Isolation, structure elucidation, and synthesis of antiplasmodial quinolones from Crinum firmifolium

Article abstract of DOI:10.1016/j.bmc.2017.06.017

Antiplasmodial bioassay guided fractionation of a Madagascar collection of Crinum firmifolium led to the isolation of seven compounds. Five of the seven compounds were determined to be 2-alkylquinolin-4(1H)-ones with varying side chains. Compounds 1 and 4 were determined to be known compounds with reported antiplasmodial activities, while 5 was believed to be a new branched 2-alkylquinolin-4(1H)-one, however, it was isolated in limited quantities and in admixture and therefore was synthesized to confirm its structure as a new antiplasmodial compound. Along with 5, two other new and branched compounds 6 and 7 were synthesized as well. Accompanying the five quinolones were two known compounds 2 and 3 which are inactive against Plasmodium falciparum. The isolation, structure elucidation, total synthesis, and biological evaluation of these compounds are discussed in this article.

Full text of DOI:10.1016/j.bmc.2017.06.017

Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Isolation, structure elucidation, and synthesis of antiplasmodial
quinolones from Crinum firmifolium
Christopher C. Presley a, Yongle Du a, Seema Dalal b, Emilio F. Merino c, Joshua H. Butler c,
Stéphan Rakotonandrasana d, Vincent E. Rasamison d, Maria B. Cassera c, David G.I. Kingston a,
⇑
a Department of Chemistry and Virginia Tech Center for Drug Discovery, M/C 0212, Virginia Tech, Blacksburg, VA 24061, United States
b Department of Biochemistry and Virginia Tech Center for Drug Discovery, M/C 0308, Virginia Tech, Blacksburg, VA 24061, United States
c Department of Biochemistry and Molecular Biology, and Center for Tropical and Emerging Global Diseases (CTEGD), University of Georgia, Athens, GA 30602, United States
d Centre National d’Application des Recherches Pharmaceutiques, B.P 702, Antananarivo 101, Madagascar
a r t i c l e i n f o
a b s t r a c t
Article history:
Antiplasmodial bioassay guided fractionation of a Madagascar collection of Crinum firmifolium led to the
isolation of seven compounds. Five of the seven compounds were determined to be 2-alkylquinolin-4
(1H)-ones with varying side chains. Compounds 1 and 4 were determined to be known compounds with
reported antiplasmodial activities, while 5 was believed to be a new branched 2-alkylquinolin-4(1H)-one,
however, it was isolated in limited quantities and in admixture and therefore was synthesized to confirm
its structure as a new antiplasmodial compound. Along with 5, two other new and branched compounds
6 and 7 were synthesized as well. Accompanying the five quinolones were two known compounds 2 and
3 which are inactive against Plasmodium falciparum. The isolation, structure elucidation, total synthesis,
and biological evaluation of these compounds are discussed in this article.
Received 10 May 2017
Revised 6 June 2017
Accepted 12 June 2017
Available online xxxx
Keywords:
Antiplasmodial
Quinolone
Crinum
Amaryllidaceae
Ó 2017 Elsevier Ltd. All rights reserved.
1. Introduction
producing antimalarial medicines such as quinine and artemisinin,
our group is investigating plant extracts from the Natural Products
The genus Crinum (Amaryllidaceae) contains around 110
accepted species with over 270 synonyms.1 Members of this genus
possess large extravagant flowers on leafless stems and are dis-
tributed in moist sites, such as forests, river edges, seasonal pools,
or saltpans, and can be found throughout the tropics of Africa, Asia,
America, Southern Africa, Madagascar, and the Mascarene and
Pacific Islands.2 Extracts from Crinum species have been used tradi-
tionally to treat a variety of ailments including fever, pain manage-
ment, swelling, sores and wounds, cancer, and malaria.3 The genus
is a rich source of norbelladine type alkaloids such as lycorine, cri-
nine, and narciclasine alkaloids.4 Alkaloids isolated from various
Crinum species have shown activity in a wide variety of assays
including analgesic, anticancer, antibacterial, antiviral, antifungal,
and antimalarial assays.3
Discovery Institute (NPDI) collection for new compounds or known
compounds with new activity against the malaria parasite
Plasmodium falciparum. Recently, our group has had success with
isolating new potently antiplasmodial compounds from an extract
of Crinum erubescens L. f. ex Aiton.6 This prompted us to investigate
the Crinum species C. firmifolium in our International Cooperative
Biodiversity Group (ICBG) collection. Its extract was found to pos-
sess weak activity against P. falciparum (IC50 ꢀ 10 mg/mL), which
had increased activity in the mid-polarity fraction after a simple
liquid-liquid extraction (IC50 5 < x < 10 mg/mL). It was thus selected
for further investigation.
2. Results and discussion
Malaria is a vector-borne disease that has a disproportionate
effect in poor and underdeveloped countries without access to
western medicine, and with the appearance of artemisinin-resis-
tant parasites in 2008 there is an urgent need for new and afford-
able medicines.5 Since plants have been successful in the past with
2.1. Isolation
The ethanolic extract of C. firmifolium (2.1 g) was dissolved in
aqueous methanol and partitioned with hexanes. The aqueous
methanol fraction was then dried and suspended in water and
exhaustively extracted with EtOAc to afford an active EtOAc frac-
tion (286 mg, IC50 5 < x < 10 mg/mL). The EtOAc fraction was sepa-
rated further into four fractions via a C18 solid phase extraction
⇑
Corresponding author.
E-mail address: dkingston@vt.edu (D.G.I. Kingston).
http://dx.doi.org/10.1016/j.bmc.2017.06.017
0968-0896/Ó 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Presley C.C., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.06.017

2
C.C. Presley et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
(SPE) with different concentrations of aqueous methanol. The sec-
ond fraction (127 mg) was found to be active (IC50 ꢀ 1.25 mg/mL)
and was separated further into the ten fractions H1–H10 by C18
HPLC using an aqueous acetonitrile solvent system. Fraction H2
was found to have potent antiplasmodial activity (IC50 <<<
1.25 mg/mL), but after multiple attempts utilizing a variety of sta-
tionary and mobile phases only compound 1 could only be isolated,
and then only as the major component of fraction H2-4 containing
a mixture of similar compounds in a yield of 0.2 mg. The weakly
active fraction H3 (IC50 1.25 < x < 2.5 mg/mL) was subjected to
HPLC separation on a C18 column using aqueous MeOH, but only
the two inactive compounds 2 and 3 could be isolated; no active
compound was obtained from this fraction. Fraction H4 (IC50 <<<
1.25 mg/mL) was separated into the ten fractions H4-1–H4-10 by
HPLC on a C18 column with elution by aqueous MeOH. Fractions
H4-6 and H4-8 were both found to be highly active (IC50 <<<
1.25 mg/mL) but the only compound that could be isolated from
fraction H4-6 was 4 as the major component of a mixture of similar
compounds, and likewise compound 5 was the only compound
obtained from fraction H4-8 as the major component of a mixture
of similar compounds. The small amount of available extract pre-
vented additional isolation studies.
the 1H NMR spectrum of H2-4 in CD3OD matched those of an
authentic sample of HHQ.
Compound 2 (0.3 mg) was found to have the composition
C15H15NO3 from its HRESIMS. The spectrum also displayed a very
stable fragment resulting from the loss of water, indicating the
presence of a hydroxyl group. Two geminally coupled benzylic pro-
tons were observed in its 1H NMR spectrum at dH 4.28 (d,
J = 12.0 Hz, 1H) and dH 4.21 (d, J = 12.0 Hz, 1H), consistent with
the protons of a benzyl alcohol. The most shielded resonance
observed in its 1H NMR spectrum was at dH 2.75 (s, 3H) for an N-
methyl group. A methylenedioxy resonance was observed at dH
6.01 (s, 2H) along with two singlet aromatic protons at dH 7.03
(s, 1H) and dH 6.69 (s, 1H) which could indicate the presence of a
2,3,5,6-substituded aromatic ring. The remaining four aromatic
protons made up a 2,3-substituted aromatic coupling pattern
resulting from two doublet-like proton resonances at dH 7.00 and
dH 6.75 and two triplet-like proton resonances at dH 7.31, and dH
6.84, which support this claim. The identity of the isolated com-
pound was determined to be the known compound 2 based on a
comparison with literature data.8
Compound 3 (0.4 mg) was found to have the composition
C
16H11NO3 from its HRESIMS. Examining the 1H NMR spectrum
revealed a signal for a methoxy methyl group at dH 4.10 (s, 3H)
as the most shielded proton resonance. The remaining observable
signals were all deshielded aromatic proton signals. Two coupled
methine pyrrole-like double bond resonances at dH 8.06 (d,
J = 3.6 Hz, 1H) and dH 6.91 (d, J = 3.6 Hz, 1H) could be observed,
as well two singlets at dH 8.04 (s, 1H) and dH 7.79 (s, Hz, 1H), indi-
cating a 2,3,5,6-substituded aromatic ring. The remaining aromatic
resonances were all coupled to one another with resonances at dH
7.96 (d, J = 7.7 Hz, 1H), dH 7.75 (d, J = 7.7 Hz, 1H) and dH 7.49 (t,
J = 7.7 Hz, 1H), indicating a 4,5,6-substituted aromatic ring moiety.
