Synthesis from norambreinolide, structure, and antimicrobial activity of dihomodrimane sesquiterpenoids with azine, hydrazide, and dihydrazide fragments

Article abstract of DOI:10.1007/s10600-016-1854-6

12,12'-Azino-di-8α-hydroxy-11-dihomodrimane, N,N’-di-(8α-hydroxy-11-dihomodrim-12-ylidene)-adipic acid dihydrazide, N,N’-di-(Δ^8,13-bicyclohomofarnesenoyl)-hydrazine, and hydrazides of Δ^8,9- and Δ^8,13-bicyclohomofarnesenoic

Full text of DOI:10.1007/s10600-016-1854-6

DOI 10.1007/s10600-016-1854-6
Chemistry of Natural Compounds, Vol. 52, No. 6, November, 2016
SYNTHESIS FROM NORAMBREINOLIDE, STRUCTURE,
AND ANTIMICROBIAL ACTIVITY OF DIHOMODRIMANE
SESQUITERPENOIDS WITH AZINE, HYDRAZIDE,
AND DIHYDRAZIDE FRAGMENTS
1*
1
1
A. N. Aricu, K. I. Kuchkova, A. N. Barba,
1
2
3
I. P. Dragalin, S. G. Shova, N. Vornicu,
1
1
1
E. K. Gorinchoi, E. S. Sekara, L. V. Lungu,
4
1
1
M. Niculaua, N. D. Ungur, and P. F. Vlad
12,12ꢀ-Azino-di-8ꢁ-hydroxy-11-dihomodrimane, N,Nꢀ-di-(8ꢁ-hydroxy-11-dihomodrim-12-ylidene)-adipic
8,13
8,9
acid dihydrazide, N,Nꢀ-di-(ꢂ
ꢂ
-bicyclohomofarnesenoyl)-hydrazine, and hydrazides of ꢂ - and
8,13
-bicyclohomofarnesenoic acids were synthesized.
Keywords: dihomodrimane sesquiterpenoids, norambreinolide, azines, hydrazides, bicyclohomofarnesenoic acid,
synthesis.
New dihomodrimanes containing pharmacophoric azine and hydrazide fragments were synthesized in order to discover
new biologically active compounds and to continue our studies on the synthesis of N-containing drimane sesquiterpenoids
[1–5]. Several azines and hydrazides are known to possess high and varied biological activity [6, 7], including antituberculosis
and antibacterial. We supposed that adding these fragments to the drimane scaffold could increase its biological potential.
The starting material for synthesizing azine 1, dihydrazide 2, and 11-dihomodriman-8ꢁ-ol-12-one (3) was prepared
from norambreinolide 4, as described previously by us [8]. Reaction of 3 with hydrazine hydrate in MeOH formed 1. Refluxing
3 with adipic acid dihydrazide in MeOH gave 2, in which two ketones were condensed through the carbonyls to adipic acid
dihydrazide (Scheme 1).
13
14
13'
14'
O
11
N
N
11'
O
17'
17
10
1
1'
O
9
9'
a
b
OH HO
OH
7
7'
3'
3
5
5'
4
3
1
16
15
15' 16'
c
13
13'
N
N
NH C(O) CH (CH ) CH C(O) NH
2 2
11'
11
9
2
2
18
19
19'
17'
14'
18'
1
1'
OH
HO
5
6'
2
16'
15'
a. CH Li, Et O, 20ꢃÑ, 15 min, 65%; b. N H 4H O, CH OH, ꢂ, 10 h, 80%; c. NH NHCO(CH ) CONHNH , CH OH, 20ꢃÑ, 48 h, ꢂ, 20 h, 31%
3
2
2
4
2
3
2
2 4
2
3
Scheme 1
1) Institute of Chemistry, Academy of Sciences of Moldova, 3 Akademicheskaya St., MD-2028, Kishinev, Moldova,
e-mail: aculina.aricu@gmail.com; 2) P. Poni Institute of Macromolecular Chemistry, 41A Aleea Grigore Ghica Vodye,
RO-700487 Iasi, Romania; 3) Mitropolitan Research Center T.A.B.O.R., 9 Closca St., Iasi, RO-700066, Romania; 4) Research
Center for Oenology, RomanianAcademy, Iasi Branch, 9Aleea M. Sadoveanu, 700490 Iasi, Romania. Translated from Khimiya
Prirodnykh Soedinenii, No. 6, November–December, 2016, pp. 885–891. Original article submitted March 29, 2016.
©
0009-3130/16/5206-1029 2016 Springer Science+Business Media New York
1029

8,9
Norambreinolide (4) yielded (in six steps) in 62% overall yield ꢂ -bicyclohomofarnesenoic acid (5). The key steps
8,9
in this synthesis were oxidation of ꢂ -bicyclohomofarnesen-12-ol (6) by P O and DMSO into aldehyde 7 and subsequent
2
5
oxidation of this aldehyde by NaClO into acid 5. Direct oxidation of 6 by Jones reagent gave acid 5 in only 42% yield.
2
Alcohol 6 was produced via dehydration of sclaradiol monoacetate 8 [9, 10] by MeSO SiMe (trimethylsilylmethanesulfonate)
3
3
in MeCN to form acetate 9 that was subsequently hydrolyzed. Reaction of acid 5 with (COCl) produced in situ acid chloride
2
10, reaction of which with N H 4H O in CH Cl afforded hydrazide 11. The main product from the reaction in EtOH was
2
4
2
2
2
8,9
ethyl ꢂ -bicyclohomofarnesenoate (12) in 55% yield (Scheme 2).
