Antineoplastic Agents. 603. Quinstatins: Exceptional Cancer Cell Growth Inhibitors

Article abstract of DOI:10.1021/acs.jnatprod.6b01006

Discovery of the exceptionally powerful anticancer drug dolastatin 10 (1), contained in the sea hare Dolabella auricularia, opened a new frontier needed for improving human cancer treatment. Subsequently, major advances have been achieved based on results of structurally modifying this unusual natural peptide while maintaining the remarkable anticancer activity necessary for preparation of successful monoclonal antibody drug conjugates (ADC). Among the first several hundred SAR products based on dolastatin 10 our group synthesized and termed auristatins was auristatin E (2a). An anticancer activity-equivalent, desmethylaurisatin E (2b), linked to a CD30 monoclonal antibody is the very successful anticancer drug Adcetris, now approved for use in 65 countries. In the present investigation, we discovered a new subset of auristatins designated quinstatins derived from dolastatin 10 by replacing the C-terminal Doe unit with a carefully designed quinoline, which led to low or subnanomolar levels of cancer cell growth inhibition required for construction of chemically unique ADC drugs. The synthesis of quinstatins 2-8 is presented along with their cancer cell line biological data.

Full text of DOI:10.1021/acs.jnatprod.6b01006

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Article
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Antineoplastic Agents. 603. Quinstatins: Exceptional Cancer Cell
Growth Inhibitors
George R. Pettit,* Noeleen Melody, and Jean-Charles Chapuis
Department of Chemistry and Biochemistry, Arizona State University, P.O. Box 871604, Tempe, Arizona 85287-1604, United States
S
* Supporting Information
ABSTRACT: Discovery of the exceptionally powerful anticancer
drug dolastatin 10 (1), contained in the sea hare Dolabella auricularia,
opened a new frontier needed for improving human cancer treat-
ment. Subsequently, major advances have been achieved based on
results of structurally modifying this unusual natural peptide while
maintaining the remarkable anticancer activity necessary for prepara-
tion of successful monoclonal antibody drug conjugates (ADC). Among the first several hundred SAR products based on
dolastatin 10 our group synthesized and termed auristatins was auristatin E (2a). An anticancer activity-equivalent, desmethyl-
aurisatin E (2b), linked to a CD30 monoclonal antibody is the very successful anticancer drug Adcetris, now approved for use in
65 countries. In the present investigation, we discovered a new subset of auristatins designated quinstatins derived from
dolastatin 10 by replacing the C-terminal Doe unit with a carefully designed quinoline, which led to low or subnanomolar levels
of cancer cell growth inhibition required for construction of chemically unique ADC drugs. The synthesis of quinstatins 2−8 is
presented along with their cancer cell line biological data.
ur discovery1a,b of the dolastatin series1c,d of cancer cell
growth inhibitors contained in the wide ranging sea hare
treatment of patients with relapsed or refractory Hodgkin’s
lymphoma has resulted in a remarkable level of complete
remissions.4d Those early clinical results have inspired a major
current research effort to link auristatin (2b) to other mono-
clonal antibodies4a,b representing a spectrum of cancer types. In
parallel, considerable research has been continued to discover
structural modifications of dolastatin 10 that would provide such
powerful cancer cell growth inhibition without a high toxic level
and hence provide a much better therapeutic range for inspired
ADC use. Toward this objective we have investigated a new
series of dolasatatin 10 modified structures termed quinstatins
that involve a quinoline foundation. This research has resulted in
a special subset of the auristatins with generally very low to
subnanomolar cancer cell growth inhibitory activities, namely,
quinstatins 2−8 (Figure 1).
O
Dolabella auricularia revealed a new and very promising vista for
anticancer drug discovery. This proved to be broadly confirmed
by the clinical development2 of dolastatins 10 (1) and 152c and
structural modifications we designated auristatins.3 By 2001,
auristatin E (2a)3b began preclinical development as a very
promising antibody drug conjugate (ADC) by linkage to a CD30
monoclonal antibody. Soon, the development continued,
employing the cancer biology equivalent desmethylauristatin E
(2b).4,5
RESULTS AND DISCUSSION
■
The synthesis of quinstatins 2 (3) and 6 (4) and the cytotoxic
activity of quinstatins 2 (3) 3−5, 6 (4), 7, and 8, described
as follows, serves as a useful illustration of this new quinoline
approach to reaching the dolasatin 10 level of potential anti-
cancer activity. Extensive experience from our prior structure−
activity relationship (SAR) studies3 of dolastatin 10 (1) sug-
gested that employing a quinoline ring base3a replacement for the
C-terminal Doe unit of 1 separated by a two-carbon linker from
the peptide could be very productive. This prediction proved to
be on target and was implemented in the manner following the
use of 2′-aminoethylquinolines to provide the necessary spacing
Special Issue: Special Issue in Honor of Phil Crews
Received: November 3, 2016
Presently, the resulting ADC anticancer drug4a−e,5 designated
Adcetris has been approved for use in 65 countries. For example,
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.6b01006
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Scheme 2. Synthesis of Quinstatin 6 (4)
Figure 1. Quinstatins 2−8.
from the peptide unit. Illustrated is the convenient synthesis of
quinstatins 2 (3), 4, 5, and 8, as summarized in Scheme 1, for
quinstatin 2 (3).
In contrast, synthesis of quinstatins 3, 6 (4), and 7 required an
alternative route. By way of an example, the synthesis of quins-
tatin 6 (4) is presented in Scheme 2. Converting the available
quinoline 6-acetic acid to an amide followed by reduction to the
corresponding amine resulted in complex mixtures of reduction
products. This was surprising and not corrected by employing a
series of amide reduction methods. However, reduction of the
carboxylic acid to the alcohol and proceeding via the bromide to
an azide to an amine (Scheme 2) proved to be very effective.
Doubling of signals in the 1H and 13C NMR spectra for the quins-
tatins was attributed to the presence of conformational
isomers,3a,7 with quinstatin 5 having one major conformation
upon examination of its NMR spectra.
Adoption of Scheme 1 or 2 led to expansion of the quinstatin
series of important cytotoxic agents with the exceptionally strong
potency (Table 1) necessary for preparation of successful cancer
monoclonal drug conjugates. Table 1 summarizes a series of
cancer cell line growth inhibition experiments performed in
parallel with very revealing and important results. These results
are also in sharp contrast to our previous study using direct
attachment of the peptide to 2- and 6-aminoquinoline,3a where
the cytotoxic potency was far weaker. Very noteworthy is the
usual cancer biology equivalence of auristatin E (2a) and
desmethylauristatin E (2b) as well as the 10 to 100 times increase
in inhibition provided by quinstatin 2 as compared to auristatin
2-AQ.3a In these experiments quinstatin 8 delivered cancer
inhibitions close to the remarkably powerful dolastatin 10 level
(0.006 nM). Presently our group is continuing research directed
at further probing requirements for increasing the therapeutic
range of dolastatin 10 while retaining its powerful anticancer
activity and a more favorable therapeutic index.
Scheme 1. Synthesis of Quinstatin 2 (3)
EXPERIMENTAL SECTION
■
General Experimental Procedures. Both N-Boc-dolaproine and
Dov-Val-Dil·TFA were synthesized as described earlier.6,7 2-(1′-Ethyl-
2′-amino)quinoline dihydrochloride and 6-(1′-acetic acid)quinoline
were purchased from J&W Pharmlab LLC and Astatech, Inc.,
B
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Table 1. Human Cancer Cell Line (GI50 μg/mL [nM]) Growth Inhibition Results from Comparison Experiments
a
cell line
compound
BXPC-3
MCF-7
SF-268
NCI-H460
KM20L2
DU-145
dolastatin 101a (1)
quinstatin 2 (3)
quinstatin 3
0.000040 [0.051]
0.0093 [12.36]
0.021 [27.9]
0.0000042 [0.005]
0.00031 [0.412]
0.00023 [0.305]
0.00030 [0.398]
0.000073 [0.097]
0.00040 [0.532]
0.000030 [0.039]
0.000040 [0.053]
0.00017 [0.232]
0.00029 [0.404]
0.0000043 [0.006]
0.00023 [0.31]
0.00018 [0.229]
0.0051 [6.77]
0.031 [41.2]
0.0000080 [0.010]
0.00033 [0.438]
0.00040 [0.532]
0.00032 [0.425]
0.00023 [0.305]
0.00039 [0.518]
0.000041 [0.054]
0.000040 [0.053]
0.00036 [0.492]
0.00043 [0.599]
0.0000052 [0.007]
0.00031 [0.412]
0.00020 [0.266]
0.00023 [0.305]
0.00013 [0.172]
0.00030 [0.399]
0.000030 [0.040]
0.000040 [0.053]
0.00031 [0.424]
0.00030 [0.418]
0.00029 [0.385]
0.00019 [0.252]
0.00011 [0.146]
0.00030 [0.399]
0.000024 [0.032]
0.000023 [0.030]
0.00036 [0.492]
0.00031 [0.432]
quinstatin 4
0.0044 [5.84]
0.0021 [2.79]
0.0020 [2.66]
0.0031 [4.12]
0.00050 [0.425]
>0.001 [>1.37]
>0.001 [>1.39]
0.0043 [5.71]
0.0048 [6.37]
0.0043 [5.71]
0.011 [14.61]
0.00090 [1.19]
0.00039 [0.533]
0.00049 [0.683]
quinstatin 5
quinstatin 6 (4)
quinstatin 7
quinstatin 8
auristatin E (2a)
desmethylauristatin E
(2b)
a
Cancer cell lines in order: pancreas (BXPC-3); breast (MCF-7); CNS (SF-268); lung (NCI-H460); colon (KM20L2); prostate (DU-145).