A search of the literature revealed two possible structures that fit
the observed data, with the difference being the location of the
methoxy group. Both possible isomers of the compound have been
isolated, and the methoxy groups, as well as the singlets for posi-
tions H-8 and H-11, all have differing chemical shifts in the two
isomers.9 The resonances observed in the isolated molecule agree
with the published literature for a methoxy methyl in position
10 and confirm the structure of the isolated compound to be that
of pratorinine (3).10
2.2. Structure elucidation
The major component of H2-4 was determined to have the
composition C16H21NO from its HRESIMS. Its 1H NMR spectrum
in CD3OD showed strong signals for an aromatic moiety with four
protons at dH 8.21 (dd, J = 8.2, 1.5 Hz, 1H), dH 7.70 (ddd, J = 8.4, 7.0,
1.5 Hz, 1H), dH 7.58 (brd, J = 8.4 Hz, 1H), and dH 7.40 (ddd, 8.2, 7.0,
1.0 Hz, 1H) indicating the presence of a 1,2-disubstituted aromatic
ring. A single isolated a-proton was observed at dH 6.24 (s, 1H). In
the aliphatic region of the spectrum, a triplet-like multiplet at dH
2.78–2.65 (m, 2H) along with a pentet at dH 1.77 (p, J = 7.8 Hz,
2H), a broad signal at dH 1.49–1.24 (m, 8H), and a triplet at dH
0.90 (t, J = 6.9 Hz, 3H) all suggested the presence of an n-heptyl
alkyl chain. Comparison of literature data for compounds with
the observed chemical formula and 1H NMR signals identified the
major component of H2-4 as the known antiplasmodial compound
2-heptylquinolin-4(1H)-one (HHQ) (1, Fig. 1).7 The major peaks in
The major compound from fraction H4-6 (0.2 mg) was approx-
imately 76% pure as judged by its 1H NMR spectrum. It had the
composition C18H23NO as deduced from its HRESIMS, and its 1H
NMR spectrum showed signals for an alkenyl chain, with vinyl pro-
ton signals at dH 5.45–5.39 (m, 2H) and signals for the same 2-
alkylquinolin-4(1H)-one moiety as in 1. Comparison of these data
with literature data identified the compound as the known com-
pound acutine (4), or 2-[(4Z)hept-4-en-1-yl]quinolin-4(1H)-one.11
Acutine has been reported to have moderate antiplasmodial activ-
ity against the chloroquine-sensitive 3D7 strain of P. falciparum.11
The major compound from fraction H4-8 (approximately 50%
by 1H NMR spectroscopy) was also isolated in very limited quanti-
ties (0.2 mg). Its HRESIMS data indicated the chemical composition
O
O
O
O
HN
OH
N
N
H
ꢀ
HO
O
ꢂ
ꢁ
O
O
N
H
5
N
H
ꢃ
O
O
C
17H23NO, and its 1H NMR spectrum showed signals for the same
2-alkylquinolin-4(1H)-one moiety observed in 1 and 4. Examina-
tion of the aliphatic region of the spectrum revealed a methylene
multiplet at dH 2.73 (m, 2H) and a 6H methyl doublet at dH 0.88
(d, J = 6.6 Hz) that could be clearly distinguished from signals of
the other components; these signals indicated the presence of a
60-methylheptyl chain. The only compound in the literature with
the chemical formula C17H23NO containing a 2-alkylquinolin-4
(1H)-one moiety is 2-octylquinolin-4(1H)-one (8), so the major
compound from fraction H4-8 must be a new compound with
the structure 5 (Fig. 1). Since 5 could not be isolated in pure form,
its structure and biological activity were confirmed by synthesis.
N
H
N
H
7
6
O
N
H
8
Fig. 1. Structures 1 to 8. Compounds 1, 2, 3, 4, and 8 are known, and 5, 6, and 7 are
new compounds.
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C.C. Presley et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
3
The new analogues 6 and 7 and the known compound 8 were also
prepared for determination of basic structure-activity
relationships.
and 19–22 to afford the desired b – enaminoates, which were
cyclized with polyphosphoric acid without isolation to afford the
2-alkylquinolin-4(1H)-one title compounds 5–8 (Scheme 1).
2.3. Synthetic studies
2.4. Analysis
There are many synthetic routes in the literature for 2-
alkylquinolin-4(1H)-ones. The published procedures include the
Conrad – Limpach synthesis,12 the InCl3 – mediated solvent free
Conrad – Limpach reaction,13 and the microwave cyclization of b
– enaminoates,14 but none of these afforded the desired 2-
alkylquinolin-4(1H)-ones in our hands. However, InCl3 was found
to rapidly catalyze the formation of the intermediate b – enamino-
ates from aniline and the corresponding b – ketoesters,15 and
cyclization of the b – enaminoates to afford the desired 2-
alkylquinolin-4(1H)-ones was finally achieved with polyphospho-
ric acid.16
The long chain b – ketoesters needed for the synthesis of the b –
enaminoates were synthesized via alkylation of ethyl acetoacetate
with the corresponding alkyl iodides.17 Alkyl iodides 14–16 were
synthesized by standard routes starting from terminal alcohols
11–13; iodides 14 and 15 were obtained as racemates. Alcohols
12 and 13 were prepared from terminal alkenes 9 and 10 by reac-
tion with borane THF solution and hydrogen peroxide to afford the
corresponding primary alcohols, and alcohol 11 was purchased.
These three primary alcohols were reacted with triphenyl phos-
phine, imidazole, and iodine to afford the primary alkyl iodides
14, 15, and 16, and primary alkyl iodide 17 was purchased. The
dianion of ethyl acetoacetate 18 was produced with two equiva-
lents of LDA and then alkylated with halides 14–17 to prepare
the desired long chain ketoesters 19–22 in low to moderate yield.
Condensation reactions were then carried out with aniline, InCl3,
Once the total syntheses of 5–8 were complete and their struc-
tures were confirmed via NMR and HRESIMS, the identities of the
isolated natural product could finally be determined. The first anal-
ysis conducted was a 1H NMR comparison of the major compound
from fraction H4-8 with that of compound 5 to determine if they
were in fact the same compound. Comparison of the aromatic pro-
tons and the alpha proton were an exact match, and comparison of
the aliphatic methylene and methyl proton signals for 10,60 – Me,
and 70 were also an exact match (Fig. 2). Comparison of the 1H
NMR spectra of 5–8 with that of the major compound from fraction
H4-8 clearly indicated that among quinolones with the composi-
tion C17H23NO, synthetic 5 is the best match to the spectrum of
the isolated compound (Table 1 and Fig. 2).
The next comparison conducted was an HPLC analysis. The
method used for the isolation of the natural products did not give
baseline separation for all four synthetic compounds, so a new
method was developed utilizing a C12 column with aqueous MeCN
with 0.1% TFA. This method was also able to separate the impurity
from the quinolones isolated from the natural source. The flow rate
of 0.3 mL/min was so slow that very subtle differences could be
observed in retention times (tR in min) on repeat injections of
the same compound. To help account for the variance in retention
times the natural product was ‘‘bracketed” with two runs of the
synthetic compound with one before and the other after the natu-
ral product run. Comparison of the retention times for the syn-
thetic compound were tR 35.49 min for the run before and tR
PPh3, imidazole,
0 oC, 10 min
2:1 Et2O/MeCN
I2, 2 hr, RT
R1 R3
R1 R3
R1 R3
1. Borane THF complex
2. NaOH, 50% H2O2, 0 o
1.