O
CONHNH
2
OR
OCOCH
OH
3
O
cc. 9, 10
a
2
2 steps
4
11
8
6, 9
6
b
9
g
6: R = H; 9: R = Ac
16
18
CO CH CH
2
2
3
CHO
11
CO H
COCl
2
15
5
1
9
12
f
c
d
h
6
14
13
7
5
10
12
e
a. MeSO SiMe , CH CN, 20ꢃC, 5 h, 72%; b. KOH, MeOH, 20ꢃC, 2 h, 99%; ñ. DMSO, P O , CH Cl , 0ꢃC, 10 min, 20ꢃC, 45 min; Et N, 0ꢃC,
3
3
3
2
5
2
2
3
10 min, 20ꢃC, 45 min, 95 %; d. NaClO , NaH PO 42H O, 2-Me-2-butene, t-BuOH, 20ꢃC, 2 h, 94 %; å. Jones reagent, (CH ) CO, 0ꢃC,
2
2
4
2
3 2
48 h, 42 %; f. (COCl) , C H , 20ꢃC, 1 h, ꢂ, 1 h; g. N H 4H O, CH Cl , 20ꢃC, 2 h, ꢂ, 7 h, 65%; h. N H 4H O, C H OH, 20ꢃC, 7 h, 55%
2
6
6
2
4
2
2
2
2
4
2
2 5
Scheme 2
8,13
Norambreinolide 4 was also used to synthesize ꢂ -bicyclohomofarnesenoic acid (13) in 60% overall yield as
8,13
before [4]. Reaction of its acid chloride 14 with hydrazine hydrate in CH Cl gave its hydrazide 15 and N,Nꢀ-di-(ꢂ
-
2
2
bicyclohomofarnesenoyl)-hydrazine (16) and hydrazide 17 with a reduced double bond. These compounds were isolated pure
by liquid chromatography (Scheme 3).
O
COCl
COOH
O
b
cc. 4
a
6 steps
4
14
13
O
O
CONHNH
11'
16'
CONHNH
H
11 CNHNHC
2
2
16
12
12'
13'
1
1'
13
9
9'
+
+
5
14'
15 14
15'
15 (25%)
17 (19%)
16 (30%)
a. (COCl) , C H , 20°C, 1 h, ꢂ, 1 h; b. N H 4H O, CH Cl , 20°C, 10 h, ꢂ, 10 h
2
6
6
2
4
2
2
2
Scheme 3
13
15
PMR, C NMR, N NMR, and IR spectroscopic data and mass spectral analysis including high-resolution in addition
to an X-ray crystal structure analysis for the initially prepared 1, 2, 5–7, 11, 12, and 15–17 agreed with the formulas given in
1
1
the schemes. H– H NOESY experiments demonstrated that the C-8 and C-10 methyls coupled so that C-8 of hydrazide 17
13
15
was assigned the S-configuration. PMR, C NMR, and N NMR spectra of 1 in CDCl were consistent with two isomers in
3
13
a 2:1 ratio. Analogously, PMR and C NMR spectra of dihydrazide 2 in CDCl showed that it also existed as two isomers in
3
an approximately 1:1 ratio. Special studies are required to establish their structures.
1030

TABLE 1. Crystal Data and Structure Refinement for 1
Empirical formula
C34H60N2O2
Formula weight
Crystal system
Space group
528.84
Monoclinic
P21
15.1323(7)
6.1227(3)
17.6171(8)
95.567(4)
1624.53(13)
2
°
à, A
°
b, A
°
ñ, A
ꢅ, deg.
° 3
V, A
Z
(calcd), g/cm3
1.081
0.499
ꢄ, mm–1
F(000)
Crystal size, mm
588
0.35 ꢆ 0.08 ꢆ 0.02
7.358–133.19
ꢇ-range, deg.
Limiting indices
Reflections collected/unique (Rint)
Number of refined parameters
GOOF
–18 ꢈ h ꢈ 18; –7 ꢈ k ꢈ 7; –20 ꢈ l ꢈ 20
19919/5473 (0.0967)
356
1.039
R- factor (I >2ꢉ(I))
R1 = 0.0569 (wR2 = 0.1252)
R1 = 0.0759 (wR2 = 0.1362)
0.14, –0.16
R-factor (whole dataset)
° –3
ꢂ
max, ꢂ min, e A
C14A
O1A
C8A
O1B
C8B
C7B
C14B
C6B
C13A
C7A
C16A
C11A
C9A
C10A
C1A
C2A
C15B
N1A
C5B
C4B
N1B
C9B
C11B
C1B
C17B
C5A
C6A
C12B
C10B
C16B
C3B
C2B
C13B
C4A
C15A
C3A
Fig. 1. Molecular structure of 1.
An X-ray crystal structure analysis of 1 found that the molecule had the trans-configuration in the single crystal.
Figure 1 shows the molecular structure of the asymmetric unit. It consisted of two chemically equivalent but crystallographically
°
independent parts designated A and B and joined by the N1A–N1B bond of 1.417(5) A.
An intermolecular H-bond with O1A–H as the proton donor and O1B–H as the proton acceptor formed in the crystal.
However, O1B–H acted as a donor in forming an intramolecular H-bond with N1B. The molecules were combined into
infinite chains as a result of these interactions.
