, 990 cm-1; 1H NMR (CD3OD, 400 MHz) δ 7.17-7.15 (m, 4H),
7.09 (d, J = 8.0 Hz, 4H), 4.78-4.72 (m, 2H), 4.72 (d, J = 8.0 Hz, 1H),
4.64 (d, J = 8.4 Hz, 1H), 4.12 (m, 1H), 4.07 (m, 1H), 3.98-3.94
(m, 2H), 3.91 (dd, J = 9.1, 2.3 Hz, 2H), 3.70 (m, 1H), 3.59 (m, 1H),
3.51-3.41 (m, 4H), 3.39 (s, 3H), 3.37 (s, 3H), 3.36-3.32 (m, 2H), 3.30
(s, 6H), 3.27 (s, 3H), 3.14 (s, 3H), 2.81-2.70 (m, 4H), 2.65 (d, J = 7.2
Hz, 1H), 2.63 (d, J = 7.2 Hz, 1H), 2.49 (d, J = 6.4 Hz, 2H), 2.31 (s, 6H),
2.29 (s, 6H), 2.29-2.22 (m, 2H), 2.08-1.87 (m, 9H), 1.81-1.68
(m, 5H), 1.43-1.36 (m, 2H), 1.17 (t, J = 5.3 Hz, 6H), 1.03-0.95
(m,28H),0.85(q,J= 7.2 Hz, 12H); 31PNMR(CD3OD, 162.0 MHz) δ-3.42.
Lithium Auristatin TP (3a). Ion-exchange chromatography of
sodium salt 3b (12 mg, 0.014 mmol) with aqueous LiOH led to 3a as a
colorless solid (11 mg, 96%): mp 263 °C (dec); IR (neat) νmax 3315,
2965, 1630, 1105, 1005, 920 cm-1; 1H NMR (CD3OD, 400 MHz) δ
7.20 (d, J = 8.0 Hz, 2H), 7.18 (d, J = 8.0 Hz, 2H), 7.04 (d, J = 8.0 Hz, 4H),
4.74-4.70 (m, 2H), 4.72 (d, J = 8.0 Hz, 1H), 4.64 (d, J = 8.8 Hz, 1H),
4.14-4.00 (m, 4H), 3.95 (m, 1H), 3.91 (dd, J = 9.0, 2.2 Hz, 1H), 3.71
(m, 1H), 3.58 (m, 1H), 3.52-3.34 (m, 6H), 3.39 (s, 3H), 3.38 (s, 3H),
3.30 (s, 6H), 3.27 (s, 6H), 3.13 (s, 3H), 2.75-2.68 (m, 4H), 2.64 (d, J =
4.8 Hz, 1H), 2.63 (d, J = 4.8 Hz, 1H), 2.51 (d, J = 6.4 Hz, 2H), 2.30 (s,
6H), 2.29 (s, 6H), 2.27-2.23 (m, 2H), 2.07-1.94 (m, 9H), 1.82-1.71
(m, 5H), 1.45-1.37 (m, 2H), 1.18 (t, J = 6.2 Hz, 6H), 1.03-0.95 (m,
28H), 0.85 (q, J = 6.9 Hz, 12H); 31P NMR (CD3OD, 162.0 MHz) δ -
0.58.
General Procedure for the Synthesis of 3e-h. The amine or
amino acid (25.0 μmol) was added to a stirred solution of acid 10b (10
mg, 12.5 μmol) in either MeOH (300 μL) or deionized H2O (for 3h),
and the mixture was stirred for 15 h. Removal of solvent yielded the
desired salt.
Quinine Auristatin TP (3e): colorless solid; mp 118-120 °C; 1H
NMR (CD3OD, 500 MHz) δ 8.67 (d, J = 4.8 Hz, 4H), 7.93 (d, J = 9.3
Hz, 4H), 7.72 (d, J = 4.8 Hz, 4H), 7.43 (d, J = 2.3 Hz, 4H), 7.40 (dd, J =
9.3, 2.3 Hz, 4H), 7.17 (t, J = 7.3 Hz, 4H), 7.03 (d, J = 7.3 Hz, 4H), 5.93 (s,
4H), 5.73 (m, 4H), 5.05 (d, J = 17.5 Hz, 4H), 4.95 (d, J = 11 Hz, 4H),
4.82-4.71 (m, 2H), 4.72 (d, J = 8.0 Hz, 1H), 4.65 (d, J = 8.5 Hz, 1H),
4.12 (m, 1H), 4.07 (m, 1H), 3.98 (s, 12H), 3.91 (d, J = 2.0 Hz, 1H), 3.89
(d, J = 2.0 Hz, 1H), 3.71-3.65 (m, 2H), 3.56 (m, 1H), 3.50 (d, J = 10.0
Hz, 1H), 3.45-3.23 (m, 12H), 3.39 (s, 3H), 3.38 (s, 3H), 3.30 (s, 3H),
3.29 (s, 3H), 3.27 (s, 3H), 3.14 (s, 3H), 3.00-2.92 (m, 8H), 2.71-2.65
(m, 6H), 2.56-2.48 (m, 8H), 2.34 (s, 6H), 2.31 (s, 6H), 2.30-2.21 (m,
2H), 2.08-1.90 (m, 24H), 1.78-1.71 (m, 10H), 1.45-1.40 (m, 6H),
1.17 (t, J = 7.0 Hz, 6H), 1.04-0.95 (m, 28H), 0.98-0.80 (m, 12H); 31
P
NMR (CD3OD, 162.0 MHz) δ -1.82.