2.
R4
HO
I
C
R2
R2
R2
1 = CH3, R2 = H, R3 = H, R4 = H, 62%
R1 = H, R2 = CH3, R3 = H, R4 = H, 67%
16 R1 = H, R2 = H, R3 = CH3, R4 = H, 68%
17 R1 = H, R2 = H, R3 = H, R4 = CH3 Purchased
14
15
11 R1 = CH3, R2 = H, R3 = H, Purchased
12
R
R1 = H, R2 = CH3, R3 = H, 63%
9 R1 = H, R2 = CH3, R3 = H
10 R1 = H, R2 = H, R3 = CH3
13 R1 = H, R2 = H, R3 = CH3, 67%
O
O
O
O
1. 2 eq LDA, THF; 0 oC; 1 h
T = -78 - 20 oC; 1 h
EtO
C
H
CH2
Li
EtO
Li
18
O
EtO
R1 R3
O
O
R1 R3
NH2
R4
R4
HN
EtO
InCl3, rt, 16 hr
R2
R2
19
20
R1 = CH3, R2 = H, R3 = H, R4 = H, 67%
R1 = H, R2 = CH3, R3 = H, R4 = H, 61%
21 R1 = H, R2 = H, R3 = CH3, R4 = H, 16%
22 R1 = H, R2 = H, R3 = H, R4 = CH3, 53%
Polyphosphoric acid (85% P2O5)
Additional P2O5, 110 oC, 24 hr
O
R1 R3
R4
N
H
R2
5
6
R1 = H, R2 = H, R3 = CH3, R4 = H, 18%
1 = CH3, R2 = H, R3 = H, R4 = H, 13%
R
7 R1 = H, R2 = CH3, R3 = H, R4 = H, 6%
8
R1 = H, R2 = H, R3 = H, R4 = CH3, 21%
Scheme 1. Synthesis of compounds 5–8.
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C.C. Presley et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Fig. 2. 1H NMR comparison of synthetic 5 with the small amount of the impure natural product 5 isolated from the plant in CD3OD.
Table 1
Key 1H and 13C NMR Chemical Shifts from 5–8 in CD3OD.
5
6
7
8
dH, mult. (J Hz)a
dC
dH, mult. (J Hz)a
dC
dH, mult. (J Hz)a
dC
dH, mult. (J Hz)a
dC
b
b
b
b
Posn
3
5
6
7
8
10
6.23, s
8.21, dd (8.2, 1.5)
7.39, ddd (8.2, 6.9, 0.9)
7.69, ddd (8.5, 6.9, 1.5)
7.58, br d (8.5)
2.75–2.69, m
–
–
108.8
126.0
125.1
133.4
119.1
35.0
–
6.23, s
8.21, dd (8.2, 1.5)
7.39, ddd (8.2, 7.0, 1.1)
7.69, ddd (8.4, 7.0, 1.5)
7.58, br d (8.4)
2.76–2.64, m
0.89, d (6.6)
–
108.8
126.0
125.1
133.4
119.1
35.3
19.9
–
6.24, s
8.21, dd (8.2, 1.5)
7.40, ddd (8.2, 6.9, 1.0)
7.69, ddd (8.4, 6.9, 1.5)
7.58, br d (8.4)
2.73 t (7.8)
–
0.84, d (6.0)
–
0.86, t (7.2)
–
108.8
126.0
125.1
133.4
119.1
35.0
–
19.5
–
11.7
–
6.24, s
8.21, brd (8.1)
7.39, brt (8.1)
7.68, ddd (8.4, 6.9, 1.4)
7.58, br d (8.4)
2.76–2.65, m
–
–
108.8
125.1
125.9
133.4
119.1
33.0
–
–
–
–
14.4
40-Me
50-Me
60-Me
70
–
0.88, d (6.6)
0.88, d (6.6)
–
23.0
23.0
–
–
–
14.7
–
–
–
0.88, t (7.1)
–
80
0.88, t (6.9)
a
Data (d) measured at 500 MHz; s = singlet, d = doublet, br d = broad doublet, t = triplet, brt = broad triplet, dd = doublet of doublets, ddd = doublet of doublets of doublets,
and m = multiplet. J values are in Hz and are omitted if the signals overlapped as multiplets.
b
Data (d) measured at 125 MHz.
35.81 min for the run after the natural product had been run, while
the natural product was found to elute at tR 35.93 min. Although
these retention times are not identical, this appears to be due to
small random variations in pumping speed. The combination of
identical key resonances in their 1H NMR spectra and identical
HPLC retention times within experimental error assures that the
isolated and synthetic compounds have the same structure.
nolones were also tested against the chloroquine-sensitive 3D7
strain of P. falciparum. Compounds 1, 5, 6, 7, and 8 had IC50 values
of 1.3 0.3 mM, 501 82 nM, 385 78 nM, 921 206 nM, and
3.8 0.6 mM respectively. The compounds were also tested against
the A2780 drug-sensitive mammalian cancer cell line. Alkaloids 2
and 3 were found to be inactive at all concentrations tested, with
IC50 values greater than 78 mM and 75 mM, respectively. The quino-
lones 1, 5, 6, 7, and 8 had IC50 values of 35 5, 34 5, 29 1,
8.7 2.1, and 12 2 mM activities, respectively (Table 2).
2.5. Bioassay data
The availability of quinolones 1, 5, 6, 7, and 8 enabled some pre-
liminary structure-activity relationships to be noted. The first
observation is that the potency of these compounds against the
drug-resistant Dd2 strain of P. falciparum was approximately the
same as their potency against the drug-sensitive 3D7 strain. The
next observation is that shorter side chains appear to give more
potent activity than longer ones. Thus compounds 1 and 5–7, all
with n-heptyl or methylated n-heptyl side chains, are all more
potent by a factor of three or more than compound 8 with an n-
octyl side chain. This observation suggests that further increases
in the length of the side chain would be unfavorable for bioactivity.
A final observation is that chain branching improves activity. Com-
pounds 1, 5, 6, and 7 all possess a heptyl side chain, and the
branched chain compounds 5, 6, and 7 all showed improved activ-
ities over the n-heptyl side chain of 1.
The isolated compounds 2 and 3 and the synthetic compounds 1
and 5–8 were tested against the chloroquine-resistant Dd2 strain
of P. falciparum. Compound 4 was not obtained completely pure,
so accurate determination of its antiplasmodial activity was not
possible. However, based on the activity of the fraction H4-6 which
contained 4 as its major component, it almost certainly is active
against the chloroquine-resistant Dd2 strain of P. falciparum. It
has been previously shown to have activity against the chloro-
quine-sensitive 3D7 strain of P. falciparum.11
Compound 1 was found to have a modest IC50 against the Dd2
strain of P. falciparum of 1.3 0.3 mM; it has also been reported to
have activity against the W2 and D6 strains of P. falciparum.7 Com-
pounds 2 and 3 were found to be inactive against the Dd2 strain of
P. falciparum at the highest concentrations tested of 39 mM and
38 mM respectively. The C17H23NO isomers 5, 6, 7, and 8 had IC50
values against the Dd2 strain of P. falciparum of 583 330 nM,
330 43 nM, 750 93 nM, and 3.7 0.5 mM respectively. The qui-
The selectivity of the quinolones can be measured via a selectiv-
ity ratio (A2780 IC50 value/Dd2 IC50 value) that is related to a ther-
apeutic index. Artemisinin has been reported to have a therapeutic
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C.C. Presley et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
5
Table 2
Biological Data for 1–8.
Plasmodium falciparum
Activity against A2780
Selectivity ratio
ovarian cancer cells (mM)
Cmpd
Dd2 (nM)
3D7 (nM)
1
2
3
5
6
7
8
1315 292
>39,000
>38,000
583 330
330 43
750 93
3652 470
1356 329
–
–
501 82
385 78
921 206
3773 575
35
5
26.6
NM
NM
57.7
89.0
11.6
3.4
>78
>75
34
29
8.7 2.1
12
5
1
2
Artemisinin was used as a positive control for the Dd2 and 3D7 strains of P. falciparum with IC50 values of 9.0 2.7 nM and 5.9 0.5 nM, respectively. Taxol was used as a
positive control for the A2780 mammalian ovarian cancer cell line assay with an IC50 value of 13.7 0.7 nM.