Compounds 1, 2, 11, 15, 16, and 17 were tested in vitro for antifungal and antibacterial activity against pure cultures
of five fungal species (Aspergillus flavus, Fusarium, Penicillium chrysogenum, P. frequentans, Alternaria alternata) and
Gram-negative (Pseudomonas aeruginosa) and Gram-positive (Bacillus sp.) bacteria. Compound 2 exhibited significant
antifungal activity with a characteristic minimum inhibiting concentration (MIC) of 0.50 ꢄg/mL, which was comparable with
the activity of the known antifungal agent caspofungin that was used as the reference drug (MIC = 0.25 ꢄg/mL), and moderate
antibacterial activity (MIC = 24 ꢄg/mL) compared with the reference antibiotic kanamycin (MIC = 3 ꢄg/mL).
Thus, new dihomodrimane sesquiterpenoids containing azine, hydrazide, and dihydrazide fragments were synthesized.
It was found that N,Nꢀ-di-(8ꢁ-hydroxy-11-dihomodrim-12-ylidene)-adipic acid dihydrazide (2) possessed significant antifungal
and antibacterial activity.
1031

EXPERIMENTAL
Melting points were measured on a Boetius apparatus; [ꢁ] values, in CHCl on a JASCO P-2000 polarimeter.
D
3
13
15
IR spectra were recorded on a PerkinElmer Spectrum 100 FTIR spectrophotometer. PMR, C NMR, and N NMR spectra
were recorded in CDCl on a Bruker Avance III 400 spectrometer (400, 100, and 40 MHz). Chemical shifts were given on the
3
ꢊ-scale in ppm relative to CD(H)Cl resonances as internal standards (resonances of ꢊ 7.24 and 77.00 ppm, respectively) in
3
13
15
13
PMR and C NMR spectra and relative to MeNO external standard in N NMR spectra. Resonances in C NMR spectra
2
1
13
1
1
1
13
1
13
1
1
were assigned using H– C DEPT, H– H COSY-45, H– C HMQC, H– C HMBC, and H– H NOESY experiments; in
15
1
15
1
15
N NMR spectra, H– N HMBC and H– N HMQC experiments. The course of reactions was monitored by TLC on
Silufol plates with detection by I vapor. Products were isolated using column chromatography over Fluka-60 silica gel and a
2
Gilson HPLC with a refraction detector. The product compositions were determined and mass spectra were recorded on an
Agilent 7890A chromatograph with an MSD 5975C VL quadrupole MS detector and an HP-5ms capillary column
(30 m ꢆ 0.25 ꢄm). The vaporizer temperature was 250°C; ionization potential 70 eV. Analysis conditions: T = 70°C (2 min),
1
5°C/min to 200°C, 20°C/min to 300°C, T = 300°C (5 min). The He flow rate was 1 mL/min. High-resolution mass spectra
2
(HR-EI-MS) were recorded on an AEI MS 902 spectrometer (EI 70 eV). Et O and EtOAc extracts were dried over anhydrous
2
MgSO . Solvents were distilled from extracts at reduced pressure. Elemental analyses of all compounds agreed with those
4
calculated.
Preparation of 12,12ꢀ-Azino-di-8ꢁ-hydroxy-11-dihomodrimane (1). A solution of 3 (100 mg, 0.37 mmol) in
MeOH (3 mL) was treated with N H 4H O (0.2 mL, 0.206 g, 4.11 mmol), stirred, and refluxed for 10 h. The MeOH was
2
4
2
removed at reduced pressure. The residue was treated with H O (5 mL) and EtOAc (30 mL) and stirred. The organic layer
2
was separated. The aqueous layer was extracted with EtOAc (10 mL). The combined EtOAc extracts were washed with H O
2
(3 ꢆ 5 mL) and dried. The EtOAc was distilled. The crystalline solid was recrystallized from MeCN to afford 1 (80 mg, 80%),
26
–1
mp 168–169ꢃC (MeCN), [ꢁ] –151.1ꢃꢋ (ñ 0.53, CHCl ). IR spectrum (ꢌ, cm ): 3381, 3222 (ÎÍ), 1636, 1619 (Ñ=N). Main
D
3
1
isomer. Í NMR spectrum (400 MHz, CDCl , ꢊ, ppm, J/Hz): 0.80 (6Í, s, ÑÍ -16, 16ꢀ), 0.82 (6H, s, ÑÍ -17, 17ꢀ), 0.87 (6H,
3
3
3
s, ÑÍ -15, 15ꢀ), 0.95* (2H, m, H-5ꢁ, 5ꢀꢁ), 1.19 (6H, s, ÑÍ -14, 14ꢀ), 1.86* (2H, m, H-9ꢁ, 9ꢀꢁ), 1.93 (6H, s, ÑÍ -13, 13ꢀ), 2.31
3
3
3
(2H, dd, J = 16.7, 6.4, H-11a, 11ꢀa), 2.50 (2H, dd, J = 16.7, 2.3, H-11b, 11ꢀb), 3.77 (2H, br.s, 2 ꢆ (OH)). (*overlapped resonances).