respectively, and were used as received. 3-(1′-Acetic acid)quinoline,
4-(1′-ethyl-2′-amino)quinoline, 5-(1′-ethyl-2′-amino)quinoline, and
7-(1′-acetic acid)quinoline were purchased from Princeton Bimolecular
Research, Inc., and 8-(1′-ethyl-2′-amino)quinoline was purchased from
Fischer Scientific; all were used as received. Other reagents including
diethyl cyanophosphonate (DEPC) and anhydrous solvents were
purchased from Acros Organics (Fisher Scientific) and Sigma−Aldrich
Chemical Co. and were used as received.
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 [α]D values are given
in 10−1 deg cm2 g−1. The 1H and 13C NMR spectra were recorded on
Varian Unity INOVA 400 and 500 and Bruker 400 instruments with
deuterated solvents. High-resolution mass spectra were obtained using a
JEOL LCMate instrument in the Arizona State University CLAS High
Resolution Mass Spectroscopy Laboratory and provided by Dr. John C.
Knight and Natalya Zolotova. For thin-layer chromatography, Analtech
silica gel GHLF Uniplates were used and visualized with short-wave UV
irradiation and an iodine chamber. For column chromatography, silica
gel (230−400 mesh ASTM) from E. Merck (Darmstadt, Germany) was
employed.
dissolved in anhydrous CH2Cl2 (5 mL), and the solution was stirred
under N2 and cooled to 0 °C. TEA (0.12 mL, 0.86 mmol) and DEPC
(0.035 mL, 0.21 mmol) were added, and the mixture was stirred under
N2 for 18 h with warming to room temperature (rt). The reaction
mixture was concentrated under reduced pressure and separated on a
silica gel column. Elution with CH2Cl2−MeOH (95:5) gave the product
as a colorless foam (74 mg, 62%): TLC Rf 0.3 (CH2Cl2−CH3OH 7%);
[α]22 −8.2 (c 0.27, CHCl3); conformational isomers are present3a,7
D
(∼2:1), and doubling of signals was observed in both the proton and
carbon NMR spectra for 3; 1H NMR (CDCl3, 400 MHz) major
conformer δ 8.08 (1H, d, J = 8.8 Hz), 8.02 (1H, d, J = 8.4 Hz), 7.79 (1H,
d, J = 7.0 Hz), 7.70 (1H, t, J = 8.0 Hz), 7.51 (1H, t, J = 6.8 Hz), 7.33 (1H,
d, J = 9.0 Hz), 7.16 (1H, m), 6.88 (1H, m), 4.80, 4.78 (1H, d, J = 6.9,
9 Hz), 4.12−4.04 (2H, m), 3.89−3.74 (3H, m), 3.43−3.25 (8H, m),
3.33, 3.27 (s, OCH3), 3.23−3.15 (3H, m), 3.00 (3H, s), 2.48−2.22 (9H,
m), 2.20−1.84 (5H, m), 1.73 (1H, m), 1.54 (1H, m) 1.43−1.32 (1H,
m), 1.25−1.20 (3H, m), 1.06−0.88 (16 H, m), 0.85−0.79 (3H, m); 13C
NMR (CDCl3, 400 MHz) two conformers, δ 174.1, 173.7, 173.4, 171.8,
170.2, 170.16, 160.5, 160.1, 147.9, 147.8, 136.9, 136.5, 129.9, 129.6,
129.0, 128.9, 127.8, 127.7, 127.0, 126.9, 126.4, 126.1, 121.9, 121.8, 86.2,
82.2, 78.4, 76.6, 61.7, 60.5, 59.2, 58.3, 58.1, 53.9, 53.7, 47.7, 46.6, 45.2,
44.2, 43.0, 38.7, 38.0, 37.9, 37.6, 37.3, 33.3, 31.1, 27.8, 26.1, 25.8, 25.1,
24.9, 23.5, 20.3, 20.0, 17.9, 17.8, 14.4, 10.9, 10.5 ppm; (+)-HRAPCIMS
m/z 753.5292 [M + H]+ (calcd for C42H69N6O6, 753.5279).
Quinstatin 2 (3). 2-(1′- Ethyl-2′-amido-Boc-Dap)quinoline (5). To
a stirred solution of Boc-Dap6 (0.06 g, 0.21 mmol) and 2-(1′-ethyl- 2′-
amino)quinoline dihydrochloride (0.08 g, 0.32 mmol) in anhydrous
CH2Cl2 (1 mL) at 0 °C under N2 were added triethylamine (TEA)
(0.2 mL, 1.43 mmol) and DEPC (0.1 mL, 0.6 mmol). The reaction mix-
ture was stirred at 0 °C with warming to rt for 6 h and then concentrated
under reduced pressure to an orange-colored residue that was purified
by chromatography on a silica gel column. Gradient elution with
hexanes 100% to 7:2 hexanes−acetone gave the product as an off-white
waxy solid, 112 mg (79%): TLC Rf 0.5 (3:4 hexanes−acetone); doubling
Quinstatin 3. 3-(1′-Ethyl-2′-hydroxy)quinoline. To a stirred
solution containing 3-(1′-acetic acid)quinoline (0.03 g, 0.16 mmol) in
anhydrous tetrahydrofuran (THF) (2 mL) at 0 °C was added dropwise
LiAlH4 (1 M solution in THF, 0.2 mL, 0.3 mmol, 1.25 equiv). The
reaction mixture was stirred at 0 °C for 1 h and dampened with the
successive addition of water (8 μL), 15% NaOH (8 μL), and again water
(24 μL), prior to being stirred for 45 min at 0−5 °C and drying
(Na2SO4). The solution was filtered to remove the precipitated solids,
and the filtrate was concentrated under reduced pressure to give a yellow
oil, which solidified on standing to a waxen solid (15.8 mg, 57%): TLC
1
13
of signals observed in the H and
C NMR data indicating con-
formational isomers in almost 1:1 ratio; 1H NMR (CDCl3, 400 MHz) δ
8.01 (1H, d, J = 8 Hz), 7.95 (1H, d, J = 8 Hz), 7.72 (1H, d, J = 8 Hz), 7.63
(1H, t, J = 8 Hz), 7.43 (1H, t, J = 8 Hz), 7.23 (1H, d, J = 8 Hz), 6.99, 6.85
(1H, br s), 3.83−3.56 (4H, m), 3.46−3.34 (1H, m), 3.28 (4H, m), 3.18−
3.01 (2H, m), 2.36−2.15 (1H, m), 1.83−1.47 (4H, m), 1.37, 1.41 (9H, s,
t-Bu), 1.12−1.09 (3H, m); 13C NMR (CDCl3, 400 MHz) δ 174.1, 173.6,
160.1 154.6, 154.3, 147.6, 136.5, 129.6, 128.7, 127.6, 126.8, 126.1, 121.7,
83.9, 82.3, 79.6, 79.0, 60.7, 60.5, 58.7, 46.8, 46.5, 44.2, 43.9, 38.3, 38.1,
37.5, 37.3, 28.5, 25.7, 25.3, 252, 24.4, 23.9, 14.1, 14.05; (+)-HRAPCIMS
m/z 442.2707 [M + H]+ (calcd for C25H36N3O4 442.2706).