NM = not meaningful.
index of 4987, and is considered to be nontoxic by most
researchers.18 Compounds 1, 5, 6, 7, and 8 have selectivity ratios
of 26.6, 57.7, 89.0, 11.6, and 3.4, respectively. Although these val-
ues are not ideal, they do indicate that chain branching enhances
selectivity for P. falciparum over A2780 mammalian cells, with
compound 6 showing not only the best activity against the Dd2
strain of P. falciparum but also showing the largest selectivity ratio.
It is thus possible that analogs with increased chain branching
might have better antiplasmodial activity and improved
selectivity.
Sorbtech C18-W silica TLC plates, w/UV254 on an aluminum back-
ing with a thickness of 150 mm.
4.2. Plant material
Leaves of Crinum firmifolium Baker were collected in the dry
deciduous forest at Solanampilana, 3 km south of the village of
Befarafara, district of Vohémar, region of SAVA, Madagascar, at
coordinates 13°0504200S, 049°3405700E on 10 Dec 2006 and an eleva-
tion of 137 m. The plant was herbaceous with a bulb 10 cm in
diameter, leaves 65 cm, inflorescence 1 m high, with 1 to 2 inflo-
rescences per bulb. It had white petals and red stamens and stalk.
Duplicate voucher specimens (Rakotonandrasana 1053) were
deposited at the Herbaria of the Centre National d’Application
des Recherches Pharmaceutiques (CNARP), of the Parc Botanique
et Zoologique de Tsimbazaza, Antananarivo, Madagascar (TAN),
of the Muséum National d’Histoire Naturelle in Paris, France (P)
and of the Missouri Botanical Garden, St. Louis, Missouri (MO).
3. Conclusions
The genus Crinum continues to produce new and biologically
active alkaloids. Four known alkaloids (1–4) were isolated along
with the new alkaloid 5, whose structure and biological activity
was confirmed through total synthesis. Unfortunately, the small
amounts and difficulty of separation of the other two C17H23NO
quinolones that could be identified prevented their structural
assignment via total synthesis or spectroscopic comparison. Along
with the synthesis of 5 the other new natural product-like quino-
lones 6 and 7 were successfully synthesized and biologically eval-
uated. The isolated and synthesized quinolones display moderate
levels of selectivity for P. falciparum over mammalian cells. At this
time the quinolone moiety has been thoroughly investigated for
antimalarial activity, with several different structure-activity
relationships from studies if the effect of substituents at the 1, 2,
and 3 positions, as well as modifying the electronics of the
aromatic ring.19–21
4.3. Plant extraction
A ground sample of C. firmifolium leaves (43 g) was extracted
with EtOH (700 mL) at room temperature to yield 4.12 g of crude
EtOH extract designated MG 4166, and a 1.0 g portion of this
extract was made available to Virginia Tech. Similar amounts were
made available to Dow AgroSciences and Eisai Inc., who were kind
enough to send their remaining supply to Virginia Tech for bioas-
say-guided isolation.
4.4. Antiplasmodial bioassay
4. Experimental section
Each fraction and isolated compound was tested against the
chloroquine/mefloquine-resistant Dd2 strain and the chloro-
quine-sensitive 3D7 strain of P. falciparum in a 72-h treatment
assay using the malaria SYBR green I-based fluorescence assay as
described previously.22,23 The antimalarial drug artemisinin was
used as a positive control for the Dd2 and 3D7 strains of P. falci-
parum, with IC50 values of 9.0 2.7 nM and 5.9 0.5 nM,
respectively.
4.1. General
All chemicals used were purchased from either Sigma-Aldrich
or the Fisher Scientific Company and were used without further
purification. 2-Heptylquinolin-4(1H)-one (HHQ) was purchased
from Sigma-Aldrich. UV spectra were measured on a Shimadzu
UV-1201 spectrophotometer. 1H and 13C NMR spectra were
obtained on a Bruker Avance 500 spectrometer in CD3OD for solu-
bility reasons. Mass spectra were obtained on an Agilent 6220 LC-
TOF-MS spectrometer and an Agilent 7890B GC system coupled
with a 5977A MSD. Semipreparative HPLC was performed using
Shimadzu LC-10AT pumps coupled with a Shimadzu SPD-M10A
diode array detector, a SCL-10A system controller, and a Phenom-
enex 5 mm, 100 Å, Luna C18(2) (250 ꢁ 10 mm) column, a Kromasil
5 mm, 60 Å, Diol (250 ꢁ 10 mm) column, or a Phenolmenex 4 mm,
90 Å, Jupiter (150 ꢁ 2 mm) column. Solid phase extractions were
conducted with Thermo Scientific hypersep C18 500 mg in 3 mL
SPE cartridges, and all reverse phase TLC was conducted with
4.5. Antiproliferative bioassay
The A2780 ovarian cancer cell line assay was performed at
Virginia Tech as previously reported.24,25 The A2780 cell line in a
drug-sensitive ovarian cancer cell line.26
4.6. Isolation of bioactive compounds
The EtOH extract of C. firmifolium (2.1 g) was dissolved in aque-
ous MeOH (95:5, 200 mL) and extracted with hexanes
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6
C.C. Presley et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
(3 ꢁ 200 mL), which generated the inactive hexanes fraction
(247 mg). The aqueous MeOH fraction was dried under reduced
pressure and then suspended in water (200 mL) and extracted with
EtOAc (3 ꢁ 200 mL). Both fractions were concentrated under
reduced pressure, and the water fraction (1.4 g) was found to be
inactive while the EtOAc fraction (286 mg) was found to possess
modest activity (IC50 5 < x < 10 mg/mL). This fraction was sepa-
rated further into four sub-fractions via a C18 solid phase extraction
(SPE) with different concentrations of aqueous methanol (20:80,
80:20, and 100:0) and a dichloromethane (DCM) flush. The second
fraction (127 mg) was found to be active (IC50 ꢀ 1.25 mg/mL) and
was separated further into ten sub-fractions (H1–H10) by C18 HPLC
with a H2O/MeCN gradient starting from 62:38 to 30:70 over
40 min followed by a maintained flow at 0:100 for 20 min. Fraction
H2 (3.4 mg, IC50 value <<< 1.25 mg/mL) proved to be difficult to
purify. The major active constituents were isolated as a mixture
of similar compounds from diol HPLC with a H2O/MeCN gradient
starting from 70:30 to 63:37 over 20 min. This procedure afforded
0.2 mg of a mixture (Fraction H2-4) of similar compounds 1 (tR
11.08 min) with an IC50 value of <<< 1.25 mg/mL and an estimated
purity of 66% as judged from its 1H NMR spectrum. HPLC fraction
H3 (2.5 mg) was found to be weakly active (IC50 value 1.25
< x < 2.5 mg/mL) and was separated into eight fractions (H3-1–
H3-8) with a H2O/MeOH gradient starting at 38:62 to 32:68 over
40 min followed by a maintained flow at 0:100 for 20 min, but
none of the resulting fractions had any significant antiplasmodial
activity. Fractions H3-4 (tR 26.49 min) and H3-6 (tR 35.18 min)
were determined to be compounds 2 (0.3 mg) and 3 (0.4 mg),
respectively. HPLC fraction H4 (3.9 mg) was highly active (IC50
value <<< 1.25 mg/mL) and was separated into ten sub-fractions
(H4-1–H4-10) using a C18 column with H2O/MeOH starting at
35:65 to 26:74 over 10 min followed by 10 min of maintained flow
at 26:74 then lastly a 5 min ramp to 0:100 followed by 10 min of
maintained flow. Final purification was attempted on fraction
H4-6 (0.6 mg; IC50 value <<< 1.25 mg/mL; tR 27.82 min) with diol
HPLC using H2O/MeCN starting at 60:40 to 59:41 over 8 min fol-
lowed by a maintained flow at 0:100 for 8 min to afford 0.2 mg
of 4 (tR 7.12 min) with an estimated purity of 76% as judged from
its 1H NMR spectrum. Final purification was also attempted on
fraction H4-8 (0.7 mg; IC50 value <<< 1.25 mg/mL; tR 30.16 min)
using the same method that produced 4 to afford 0.2 mg of 5 (tR
8.00 min) with an estimated purity of 50% as judged from its 1H
NMR spectrum.
