Journal of Natural Products
Article
+
3
.0 mg; 3, 4.5 mg; 4, 5.0 mg). The remaining crude product from each
[M + Na] ; HRESIMS m/z 496.3901 (calcd for C H N O ,
31 50 3 2
reaction was used as such in the next step.
496.3902).
1
1
Farnesoyl Guanidine (2): H NMR (CDCl , 400 MHz) δ 5.85
1H, s, H-2), 5.10 (1H, s, H-6), 5.09 (1H, s, H-10), 2.18 (3H, s, H3-
N,N′-Digeranoyl Guanidine (6): H NMR (CDCl , 400 MHz) δ
3
H
3
H
(
5.78 (2H, s, H-2 and H-2″), 5.08 (2H, m, H-6 and H-6″), 2.19 (6H, s,
1
5), 2.17 (2H, m, H -9), 2.15 (2H, m, H -4), 2.03 (2H, m, H -5), 1.98
H -10 and H -10″), 2.17 (8H, m, H -4, H -5, H -4″, and H -5″), 1.69
2
2
2
3
3
2
2
2
2
1
3
(
2H, m, H -8), 1.67 (3H, m, H -12), 1.61 (3H, m, H -14), 1.60 (3H,
(6H, m, H
3
-8 and H
3
-8″), 1.61 (6H, m, H
3
-9 and H -9″); C NMR
3
2
3
3
13
m, H -13); C NMR (CDCl , 400 MHz, indirect detection from 2D
(CDCl
experiments) δ
(C, C-7 and C-7″), 123.0 (CH, C-6 and C-6″), 119.8 (CH, C-2 and C-
2″), 41.4 (CH , C-4 and C-4″), 26.3 (CH , C-5 and C-5″), 25.3 (CH
C-8 and C-8″), 18.8 (CH , C-10 and C-10″), 17.8 (CH , C-9 and C-
9″); LRESIMS m/z 360 [M + H] , 382 [M + Na] ; HRESIMS m/z
360.2647 (calcd for C21 , 360.2651).
N,N′-Disenecioyl Guanidine (7): H NMR (CDCl
5.77 (2H, s, H-2 and H-2″), 2.22 (6H, s, H -4 and H -4″), 1.96 (6H, s,
, 400 MHz, indirect detection
from 2D heteronuclear experiments) δ 166.9 (C, C-1 and C-1″),
158.8 (C, C-3 and C-3″), 117.2 (CH, C-2 and C-2″), 27.9 (CH , C-4
3
, 400 MHz, indirect detection from 2D heteronuclear
3
3
heteronuclear experiments) δ 175.4 (C, C-1), 159.0 (C, C-3), 136.3
C
175.4 (C, C-1), 160.0 (C, C-3 and C-3″), 133.0
C
(
(
9
1
C, C-7), 131.4 (C, C-11), 124.0 (CH, C-10), 123.2 (CH, C-6), 121.5
CH, C-2), 41.0 (CH , C-4), 40.0 (CH , C-8), 26.3 (CH , C-6 and C-
2
2
,
3
2
2
2
), 25.8 (CH , C-12), 19.0 (CH , C-15), 18.1 (CH , C-13 or C-14),
3
3
3
3
3
+
+
+
6.6 (CH , C-14 or C-13); LRESIMS m/z 278 [M + H] , 300 [M +
3
+
Na] ; HRESIMS m/z 278.2224 (calcd for C H N O, 278.2227).
H N O
34 3 2
16
28
3
1
1
Geranoyl Guanidine (3): H NMR (CDCl , 400 MHz) δ 5.78
, 400 MHz) δ
3 H
3
H
(
1H, s, H-2), 5.06 (1H, s, H-6), 2.24−2.18 (overlapped signals, m, 7H,
3
3
13
13
H -4, H -5, H -10), 1.68 (3H, m, H -8), 1.60 (3H, m, H -9);
C
H
3
-5 and H
3
-5″); C NMR (CDCl
3
2
2
3
3
3
NMR (CDCl , 400 MHz, indirect detection from 2D heteronuclear
C
3
experiments) δ 174.6 (C, C-1), 158.4 (C, C-3), 133.6 (C, C-7), 122.0
3
C
+
(
(
CH, C-6), 115.6 (CH, C-2), 41.6 (CH , C-4), 26.2 (CH , C-5), 25.4
and C-4″), 20.9 (CH , C-5 and C-5″); LRESIMS m/z 224 [M + H] ,
246 [M + Na] ; HRESIMS m/z 224.1394 (calcd for C11H N O ,
224.1399).