1
Rf 0.17 (CH2Cl2−MeOH 3%); H NMR (CDCl3, 400 MHz) δ 8.71
(1H, s), 8.01 (1H, d, J = 7.5 Hz), 7.96 (1H, s), 7.70 (1H, d, J = 8.4 Hz),
7.62 (1H, t, J = 7.5 Hz), 7.48 (1H, t, J = 7.5 Hz), 3.97 (2H, t, J = 6.4 Hz),
3.01 (2H, t, J = 6.4 Hz); 13C NMR (CDCl3, 100 MHz) δ 151.9, 146.6,
135.5, 131.8, 128.9, 128.8, 128.0, 127.4, 126.7, 62.9, 36.5.
3-(1′-Ethyl-2′-bromo)quinoline. A stirred solution of 3-(1′-ethyl-2′-
hydroxy)quinoline (0.15 g, 0.86 mmol) in anhydrous benzene
(20.0 mL) was heated gently to effect a solution and then cooled to
0 °C. Phosphorus tribromide (0.2 mL, 2.4 g, 2.1 mmol, 2.4 equiv) in
benzene (1.5 mL) was added, and the mixture heated to 60 °C for
45 min, then cooled to 0 °C. Saturated aqueous NaHCO3 (6 mL) was
added until neutral pH. The mixture was extracted with ethyl acetate
(3 × 10 mL), and the combined organic extracts were washed with
water, dried (Na2SO4), and concentrated to give a yellow residue,
which was separated using silica gel column chromatography by
gradient elution of CH2Cl2 (100%) → CH2Cl2 (96%)−CH3OH (4%),
to yield the product as a yellow oil (68 mg, 34% yield): TLC Rf 0.54
2-(1′-Ethyl-2′-amido-Dap)quinoline trifluoroacetate salt (6). To a
stirred solution of compound 5 (0.070 g, 0.16 mmol) in CH2Cl2 (4 mL)
at 0 °C under N2 was added trifluoroacetic acid (TFA) (1 mL), and
stirring was continued for 1.5 h at 0 °C. The reaction was stopped and
concentrated under reduced pressure to remove CH2Cl2 and excess
TFA to yield a yellow oil. This material was used without further
purification for the next reaction.
2-(1′-Ethyl-2′-amido-Dap-Dil-Val-Dov)quinoline [quinstatin 2
(3)]. Amide 6 and Dov-Val-Dil·TFA7 (90 mg, 0.16 mmol) were
C
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(CH2Cl2 (96%)−CH3OH (4%); 1H NMR (CDCl3, 400 MHz) δ 8.79
(1H, s), 8.09 (1H, d, J = 8.8 Hz), 8.00 (1H, s), 7.80 (1H, d, J = 8.0 Hz)
7.63 (1H, t, J = 7.8 Hz) 7.54 (1H, t, J = 8 Hz), 3.66 (2H, t, J = 7.8 Hz),
3.36 (2H, t, J = 7.2 Hz); 13C NMR (CDCl3, 100 MHz) δ 151.6 (x2),
135.4, 131.7, 129.5, 129.4, 128.1, 127.7, 127.1, 36.6, 32.5.
3-(1′-Ethyl-2′-azido)quinoline. To a stirred solution containing
3-(1′-ethyl-2′-bromo)quinoline (0.023 g, 0.1 mmol) in anhydrous
dimethylformamide (DMF) (0.5 mL) was added NaN3 (15 mg,
0.23 mmol, 2.3 equiv). The reaction mixture was heated to 90 °C for
30 min, cooled, diluted with ethyl acetate (3 mL), washed with water
(3 mL) and brine (3 mL), dried (MgSO4), and concentrated to give an
azide as a brown oil, 15.5 mg, 79% yield: TLC Rf 0.50 (CH2Cl2 (97%)−
CH3OH (3%)); 1H NMR (CDCl3, 400 MHz) δ 8.79 (1H, s), 8.13 (1H,
d, J = 8 Hz), 8.07 (1H, d, J = 8 Hz), 7.67 (1H, s), 7.59 (1H, d, J = 8.8 Hz),
7.41 (1H, dd, J = 8, 4 Hz), 3.63 (2H, t, J = 7 Hz), 3.09 (1H, t, J = 7 Hz);
13C NMR (CDCl3, 100 Hz) δ 150.2, 147.4, 137.8, 136.4, 135.7, 130.6,
129.8, 127.1, 121.3, 52.2, 35.3.
dichloromethane and washing with water, drying (Na2SO4), and
concentrating to an off-white glass solid (powder when scratched),
10 mg (29% yield): TLC Rf 0.14 (hexanes−acetone, 1:1); [α]22D −22.6
1
(c 0.5, CHCl3); H NMR (CDCl3, 400 MHz) δ 8.75 (1H, br s), 8.03
(1H, d, J = 8 Hz), 7.98 (1H, s), 7.74 (1H, d, J = 8 Hz), 7.63 (1H, t, J =
8 Hz), 7.50 (1H, t, J = 7.6 Hz), 7.27 (1H, m), 6.90 (1H, nm), 4.73 (1H, t,
J = 8 Hz), 3.95 (1H, m), 3.84 (1H, m), 3.78 (1H, d, J = 9 Hz), 3.75−3.47
(2H, m), 3.38−3.14 (8H, m), 3.38, 3.21 (s, OCH3), 3.13−2.91 (6H, m),
2.64−2.47 (6H, br s), 2.28 (1H, m), 2.17−1.70 (6H, m), 1.64 (2H, m),
1.35−1.11 (6H, m), 1.07−0.81 (16H, m), 0.81−0.68 (3H, t, J = 7.2 Hz);
13C NMR (CDCl3, 100 MHz) δ 174.7, 173.0, 170.6, 152.2, 147.0, 135.5,
135.47, 132.4, 131.l, 129.2, 129.1, 128.3, 127.7, 127.3, 127.2, 127.0, 81.8,
70.0, 77.43, 61.0, 59.9, 58.5, 58.1, 54.5, 47.9, 46.0, 45.0, 40.5, 37.5, 33.2,
32.9, 31.2, 30.6, 29.9, 28.2, 26.0, 25.2, 24.9, 19.9, 19.6, 18.6, 18.4, 16.1,
15.5, 11.0, 8.8; (+)-HRAPCIMS mlz 753.5282 [M + H]+ (calcd for
C42H69 N6O6, 753.5279).
Quinstatin 4. 4-(1′-Ethyl-2′-amido-Boc-Dap)quinoline. To a
stirred solution of Boc-Dap6 (0.1 g, 0.35 mmol) and 4-(1′-ethyl-2′-
amino)quinoline (0.06 mL, 0.38 mmol) in anhydrous CH2Cl2 (2 mL) at
0 °C under N2 was added TEA (0.15 mL, 1.08 mmol) and DEPC
(0.06 mL, 0.38 mmol, l. l equiv). The reaction mixture was stirred at 0 °C
with warming to rt for 18 h and then concentrated under reduced
pressure to a brown oil, which was purified by chromatography on silica
gel (eluents: CH2Cl2−MeOH 2−4%) to provide the desired product as
a colorless, waxy solid (0.096 g, 62%): TLC Rf 0.2 (CH2Cl2−MeOH
3-(1′-Ethyl-2′-amino)quinoline. To a stirred solution of the
preceding azide (0.015 g, 0.075 mmol) in anhydrous THF (1 mL)
cooled to 0 °C under N2 was added dropwise LiAlH4 (1 M solution in
THF, 0.1 mL, 0.1 mmol, 1.3 equiv). Stirring at 0 °C was continued for
45 min. The reaction was completed by the dropwise addition of H2O
(4 μL), 15% NaOH (4 μL), and H2O (12 μL) successively, and stirring
was continued for 30 min. The mixture was diluted with ethyl acetate
(5 mL) and dried (Na2SO4), and the product solution filtered. The filter
cake was washed with ethyl acetate (5 mL), and the organic layers were
combined, concentrated, and dried under reduced pressure to give a
1
4%); H NMR (CDCl3, 400 MHz) δ 9.14 (1H, s), 7.89 (1H, d, J =
8.4 Hz), 7.69 (1H, d, J = 8.4 Hz), 7.62 (1H, t, J = 6.4 Hz), 7.50 (1H, t, J =
7.6 Hz), 7.47 (1H, s), 6.76, 6.54 (1H, br s, NH), 3.86−3.57 (4H, m),
3.45−3.22 (4H, m), 3.17−3.02 (3.5H, m), 2.77 (0.5 H, br s), 2.33−2.14
(l H, m), 1.83−1.33 (13H, m), 1.15 (3H, m); 13C NMR (CDCl3,
100 MHz) δ 174.2, 173.6, 154.8, 154.5, 152.8, 152.2, 136.5, 130.8, 130.6,
127.6, 127.4, 127.1, 126.9,126.3, 119.3, 84.0, 82.3, 79.9, 79.3, 60.9, 60.7,
59.1, 58.8, 47.0, 46.6, 44.3, 39.5, 39.4, 36.9, 36.7, 28.8, 28.7, 25.8, 25.4,
24.6, 24.1, 14.4; (+)-HRAPCIMS m/z 442.2716 [M + H]+ (calcd for
C25H36N3O4, 442 0.2706).