6.73–6.67 (m, 2H), 6.59 (s, 1H), 5.97 (d, 1.8 Hz, 2H), 4.25 (d,
12.8 Hz, 1H), 4.21 (d, 12.8 Hz, 1H), 2.72 (s, 3H).
4.6.3. 9-Hydroxy-10-methoxy-7H-pyrrolo[3,2,1-de]phenanthrid-in-7-
one (3)
HRESIMS [2M+Na]+ m/z 553.1351 (calcd for C32H22N2NaO+6
553.1370), [2M+H]+ m/z 531.1533 (calcd for C32H23N2O6+
531.1551), [M+Na]+ m/z 288.0613 (calcd for C16H11NNaO+3
288.0631), [M+H]+ m/z 266.0803 (calcd for C16H12NO+3 266.0812).
1H NMR (500 MHz, CDCl3) d 8.06 (d, J = 3.6 Hz, 1H), 8.04 (s, 1H),
7.96 (d, J = 7.7 Hz, 1H), 7.79 (s, 1H), 7.75 (d, J = 7.7 Hz, 1H), 7.49
(t, J = 7.7 Hz, 1H), 6.91 (d, J = 3.6 Hz, 1H), 4.10 (s, 3H). 1H NMR
(500 MHz, CD3OD) 8.07 (d, J = 7.7 Hz, 1H), 8.03 (d, J = 3.6 Hz, 1H),
7.95 (s, 1H), 7.78 (d, 7.7 Hz, 1H), 7.74 (s, 1H), 7.50 (t, J = 7.7 Hz,
1H), 6.98 (d, J = 3.6 Hz, 1H), 4.03 (s, 3H).
4.6.4. (Z)-2-(Non-3-en-1-yl)quinolin-4(1H)-one (4)
HRESIMS [2M+Na]+ m/z 561.3430 (calcd for C36H46N2NaO+2
561.3451), [2M+H]+ m/z 539.3638 (calcd for C36H47N2O2+
539.3632), [M+Na]+ m/z 292.1665 (calcd for C18H23NNaO+
292.1672), [M+H]+ m/z 270.1859 (calcd for C18H24NO+ 270.1852).
1H NMR (500 MHz, CD3OD) d 8.21 (brd, J = 7.2 Hz, 1H), 7.69 (ddd,
J = 8.5, 6.9, 1.5 Hz, 1H), 7.58 (brd, J = 8.3 Hz, 1H), 7.40 (ddd,
J = 8.2, 7.0, 1.1 Hz, 1H), 6.23 (s, 1H), 5.45–5.39 (m, 2H), 2.78 (t,
J = 7.2 Hz, 2H), 0.80–0.76 (m, 3H).
4.6.5. 2-(6-Methylheptyl)quinolin-4(1H)-one (5) (Isolated natural
product)
HRESIMS [2M+Na]+ m/z 537.3453 (calcd for C34H46N2NaO+2
537.3451), [2M+H]+ m/z 515.3650 (calcd for C34H47N2NaO+2
515.3632), [M+Na]+ m/z 280.1674 (calcd for C17H23NNaO+
280.1672), [M+H]+ m/z 258.1865 (calcd for C17H24NO+ 258.1852).
1H NMR (500 MHz, CD3OD) d 8.21 (brd, J = 8.7 Hz, 1H), 7.70 (m,
1H), 7.58 (brd, J = 8.3 Hz, 1H), 7.40 (brt, J = 7.3 Hz, 1H), 6.24 (s,
1H), 2.74–2.71 (m, 2H), 0.88 (d, J = 6.6 Hz, 3H).
4.7. Synthesis
4.7.1. Synthesis of alcohols 12 and 13
An oven dried 2-necked round bottom flask, vacuum arm adap-
tor, magnetic stir bar, and rubber septum were assembled hot and
cooled under vacuum and purged with nitrogen. Freshly distilled
THF (5 mL) was added and cooled to 0 °C and 1 M borane THF com-
plex was added dropwise. Alkene was then added dropwise and
the reaction was allowed to warm up to room temperature and
stirred for 24 h. The solution was hydrolyzed with 3 M NaOH and
50% hydrogen peroxide and stirred for an additional 3 h. The reac-
tion mixture was extracted with EtOAc (3 ꢁ 50 mL). The combined
organic layers were washed with brine then concentrated under
reduced pressure. The resulting oil was chromatographed with sil-
ica gel using 80:20 hexane/EtOAc to afford the primary alcohols 12
and 13. This procedure was adopted from Hajare et al.27
4.6.1. 2-Heptylquinolin-4(1H)-one (1)
HRESIMS [2M+Na]+ m/z 509.3155 (calcd for C32H42N2NaO+2
509.3138), [2M+H]+ m/z 487.3339 (calcd for C32H43N2O2+
487.3319), [M+Na]+ m/z 266.1522 (calcd for C16H21NNaO+
280.1515), [M+H]+ m/z 244.1705 (calcd for C16H22NO+ 244.1696).
1H NMR (500 MHz, CD3OD) d 8.21 (dd, J = 8.2, 1.5 Hz, 1H), 7.69
(ddd, J = 8.4, 7.0, 1.5 Hz, 1H), 7.58 (brd, J = 8.4 Hz, 1H), 7.40 (ddd,
J = 8.2, 7.0, 1.0 Hz, 1H), 6.24 (s, 1H), 2.78–2.65 (m, 2H), 1.77 (p,
J = 7.8 Hz, 2H), 1.49–1.24 (m, 8H), 0.90 (t, J = 6.9 Hz, 3H).
4.6.2. (6-(2-(Methylamino)phenyl)benzo[d][1,3]dioxol-5-yl) methanol
(2)
4.7.2. 4-Methyl-1-hexanol (12)
The compound was obtained from 3.17 g (32.3 mmol) of 9 (4-
methyl-1-hexene) and a large excess of 1 M borane THF solution
(2 eq. or 65 mL or 65 mmol), and 12 mL of 3 M NaOH and 12 mL
of 50% hydrogen peroxide were used for the hydrolysis. Silica gel
column chromatography afforded 2.50 g of 12 (67% yield) as a clear
liquid. GC–MS m/z 115.0. 1H NMR (500 MHz, CDCl3) 3.59 (t,
J = 6.7 Hz, 2H), 1.90 (brs, 1H), 1.64–1.44 (m, 2H), 1.39–1.26 (m,
3H), 1.20–1.05 (m, 2H), 0.90–0.77 (m, 6H). 13C NMR (125 MHz,
CDCl3) 63.4, 34.4, 32.6, 30.4, 29.5, 19.2, 11.5. This compound has
been previously reported.28
HRESIMS [2M+Na]+ m/z 537.1984 (calcd for C30H30N2NaO+6
537.1996), [M+Na]+ m/z 280.0929 (calcd for C15H15NNaO+3
280.0944), [M+H]+ m/z 258.1120 (calcd for C15H16NO+3 258.1125),
[M-H2O+H]+ m/z 240.1014 (calcd for C15H14NO+2 240.1019). 1H
NMR (500 MHz, CDCl3) d 7.31 (ddd, J = 8.7, 7.5, 1.6 Hz, 1H), 7.03
(s, 1H), 7.00 (dd, J = 7.5, 1.6, 1H), 6.85–6.82 (m, 1.0 Hz, 1H), 6.75
(brd, J = 8.1 Hz, 1H), 6.69 (s, 1H), 6.01 (s, 2H), 4.28 (d, J = 12.0 Hz,
1H), 4.21 (d, J = 12.0 Hz, 1H), 2.75 (s, 3H). 1H NMR (500 MHz, CD3-
OD) 7.22 (td, 7.8, 1.6 Hz, 1H), 7.06 (s, 1H), 6.90 (dd, 7.3, 1.7 Hz, 1H),
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C.C. Presley et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
7
4.7.3. 5-Methyl-1-hexanol (13)
added dropwise. Once the addition was completed the mixture
was warmed to 0 °C and stirred for 30 min. The mixture was
returned to ꢂ78 °C and 1 eq. of ethyl acetoacetate were added
dropwise and then the mixture was stirred for 1 h. Lastly, 1.1 eq.