1
TRIS Auristatin TP (3f): colorless solid; mp 122-123 °C; H
NMR (D2O, 500 MHz) δ 7.21-7.12 (m, 1H), 4.73-4.64 (m, 4H), 4.17
(m, 1H), 4.11 (m, 1H), 3.92-3.86 (m, 2H), 3.74-3.62 (m, 2H), 3.67 (s,
24H), 3.59-3.38 (m, 6H), 3.44 (s, 3H), 3.42 (s, 3H), 3.33 (s, 3H), 3.33
(s, 3H), 3.26 (s, 3H), 3.18 (s, 3H), 3.12 (d, J = 8.5 Hz, 3H), 3.02 (d, J =
9.5 Hz, 1H), 2.87-2.75 (m, 2H), 2.67-2.62 (m, 2H), 2.58-2.54 (m,
2H), 2.52 (s, 6H), 2.45 (s, 6H), 2.36-2.27 (m, 2H), 2.22-2.09 (m,
2H), 2.08-2.01 (m, 2H), 1.99-1.72 (m, 9H), 1.68-1.61 (m, 1H),
1.39-1.30 (m, 2H), 1.20 (d, J = 6.5 Hz, 3H), 1.16 (d, J = 7.0 Hz, 3H),
1.05-0.97 (m, 28H), 0.88-0.84 (m, 12H); 31P NMR (CD3OD, 162.0
MHz) δ -0.01.
Serine Auristatin TP (3g): colorless solid; mp 158 °C (dec); 1H
NMR (D2O, 500 MHz) δ 7.26 (d, J = 8.5 Hz, 2H), 7.23 (d, J = 8.5 Hz,
2H), 7.14 (d, J = 8.5 Hz, 2H), 7.09 (d, J = 8.5 Hz, 2H), 4.75 (d, J = 9.5 Hz,
1H), 4.73-4.68 (m, 2H), 4.66 (d, J = 9.5 Hz, 1H), 4.18 (m, 1H), 4.11
(m, 1H), 4.01-3.93 (m, 10H), 3.86-3.83 (m, 6H), 3.79 (t, J = 5.8 Hz,
2H), 3.74-3.68 (m, 2H), 3.62-3.51 (m, 4H), 3.47-3.36 (m, 4H), 3.44
(s, 3H), 3.39 (s, 3H), 3.33 (s, 3H), 3.32 (s, 3H), 3.24 (s, 3H), 3.18
(s, 3H), 2.97 (s, 6H), 2.95 (s, 6H), 2.92-2.79 (m, 4H), 2.67-2.44
(m, 6H), 2.33 (m, 1H), 2.24 (m, 1H), 2.12-2.02 (m, 2H), 1.97-1.66
(m, 9H), 1.51 (m, 1H), 1.38-1.32 (m, 2H), 1.20 (d, J = 7.0 Hz, 3H),
Potassium Auristatin TP (3c). Ion-exchange chromatography of
acid 10b with aqueous KOH led to 3c as a colorless solid (4 mg, 64%):
mp 198 °C; IR (neat) νmax 3230, 2965, 1620, 1100, 980, 885 cm-1; 1H
NMR (CD3OD, 400 MHz) δ 7.21 (d, J = 8.2 Hz, 2H), 7.19 (d, J = 8.2
Hz, 2H), 7.04 (d, J = 8.2 Hz, 4H), 4.75-4.72 (m, 2H), 4.72 (d, J = 8.0
Hz, 1H), 4.64 (d, J = 6.6 Hz, 1H), 4.14-4.01 (m, 4H), 3.95 (m, 1H),
3.91 (dd, J = 8.8, 2.4 Hz, 1H), 3.71 (m, 1H), 3.58 (m, 1H), 3.52-3.35
(m, 6H), 3.39 (s, 3H), 3.38 (s, 3H), 3.30 (s, 6H), 3.26 (s, 3H), 3.13 (s,
3H), 2.75-2.68 (m, 4H), 2.64 (d, J = 8.8 Hz, 1H), 2.63 (d, J = 9.2 Hz,
1H), 2.49 (d, J = 5.6 Hz, 2H), 2.30 (s, 6H), 2.29 (s, 6H), 2.27-2.23 (m,
2H), 2.07-1.94 (m, 9H), 1.83-1.72 (m, 5H), 1.45-1.37 (m, 2H), 1.18
1.14 (d, J = 7.0 Hz, 3H), 1.05-0.95 (m, 30H), 0.92-0.82 (m, 10H); 31
NMR (CD3OD, 162.0 MHz) δ -4.07.