4-(1′-Ethyl-2′-amido-Dap)quinoline trifluoroacetate salt. To a
stirred solution of the preceding amide (0.070 g, 0.16 mmol) in CH2Cl2
(2 mL) at 0 °C under N2 was added TFA (0.5 mL), and stirring was
continued for 2 h at 0 °C. The reaction mixture was concentrated under
reduced pressure to yield a yellow oil. This was dried under high vacuum
for a further 18 h and used without further purification for the next
reaction.
4-(1′-Ethyl-2′-amido-Dap-Dil-Val-Dov)quinoline (quinstatin 4).
The preceding TFA salt and Dov-Val-Dil.TFA7 (83 mg, 0.16 mmol)
were dissolved in anhydrous CH2Cl2 (2 mL), and the solution was
stirred under N2 and cooled to 0 °C. TEA (0.12 mL, 0.86 mmol) and
DEPC (0.04 mL, 0.26 mmol, 1.7 equiv) were added, and the mixture was
stirred under N2 at 0 °C for 8 h. The reaction mixture was concentrated
under reduced pressure and separated by column chromatography
(eluent: gradient elution CH2Cl2−MeOH 5−10%) to give the product
as a colorless glass, 48 mg (42%): TLC Rf 0.075 (CH2Cl2−CH3OH 5%);
[α]22D −6.9 (c 0.36, CHCl3); 1H NMR (CDC13, 400 MHz) δ 9.15 (1H,
br s), 7.90 (1H, d, J = 8 Hz), 7.70 (1H, d, J = 8 Hz), 7.63 (1H, m), 7.55−
7.48 (2H, m), 6.95−6.88 (2H, m), 4.88, 4.74 (1H, dd, J = 8.4, 6 Hz), 4.02
(1H, m), 3.82 (1H, dd, J = 8, 2 Hz), 3.74−3.60 (3H, m), 3.32 (2H, d, J =
4 Hz), 3.29 (3H, OCH3), 3.23 (3H, OCH3), 3.15−3.10 (3H, m), 2.96
(3H, br s), 2.43−2.30 (3H, m), 2.23 (6H, s), 2.16−1.86 (5H, m), 1.64
(2H, m), 1.43−1.25 (2H, m), 1.21−1.03 (4H, m), 1.05−0.82 (16 H, m),
0.81−0.76 (3H, m); 13C (CDCl3, 100 MHz), δ 174.3, 173.9, 170.3,
170.2, 153.0, 152.7, 152.4, 152.3, 136.6, 130.9, 130.6, 127.6, 127.5, 127.4,
127.2, 126.9, 126.3, 119.4, 119.3, 86.2, 82.1, 76.6, 61.8, 60.6, 59.4, 59.2,
58.3, 58.1, 53.9, 47.8, 46.7, 45.2, 44.4, 42.9, 39.6, 39.2, 37.6, 37.2, 36.5
36.0, 33.3, 31.1, 31.06, 29.8, 27.8, 26.0, 25.9, 25.1, 24.9, 23.5, 20.3, 20.0,
17.97, 17.94, 16.0, 15.5, 14.7, 10.9, 10.5; (+)-HRAPCIMS m/z 753.5275
[M + H]+ (calcd for C42H69N60O6, 753.5279).
1
yellow oil (11 mg, 84%): H NMR (CDCl3, 400 MHz) indicated a
mixture of the desired amine along with a dihydroquinoline based on the
observed upfield shift of the aromatic ring signals in the proton
spectrum. The material was carried forward as this mixture to coupling
with Boc-Dap.
3-(1′-Ethyl-2′-amido-Boc-Dap)quinoline. To a solution of the
preceding amine mixture (0.04 g, 0.23 mmol) in anhydrous CH2Cl2
(1 mL) was added a solution of Boc-Dap6 (0.06 g, 0.22 mmol) in
anhydrous CH2Cl2 (1 mL), and the resulting mixture cooled to 0 °C.
Triethylamine (0.2 mL, 1.43 mmol) and DEPC (0.12 mL, 0.8 mmol)
were added, and the solution was stirred at 0 °C with warming to rt for
6 h. Concentration under reduced pressure led to a dark orange oil,
which was separated by silica gel chromatography. Elution with the
gradient 100% hexanes → 1:1 → 2:3 hexanes−acetone yielded a dark
orange oil, 100 mg. Purification was achieved using flash silica gel
chromatography by gradient elution of hexanes 100% → hexanes−
1
acetone (1:4) to give a frothy product (20 mg, 21% yield): H NMR
(CDCl3, 400 MHz) δ 8.77 (1H, s), 8.06 (1H, d, J = 8.6 Hz), 7.98 (1H, br
s), 7.74 (1H, d, J = 8 Hz), 7.66 (1H, t, J = 7.4 Hz), 7.52 (1H, t, J =
7.4 Hz), 6.59 (1H, br s), 5.85 (1H, br s), 3.81−3.41 (4H, m), 3.34 (3H,
s), 3.20−3.08 (2H, m), 3.06−2.99 (2H, m), 2.28 (1H, m), 1.83−1.70
(2H, m), 1.64−1.54 (2H, m), 1.50−1.38 (9H, m), 1.22−1.13 (3H, m);
13C NMR (100 MHz, CDCl3) δ 174.8, 174.2, 155.1, 151.9, 147.1, 135.4,
132.2, 129.3, 128.2, 127.6, 127.0, 84.1, 82.2, 80.0, 79.7, 61.0, 59.4, 58.7,
53.7, 47.1, 46.7, 44.7, 44.2, 33.0, 28.8, 26.0, 25.2, 24.8, 24.3, 15.1, 14.3;
(+)-HRAPCIMS m/z 442.2707 [M + H]+ (calcd for C25H36N3O4,
442.2706).
3-(1′-Ethyl-2′-amido-Dap)quinoline trifluoroacetate salt. To a
stirred solution of the preceding amide (0.02 g, 0.04 mmol) in
anhydrous CH2Cl2 (1.5 mL) at 0 °C under N2 was added TFA (0.3 mL,
0.2 g, 1.76 mmol, 44 equiv). The solution was stirred for 1.5 h at 0 °C
and then concentrated and dried under reduced pressure to give a
residue, which was used immediately in the next reaction.
3-(1′-Ethyl-2′-amido-Dap-Dil-Val-Dov)quinoline (quinstatin 3).