3
2
2
+
CH , C-8), 19.6 (CH , C-10), 17.5 (CH , C-9); LRESIMS m/z 210
18 3 2
3
3
3
+
+
[
M + H] , 232 [M + Na] ; HRESIMS m/z 210.1600 (calcd for
1
N-Geranoyl-N′-senecioyl Guanidine (8): H NMR (CDCl , 400
C H N O, 210.1601).
Senecioyl Guanidine (4): H NMR (CDCl , 400 MHz) δ 5.69
3
11
20
3
1
MHz) δ
5.78 (1H, s, H-2″), 5.75 (1H, s, H-2), 5.07 (1H, m, H-6),
3
H
H
1
3
2
.20 (6H, s, H -10 and H -5″), 2.17 (4H, m, H -4, H -5), 1.92 (3H, m,
(
1H, s, H-2), 2.14 (3H, s, H -4), 1.89 (3H, s, H -5); C NMR
3
3
2
2
3
3
1
3
H -4″), 1.69 (3H, m, H -8), 1.61 (3H, m, H -9); C NMR (CDCl ,
(
CDCl , 400 MHz, indirect detection from 2D heteronuclear
3
3
3
3
3
4
00 MHz, indirect detection from 2D heteronuclear experiments) δ
experiments) δC 170.9 (C, C-1), 156.9 (C, C-3), 116.1 (CH, C-2),
C
+
1
72.4 (C, C-1 and C-1″), 161.3 (C, C-3), 157.1(C, C-3″), 132.6 (C,
2
7.2 (CH , C-4), 20.3 (CH , C-5); LRESIMS m/z 142 [M + H] , 164
3
3
+
C-7), 122.4 (CH, C-6), 120.0 (CH, C-2″), 119.4 (CH, C-2), 41.3
[
M + Na] ; HRESIMS m/z 142.0977 (calcd for C H N O,
6 12 3
(
2
2
CH , C-4), 27.8 (CH , C-4″), 26.0 (CH , C-5), 25.9 (CH , C-8),
142.0975).
2
3
2
3
0.3 (CH , C-5″), 18.6 (CH , C-10), 17.6 (CH , C-9); LRESIMS m/z
Syntheses of Diacylguanidines 1 and 5−9. Actinofide (1) and
3
3
3
+ +
92 [M + H] , 314 [M + Na] ; HRESIMS m/z 292.2019 (calcd for
diacylguanidine analogues (compounds 5−9) were prepared following
the synthetic scheme described in ref 17 by combining mono-
acylguanidines 2−4 with either farnesoic or geranoic or senecioic acid.
C H N O , 292.2025).
16 26
3
2
1
N-Farnesoyl-N′-geranoyl Guanidine (9): H NMR (CDCl , 400
3
17
MHz) δ 5.71 (2H, s, H-2 and H-2″), 5.09 (4H, m, H-6, H-10, H-6″,
According to Serra et al., the general procedure was as follows: 2-
chloro-1-methylpyridinium iodide (64.0 mg, 0.25 mmol) was added to
a stirred solution of the terpenoic acid (0.20 mmol) in N-
methylpyrrolidone (1.5 mL). The solution was stirred for 30 min at
rt and then for 1 h at 50 °C. After the reaction was cooled to rt, the
monoacylguanidine derivative (0.20 mmol) dissolved in N-methyl-
pyrrolidone (0.5 mL) and DIEA (0.5 mL) was added in one portion to
the solution. The obtained mixture was stirred for 12 h. The reaction
H
and H-10″), 2.21−2.17 (overlapped signals, 14H, H -4, H -5, H -15,
2
2
3
H -4″, H -5″, and H -10″), 2.06 (2H, m, H -9), 1.99 (2H, m, H -8),
2
2
3
2
2
1
.68 (6H, m, H -12 and H -8″), 1.605 (3H, m, H -14), 1.602 (6H, m,
3
3
3
13
H -13 and H -9″); C NMR (CDCl , 400 MHz, indirect detection
from 2D heteronuclear experiments) δ 170.9 (C, C-1), 162.2 (C, C-3
3
3
3
C
and C-3″), 136.3 (C, C-7), 131.2 (C, C-11 and C-7″), 123.6 (CH, C-
6
3
, C-10, C-6″), 114.7 (CH, C-2 and C-2″), 41.1 (CH , C-4 and C-4″),
2
9.2 (CH , C-8), 26.4 (CH , C-9), 25.9 (CH , C-5 and C-5″), 25.5
was quenched by addition of H O (4 mL) and extracted with EtOAc
2
2
2
2
(
CH , C-12 and C-8″), 19.0 (CH , C-15 and C-10″), 17.5 (CH , C-13
(
3 × 6 mL). The organic phases were combined and concentrated
under reduced pressure to give crude diacyl derivatives (1, 50 mg; 5,
0 mg; 6, 50 mg; 7, 13 mg; 8, 40 mg; 9, 60 mg). An aliquot (10−15
3
3
3
+
and C-9″), 16.4 (CH , C-14); LRESIMS m/z 428 [M + H] , 450 [M
+
3
+
Na] ; HRESIMS m/z 428.3272 (calcd for C H N O 428.3277).