P
Nitroarginine Auristatin TP (3h): colorless solid; mp 157-
158 °C (dec); IR (neat) νmax 3295, 2965, 1625, 1360, 1270, 1095 cm-1
;
1H NMR (D2O, 500 MHz) δ 7.21 (d, J = 7.8 Hz, 2H), 7.18 (d, J = 7.8 Hz,
2H), 7.10 (d, J = 7.8 Hz, 2H), 7.05 (d, J = 7.8 Hz, 2H), 4.71 (d, J = 9.0 Hz,
1H), 4.70-4.64 (m, 2H), 4.62 (d, J = 8.5 Hz, 1H), 4.14 (m, 1H), 4.06 (m,
1H), 3.81-3.72 (m, 4H), 3.74 (t, J = 6.5 Hz, 4H), 3.69-3.63 (m, 2H),
966
dx.doi.org/10.1021/np1007334 |J. Nat. Prod. 2011, 74, 962–968

Journal of Natural Products
ARTICLE
3.59-3.45 (m, 4H), 3.43-3.33 (m, 2H), 3.40 (s, 3H), 3.35 (s, 3H), 3.30 (t,
J = 6.5 Hz, 8H), 3.29 (s, 3H), 3.28 (s, 3H), 3.20 (s, 3H), 3.14 (s, 3H), 2.93
(s, 6H), 2.90 (s, 6H), 2.88-2.75 (m, 4H), 2.63-2.48 (m, 5H), 2.44-2.39
(m, 1H), 2.29 (m, 1H), 2.20 (m, 1H), 2.08-1.97 (m, 2H), 1.95-1.61 (m,
25H), 1.47 (m, 1H), 1.34-1.27 (m, 2H), 1.16 (d, J = 6.5 Hz, 3H), 1.10 (d,
J = 7.0 Hz, 3H), 1.01-0.96 (m, 18H), 0.93-0.90 (m, 12H), 0.86-0.81 (m,
10H); 31P NMR (CD3OD, 162.0 MHz) δ -3.56.
N-Boc-Dap-2-aminoquinoline (11). To a solution of Boc-
Dap15 (6, 0.172 g; 0.6 mmol) in CH2Cl2 (3 mL) was added 2-amino-
quinoline (82.8 mg; 0.57 mmol), and the mixture was stirred and cooled
to 0 °C under argon. Triethylamine (TEA, 0.3 mL; 2.1 mmol) and
diethylcyanophosphonate (DEPC; 0.2 mL; 1.2 mmol) were added, and
the resultant yellow solution was allowed to warm to room temperature
(rt) and was stirred under argon for 6 h. Removal of solvent yielded a
dark orange-brown residue, which was fractioned under pressure on
silica gel [eluent: hexane-acetone (7:2 to 2:3)] to give the product as a
colorless solid (90.8 mg, 0.22 mmol, 36.6%, based on recovery of starting
material): 1H NMR (CDCl3, 300 MHz) δ 8.43 (1H, dd, J = 8.7. 1.5 Hz),
8.16 (1H, d, J = 8.7 Hz), 7.83 (1H, d, J = 8.7 Hz), 7.72 (1H, d, J = 8.4 Hz),
7.66 (1H, t, J = 7.5 Hz), 7.44 (1H, t, J = 7.5 Hz), 4.05-3.92 (2H, m,
NCH, OCH), 3.53 (3H, s, OCH3), 3.44 (2H, br d, J = 13 Hz, NCH2),
2.60-2.80 (1H, m, CHCH3), 1.74-1.98 (4H, m, 2 ꢀ CH2), 1.52 (9H,
s, C(CH3)3), 1.45 (3H, d, J = 9.3 Hz, CHCH3)); MS (APCIþ) m/z
414.2373 [M þ H]þ (calcd for C23H32N3O4, 414.2393).