To a stirred solution of the preceding TFA salt (0.015 g, 0.04 mmol)
and Dov-Val-Dil TFA7 (0.025 g, 0.04 mmol) in anhydrous CH2Cl2
(3 mL) at 0 °C under N2 was added TEA (0.03 mL) followed by DEPC
(0.011 mL), and the solution was stirred at 0 °C for 3 h. The solvent was
removed at room temperature under reduced pressure to give a yellow
oil, which was separated by chromatography on a silica gel column
eluting with a gradient of CH2Cl2−MeOH 4% to CH2Cl2−MeOH 12%,
which gave the product in the final fractions as an oil, 30 mg, containing
contaminants. These were removed by extracting the product into
Quinstatin 5. 5-(1′-Ethyl-2′-amido-Boc-Dap)quinoline. To a
stirred solution of Boc-Dap6 (0.1 g, 0.35 mmol) and 5-(1′-ethyl-2′-
amino)quinoline (0.06 mL, 0.38 mmol) in anhydrous CH2Cl2 (2 mL) at
0 °C under N2 were added TEA (0.15 mL, 1.08 mmol) and DEPC
(0.06 mL, 0.38 mmol, 1.1 equiv). The reaction mixture was stirred at
D
DOI: 10.1021/acs.jnatprod.6b01006
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Journal of Natural Products
Article
0 °C with warming to rt for 18 h and then concentrated under reduced
pressure to a light brown oil, which was purified by chromatography on
silica gel (eluent: CH2Cl2−CH3OH 2−4%) to provide the desired
6-(1′-Ethyl-2′-azido)quinoline (9). To a stirred solution of bromide
8 (0.12 g, 0.5 mmol) in anhydrous DMF (3 mL) was added NaN3
(0.12g, 1.84 mmol, 3.7 equiv). The reaction mixture was heated to 90 °C
for 30 min, cooled, diluted with ethyl acetate (30 mL), washed with
water (30 mL) and brine (30 mL), dried (MgSO4), and concentrated to
product as a colorless waxen solid (0.101 g, 66% yield): [α]22 −3.5
D
(c 4.5, CH2Cl2); TLC Rf 0.38 (CH2Cl2−MeOH 5%); 1H NMR (CDCl3,
400 MHz) δ 8.81 (1H, s), 8.50 (1H, d, J = 9.2 Hz), 7.91(1H, d, J =
8.4 Hz), 7.54 (1H, t, J = 7.1 Hz), 7.39−7.29 (2H, m), 6.79, 6.28 (0.5 H,
br s, NH), 3.79−3.65 (2H, m), 3.6−3.4 (2.5H, m), 3.34−3.09 (7H, m),
2.69 (1.5H, m), 2.3 (1H, m), 1.89−1.52 (4.5H, m), 1.50−1.33 (10H,
m), 1.25−1.09 (3.5H); 13C NMR (CDCl3, 100 MHz) confomers
observed, δ 174.8, 174.3, 155.0, 150.1, 148.7, 136.0, 135.8, 132.7, 129.1,
128.6, 128.4, 127.3, 127.2, 121.2, 84.1, 82.2, 79.9, 79.6, 60.9, 59.4, 59.35,
58.7, 47.0, 46.6, 44.6, 43.9, 40.7, 32.3, 32.2, 28.7, 28.6, 26.0, 25.3, 24.7,
24.2, 14.8, 14.4; (+)-HRAPCIMS m/z 442.2701 [M + H]+ (calcd for
C25H36N3O4, 442.2706).
1
give the azide 9 as a brown oil, 91 mg, 91% yield: H NMR (CDCl3,
400 MHz) δ 8.91 (1H, s), 8.13 (1H, d, J = 8 Hz), 8.07 (1H, d, J = 8 Hz),
7.67 (1H, s), 7.59 (1H, d, J = 8.8 Hz), 7.41 (1H, dd, J = 8, 4 Hz), 3.63
(2H, t, J = 7 Hz), 3.09 (1H, t, J = 7 Hz); 13C NMR (CDCl3, 100 Hz) δ
150.2, 147.4, 137.8, 136.4, 135.7, 130.6, 129.8, 127.1, 121.3, 52.2, 35.3.
6-(1′-Ethyl-2′-amino)quinoline (10). To a stirred solution of azide 9
(0.089 g, 0.45 mmol) in anhydrous THF (3 mL), cooled to 0 °C under
N2, was added dropwise LiAlH4 (1 M solution in THF, 0.75 mL,
0.75 mmol, 1.65 equiv). Stirring at 0 °C was continued for 45 min. The
reaction was completed by the dropwise addition of H2O (30 μL), 15%
NaOH (30 μL), and H2O (90 μL) successively, with stirring continued
for 20 min. The mixture was diluted with ethyl acetate (10 mL) and
dried (Na2SO4), and the solution filtered. The solid phase was washed
with ethyl acetate (5 mL), and the organic layers were combined,
concentrated, and dried under reduced pressure to give amine 8 as a
5-(1′-Ethyl-2′-amido-Dap)quinoline trifluoroacetate salt. To a
stirred solution of the preceding amide (0.070 g, 0.16 mmol) in CH2Cl2
(2 mL) at 0 °C under N2 was added TFA (0.5 mL), and stirring was
continued for 2 h at 0 °C. The reaction was stopped and concentrated
under reduced pressure to yield a yellow oil. After drying under high
vacuum for a further 18 h the TFA salt was used without further
purification in the next reaction.
1
yellow oil (quantitative yield): H NMR (CDCl3, 500 MHz) δ 8.88
(1H, m), 8.11 (1H, d, J = 8.3 Hz), 8.06 (1H, d, J = 8.7 Hz), 7.63 (1H, s),
7.59 (1H, d, J = 8.5 Hz), 7.39 (1H, dd, J = 8, 4 Hz), 3.09 (2H, t, J = 7 Hz),
2.95 (2H, t, J = 7 Hz), 1.81 (2H, br s); 13C NMR (CDCl3, 120 Hz) δ
149.9, 147.3, 137.6, 135.6, 131.0, 129.6, 126.9, 121.2, 43.2, 39.9.
6-(1′-Ethyl-2′-amido-Boc-Dap)quinoline (11). A solution of Boc-
Dap6 (0.153 g, 0.5 mmol) in anhydrous CH2Cl2 (1 mL) was added to a
solution of the amine 10 (0.083 g, 0.5 mmol) in anhydrous CH2Cl2
(3 mL), and the resulting mixture cooled to 0 °C. Triethylamine
(0.3 mL, 2.1 mmol) and DEPC (0.2 mL, 1.2 mmol) were added, and the
solution was stirred at 0 °C for 2 h. Concentration under reduced
pressure led to a dark orange oil, which was separated on a silica gel
column. Elution with a gradient 3:2 → 1:1 → 2:3 of hexanes−acetone
gave the desired amide as a colorless oil, 111 mg, 54% yield: TLC Rf 0.5
(hexanes−acetone, 1:1); 1H NMR (CDCl3, 400 MHz) δ 8.88 (1H, s),
8.10−8.04 (2H, m), 7.65−7.58 (2H, m), 7.39 (1H, m), 6.42, 5.73
(1H, br s, NH), 3.85−3.42 (4H, m), 3.35 (3H, s), 3.37−3.27 (1H, m),
3.19−3.10 (1H, m), 3.09−3.01 (2H, m), 2.40−2.21 (1H, m), 1.84−1.71
(3H, m), 1.53−1.42 (10H, m), 1.19 (3H, nm); (+)-HRAPCIMS m/z
442.2695 (M + H)+ (calcd for C25H36N3O4, 442.2706).
5-(1′-Ethyl-2′-amido-Dap-Dil-Val-Dov)quinoline (quinstatin 5).
The above TFA salt and Dov-Val-Dil.TFA7 (83 mg, 0.16 mmol) were
dissolved in anhydrous CH2Cl2 (2 mL), and the solution was stirred
under N2 and cooled to 0 °C. TEA (0.12 mL, 0.86 mmol) and DEPC
(0.04 mL, 0.26 mmol, 1.7 equiv) were added, and the mixture was stirred
under N2 at 0 °C for 7 h. The reaction mixture was concentrated under
reduced pressure and separated by column chromatography (eluent:
gradient elution CH2Cl2−MeOH 5−10%) to give the product as a
colorless glass, 41 mg (35%): TLC Rf 0.075 (CH2Cl2−CH3OH 5%);
[α]20D −34 (c 0.20, CHCl3); 1H NMR (CDCl3, 400 MHz) δ 8.86 (1H,
m), 8.55 (1H, d, J = 8.4 Hz), 7.94 (1H, d, J = 8 Hz), 7.57 (1H, t, J =
8 Hz), 7.43−7.37 (2H, m), 6.90 (2H, m), 4.73 (1H, dd, J = 8, 6.4 Hz),
4.06−3.94 (1H, m), 3.79 (1H, d, J = 8.6 Hz), 3.66−3.54 (1H, m), 3.53−
3.36 (2H, m), 3.35−3.21 (10H, m), 3.32, 3.25 (s, OCH3), 2.98 (3H, s),
2.47−2.29 (5H, m), 2.21 (6H, s), 2.09−1.83 (5H, m), 1.78 (3H, m),
1.37−1.26 (1H, m), 1.20 (3H, m), 1.01−0.82 (18H, m), 0.78 (3H, t, J =
5.2 Hz); 13C NMR (CDCl3, 100 MHz) δ 174.7, 171.9, 170.7, 150.2,
148.8, 136. l, 132.8, 129.1, 128.9, 128.6, 127.5, 127.3, 121.2, 82.0, 76.6,
60.9, 59.7, 58.1, 54.0, 47.9, 44.8, 43.0, 40.8, 37.5, 33.1, 32.3, 31.1, 27.8,
25.8, 25.1, 20.3, 17.9, 16.0, 15.1, 10.8; (+)-HRAPCIMS m/z 753.5274
[M + H]+ (calcd for C42H69N6O6, 753.5279).