13
C NMR spectrum (100 MHz, CDCl , ꢊ, ppm): 15.49 (C-17, 17ꢀ), 18.47 (C-2, 2ꢀ), 18.71 (C-13, 13ꢀ), 20.44 (C-6, 6ꢀ), 21.44
3
(C-16, 16ꢀ), 24.13 (C-14, 14ꢀ), 33.35 (C-4, 4ꢀ), 33.38 (C-15, 15ꢀ), 34.54 (C-11, 11ꢀ), 38.81 (C-10, 10ꢀ), 39.63 (C-1, 1ꢀ), 41.84
15
(C-3, 3ꢀ), 44.24 (C-7, 7ꢀ), 56.11 (C-5, 5ꢀ), 57.19 (C-9, 9ꢀ), 73.12 (C-8, 8ꢀ), 168.54 (C-12, 12ꢀ). N NMR spectrum (40 MHz,
1
CDCl , ꢊ, ppm): 336, 329 (2 ꢆ (C=N)). Minor isomer. Í NMR spectrum (400 MHz, CDCl , ꢊ, ppm, J/Hz): 0.77 (6H, s,
3
3
ÑÍ -17, 17ꢀ), 0.80 (6H, s, ÑÍ -16, 16ꢀ), 0,87 (6H, s, ÑÍ -15, 15ꢀ), 0.95* (2H, m, H-5ꢁ, 5ꢀꢁ), 1.27 (6H, s, ÑÍ -14, 14ꢀ), 1.80*
3
3
3
3
15
(2H, m, H-11a, 11ꢀa), 2.09 (6H, s, ÑÍ -13, 13ꢀ), 2.70* (2H, m, H-11b, 11ꢀb), 5.48 (2H, br.s, 2 ꢆ (OH)). N NMR spectrum
(40 MHz, CDCl , ꢊ, ppm): 338, 331 (2 ꢆ (C=N)). Mass spectrum (EI, 70 eV), m/z (I , %): 495 (Ì – 34 (2 ÎÍ), 7), 405 (4),
3
+
3
rel
355 (4), 315 (9), 281 (41), 246 (52), 207 (100), 191 (82), 175 (27), 137 (29), 121 (40), 109 (71), 95 (48), 81 (47), 69 (59), 55
(48), 43 (68). HR-EI-MS: found 529.4709. C H N O . Calcd 528.8602.
34 60
2 2
Preparation of N,Nꢀ-Di-(8ꢁ-hydroxy-11-dihomodrim-12-ylidene)-adipic Acid Dihydrazide (2). Adipic acid
dihydrazide (32 mg, 0.18 mmol) was dissolved with heating in MeOH (16 mL). The solution was stirred, cooled to room
temperature, treated with ketone 3 (100 mg, 0.37 mmol), stirred at 20°C for 48 h, and refluxed for 20 h. The MeOH was
removed at reduced pressure. The residue was dried over P O in a vacuum desiccator, treated with CHCl (5 mL), and stirred
2
5
3
for 1 h. The insoluble precipitate (15 mg) was filtered off and rinsed with CHCl . The filtrates were evaporated. The residue
3
was dried over P O to afford a product (136 mg) that was recrystallized from hexane. The crystalline precipitate was filtered
2
5
off and rinsed with hexane to afford a mixture (80 mg) of the reaction product and starting ketone 3. The hexane filtrate was
evaporated to give unreacted 3 (37 mg). Target compound 2 was separated from its mixture (80 mg) with 3 by column
chromatography over silica gel (4 g). Ketone 3 (9 mg) was eluted by CHCl ; dihydrazide 2 (31%; considering unreacted 3, the
3
yield of dihydrazide 2 was 57%), by CHCl –MeOH (24:1). Dihydrazide 2. White crystals, mp 101–102ꢃC (hexane),
3
26
–1
15
[ꢁ] –60.6ꢃ (ñ 1.35, CHCl ). IR spectrum (ꢌ, cm ): 3400, 3227, 2923, 1662, 1534, 1454, 1387, 1250, 1067, 938. N NMR
spectrum (40 MHz, CDCl , ꢊ, ppm): 169 (NÍ), 299, 301, 302, 305 (C=N). Main isomer. Í NMR spectrum (400 MHz,
D
3
1
3
CDCl , ꢊ, ppm, J/Hz): 0.80 (6H, s, ÑÍ -15, 15ꢀ), 0.83 (6H, s, ÑÍ -17, 17ꢀ), 0.84 (6H, s, ÑÍ -16, 16ꢀ), 1.22, 1.25 (6H, s,
3
3
3
3
13
ÑÍ -14, 14ꢀ), 2.11, 2.13 (6H, s, ÑÍ -13, 13ꢀ), 3.0 (2H, br.s, 2 ꢆ (OH)), 8.27, 8.37 (2H, br.s, 2 ꢆ NH). C NMR spectrum
3
3
(100 MHz, CDCl , ꢊ, ppm): 15.20, 15.26 (C-17, 17ꢀ), 18.43, 18.50 (C-6, 6ꢀ), 20.36 (C-2, 2ꢀ), 21.39, 21.51 (C-16, 16ꢀ), 23.99,
3
24.16 (C-14, 14ꢀ), 24.05, 24.49 (C-20, 20ꢀ), 26.11, 26.19 (C-13, 13ꢀ), 28.29 (C-11, 11ꢀ), 32.04, 32.67 (C-19, 19ꢀ), 33.30 (C-4, 4ꢀ),
1032

33.41 (C-15, 15ꢀ), 38.70, 38.76 (C-10, 10ꢀ), 39.72, 39.87 (C-1, 1ꢀ), 41.72, 41.80 (C-3, 3ꢀ), 43.96, 44.11 (C-7, 7ꢀ), 56.24
(C-5, 5ꢀ), 57.22, 57.27 (C-9, 9ꢀ), 74.96, 75.29 (C-8, 8ꢀ), 156.61, 157.85 (C-12, 12ꢀ), 175.07, 175.26 (C-18, 18ꢀ).