(1H, br s), 8.11 (1H, d, J = 8.1 Hz), 8.04 (1H, d, J = 9.3 Hz), 7.72 (1H,
br), 7.37 (1H, dd, J = 8.4, 4.2 Hz), 4.15-3.90 (2H, m, NCH, OCH), 3.55
(3H, s, OCH3), 3.43 (m, 1H), 3.27 (m, 1H), 2.72 (1H, m), 2.06-1.76
(4H, m), 1.50 (9H, s), 1.39 (3H, m); MS (APCIþ) m/z 414.2408 [M þ
H]þ (calcd for C23H32N3O4, 414.2393).
Method B. Intermediate acid fluoride 16 was first prepared by
successive addition of pyridine (0.05 mL) and cyanuric fluoride (15, 0.15
mL, 1.75 mmol) to a solution of Boc-Dap (6, 76.3 mg, 0.27 mmol) that was
stirring under argon at 0 °C, with continued stirring for 20 h and warming to
rt. Next, CH2Cl2 (10 mL) and ice were added, followed by cold H2O. The
organic phase was removed and the aqueous layer was further extracted with
CH2Cl2. The combined organic extract was washed with cold H2O and
dried to give a dark orange, oily solid (65.6 mg) that by TLC comprised
product 16 and a trace of Boc-Dap (6). Without further purification, the
crude product was dissolved in CH2Cl2 and was treated with pyridine (0.1
mL) followed by 6-AQ (34.8 mg, 0.24 mmol). The mixture was stirred for
21 h and was then extracted with CH2Cl2 (10 mL). The solution was
washed with 10% citric acid solution, followed by H2O. Drying over Na2SO4
and removal of solvent yielded a pale brown oil (52.8 mg), which was
separated by column chromatography [eluent: toluene-acetone (2:1)] to
give product 14 (30.3 mg, 0.07 mmol, 26%).
Dap-6-aminoquinoline Trifluoroacetate (17). To a solution
of N-Boc-Dap-6-AQ (14, 49.3 mg, 0.12 mmol) in CH2Cl2 (2 mL) that
was stirring at 0 °C under argon was added trifluoroacetic acid (TFA, 2
mL). Stirring was continued for 3 h with warming to rt. The solvent was
removed under vacuum, toluene being used to form an azeotrope with
the remaining TFA, to give a green-tinged oily solid (17; quantitative),
which was used immediately in the next reaction.
Dap-2-aminoquinoline Trifluoroacetate (12). To a solution
of N-Boc-Dap-2-AQ (11, 68.0 mg, 0.16 mmol) in CH2Cl2 (4 mL) that
was stirring at 0 °C under argon was added trifluoroacetic acid (TFA,
2 mL), and stirring was continued for 2 h with warming to rt. The solvent
was removed under vacuum, toluene being used to form an azeotrope
with the remaining TFA. The residue, a yellow oil, was allowed to stand
under diethyl ether for 1 h. Removal of the ether left a yellowish oily
solid, to which hexane was added and removed under vacuum until a
constant weight was reached (99.4 mg; quantitative), and this material
was used immediately in the next reaction.
Dov-Val-Dil-Dap-6-aminoquinoline (Auristatin 6-AQ, 5).
Dap-6-AQ salt 17 (0.12 mmol) and Dov-Val-Dil TFA16 (8, 70.0 mg;
3
0.13 mmol) were dissolved in CH2Cl2 (2 mL), and the solution was stirred
under argon and cooled to 0 °C. Next were added TEA (0.11 mL; 0.79
mmol) and DEPC (0.03 mL; 0.18 mmol), and the mixture was stirred under
argon for 18 h with warming to rt. Removal of solvent and separation on silica
gel under pressure [eluent: hexane-acetone (5:2 to 2:3)] gave the crude
product 5 (48.6 mg), of which a 19.1 mg sample was further purified by
column chromatography in CH2Cl2-MeOH (19:1) to give auristatin 6-AQ
(5) as a pale yellow, glassy oil (powder when scratched): 1H NMR (CDCl3,
300 MHz) δ 9.04 (1H, br s), 8.81 (1H, br d, J = 3 Hz), 8.47 (1H, s), 8.11
(1H, d, J = 8.4 Hz), 8.02 (1H, d, J = 9.3 Hz), 7.76 (1H, br d, J = 8.7 Hz), 7.36
(1H, dd, J = 8.4, 3.8 Hz), 6.96 (1H, br), 4.79 (2H, m), 4.30-4.07 (3H, m),
3.51 (3H, s), 3.50-3.26 (2H, m), 3.35 (3H, s), 3.05 (s, 3H), 2.71 (1H, m),
2.54-2.42 (2H, m), 2.32-2.22 (1H, m), 2.28 (6H, s), 2.12-2.05 (2H, m),
1.82 (2H, m), 1.42-1.26 (5H, m), 1.08-0.80 (21H, m); MS (APCIþ)
m/z 725.4907 [M þ H]þ (calcd for C40H65N6O6, 725.4966).