Quinstatin 6 (4). 6-(1′-Ethyl-2′-hydroxy)quinoline (7). To a stirred
solution containing 6-(1′-acetic acid)quinoline (0.09 g, 0.48 mmol) in
anhydrous THF (9 mL) at 0 °C was added dropwise LiAlH4 (1 M
solution in THF, 0.6 mL, 0.6 mmol, 1.25 equiv).8 The reaction mixture
was stirred at 0 °C for 1 h and terminated by addition of H2O (0.02 mL),
15% NaOH (0.02 mL), and again H2O (0.06 mL), prior to stirring for
45 min at 0 °C and drying (Na2SO4). The solids were removed by
filtration, and the organic filtrate was concentrated and dried further
under reduced pressure to produce alcohol 7, a yellow oil, 72 mg, 86%
yield: 1H NMR (CDCl3, 400 MHz) δ 8.82 (1H, dd J = 1.6, 4.3 Hz), 8.08
(1H, d, J = 8 Hz), 8.00 (1H, d, J = 8 Hz), 7.64 (1H, br s), 7.58 (1H, dd,
J = 8, 1.8 Hz), 7.36 (1H, dd, J = 8.8, 4.4 Hz), 3.98 (2H, t, J = 6.5 Hz), 3.06
(1H, t, J = 6.5 Hz); 13C NMR (CDCl3, 100 MHz) δ 150.3, 147.4, 137.5,
135.9, 131.3, 129.7, 127.4, 125.7, 121.4, 63.4, 39.4.
6-(1′-Ethyl-2′-amido-Dap)quinoline trifluoroacetate salt (12). To
a stirred solution of amine 11 (0.1 g, 0.23 mmol) in anhydrous CH2Cl2
at 0 °C under N2 was added TFA (1 mL). The solution was stirred for
2 h at 0 °C and then concentrated under reduced pressure to give a
residue, which was dissolved in toluene and reconcentrated (×2). The
oily TFA salt was dried under reduced pressure and used immediately
for the next reaction.
6-(1′-Ethyl-2′-amido-Dap-Dil-Val-Dov)quinoline [quinstatin 6
(4)]. To a stirred solution of TFA salt 12 (0.96 mg) and Dov-Val-Dil
TFA7 (0.114 g, 0.21 mmol) in anhydrous CH2Cl2 (3 mL) at 0 °C
under N2 was added TEA (0.12 mL, 0.86 mmol) followed by DEPC
(0.035 mL, 0.21 mmol), and the solution stirred at 0 °C for 2 h. The
solvent was removed at room temperature under reduced pressure to
give a yellow oil, which was separated by chromatography on a short
silica gel column (7 × 3 cm), by eluting slowly with hexanes−acetone
(1:1), to give quinstatin 6 (4) as a yellow froth, 78 mg (47%): TLC Rf
1
0.23 (CH2Cl2−MeOH 6%); [α]22 −6.5 (c 0.34, CHCl3); H NMR
D
6-(1′-Ethyl-2′-bromo)quinoline (8).9 A solution of phosphorus
tribromide (0.9 mL, 5.6 mmol, 8 equiv) anhydrous in benzene
(1.0 mL) was added to a stirred solution of alcohol 7 (0.12 g, 0.67
mmol) in anhydrous benzene (2.0 mL) at 0 °C. The mixture was heated
to 60 °C for 45 min, then cooled to rt, and saturated aqueous NaHCO3
(100 mL) was added until a neutral pH was achieved. The mixture was
extracted with ethyl acetate (3 × 20 mL), and the combined organic
extracts were washed with water, dried (Na2SO4), and concentrated to
give the bromide as a yellow oil, 0.12 mg, 76% yield: 1H NMR (CDCl3,
400 MHz) δ 8.90 (1H, s), 8.13 (1H, d, J = 8.4 Hz), 8.08 (1H, d, J =
8.4 Hz), 7.66 (1H, s), 7.58 (1H, d, J = 8.4 Hz), 7.39 (1H, m), 3.65 (2H, t,
J = 7.2 Hz), 3.34 (1H, t, J = 7.2 Hz); 13C NMR (CDCl3, 400 MHz)
150.2, 147.4, 137.2, 135.7 × 2, 130.5, 129.8, 127.1, 121.3, 39.1, 32.5.
(CDCl3, 400 MHz) δ 8.88 (1H, dd, J = 4.4, 1.6 Hz), 8.10 (1H, d, J =
8 Hz), 8.03 (1H, d, J = 8.8 Hz), 7.66 (1H, s), 7.61 (1H, dd, J = 8, 2 Hz),
7.39 (1H, dd, J = 8.0, 4.0 Hz), 6.88 (1H, d, J = 10 Hz), 6.72 (1H, t, J =
4.4 Hz), 4.79 (1H, dd, J = 10, 6.8 Hz), 4.09−4.03 (1H, m), 3.98 (1H, m),
3.82 (1H, dd, J = 8.8 Hz, 1.6 Hz), 3.71 (1H, m), 3.59 (1H, m), 3.43−3.35
(2H, m), 3.33 (3H, s, OCH3), 3.28 (3H, s, OCH3), 3.07 (2H, t, J =
8 Hz), 3.03 (3H, s), 2.49−2.43 (1H, m), 2.40−2.31(2H, m), 2.28−2.24
(7H, m), 2.14−1.84 (5H, m), 1.66 (2H, m), 1.39−1.26 (2H, m), 1.24−
1.14 (4H, m), 1.06−0.92 (16H, m), 0.85−0.82 (3H, m); 13C NMR
(CDC13, 100 MHz) δ 174.4, 174.1, 173.5, 171.8, 170.5, 170.2, 150.l,
147.4, 137.9, 135.7, 131.3, 130.7, 129.9, 129.5, 128.4, 127.l, 121.6, 121.3,
86.0, 81.8, 78.6, 76.6, 61.7, 60.7, 59.6, 59.2, 58.3, 58.0, 59.9, 47.8, 45.0,
44.7, 43.0, 40.6, 37.5, 35.4, 33.1, 31.2, 27.8, 25.8, 25.0, 23.5, 20.3, 20.0,
E
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Journal of Natural Products
Article
19.6, 18.4, 17.9, 16.0, 15.7, 15.2, 10.9, 10.6; (+)-HRAPCIMS m/z
753.5279 [M + H]+ (calcd for C42H69N6O6, 753.5279).
Quinstatin 7. The general experimental procedure summarized
above for the synthesis of the 2′-ethylamine intermediate from the
corresponding 3- and 6-quinoline acetic acids was employed for
preparation of the amine intermediate required for the synthesis of
quinstatin 7.
7-(1′-Ethyl-2′-hydroxy)quinoline: yellow oil that solidified on
standing to a waxy solid, yield 86%; TLC Rf 0.25 (CH2Cl2−CH3OH
3%); 1H NMR (CDCl3, 400 MHz) δ 8.81 (1H, dd, J = 4.6, 1.7 Hz), 8.09
(1H, d, J = 8.5 Hz), 7.93 (1H, s), 7.73 (1H, d, J = 8.8 Hz), 7.42 (1H, dd,
J = 8.8, 0.16 Hz), 7.32 (1H, dd, J = 8.5, 4.6 Hz), 3.99 (2H, t, J = 6.0 Hz),
3.07 (2H, t, J = 6.0 Hz); 13C NMR (CDCl3, 100 MHz) δ 150.3, 140.7,
135.9 (×2C), 128.4, 127.8, 126.9, 120.6, 63.2, 39.4.
(3H, s), 3.02 (2H, t, J = 7.6 Hz), 2.97 (3H, br s), 2.40 (1H, d, J = 7.1 Hz),
2.34−2.26 (2H, m), 2.20 (7H, s), 2.09−1.71 (5H, m), 1.62 (2H, m),
1.30 (1H, m), 1.16 (3H, d, J = 7.2 Hz), 0.99−0.84 (16H, m), 0.76 (3H, t,
J = 7.9 Hz); 13C NMR (CDCl3, 100 MHz) δ 174.4, 173.6, 171.9, 171.6,
170.6, 170.2, 150.6, 148.5, 141.3, 140.6, 139.2, 135.9, 129.0, 128.7, 128.3,
128.0, 127.1,120.8, 81.9, 78.5, 76.6, 61.7, 60.7, 59.6, 58.1, 54.0, 47.8, 46.7,
44.6, 43.0, 42.9, 40.5, 37.6, 35.9, 33.2, 31.1, 27.8, 25.9, 25.0, 20.3, 19.9,
19.7, 18.4, 18.1, 17.9, 16.0, 15.7, 15.1, 10.9; (+)-HRAPCIMS m/z
753.5292 [M + H]+ (calcd for C42H69N6O6, 753.5279).