1
Minor isomer. Í NMR spectrum (400 MHz, CDCl , ꢊ, ppm, J/Hz): 0.86 (6Í, s, ÑÍ -15, 15ꢀ), 0.88 (6H, s, ÑÍ -16, 16ꢀ), 0.89
3
3
3
(6H, s, ÑÍ -17, 17ꢀ), 1.21 (6H, s, ÑÍ -14, 14ꢀ), 1.85, 1.89 (6H, s, ÑÍ -13, 13ꢀ), 3.0 (2H, br.s, 2 ꢆ (OH)), 11.44, 11.46 (2H,
3
3
3
13
br.s, 2 ꢆ NH). C NMR spectrum (100 MHz, CDCl , ꢊ, ppm): 15.43, 15.48 (C-17, 17ꢀ), 15.65, 16.07 (C-13, 13ꢀ), 18.22, 18.26
3
(C-6, 6ꢀ), 20.36 (C-2, 2ꢀ), 21.43, 21.47 (C-16, 16ꢀ), 24.05, 24.49 (C-20, 20ꢀ), 24.24 (C-14, 14ꢀ), 32.04, 32.67 (C-19, 19ꢀ), 33.20
(C-4, 4ꢀ), 33.28 (C-15, 15ꢀ), 34.59, 34.70 (C-11, 11ꢀ), 38.80, 38.87 (C-10, 10ꢀ), 40.03, 40.20 (C-1, 1ꢀ), 41.45, 41.51 (C-3, 3ꢀ),
44.36, 44.50 (C-7, 7ꢀ), 55.84, 55.90 (C-5, 5ꢀ), 56.85, 56.91 (C-9, 9ꢀ), 73.55, 73.97 (C-8, 8ꢀ), 154.68, 155.61 (C-12, 12ꢀ),
169.70, 170.23 (C-18, 18ꢀ). HR-EI-MS: found 671.5471. Ñ Í N Î . Calcd 671.0174.
40 70
4 4
8,9
Preparation of 12-Acetoxy-ꢂ -bicyclohomofarnesen-12-ol (9). A solution of sclaradiol monoacetate (8, 2 g,
6.75 mmol) [9, 10] in anhydrous MeCN (18 mL) was treated with Me SiSO Me (5.3 mL, 5.78 g, 34.3 mmol), stirred for 5 h
3
3
at 20°C, diluted with H O (100 mL) and Et O (300 mL), and stirred. The aqueous layer was separated. The Et O layer was
2
2
2
washed with NaHCO solution (3 ꢆ 30 mL) and H O (3 ꢆ 30 mL) and dried. The Et O was evaporated to afford a mixture of
3
2
2
8,9 7,8
8,13
the ꢂ , ꢂ , and ꢂ
isomers (1.6 g, 85%) in an 85:11:2 ratio (from PMR spectra and GC-MS analysis). The mixture (1 g)
of isomers was chromatographed over a column of silica gel (100 g). Elution by petroleum ether–Et O (98.5:1.5) eluted the
2
13
isomer (0.8 g) with the double bond in the C –C position (9). PMR and C NMR and IR spectroscopic data and mass
8
9
spectral analysis of this compound agreed fully with those in the literature [11].
8,9
Preparation of ꢂ -Bicyclohomofarnesen-12-ol (6). Acetate 9 (0.46 g, 16.5 mmol) was treated with a solution of
KOH (0.46 g, 82.0 mmol) in MeOH (14 mL) and stirred for 2 h at 20°C. The MeOH was distilled at reduced pressure. The
residue was treated with H O (25 mL) and Et O (50 mL) and stirred. The aqueous layer was extracted with Et O (3 ꢆ 15 mL).
2
2
2
The combined Et O extracts were washed with H O (5 ꢆ 10 mL) and dried. The Et O was evaporated to afford 6 (386 mg,
2
2
2
16
–1
1
99%). Ñ Í Î, [ꢁ] +95.2ꢃ (ñ 1.39, CHCl ). IR spectrum (ꢌ, cm ): 3306, 1036, 1019 (ÎÍ). Í NMR spectrum (400 MHz,
16 28
D
3
CDCl , ꢊ, ppm, J/Hz): 0.83 (3H, s, ÑÍ -15), 0.88 (3H, s, ÑÍ -14), 0.95 (3H, s, ÑÍ -16), 1.09 (1H, dd, J = 12.6, 6.1, H-5ꢁ),
3
3
3
3
1.61 (3H, s, ÑÍ -13), 1.96 (1H, dd, J = 17.7, 6.5, H-7a), 2.05 (1H, td, J = 17.7, 10.9, 7.0, H-7b), 2.24 (1H, td, J = 13.3, 9.7, 6.2,
3
H-11a), 2.39 (1H, td, J = 13.3, 9.8, 6.9, H-11b), 3.59 (1H, td, J = 16.5, 9.7, 6.2, H-12a), 3.62 (1H, td, J = 16.5, 9.8, 6.9, H-12b).
13
C NMR spectrum (100 MHz, CDCl , ꢊ, ppm): 19.00 (C-2), 19.02 (C-6), 19.94 (C-13), 20.09 (C-16), 21.70 (C-15), 31.50
3
(C-11), 33.32 (C-14), 33.34 (C-4), 33.66 (C-7), 37.15 (C-1), 38.68 (C-10), 41.73 (C-3), 51.69 (C-5), 62.64 (C-12), 128.56
+
(C-8), 136.19 (C-9). Mass spectrum (EI, 70 eV), m/z (I , %): 236 (Ì , 40), 221 (63), 203 (26), 191 (97), 177 (42), 163 (34),
rel
151 (41), 135 (43), 121 (85), 107 (88), 95 (100), 79 (61), 69 (55), 55 (54), 41 (69).
