Journal of Natural Products
Article
a
Table 2. In Vitro Antiprotozoal Activities of Compounds 1−3
b
c
IC50 (IC90 ), μg/mL
compound
L. donovani promastigote
L. donovani amastigote
L. donovani amastigote/THP
T. brucei
THP-1
1
5.7 0.6 (7.9 0.8)
6.7 0.7 (9.0 1.1)
5.0 0.3 (8.3 0.7)
0.2 0.03 (0.5 0.04)
>10 (>10)
3.1 0.2 (>10)
2.7 0.2 (3.6 0.03)
4.0 0.3 (6.8 0.03)
3.5 0.2 (6.5 0.04)
>10 (>10)
>10 (>10)
>10 (>10)
>2 (>2)
2
>10 (>10)
3.4 0.1 (8.8 0.4)
>10 (>10)
3
>10 (>10)
d
AMB
DFMO
0.9 0.05 (1.4 0.07)
0.2 0.02 (0.4 0.04)
nt
d
d
d
d
nt
nt
nt
6.1 0.7 (12.1 1.3)
nt
a
Data are mean values with standard deviations from two independent experiments, each with two replicates. The highest test concentration for 1−
b
c
3, amphotericin B (AMB), and α-difluoromethylornithine (DFMO) are 10, 2, and 20 μg/mL, respectively. 50% inhibitory concentration. 90%
inhibitory concentration. Not tested.
d
Boc-piperidinone (8, 1 equiv) in THF at −30 °C for 9 and 10 or at 0
°C for 11, under a nitrogen atmosphere. After stirring at 25 °C for 10
h, 2 M HCl was added to quench the reaction, and the mixture was
extracted with CH2Cl2 (3 × 5 mL). The organic phase was dried over
anhydrous Na2SO4. After workup, the products were separated by
column chromatography on silica gel using PE−EtOAc (15:1) as
eluent to give the corresponding N-Boc-aminoketone as a white solid.
tert-Butyl 6-oxohenicosyl-2-carbamate (9): yield 82% from n-
2-Methyl-6-tetradecyl-Δ1,6-piperideine (2): yield 98% from 10,
white solid; IR (neat) 2917, 2850, 1692, 1463, 1262, 1064, 1018, 780,
1
729 cm−1; H NMR (500 MHz, CDCl3) δH 0.88 (t, 3H, CH3), 1.30
(m, 22H, CH2), 1.42 (d, 3H, CH3), 1.53−1.80 (m, 6H, CH2), 2.37−
2.48 (m, 4H, CH2), 3.75 (m, 1H, CH); 13C NMR (125 MHz, CDCl3)
δC 14.4 (CH3), 18.9 (CH3), 19.7 (CH2), 22.9 (CH2), 24.1 (CH2), 29.5
(CH2), 29.6 (CH2), 29.7 (CH2), 29.8 (CH2), 29.89 (CH2), 29.92
(CH2), 32.2 (CH2), 34.2 (CH2), 42.1 (CH2), 43.2 (CH2), 48.5 (CH2),
63.3 (CH), 172.3 (CN); HREIMS m/z 293.3087 (calcd for
C20H39N, 293.3083).
1
C15H31MgBr, white solid; H NMR (500 MHz, CDCl3) δH 0.88 (t,
3H, CH3), 1.11 (d, 3H, CH3), 1.25 (m, 26H, CH2), 1.44 (s, 9H, CH3
× 3), 1.54−1.61 (m, 4H, CH2), 2.36−2.49 (m, 4H, CH2), 3.64 (m,
1H, CH), 4.34(s, br, 1H, NH); 13C NMR (125 MHz, CDCl3) δC 14.4
(CH3), 20.4 (CH3), 21.5 (CH2), 22.9 (CH2), 24.1 (CH2), 28.7 (CH3
× 3), 29.5 (CH2), 29.6 (CH2), 29.67 (CH2), 29.73 (CH2), 29.86
(CH2), 29.90 (CH2), 29.93 (CH2), 32.2 (CH2), 36.9 (CH2), 42.5
(CH2), 43.1 (CH2), 46.4 (CH), 79.2 (C), 155.7 (NHCO), 211.5
(CO).
2-Methyl-6-hexadecyl-Δ1,6-piperideine (3): yield 96% from 11,
white solid; IR (neat) 2917, 2850, 1705, 1467, 1259, 1068, 1019, 800,
1
721 cm−1; H NMR (500 MHz, CDCl3) δH 0.88 (t, 3H, CH3), 1.25
(m, 26H, CH2), 1.34 (d, 3H, CH3), 1.54−1.95 (m, 6H, CH2), 2.32−
2.45 (m, 4H, CH2), 3.68 (m, 1H, CH); 13C NMR (125 MHz, CDCl3)
δC 14.4 (CH3), 18.8 (CH3), 19.7 (CH2), 22.9 (CH2), 24.1 (CH2), 29.5
(CH2), 29.6 (CH2), 29.7 (CH2), 29.75 (CH2), 29.79 (CH2), 29.85
(CH2), 29.88 (CH2), 29.91 (CH2), 32.1 (CH2), 34.4 (CH2), 42.1
(CH2), 43.2 (CH2), 48.5 (CH2), 63.3 (CH), 172.3 (CN); HREIMS
m/z 321.3396 (calcd for C22H43N, 321.3396).
In Vitro Antifungal Assay. A modified version of the CLSI
(formerly NCCLS) method was used for susceptibility testing.