Quinstatin 8. 8-(1′-Ethyl-2′-amido-Boc-Dap)quinoline.
8-(1′-Ethyl-2′-amino)quinoline (0.1 mL, 0.7 mmol) was added to a
solution of Boc-Dap6 (0.2 g, 0.7 mmol) in anhydrous CH2Cl2 (3 mL),
and the reaction mixture was stirred at 0 °C. Next, TEA (0.3 mL,
2.15 mmol, 3 equiv) and DEPC (0.15 mL, 0.16 g, 0.98 mmol, 1.4 equiv)
were added. The reaction mixture was stirred at 0 °C for 7 h and
concentrated to an orange-colored oil, which was separated by silica gel
flash chromatography using CH2Cl2−MeOH 3.5%; column size 33 cm
× 2 cm. Fractions were combined according to TLC data. The product
was obtained as a colorless oil (139 mg, 0.32 mmol, 45%): TLC Rf 0.26
(CH2Cl2−CH3OH 3%); 1H NMR (400 MHz, CDCl3) δ 8.82 (l H, dd,
J = 4.0, 1.6 Hz), 8.06 (1H, d, J = 7.2 Hz), 7.61 (1H, d, J = 7.4 Hz), 7.49
(1H, d, J = 7.0 Hz), 7.37 (1H, t, J = 8.0 Hz), 7.31 (1H, m), 6.91 (1H, m),
3.70 (1H, m), 3.57 (3H, m), 3.44−3.30 (3H, m), 3.23 (3H, s, OCH3),
3.05 (1H, m), 2.22−1.96 (1H, m), 1.64 (2H, m), 1.54−1.30 (11H, m),
1.01 (3H, m); 13C NMR (100 MHz, CDCl3) (two conformers
observed) δ 174.2, 173.6, 154.6, 154.3, 149.4, 147.1, 138.4, 136.7, 136.6,
130.2, 128.5, 126.9, 126.8, 126.6, 121.0, 83.9, 82.3, 79.6, 79.0, 60.9, 60.8,
58.8, 53.5, 46.9, 46.5, 44.5, 44.0, 41.5, 41.3, 30.8,28.6, 25.6, 25.3, 24.4,
24.0, 14.3, 14.0; (+)-HRAPCIMS m/z 442.2703 [M + H]+ (calcd. for
C25H36N3O4, 442.2706).
7-(1′-Ethyl-2′-bromo)quinoline: colorless oil (66% yield); TLC Rf =
0.6 CH2Cl2−MeOH 3%; 1H NMR (CDCl3 400 MHz) δ 8.89 (1H, dd,
J = 4.0, 1.2 Hz), 8.81 (1H, d, J = 8.7 Hz), 7.98 (1H, s), 7.79 (1H, d, J = 8.7
Hz), 7.41 (2H, m), 3.65 (2H, t, J = 7.9 Hz), 3.36 (2H, t, J = 7.6 Hz); 13C
NMR (CDCl3, 400 MHz) δ 149.9, 147.3, 141.1, 136.8, 128.1, 128.06,
127.8, 127.2, 120,.9, 39.2, 32.2.
7-(1′-Ethyl-2′-azido)quinoline: brown oil, quantitative yield; 1H
NMR (CDCl3, 400 MHz) δ 8.88 (1H, dd, J = 4.0, 1.6 Hz), 8.11 (1H, d,
J = 8 Hz), 7.93 (1H, s), 7.76 (1H, d, J = 8 Hz), 7.40 (1H, dd, J = 8.8, 2
Hz), 7.35 (1H, dd, J = 8, 4 Hz), 3.61 (2H, t, J = 7.4 Hz), 3.09 (2H, t, J =
7.1 Hz); 13C NMR (CDCl3, 100 MHz) δ 150.9, 148.5, 139.9, 136.0,
128.9, 128.3, 128.0, 127.3, 121.1, 52.3, 35.7.
7-(1′-Ethyl-2′-amino)quinoline: yellow residue (91% yield); 1H
NMR (CDCl3, 400 MHz) δ 8.86 (1H, dd, J = 4.4, 1.8 Hz), 8.11 (1H, dd,
J = 8, 1.2 Hz), 7.9 (1H, s), 7.74 (1H, d, J = 8 Hz), 7.39 (1H, dd, J = 8,
1.5 Hz), 7.34 (1H, dd, J = 8, 4.4 Hz), 3.08 (2H, t, J = 6.8 Hz), 2.96 (2H, t,
J = 6.8 Hz), 1.63 (br s, NH2).
8-(1′-Ethyl-2′-amido-Dap)quinoline trifluoroacetate salt. A solu-
tion of the preceding amide (0.3 g, 0.68 mmol) in dry CH2Cl2 (10 mL)
was stirred at 0 °C under N2. TFA (1.0 mL, 1.49 g, 13 mmol, 19 equiv)
was added, and the reaction mixture stirred at 0 °C for 3 h and
monitored by TLC using CH2Cl2−MeOH 5% as solvent. The solvent
was removed under reduced pressure and with toluene as an azeotrope,
then dried using high vacuum for 16 h to yield a brown oil, which was
used without further purification for the next step.
7-(1′-Ethyl-2′-amido-Boc-Dap)quinoline. To a solution of the
preceding amine (0.06 g, 0.35 mmol) in anhydrous CH2Cl2 was
added a solution of Boc-Dap6 (0.1 g, 0.35 mmol) in anhydrous CH2Cl2
(1 mL), and the resulting mixture cooled to 0 °C. TEA (0.2 mL, 1.43
mmol, 4 equiv) and DEPC (0.185 mL, 0.199 g, 1.22 mmol, 3.5 equiv)
were added. The mixture was stirred at 0 °C for 8 h, then overnight at rt
for 24 h. The reaction mixture was next concentrated to a dark brown
solid, extracted with water, dried (Na2SO4), and concentrated.
Purification on silica gel flash chromatography using CH2Cl2−MeOH
5% gave the product as a yellow oil (65 mg, 43% yield based on Boc-
Dap): TLC Rf 0.4 (CH2Cl2−MeOH 5%); 1H NMR (CDCl3, 400 MHz)
δ 8.82 (1H, br s), 8.08 (1H, br d, J = 9 Hz), 7.87 (1H, s), 7.71 (1H, d, J =
8 Hz), 7.39 (1H, m), 7.31 (1H, m), 6.51, 5.99 (1H, br s, NH), 3.77 (1H,
m), 3.70 (1H, m), 3.66−3.52 (3H, m), 3.43−3.35 (1H, m), 3.29 (3H, s)
3.16−2.96 (2H, m), 2.33−2.15 (1H, m), 1.84−1.64 (2H, m), 1.63−1.51
(2H, m), 1.43, 1.39 (9H, s), 1.16−1.08 (3H, m); (+)-HRAPCIMS m/z
442.2696 [M + H]+ (calcd for C25H36N3O4, 442.2706).
7-(1′-Ethyl-2′-amido-Dap)quinoline trifluoroacetate salt. A solu-
tion of the preceding ethylamide (0.06 g, 0.136 mmol) in dry CH2Cl2
was stirred and cooled to 0 °C under N2. TFA (0.15 mL) was added, and
the solution stirred at 0 °C for 3 h. The solvent was removed under
reduced pressure using toluene as an azeotrope, to yield a residue, which
was further dried under high vacuum for 16 h. The salt was used as-is in
the next step.
7-(1′-Ethyl-2′-amido-Dap-Dil-Val-Dov)quinoline (quinstatin 7).