Dov-Val-Dil-Dap-2-aminoquinoline (Auristatin 2-AQ, 4).
The Dap-2-AQ salt 12 and Dov-Val-Dil TFA16 (8, 87.0 mg; 0.16 mmol)
3
were dissolved in CH2Cl2 (5 mL), and the solution was stirred under
argon and cooled to 0 °C. Next, TEA (0.12 mL; 0.86 mmol) and DEPC
(0.035 mL; 0.21 mmol) were added, and the mixture was stirred under
argon for 7 h with warming to rt. Removal of solvent yielded a yellowish
oil (310 mg), which was separated on silica gel under pressure [eluent:
hexane-acetone (5:2 to 3:2)] to give the product as a colorless glass
(powder when scratched) (64 mg; 0.09 mmol): 1H NMR (CDCl3, 300
MHz) δ 8.43 (1H, dd, J = 8.7. 1.5 Hz), 8.14 (1H, d, J = 8.7 Hz), 7.80-
7.41 (4H, m), 6.90 (1H, t, J = 9.3 Hz), 6.73 (1H, d, J = 9.0 Hz), 4.86 (1H,
m), 4.75 (1H, m), 4.26 (1H, m), 4.14 (1H, m), 4.04 (1H, m), 3.51 and
3.44 (3H, s), 3.35 and 3.32 (3H, s), 3.38-3.19 (2H, m), 3.02 (3H, s),
2.42 (3H, m), 2.23 (6H, s), 2.23 (1H, m), 2.08-1.98 (5H, m), 1.95-
1.74 (1H, m), 1.43-1.33 (2H, m), 0.80-1.06 (22H, m); MS (APCIþ)
m/z 725.4997 [M þ H]þ (calcd for C40H65N6O6, 725.4966).
’ ASSOCIATED CONTENT
Supporting Information. 1H NMR spectra of com-
S
b
pounds 3b, 4, and 5. This material is available free of charge
via the Internet at http://pubs.acs.org.
N-Boc-Dap-6-aminoquinoline (14). Method A. To a stirring
solution of Boc-Dap15 (6, 87.2 mg; 0.3 mmol) in DMF (2 mL) and
pyridine (0.1 mL) was added Boc2O (0.183 g; 0.84 mmol). After 10 min,
6-aminoquinoline (6-AQ; 50.4 mg; 0.35 mmol) was added to the
solution, and stirring was continued for 64 h, at which time starting
material was still present. Solvent was removed from the mixture, and the
residue was fractionated by column chromatography in hexane-acetone
(5:1 to 2:1 gradient). The first fractions to elute contained Boc-6-AQ
(35 mg): 1H NMR (CDCl3, 300 MHz) δ 8.79 (1H, dd, J = 4.5, 1.5 Hz),
8.01-8.12 (3H, m), 7.48 (1H, dd, J = 9, 2.7 Hz), 7.36 (1H, dd, J = 7.1,
4.2 Hz), 7.03 (1H, br s), 1.55 (9H, s).
’ AUTHOR INFORMATION
Corresponding Author
*Tel: (480) 965-3351. Fax: (480) 965-2747. E-mail: bpettit@asu.edu.
’ ACKNOWLEDGMENT
We are pleased to acknowledge the very necessary financial
support from grants R01 CA 90441-01-05, 2R56 CA 090441-06A1,
and 5-R01 CA 90441-07-08 from the Division of Cancer Treatment
and Diagnosis, NCI, DHHS; the Arizona Disease Control Research
Commission; the Fannie E. Rippel Foundation; and the Robert B.
Following the elution of the remaining Boc-Dap (6), compound 14
(29.7 mg, 0.07 mmol, 23% yield, or 28% based on 14.9 mg recovery of
6-AQ) was collected: 1H NMR (CDCl3, 300 MHz) δ 8.82 (2H, m), 8.45
967
dx.doi.org/10.1021/np1007334 |J. Nat. Prod. 2011, 74, 962–968

Journal of Natural Products
ARTICLE
Dalton Endowment Fund. Other very helpful assistance was
provided by Drs. J.-C. Chapuis and J. C. Knight and by M. Dodson
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