8,9
Preparation of ꢂ -Bicyclohomofarnesen-12-al (7). A solution of 6 (0.24 g, 1.01 mmol) in CH Cl (10 mL) was
2
2
stirred, cooled in an ice bath, and treated with anhydrous DMSO (0.6 mL, 0.66 g, 8.45 mmol) and P O (0.97 g, 6.83 mmol).
2
5
After 10 min, the flask was removed from the ice bath. The solution was stirred for another 45 min at room temperature,
treated with Et N (0.72 mL, 0.52 g, 5.14 mmol) with cooling in ice, stirred in the ice bath for 10 min and at room temperature
3
for 45 min, treated dropwise with H O (3 mL) and HCl (5%, 3 mL) with cooling in the ice bath, and extracted with CH Cl
2
2
2
(3 ꢆ 25 mL). The extract was washed with H O (2 ꢆ 8 mL) and dried. The solvent was evaporated to afford 7 (0.23 g, 95%
2
16
–1
1
according to GC-MS analysis). Ñ Í Î, [ꢁ] +138.6ꢃ (ñ 1.40, CHCl ). IR spectrum (ꢌ, cm ): 1723 (CHO). Í NMR
16 26
D
3
spectrum (400 MHz, CDCl , ꢊ, ppm, J/Hz): 0.84 (3H, s, ÑÍ -15), 0.90 (3H, s, ÑÍ -14), 0.94 (3H, s, ÑÍ -16), 1.55 (3H, s,
3
3
3
3
13
ÑÍ -13), 1.19* (1H, m, H-5ꢁ), 3.05, 3.11 (each 1Í, AB system, J = 17.1, H-11a, 11b), 9.53 (1H, t, J = 2.3, H-12). C NMR
3
spectrum (100 MHz, CDCl , ꢊ, ppm): 18.89 (C-2, 6), 19.77 (C-16), 19.93 (C-13), 21.58 (C-15), 33.19 (C-14), 33.29 (C-4),
3
33.86 (C-7), 37.22 (C-1), 38.46 (C-10), 41.49 (C-3), 43.18 (C-11), 51.70 (C-5), 131.25 (C-8), 132.15 (C-9), 201.43 (C-12).
+
Mass spectrum (EI, 70 eV), m/z (I , %): 234 (Ì , 17), 219 (32), 201 (43), 190 (95), 175 (100), 163 (23), 149 (50), 133 (21),
rel
123 (43), 105 (60), 91 (55), 79 (36), 69 (39), 55 (35), 41 (47).
8,9
Preparation of ꢂ -BicyclohomofarnesenoicAcid (5). A solution of 7 (0.23 g, 0.98 mmol) in t-BuOH (23 mL) and
2-methyl-2-butene (5 mL) was stirred, treated over 10 min with a solution of NaClO (1.13 g, 12.5 mmol) and NaH PO 42H O
2
2
4
2
(1.15 g, 7.37 mmol) in H O (10 mL), and stirred for 2 h at 20°C. The solvents were evaporated at reduced pressure. The
2
residue was treated with H O (25 mL), acidified with HCl (10%), and extracted with Et O (3 ꢆ 50 mL). The Et O extract was
2
2
2
washed with H O (3 ꢆ 15 mL) and dried. The Et O was evaporated to afford 5 (0.24 g, 97% according to GC-MS analysis).
2
2
17
–1
1
Ñ Í Î , [ꢁ] +70.2ꢃ (ñ 1.44, CHCl ). IR spectrum (ꢌ, cm ): 2670, 1703 (COOH). Í NMR spectrum (400 MHz, CDCl ,
16 26
2
D
3
3
ꢊ, ppm, J/Hz): 0.83 (3H, s, ÑÍ -15), 0.89 (3H, s, ÑÍ -14), 0.93 (3H, s, ÑÍ -16), 1.21 (1H, dd, J = 12.6, 1.90, H-5ꢁ), 1.59 (3H,
3
3
3
s, ÑÍ -13), 2.02 (1H, dd, J = 18.6, 6.2, H-7a), 2.13 (1H, dd, J = 18.6, 11.3, H-7b), 3.01 (1H, d, J = 17.3, H-11a), 3.14 (1H, d,
3
13
J = 17.3, H-11b), 9.58 (1H, br.s, OH). C NMR spectrum (100 MHz, CDCl , ꢊ, ppm): 18.86 (C-2), 18.89 (C-6), 19.73 (C-16),
3
1033

20.10 (C-13), 21.61 (C-15), 32.94 (C-11), 33.13 (C-14), 33.27 (C-4), 33.56 (C-7), 36.26 (C-1), 38.58 (C-10), 41.48 (C-3),
+
51.38 (C-5), 130.78 (C-8), 133.42 (C-9), 178.85 (C-12). Mass spectrum (EI, 70 eV), m/z (I , %): 250 (Ì , 35), 235 (66), 190
rel
(100), 175 (96), 165 (79), 153 (27), 139 (67), 119 (77), 105 (77), 91 (72), 79 (46), 69 (42), 55 (39), 41 (66).