The preceding TFA salt (0.06 g) and Dov-Val-Dil-TFA7 (0.078 g,
0.143 mmol, 1 equiv) in anhydrous CH2Cl2 were stirred at 0 °C. TEA
(0.1 mL, 0.72 mmol, 5 equiv) and DEPC (0.022 mL, 1.43 mmol) were
added in succession, and the solution was stirred under argon for 4 h at
0 °C. The solvent was removed under reduced pressure, and the residue
separated by chromatography using flash silica gel, eluting with
CH2Cl2−MeOH 5%; column size 20 cm × 2 cm. The product was
obtained as a light yellow powder, 65 mg (61% yield based on the TFA
salt precursor): TLC Rf 0.27 (CH2Cl2−MeOH 5%); [α]23D −4.5 (c 0.2,
CH3OH); 1H NMR (CDCl3, 400 MHz) δ 8.82 (1H, br s), 8.07 (1H, d,
J = 8 Hz), 7.85 (1H, s), 7.71 (1H, d, J = 8.8 Hz), 7.41(1H, d, J = 8.3 Hz),
7.31(1H, dd, J = 8, 4.7 Hz), 6.88 (1H, d, J = 8.6 Hz), 6.71 (1H, m), 4.73
(1H, dd, J = 9.6, 6.8 Hz), 4.02 (1H, m), 3.95 (1H, m), 3.78 (1H, dd, J =
8.7, 1.6 Hz), 3.71−3.48 (3H, m), 3.33−3.28 (2H, m), 3.27 (3H, s), 3.22
8-(1′-Ethyl-2′-amido-Dap-Dil-Val-Dov)quinoline (quinstatin 8).
The preceding TFA salt (0.3 g, 0.66 mmol) was dissolved in dry
CH2Cl2 (10 mL) and stirred at 0 °C. Dov-Val-Dil-TFA7 (0.37 g,
0.66 mmol, 1 equiv) was added followed by TEA (0.5 mL, 3.6 mmol,
5 equiv) and DEPC (0.11 mL, 7.15 mmol, 11 equiv). The reaction
mixture was stirred under N2 for 4 h at 0 °C. Solvent was removed under
reduced pressure, and the residue was dried under high vacuum.
Chromatographic separation was achieved using flash silica gel, eluting
with CH2Cl2−MeOH 5%; column size 20 cm × 4 cm. This gave the
desired product as a light yellow powder (0.32 g, 64% yield): TLC Rf 0.4
(CH2Cl2−MeOH 5%); [α]23 −12.2 (c 0.5, CH3OH); 1H NMR
D
(CDCl3, 400 MHz), doubling of signals in the proton and carbon
spectra indicating the presence of two isomers, a pattern observed in
dolastatin 10 and discovered to be due to conformational isomers arising
from cis−trans isomerism at the Dil-Dap bond,3b δ 8.89 (1H, s), 8.16,
8.12 (1H, d, J = 8 Hz), 7.70, 7.66 (1H, d, J = 8 Hz), 7.57 (1H, d, J =
6.8 Hz), 7.48, 7.44 (1H, d, J = 8 Hz), 7.46−7.36 (1H, m), 6.96 (1H, t, J =
4.4 Hz), 6.86 (1H, d, J = 8.8 Hz), 4.85, 4.74 (1H, dd, J = 6.4, 9.6 Hz), 4.0
(1H, m), 3.80 (1H, dd, J = 7.6, 2.9 Hz), 3.72−3.56 (2H, m), 3.53−3.19
(11H, m), 3.30 (3H, s, OCH3), 3.27 (3H, s, OCH3), 2.96 (3H, br s),
2.50−2.32 (2H, m), 2.23−2.20 (7H, br s), 2.08−1.77 (5H, m), 1.69−
1.51 (2H, m), 1.31 (1H, m), 1.10 (2H, d, J = 7 Hz), 1.0−0.83 (17H, m),
0.77 (3H, t, J = 7.6 Hz); 13C NMR (CDCl3, 100 MHz) 174.1, 173.7,
173.4, 171.9, 170.2, 149.6, 149.59, 147.3, 147.2, 138.6, 138.5, 137.1,
136.8., 130.6, 130.3, 128.6, 127.2, 127.0, 126.9, 126.7, 121.3, 121.2, 86.3,
82.3, 78.2,76.6, 61.8, 60.5, 59.2, 59.1, 58.2, 58.1, 53.9, 47.7, 46.6, 44.3,
43.0, 41.9, 41.3, 37.7, 33.3, 31.1, 31.0, 30.9, 27.8, 25.9, 25.1, 24.8, 23.6,
20.3, 20.0, 19.7, 18.0, 17.9, 15.4, 14.2, 10.9, 10.4; (+)-HRAPCIMS m/z
753.5285 (M + H)+ (calcd for C42H69N6O6, 753.5279).
Cancer Cell Line Procedures. Inhibition of human cancer cell
growth was assessed using the standard sulforhodamine B assay of the
U.S. National Cancer Institute, as previously described.10 In summary,
F
DOI: 10.1021/acs.jnatprod.6b01006
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
cells in a 5% fetal bovine serum/RPMI1640 medium were inoculated in
96-well plates and incubated for 24 h. Next, serial dilutions of the
compounds were added. After 48 h, the plates were fixed with
trichloroacetic acid, stained with sulforhodamine B, and read with an
automated microplate reader. A growth inhibition of 50% (GI50 or the
drug concentration causing a 50% reduction in the net protein increase)
was calculated from optical density data with Immunosoft software.
K. P. M.; Boyd, M. R.; Bai, R.; Hamel, E.; Schmidt, J. M.; Chapuis, J.-C.
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A. H.; Shanker, S.; Song, C.; Sukuru, S. C. K.; Farley, K. A.; Wagenaar, M.
M.; Shapiro, M. J.; Musto, S.; Lam, M.-H.; Loganzo, F.; O’Donnell, C. J.
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website at DOI: 10.1021/acs.jnatprod.6b01006.
■
S
1H and 13C NMR spectra of quinstatins 2 (3), 3−5, 6 (4),
7, and 8 along with the HRMS of 6-(1′-ethyl-2′-amido-
Boc-Dap)quinoline (11) and quinstatin 8 (PDF)
AUTHOR INFORMATION
Corresponding Author
*Tel: (480) 965-3351. Fax: (480) 965-2747. E-mail: bpettit@
asu.edu.
(5) Doronina, S. O.; Toki, B. E.; Torgov, M. Y.; Mendelsohn, B. A.;
Cerveny, C. G.; Chace, D. F.; DeBlanc, R. L.; Gearing, R. P.; Bovee, T.
D.; Siegall, C. B.; Francisco, J. A.; Wahl, A. F.; Meyer, D. L.; Senter, P. D.
Nat. Biotechnol. 2003, 21 (7), 778−784.
(6) Pettit, G. R.; Singh, S. B.; Herald, D. L.; Lloyd-Williams, P.;
Kantoci, D.; Burkett, D. D.; Barkoczy, J.; Hogan, F.; Wardlaw, T. R. J.
Org. Chem. 1994, 59, 6287−6295. (b) Pettit, G. R.; Grealish, M. P. J. Org.
Chem. 2001, 66, 8640−8642. (c) Mordant, C.; Reymond, S.; Tone, H.;
Lavergne, D.; Touati, R.; Ben Hassine, B.; Ratovelomanana-Vidal, V.;
Genet, J.-P. Tetrahedron 2007, 63, 6115−6123.
(7) 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, 859−863.
■
ORCID
George R. Pettit: 0000-0001-8706-8929
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
With appreciation, we acknowledge and thank for financial
support the Arizona Biomedical Research Commission, the J.W.
Kiecknefer Foundation, and the Margaret T. Morris Foundation.
(8) (a) Micovic, V. M.; Mihailovic, M. L. J. Org. Chem. 1953, 18, 1190−
1200. (b) Newman, M. S.; Fukunaga, T. J. Am. Chem. Soc. 1960, 82,
693−696.
(9) (a) Albrecht, B. K.; Bellon, S.; Booker, S.; Cheng, A. C.; D’Amico,
D.; D’Angelo, N.; Kim, T.-S.; Liu, L.; Norman, M. H.; Siegmun, A. C.;
Stec, M.; Xi, N.; Yang, K.; WO 2008103277, 2008. (b) Baba, A.;
Kawamura, N.; Makino, H.; Ohta, Y.; Taketomi, S.; Sohda, T. J. Med.
Chem. 1996, 39, 5176−5182.
(10) Monks, A.; Scudiero, D.; Skehan, P.; Shoemaker, R.; Paull, K.;
Vistica, D.; Hose, C.; Langley, J.; Cronise, P.; Viagro-Wolff, A.; Gray-
Goodrich, M.; Campbell, H.; Mayo, J.; Boyd, M. J. Natl. Cancer Inst.
1991, 83, 757−766.
DEDICATION
■
In memory of Dr. Carl Djerassi (1923−2015), a most extra-
ordinarily productive professor of chemistry, humanitarian,
and mentor as well as discoverer of the first successful oral and
dedicated to Professor Phil Crews, of the University of California,
Santa Cruz, for his pioneering work on bioactive natural
products.
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