7
26 42 3 2,
In Vitro Growth Inhibitory Measurements. The six cancer cell
lines studied were obtained either from the American Type Culture
Collection (ATCC), the European Collection of Cell Culture
mg) of the crude diacylguanidine obtained from each reaction was
filtered on SiO2 and purified on reversed-phase HPLC [Supelco
Discovery C18 (25 cm × 10 mm, 5 μm) column, 35 min gradient from
(ECACC), or the Deutsche Sammlung von Mikroorganismen and
5
0 to 100% MeOH in H O with 0.1% of TFA, flow 2 mL/min, UV
2
Zellkulturen (DSMZ). The origin and histological type of each cell line
analyzed are detailed in the legend to Table 1.
detector at λ = 210 and 254 nm] to give the corresponding pure
diacylguanidine derivatives (1, 3.1 mg; 5, 2.5 mg; 6, 2.0 mg; 7, 1.8 mg;
The various cancer cell lines were cultured in RPMI culture medium
(Lonza code BE12-115F) supplemented with 10% heat-inactivated
fetal bovine serum (Life Technologies code 10270106), 4 mM
glutamine (Lonza code BE17-605E), 100 μg/mL gentamicin (Lonza
code BE17-518Z), and penicillin−streptomycin (200 units/mL and
200 mg/mL) (Lonza code BE17-602E). The in vitro growth inhibitory
activity of the various compounds was determined by means of the
8
, 3.5 mg; 9, 1.5 mg), as TFA salts.
Actinofide (1): H and C NMR data of synthetic 1 were identical
1
13
+
to those of the natural compound; LRESIMS m/z 360 [M + H] , 382
+
[
M + Na] ; HRESIMS m/z 360.2643 (calcd for C H N O ,
21 34 3 2
3
60.2651).
1
N,N′-Difarnesoyl Guanidine (5): H NMR (CDCl , 400 MHz) δ
.85 (2H, s, H-2 and H-2″), 5.09 (4H, m, H-6, H-10, H-6″, and H-
3
H
5
1
8−20
MTT colorimetric assay as detailed previously.
1
0″), 2.20 (6H, s, H -15 and H -15″), 2.18 (8H, m, H -4, H -5, H -4″,
3
3
2
2
2
and H -5″), 2.06 (4H, m, H -9 and H -9″), 1.99 (4H, m, H -8 and H -
2
2
2
2
2
8
1
″), 1.68 (6H, m, H -12 and H -12″), 1.605 (6H, m, H -14 and H -
ASSOCIATED CONTENT
3
3
3
3
■
13
4″), 1.599 (6H, m, H -13 and H -13″); C NMR (CDCl , 400 MHz,
3
3
3
*
S
Supporting Information
indirect detection from 2D heteronuclear experiments) δ 174.4 (C,
C
C-1), 159.8 (C, C-3 and C-3″), 135.7 (C, C-7 and C-7″), 131.1 (C, C-
1
1 and C-11″), 123.6 (CH, C-6, C-10, C-6″, and C-10″), 120.5 (CH,
C-2 and C-2″), 41.7 (CH , C-4 and C-4″), 39.5 (CH , C-8 and C-8″),
2
2
2
1
1
6.7 (CH , C-9 and C-9″), 25.9 (CH , C-5 and C-5″), 25.3 (CH , C-
2
2
3
1
13
H, C, 2D NMR, and MS spectra of compounds 1−10
2 and C-12″), 19.0 (CH , C-15 and C-15″), 17.4 (CH , C-13 and C-
3
3
+
3″), 16.3 (CH , C-14 and C-14″); LRESIMS m/z 496 [M + H] , 518
3
F
J. Nat. Prod. XXXX, XXX, XXX−XXX