of iodinated alkane were added dropwise and the mixture was
allowed to reach room temperature while stirring for 18 h. The
reaction was quenched with 3 M HCl and extracted 3 ꢁ 50 mL with
diethyl ether. The combined organics were dried over sodium sul-
fate and concentrated under reduced pressure. The crude yellow
oil was then purified with silica gel chromatography 95:5 hex-
ane/diethyl ether to afford the long chain ketoesters. This proce-
dure was adopted from Lu et al.34
Compound 13 was obtained from 2.8 g (28.2 mmol) of 10 (5-
methyl-1-hexene) and 56.4 mL of 1 M borane THF solution (2 eq.
or 56.4 mmol were used), and 11 mL of 3 M NaOH and 11 mL of
50% hydrogen peroxide were used for the hydrolysis. Silica gel col-
umn chromatography afforded 1.96 mg (60% yield) of product as a
clear liquid. GC–MS m/z 115.1. 1H NMR (500 MHz, CDCl3) 3.62 (t,
J = 6.7 Hz, 2H), 1.61–1.46 (m, 4H), 1.39–1.28 (m, 2H), 1.24–1.11
(m, 2H), 0.86, (d, J = 6.6 Hz, 6H). 13C NMR (125 MHz, CDCl3) 63.2,
38.9, 33.2, 28.1, 23.7, 22.7 (double intensity). This compound has
been previously reported.29
4.7.4. General iodination procedure
An oven dried two-necked flask, vacuum arm adaptor, magnetic
stir bar, and rubber septum were assembled warm and cooled
under vacuum and purged with nitrogen. 1.3 eq of triphenylphos-
phine (PPh3) and 1.4 eq of imidazole were added to the flask under
constant N2 flushing, sealed and pumped down and purged with N2
three times. The solids were dissolved in freshly distilled diethyl
ether and acetonitrile (2:1) and cooled to 0 °C. The alcohols were
added dropwise and stirred for 10 min. Under constant N2 flushing
3 eq of iodine were added in portions, and stirred for another 2 h.
The reaction mixture was diluted with 25 mL of n-pentane and
washed with saturated Na2S2O3 (3 ꢁ 25 mL), saturated CuSO4
(3 ꢁ 25 mL), and lastly brine. The mixture was then concentrated
under reduced pressure with minimal heat. The mixture was then
chromatographed on silica gel with 100% n-pentane to afford the
desired primary halides. This procedure was adopted from Tate
et al.30
4.7.9. Ethyl 7-methyl-3-oxodecanoate (19)
LDA (2 equiv.) was prepared from 4.46 mmol diisopropylamine
and 1.8 mL (4.46 mmol) of 2.5 M n-butyllithium in hexanes. The
LDA solution was treated with ethyl acetoacetate (2.23 mmol) to
give dianion (18), which was treated with 14 (1.1 eq. or
2.48 mmol) for the alkylation. Column chromatography with 5:1
Hex/Et2O afforded 305 mg (61% yield) of product as a yellow oil.
HRESIMS [2M+H]+ m/z 457.3539 (calcd for C26H49O+6 457.3524),
[M+Na]+ m/z 251.1618 (calcd for C13H24NaO+3 251.1618). 1H NMR
(500 MHz, CDCl3) 4.18 (q, J = 7.1 Hz, 2H), 3.41 (s, 2H), 2.50 (t,
J = 7.4 Hz, 2H), 1.67–1.48 (m, 2H), 1.43–1.17 (m, 8H), 1.15–1.02
(m, 2H), 0.89–0.80 (m, 6H). 13C NMR (125 MHz, CDCl3) 203.2,
167.4, 61.4, 49.4, 43.5, 39.2, 36.4, 32.4, 21.1, 20.2, 19.6, 14.5, 14.2.
4.7.10. Ethyl 8-methyl-3-oxodecanoate (20)
LDA (2 equiv.) was prepared from 8.75 mmol diisopropylamine
and 3.5 mL (8.75 mmol) of 2.5 M n-butyllithium in hexanes. The
LDA was treated with 4.37 mmol of ethyl acetoacetate to give dian-
ion 18, to which was added 1.1 eq. (4.82 mmol) 1-iodo-4-methyl-
hexane (15). Column chromatography with 5:1 Hex/Et2O
afforded 674 mg (67% yield) of product as a yellow oil. HRESIMS
[M+Na]+ m/z 251.1611 (calcd for C13H24NaO+3 251.1618). 1H NMR
(500 MHz, CDCl3) 4.17 (q, J = 7.1 Hz, 2H), 3.41 (s, 2H), 2.52 (t,
J = 7.4 Hz, 2H), 1.62–1.49 (m, 2H), 1.36–1.18 (m, 8H), 1.14–1.03
(m, 2H), 0.87–0.77 (m, 6H). 13C NMR (125 MHz, CDCl3) 203.1,
167.4, 61.4, 49.4, 43.2, 36.4, 34.3, 29.5, 26.6, 23.9, 19.2, 14.2, 11.5.
4.7.5. 1-Iodo-3-methylhexane (14)
Iodide 14 was obtained from 664 mg (5.7 mmol) of dry 11 (3-
methyl-1-hexanol), 1.95 g (7.4 mmol) PPh3, 544 mg (8.0 mmol)
imidazole, and 4.36 g (17.2 mmol) iodine to afford 797.9 mg
(62%) of product. GC–MS m/z 226.1. 1H NMR (500 MHz, CDCl3)
3.28–3.13 (m, 2H), 1.86 (dddd, J = 13.8, 8.5, 7.2, 5.3 Hz, 1H), 1.70–
1.49 (m, 2H), 1.42–1.07 (m, 4H), 0.91–0.85 (m, 6H). 13C NMR
(125 MHz, CDCl3) 41.1, 38.6, 33.7, 20.0, 18.8, 14.4, 5.5. This com-
pound has been previously reported.31
4.7.6. 1-Iodo-4-methylhexane (15)
4.7.11. Ethyl 9-methyl-3-oxodecanoate (21)
Iodide 15 was obtained from 2.0 g (17.2 mmol) of dry 12 (4-
methyl-1-hexanol), 5.87 g (22.4 mmol) PPh3, 1.64 g (24.1 mmol)
imidazole, and 13.11 g (51.7 mmol) iodine to afford 2.59 g (67%)
of product. GC–MS m/z 226.1. 1H NMR (500 MHz, CDCl3) 3.22–
3.12 (m, 2H), 1.91–1.71 (m, 2H), 1.48–1.27 (m, 3H), 1.24–1.09
(m, 2H), 0.89–0.83 (m, 6H). 13C NMR (125 MHz, CDCl3) 37.5, 33.8,
31.4, 29.4, 19.3, 11.5, 7.7. This compound has been previously
reported.32
LDA (2 equiv.) was prepared from 4.78 mmol diisopropylamine
and 3.8 mL (4.78 mmol) of 2.5 M n-butyllithium in hexanes. The
LDA was treated with 2.39 mmol of ethyl acetoacetate to give dian-
ion 18, and to this was added 2.65 mmol (1.1 eq.) of 1-iodo-5-
methylhexane (16). Column chromatography with 5:1 Hex/Et2O
afforded 85.4 mg (16% yield) of product as a yellow oil. HRESIMS
[2M+K]+ m/z 495.3093 (calcd for C26H48KO+6 495.3082), [M+Na]+
m/z 251.1620 (calcd for C13H24NaO+3 251.1618). 1H NMR
(500 MHz, CDCl3) 4.19 (q, J = 7.2 Hz, 2H), 3.42 (s, 2H), 2.52 (t,
J = 7.4 Hz, 2H), 1.59 (m, J = 7.4 Hz, 2H), 1.56–1.44 (m, 1H) 1.32–
1.23 (m, 7H), 1.18–1.11 (m, 2H), 0.85 (d, J = 6.6 Hz, 6H). 13C NMR
(125 MHz, CDCl3) 203.2, 167.4, 61.5, 49.5, 43.2, 38.9, 29.4, 28.0,
27.2, 23.6, 22.7 (double intensity), 14.2.