8,9
Preparation of ꢂ -BicyclohomofarnesenoicAcid Hydrazide (11). Asolution of 5 (100 mg, 0.40 mmol) in anhydrous
benzene (2 mL) was treated with a solution of (COCl) (0.4 mL, 0.58 g, 4.58 mmol) in benzene (1 mL), stirred at 20°C for
2
1 h and refluxed for 1 h. The benzene and excess of (COCl) were evaporated at reduced pressure. The residue was treated
2
with CH Cl (2 mL) and N H 4H O (1 mL), stirred for 2 h at 20°C, refluxed for 7 h, and diluted with CH Cl (2 mL). The
2
2
2
4
2
2
2
excess of N H 4H O was separated. The organic layer was washed with H O (3 ꢆ 1 mL) and dried. The solvent was evaporated
2
4
2
2
to afford a product (89 mg) that was chromatographed over a column of silica gel (2.7 g) (CHCl –MeOH, 49:1) to afford 11
3
20
–1
(76 mg, 65%). Hydrazide 11, Ñ Í N Î, [ꢁ] +22.1ꢃ (ñ 1.3, CHCl ). IR spectrum (ꢌ, cm ): 3425, 3291 (NH ), 1655, 1626
(CONH). Í NMR spectrum (400 MHz, CDCl , ꢊ, ppm, J/Hz): 0.83 (3H, s, ÑÍ -15), 0.90 (3H, s, ÑÍ -14), 0.94 (3H, s,
16 28
2
D
3
2
1
3
3
3
ÑÍ -16), 0.95*–1.80* (8H, m), 1.14 (1H, dd, J = 21.0, 12.7, H-5ꢁ), 1.59 (3H, s, ÑÍ -13), 2.07 (2H, m, H-7a, 7b), 2.94 (1H, d,
3
3
13
J = 17.4, H-11a), 3.09 (1H, d, J = 17.4, H-11b), 3.86 (2H, br.s, NH ), 7.03 (1H, br.s, NH). C NMR spectrum (100 MHz, CDCl ,
2
3
ꢊ, ppm): 18.74 (C-2), 18.77 (C-6), 19.84 (C-16), 20.15 (C-13), 21.58 (C-15), 33.19 (C-14), 33.32 (C-4), 33.50 (C-7), 34.37 (C-11),
15
36.01 (C-1), 38.80 (C-10), 41.47 (C-3), 51.87 (C-5), 131.68 (C-7), 134.73 (C-8), 172.16 (C-12). N NMR spectrum (40 MHz, CDCl ,
3
+
ꢊ, ppm): 127 (NH). Mass spectrum (EI, 70 eV), m/z (I , %): 264 (Ì , 23), 249 (31), 232 (38), 190 (100), 175 (81), 163 (50), 149 (35),
rel
135 (31), 121 (50), 105 (59), 91 (52), 69 (41), 55 (39), 41 (47).
8,9
Preparation of Ethyl ꢂ -Bicyclohomofarnesenoate (12). A solution of 5 (100 mg, 0.40 mmol) in anhydrous
benzene (2 mL) was treated with a solution of (COCl) (0.4 mL, 0.58 g, 4.58 mmol) in benzene (1 mL), stirred at 20°C for
2
1 h, and refluxed for 1 h. The benzene and excess of (COCl) were evaporated at reduced pressure. The residue was treated
2
with anhydrous EtOH (2 mL) and NH NH 4H O (1 mL, 1.03 g, 20.5 mmol), stirred at 20°C for 7 h, diluted with H O (10 mL),
2
2
2
2
and extracted with EtOAc (3 ꢆ 15 mL). The extract was washed with H O (3 ꢆ 5 mL) and dried. The EtOAc was evaporated
2
to afford a product (97 mg) that was chromatographed over a column of silica gel (2.9 g). Ethyl ester 12 (60 mg, 55%) was
17
–1
eluted by petroleum ether–Et O (19:1). Ñ Í Î , [ꢁ] +79.8ꢃ (ñ 1.41, CHCl ). IR spectrum (ꢌ, cm ): 1738, 1154 (CO Et).
2
18 30
2
D
3
2
1
Í NMR spectrum (400 MHz, CDCl , ꢊ, ppm, J/Hz): 0.83 (3H, s, ÑÍ -15), 0.89 (3H, s, ÑÍ -14), 0.93 (3H, s, ÑÍ -16), 1.25
3
3
3
3
(3H, t, J = 7.3, ÑÍ -18), 1.57 (3H, s, ÑÍ -13), 2.95 (1H, d, J = 16.8, H-11a), 3.07 (1H, d, J = 16.8, H-11b), 4.12 (2H, m,
3
3
13
2 ꢆ H-17). C NMR spectrum (100 MHz, CDCl , ꢊ, ppm): 14.24 (C-18), 18.93 (C-2), 18.94 (C-6), 19.74 (C-16), 20.07 (C-13),
3
21.63 (C-15), 33.13 (C-11), 33.17 (C-14), 33.27 (C-4), 33.61 (C-7), 36.28 (C-1), 38.54 (C-10), 41.54 (C-3), 51.39 (C-5), 60.30
+
(C-17), 130.14 (C-8), 133.92 (C-9), 172.96 (C-12). Mass spectrum (EI, 70 eV), m/z (I , %): 278 (Ì , 18), 263 (25), 217 (10), 205
rel
(14), 190 (100), 175 (91), 163 (28), 147 (16), 133 (22), 119 (42), 105 (46), 91 (34), 79 (21), 55 (21), 41 (27).
8,13
Reaction of ꢂ -Bicyclohomofarnesenoic Acid (13) with N H 4H O. A solution of 13 (100 mg, 0.40 mmol) in
2
4
2
anhydrous benzene (2 mL) was treated with a solution of (COCl) (0.4 mL, 0.58 g, 4.58 mmol) in benzene (1 mL), stirred for
2
1 h at 20°C, and refluxed for 1 h. The benzene and excess of (COCl) were evaporated at reduced pressure. The residue was
2
treated with CH Cl (2 mL) and N H 4H O (1 mL), stirred for 10 h at room temperature, refluxed for 10 h, and diluted with
2
2
2
4
2
CH Cl (2 mL). The excess of N H 4H O was separated. The organic layer was washed with H O (3 ꢆ 1 mL) and dried. The
2
2
2
4
2
2
solvent was evaporated to afford crude product (107 mg) that was chromatographed over a column of silica gel (3.2 g).