Organisms (C. neoformans ATCC 90113, C. albicans ATCC 90028,
and A. fumigatus ATCC 204305) were obtained from the American
Type Culture Collection (ATCC). Amphotericin B (ICN Biomed-
icals) was used as a positive control. The detailed procedure has been
described previously.12
In Vitro Antibacterial Assay. Microorganisms were obtained
from ATCC including methicillin-resistant S. aureus ATCC 1708,
vancomycin-resistant E. faecium ATCC 700221, E. coli ATCC 2452,
and P. aeruginosa ATCC 2108. The positive control drugs included
methicillin and vancomycin (from ICN Biomedicals). All organisms
were tested using a modified version of the CLSI method, which has
been described previously.19
Antiprotozoal Assay. The in vitro antileishmanial and anti-
trypanosomal assays were done on cell cultures of L. donovani
promastigotes, axenic amastigotes, THP1-amastigotes, and T. brucei
trypomastigotes by Alamar Blue assays as described earlier.20
Compounds with appropriate dilution were added to the test
organism (2 × 106 cells/mL) in clear flat-bottom 384-well microplates,
which were incubated at 26 °C for 72 h. IC50 and IC90 values were
calculated from the growth inhibition curve using XLFit. Amphotericin
B and alpha-difluoromethylornithine were used as positive controls.
tert-Butyl 6-oxoicosyl-2-carbamate (10): yield 86% from n-
1
C14H29MgCl, white solid; H NMR (500 MHz, CDCl3) δH 0.88 (t,
3H, CH3), 1.11 (d, 3H, CH3), 1.25 (m, 24H, CH2), 1.44 (s, 9H, CH3
× 3), 1.55−1.61 (m, 4H, CH2), 2.36−2.42 (m, 4H, CH2), 3.63 (m,
1H, CH), 4.33 (s, br, 1H, NH); 13C NMR (125 MHz, CDCl3) δC 14.4
(CH3), 20.3 (CH3), 21.4 (CH2), 22.9 (CH2), 24.1 (CH2), 28.6 (CH3
× 3), 29.5 (CH2), 29.58 (CH2), 29.65 (CH2), 29.70 (CH2), 29.84
(CH2), 29.88 (CH2), 29.89 (CH2), 29.91 (CH2), 32.2 (CH2), 36.8
(CH2), 42.5 (CH2), 43.1 (CH2), 46.3 (CH), 79.2 (C), 155.6
(NHCO), 211.5 (CO).
tert-Butyl 6-oxodocosyl-2-carbamate (11): yield 56% from n-
1
C16H33MgBr, white solid; H NMR (500 MHz, CDCl3) δH 0.88 (t,
3H, CH3), 1.11 (d, 3H, CH3), 1.25 (m, 28H, CH2), 1.44 (s, 9H, CH3
× 3), 1.55−1.61 (m, 4H, CH2), 2.36−2.42 (m, 4H, CH2), 3.64 (m,
1H, CH), 4.33 (s, br, 1H, NH); 13C NMR (125 MHz, CDCl3) δC 14.4
(CH3), 20.3 (CH3), 21.5 (CH2), 22.9 (CH2), 24.1 (CH2), 28.7 (CH3
× 3), 29.5 (CH2), 29.61 (C CH2), 29.67 (CH2), 29.73 (CH2), 29.86
(CH2), 29.90 (CH2), 29.92 (CH2), 29.94 (CH2), 32.2 (CH2), 36.9
(CH2), 42.5 (CH2), 43.1 (C CH2), 46.4 (CH), 79.3 (C), 155.7
(NHCO), 211.5 (CO).
General Procedure for Preparation of 2-Methyl-6-alkyl-Δ1,6
-
piperideines (1−3). To a solution of N-Boc-aminoketone (9−11)
(100 mg) in CH2Cl2 (2 mL) at 0 °C was added 12 M HCl (1 mL)
dropwise over 5 min. After stirring for 5 h at 0 °C, the reaction mixture
was basified with 2 M NaOH and then extracted with CH2Cl2 (3 × 5
mL). The organic phase was dried over anhydrous Na2SO4 to get the
2-methyl-6-alkyl-Δ1,6-piperideines as white solids.
2-Methyl-6-pentadecyl-Δ1,6-piperideine (1): yield 97% from 9,
white solid; IR (neat) 2917, 2850, 1705, 1508, 1470, 1379, 1261, 1220,
ASSOCIATED CONTENT
■
1
1069, 1019, 803, 721 cm−1; H NMR (500 MHz, CDCl3) δH 0.88 (t,
S
* Supporting Information
3H, CH3), 1.25 (m, 24H, CH2), 1.33 (d, 3H, CH3), 1.43−1.99 (m,
6H, CH2), 2.54−2.68 (m, 4H, CH2), 3.90 (m, 1H, CH); 13C NMR
(125 MHz, CDCl3) δC 14.4 (CH3), 18.9 (CH3), 19.7 (CH2), 22.9
(CH2), 24.1 (CH2), 29.5 (CH2), 29.6 (CH2), 29.7 (CH2), 29.8 (CH2),
29.89 (CH2), 29.92 (CH2), 29.93 (CH2), 32.2 (CH2), 34.4 (CH2),
42.1 (CH2), 43.2 (CH2), 48.6 (CH2), 63.3 (CH), 172.3 (CN);
HREIMS m/z 307.3237 (calcd for C21H41N, 307.3239).
The Supporting Information is available free of charge on the
1H and 13CNMR, MS, and IR spectra of intermediates
C
J. Nat. Prod. XXXX, XXX, XXX−XXX