4.7.7. 1-Iodo-5-methylhexane (16)
The compound was obtained from 655.8 mg (5.7 mmol) of dry
13 (5-methyl-1-hexanol), with 1.9 g (7.4 mmol) PPh3, 540 mg
(7.9 mmol) imidazole, and 4.3 g (16.9 mmol) iodine. Yield
809.2 mg (62%). GC–MS m/z 226.1. 1H NMR (500 MHz, CDCl3)
3.19 (t, J = 7.0 Hz, 2H), 1.80 (p, J = 7.2 Hz, 2H), 1.60–1.47 (m, 1H),
1.45–1.32 (m, 2H), 1.25–1.13 (m, 2H), 0.88 (d, J = 6.6 Hz, 6H). 13C
NMR (125 MHz, CDCl3) 37.9, 33.9, 28.5, 28.0, 22.7 (double inten-
sity), 7.5. This compound has been previously reported.33
4.7.12. Ethyl 3-oxoundecanoate (22)
LDA prepared from 8.76 mmol diisopropylamine and 3.5 mL
(8.76 mmol) of 2.5 M n-butyllithium in hexanes was treated with
4.38 mmol of ethyl acetoacetate followed by 4.38 mmol (1.1 eq.)
of 1-iodoheptane (17). Column chromatography with 5:1 Hex/
Et2O afforded 530 mg (53% yield) of product as a yellow oil. HRE-
SIMS [2M+K]+ m/z 495.3060 (calcd for C26H48KO6+ 495.3082), [2 M
+Na]+ m/z 479.3356 (calcd for C26H48NaO6+ 479.3343), [M+Na]+ m/
4.7.8. General ketoester procedure
An oven dried two-necked flask, vacuum arm adaptor, magnetic
stir bar, and rubber septum were assembled hot and cooled under
vacuum and purged with N2. The apparatus was charged with
freshly distilled THF and 2.5 eq. of diisopropyl amine and cooled
to ꢂ78 °C. While stirring, 2.5 eq. of 2.5 M n-butyllithium were
z
251.1623 (calcd for C
13H24NaO+3 251.1618), [M+H]+ m/z
251.1800 (calcd for C13H25O+3 229.1798). 1H NMR (500 MHz, CDCl3)
4.11 (q, J = 7.2 Hz, 2H), 3.35 (s, 2H), 2.46 (t, J = 7.4 Hz, 2H), 1.51 (p,
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C.C. Presley et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
J = 7.4 Hz, 2H), 1.27–1.11 (m, 14H), 0.80 (d, J = 6.9 Hz, 3H). 13C NMR
(125 MHz, CDCl3) 202.9, 167.2, 61.2, 49.2, 43.0, 31.8, 29.3, 29.1,
29.0, 23.4, 22.6, 14.1, 14.0. This compound has been previously
reported.17
duct was then recrystallized from EtOAc to afford 23.8 mg (13%
yield) of 6. HRESIMS [2 M+Na]+ m/z 537.3444 (calcd for C34H46N2-
NaO+2 537.3451), [2M+H]+ m/z 515.3617 (calcd for C34H47N2NaO+2
515.3632), [M+Na]+ m/z 280.1668 (calcd for C17H23NNaO+
280.1672), [M+H]+ m/z 258.1847 (calcd for C17H24NO+ 258.1852).
1H NMR (500 MHz, CD3OD) d 8.21 (dd, J = 8.2, 1.5 Hz, 1H), 7.69
(ddd, J = 8.5, 7.0, 1.5 Hz, 1H), 7.58 (brd, J = 8.2 Hz, 1H), 7.39 (ddd,
J = 8.2, 7.0, 1.1 Hz, 1H), 6.23 (s, 1H), 2.76–2.64 (m, 2H), 1.88–1.67
(m, 2H), 1.52–1.37 (m, 2H), 1.35–1.19 (m, 4H), 1.17–1.05 (m,
1H), 0.93–0.84 (m, 6H). 13C NMR (125 MHz, CD3OD) d 180.6,
157.1, 141.6, 133.4, 126.0, 125.5, 125.1, 119.1, 108.9, 40.4, 37.5,
35.3, 33.5, 27.7, 21.1, 19.9, 14.7. 1H NMR (500 MHz, CDCl3) d
11.56 (s, 1H) 8.36 (dd, J = 8.2, 1.5 Hz, 1H), 7.69 (ddd, J = 8.5,
1.1 Hz, 1H), 7.58 (ddd, J = 8.5, 7.0, 1.5 Hz, 1H), 7.32 (ddd, J = 8.2,
7.0, 1.1 Hz, 1H), 6.23 (s, 1H), 2.72–2.61 (m, 2H), 1.81–1.62 (m,
2H), 1.39–1.07 (m, 6H), 1.05–0.94 (m, 1H), 0.80 (t, J = 7.1 Hz, 3H),
0.76 (d, J = 6.5 Hz, 3H). 13C NMR (125 MHz, CDCl3) d 179.1, 154.9,
140.6, 131.9, 125.6, 125.2, 123.7, 118.4, 108.5, 39.3, 36.7, 34.9,
32.5, 26.7, 20.2, 19.6, 14.5.
4.7.13. General b–enamino ester and 2-alkylquinolin-4(1H)-one
procedure
An oven dried conical 5 mL vial and magnetic spin vane were
cooled in a desiccator. Once cool the spin vane, a long chain ketoe-
ster, and aniline were added to the vial neat and stirred. A catalytic
amount of InCl3 was added and the vial was sealed with a septum,
flushed with N2, and stirred for 14 h. The reaction was then diluted
with EtOAc and H2O, and the aqueous layer extracted two more
times with EtOAc. The organic layers were combined, dried with
anhydrous sodium sulfate, and concentrated under reduced pres-
sure to give the crude b–enamino esters.15 The crude b–enamino
esters were carried forward without purification to the next step.
An oven dried two-necked round bottom flask, vacuum arm adap-
tor, magnetic stir bar, and rubber septum were assembled hot and
cooled under vacuum. Once the apparatus had cooled to room tem-
perature ꢀ 1 g of polyphosphoric acid (84% P2O5) was added under
constant N2 flushing. The apparatus was sealed and warmed to
110 °C using a sand bath under reduced pressure. Once stirring
was possible, the system was purged with N2, cooled to room tem-
perature, and a spatula tip full of P2O5 was added and the process
was repeated two more times. After the 3rd pump-purge the sys-
tem was left at 110 °C under N2 and crude b–enamino ester was
added via syringe. The mixture stirred for 24 h at 110 °C, allowed
to cool to rt, and ice cold water was added and stirred by hand with
a glass rod. The mixture was transferred to a larger container and
the pH was adjusted to 7 with solid sodium bicarbonate. The aque-
ous solution was then extracted with 3 ꢁ 25 mL of EtOAc, which
was then dried over sodium sulfate and concentrated under
reduced pressure.16
4.7.16. 2-(5-Methylheptyl)quinolin-4(1H)-one (7)
The condensation reaction and cyclization were conducted with
1.32 mmol of 20 and aniline. The crude product was then subjected
to chromatography on a silica gel column with elution with 95:5
DCM/CH3OH, and the semi-pure product was then recrystallized
from EtOAc to afford 20.0 mg (6% yield) of 7. UV (MeOH) kmax
(log
for C34H46N2NaO+2 537.3451), [2 M+H]+ m/z 515.3612 (calcd for
34H47N2NaO+2 515.3632), [M+Na]+ m/z 280.1670 (calcd for C17H23
e
) 235 (5.22) nm; HRESIMS [2 M+Na]+ m/z 537.3452 (calcd
C
-
NNaO+ 280.1672), [M+H]+ m/z 258.1853 (calcd for C17H24NO+
258.1852). 1H NMR (500 MHz, CD3OD) d 8.20 (dd, J = 8.1, 1.5 Hz,
1H), 7.67 (ddd, J = 8.4, 6.9, 1.5 Hz, 1H), 7.57 (dt, J = 8.4, 1.0 Hz,
1H), 7.38 (ddd, J = 8.1, 6.9, 1.0 Hz, 1H), 6.22 (s, 1H), 2.70 (t,
J = 7.8 Hz, 2H), 1.73 (m, 2H), 1.36 (m, 5H), 1.15 (m, 2H), 0.86 (m,
6H). 13C NMR (125 MHz, CD3OD) d 180.6, 157.2, 141.6, 133.4,
126.0, 125.5, 125.1, 119.1, 108.8, 37.4, 35.6, 35.0, 30.5, 27.7, 19.5,
11.7. 1H NMR (500 MHz, CDCl3) d 10.23 (s, 1H), 8.35 (dd, J = 8.2,
1.5 Hz, 1H), 7.58 (ddd, J = 8.4, 6.9, 1.5 Hz, 1H), 7.53 (dd, J = 8.4,
1.2 Hz, 1H), 7.32 (ddd, J = 8.2, 6.9, 1.2 Hz, 1H), 6.21 (s, 1H), 2.65
(t, J = 7.8 Hz, 2H), 1.71 (m, 2H), 1.27 (m, 5H), 1.07 (m, 2H), 0.80
(m, 6H). 13C NMR (125 MHz, CDCl3) d 179.1, 154.0, 140.2, 132.0,
125.9, 125.2, 123.7, 117.8, 108.8, 36.4, 34.7, 34.4, 29.5, 29.2, 26.8,
19.3, 11.5.
4.7.14. 2-(6-Methylheptyl)quinolin-4(1H)-one (5) (Synthetic natural
Product)
The condensation reaction and cyclization were conducted with
3.01 ꢁ 10ꢂ4 mol of 21 and aniline. The crude product was then sub-
jected to chromatography on a silica gel column with elution with
95:5 DCM/CH3OH and the semi-pure product was then recrystal-
lized from EtOAc to afford 13.6 mg (18% yield) of 5. UV (MeOH)
kmax (log
e
) 235 (5.23) nm; HRESIMS [2M+Na]+ m/z 537.3451
(calcd for C34H46N2NaO+2 537.3451), [M+Na]+ m/z 280.1668 (calcd
for C17H23NNaO+ 280.1672), [M+H]+ m/z 258.1849 (calcd for
C
17H24NO+ 258.1852). 1H NMR (500 MHz, CD3OD) d 8.21 (dd,
4.7.17. 2-Octylquinolin-4(1H)-one (8)
J = 8.2, 1.5 Hz, 1H), 7.68 (ddd, J = 8.5, 7.0,1.5 Hz, 1H), 7.57 (dd,
J = 8.5, 1.1 Hz, 1H), 7.38 (ddd, J = 8.2, 7.0, 1.1 Hz, 1H), 6.22 (s, 1H),
2.75–2.69 (m, 2H), 1.76 (p, J = 7.5 Hz, 2H), 1.52 (dp, J = 13.3,
6.7 Hz, 1H) 1.47–1.42 (m, 4H), 1.24–1.15 (m, 2H), 0.87 (d,
J = 6.7 Hz, 6H). 13C NMR (125 MHz, CD3OD) d 180.6, 157.2, 141.6,
133.4, 126.0, 125.5, 125.1, 119.1, 108.8, 40.0, 35.0, 30.5, 30.2,
29.1, 28.2, 23.0 (double intensity). 1H NMR (500 MHz, CDCl3) d
11.02 (s, 1H), 8.36 (dd, J = 8.2, 1.5 Hz, 1H), 7.63 (dd, J = 8.4,
1.2 Hz, 1H), 7.58 (ddd, J = 8.4, 6.8, 1.5 Hz, 1H), 7.32 (ddd, J = 8.2,
6.8, 1.2 Hz, 1H), 6.22 (s, 1H), 2.73–2.56 (m, 2H), 1.78–1.61 (m,
4H), 1.44 (dp, J = 13.3, 6.6 Hz, 1H), 1.34–1.18 (m, 4H), 1.14–1.02
(m, 2H), 0.80 (d, J = 6.6 Hz, 6H). 13C NMR (125 MHz, CDCl3) d
179.1, 154.5, 140.4, 131.9, 125.7, 125.2, 123.7, 118.1, 108.6, 38.9,
34.6, 29.6, 29.1, 28.0, 27.2, 22.7 (double intensity).
The condensation reaction and cyclization were conducted with
1.69 mmol of 22 and aniline. The crude product was then separated
on a short C18 column with 75% aqueous methanol and the semi
pure product was recrystallized from EtOAc to afford 91.3 mg
(21% yield) of 8. HRESIMS [2 M+Na]+ m/z 537.3453 (calcd for C34
-
H
46N2NaO+2 537.3451), [2 M+H]+ m/z 515.3642 (calcd for C34H47N2-
NaO+2 515.3632), [M+Na]+ m/z 280.1677 (calcd for C17H23NNaO+
280.1672), [M+H]+ m/z 258.1864 (calcd for C17H24NO+ 258.1852).
1H NMR (500 MHz, CD3OD) d 8.21 (brd, J = 8.1 Hz, 1H), 7.68 (ddd,
J = 8.4, 6.9, 1.4 Hz, 1H), 7.58 (brd, J = 8.4 Hz, 1H), 7.39 (brt,
J = 7.5 Hz, 1H), 6.24 (s, 1H), 2.76–2.65 (m, 2H), 1.76 (p, J = 7.5 Hz,
2H), 1.49–1.20 (m, 10H), 0.88 (t, J = 6.9 Hz, 3H). 13C NMR
(125 MHz, CD3OD) d 180.5, 157.2, 141.6, 133.4, 125.9, 125.4,
125.1, 119.1, 108.8, 35.0, 33.0, 30.4, 30.3, 30.2, 30.2, 23.7, 14.4.
1H NMR (500 MHz, DMSO-d6) d 11.47 (s, 1H), 8.02 (dd, J = 8.1,
1.4 Hz, 1H), 7.60 (ddd, J = 8.4, 6.8, 1.6 Hz, 1H), 7.52 (brd,
J = 8.2 Hz, 1H), 7.26 (brt, J = 7.4 Hz, 1H), 5.91 (brd, J = 1.5 Hz, 1H),
2.57 (t, J = 7.6 Hz, 2H), 1.66 (p, J = 7.6 Hz, 2H), 1.27 (m, 10H), 0.84
(t, J = 7.1 Hz, 3H). 13C NMR (125 MHz, DMSO-d6) d 177.0, 153.9,
140.2, 131.6, 124.8, 124.6, 122.9, 118.0, 107.7, 33.3, 31.3, 28.8,
4.7.15. 2-(4-Methylheptyl)quinolin-4(1H)-one (6)
The condensation reaction and cyclization were conducted with
7.39 ꢁ 10ꢂ4 mol of 19. and 7.39 ꢁ 10ꢂ4 mol of aniline. The crude
product was then subjected to chromatography on a silica gel col-
umn with elution with 95:5 DCM/CH3OH, and the semi-pure pro-
Please cite this article in press as: Presley C.C., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.06.017

C.C. Presley et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
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Acknowledgments
This project was supported by the National Center for Comple-
mentary and Integrative Health under award 1 R01 AT008088 and
by the Fogarty International Center, the National Cancer Institute,
the National Institute of Allergy and Infectious Diseases, the
National Institute of Mental Health, the National Institute on Drug
Abuse, the National Heart Lung and Blood Institute, the National
Center for Complementary and Alternative Medicine, the Office
of Dietary Supplements, the National Institute of General Medical
Sciences, the Biological Sciences Directorate of the National Science
Foundation, and the Office of Biological and Environmental
Research of the U.S. Department of Energy under Cooperative
Agreement U01 TW00313 with the International Cooperative Bio-
diversity Groups. This project was also supported by the National
Research Initiative of the Cooperative State Research, Education
and Extension Service, USDA, Grant #2008-35621-04732. These
supports are gratefully acknowledged. This work was also sup-
ported by the National Science Foundation under Grant No. CHE-
0619382 for purchase of the Bruker Avance 500 NMR spectrometer
and Grant No. CHE-0722638 for the purchase of the Agilent 6220
mass spectrometer. We thank Mr. B. Bebout for obtaining the mass
spectra. We also thank Dr. Youngchun Shen at Eisai Inc. and Dr.
Don Hahn at Dow AgroSciences for supplies of additional extract.
The authors thank Elizabeth Childress, Dr. Gary Richoux, and Kiaya
Sulier for their help with synthetic techniques, and Russell Snead
for help in obtaining the GCMS spectroscopic data.
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Please cite this article in press as: Presley C.C., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.06.017