A product (30 mg, 30%) that was recrystallized from MeOH to afford disubstituted hydrazine 16 (25 mg) was eluted by
CHCl . Then, a mixture (46 mg, 43%) of hydrazides 15 and 17 (1.5:1 ratio, PMR data and GC-MS analysis) was eluted
3
TM
(CHCl –MeOH, 99:1). The mixture (15 mg) of 15 and 17 was separated using HPLC (semi-preparative ꢄBondapak C18
3
°
column, 10 ꢄm, 125 A, MeOH–H O, 9:1, 0.38 psi, flow rate 1 mL/min) to afford 15 (5.2 mg, 4.9%) and 17 (5 mg, 4.7%).
2
8,13
17
ꢂ
-Bicyclohomofarnesenoic Acid Hydrazide (15). Ñ Í N Î, [ꢁ] –15.8ꢃ (ñ 0.31, CHCl ). IR spectrum
16 28 2 D 3
–1
1
(ꢌ, cm ): 3294, 3198 (NH ), 1641, 1616 (CONH). Í NMR spectrum (400 MHz, CDCl , ꢊ, ppm, J/Hz): 0.69 (3H, s, ÑÍ -16),
2
3
3
0.81 (3H, s, ÑÍ -15), 0.89 (3H, s, ÑÍ -14), 1.2* (1H, m, H-5ꢁ), 2.10 (1H, dt, J = 12.9, 5.2, H-7a), 2.22 (1H, dd, J = 15.9, 10.8,
3
3
H-11a), 2.37* (1H, m, H-7b), 2.39* (1H, m, H-11b), 3.88 (2H, br.s, NH ), 4.47 (1H, br.s, H-13a), 4.79 (1H, br.s, H-13b), 7.04
2
13
(1H, br.s, NH). C NMR spectrum (100 MHz, CDCl , ꢊ, ppm): 14.52 (C-16), 19.27 (C-2), 21.70 (C-15), 24.06 (C-6), 30.44
3
(C-11), 33.54 (C-4), 33.56 (C-14), 37.67 (C-7), 38.91 (C-1), 39.19 (C-10), 41.96 (C-3), 52.09 (C-9), 55.12 (C-5), 106.48
15
(C-13), 149.19 (C-8), 174.01 (C-12). N NMR spectrum (40 MHz, CDCl , ꢊ, ppm): 128 (NH). Mass spectrum (EI, 70 eV),
3
+
m/z (I , %): 264 (Ì , 12), 249 (94), 233 (48), 217 (28), 190 (95), 175 (47), 163 (24), 137 (64), 129 (25), 123 (52), 121 (72),
rel
119 (49), 113 (84), 109 (65), 95 (84), 91 (87), 81 (89), 69 (83), 55 (72), 41 (100).
17
–1
Bicyclohomofarnesenoic Acid Hydrazide (17). Ñ Í N Î. [ꢁ] +6.40ꢃ (ñ 0.30, CHCl ). IR spectrum (ꢌ, cm ):
16 30
2
D
3
1
3287, 3198 (NH ), 1645, 1616 (CONH). Í NMR spectrum (400 MHz, CDCl , ꢊ, ppm, J/Hz): 0.81 (3H, s, ÑÍ -15), 0.84 (3H,
2
3
3
1034

s, ÑÍ -16), 0.86 (3Í, s, ÑÍ -14), 0.94 (3H, d, J = 7.1, ÑÍ -13), 0.98* (1H, m, H-5ꢁ), 1.60* (2H, m, H-7a, 7b), 1.82* (2H, m,
3
3
3
H-8ꢁ, 9ꢁ), 2.01 (1H, dd, J = 14.2, 9.7, H-11a), 2.32 (1H, dd, J = 14.2, 3.9, H-11b), 3.90 (2H, br.s, NH ), 6.70 (1H, br.s, NH).
2
13
C NMR spectrum (100 MHz, CDCl , ꢊ, ppm): 15.88 (C-13), 16.54 (C-16), 17.40 (C-6), 18.38 (C-2), 21.54 (C-15), 31.27
3
(C-8), 32.72 (C-11), 33.34 (C-4), 33.45 (C-14), 34.44 (C-7), 38.13 (C-10), 39.39 (C-1), 42.04 (C-3), 50.41 (C-9), 56.45
15
(C-5), 174.51 (C-12). N NMR spectrum (40 MHz, CDCl , ꢊ, ppm): 128 (NH). Mass spectrum (EI, 70 eV), m/z (I , %):
3
rel
+
266 (Ì , 0.9), 264 (2), 251 (6), 249 (6), 235 (7), 217 (7), 193 (13), 177 (9), 163 (3), 149 (4), 137 (22), 123 (33), 109
(33), 95 (33), 81 (36), 74 (100), 69 (34), 55 (35), 41 (36).
8,13
16
N,Nꢀ-Di-(ꢂ -bicyclohomofarnesenoyl)-hydrazine (16). White crystals, mp 273–274ꢃC (ÌåÎÍ), [ꢁ] –3.83ꢃ
D
–1
1
(ñ 0.12, CHCl ). IR spectrum (ꢌ, cm ): 3440, 3258 (NH), 1645, 1616 (CONH). Í NMR spectrum (400 MHz, CDCl , ꢊ,
3
3
ppm, J/Hz): 0.70 (6H, s, ÑÍ -16, 16ꢀ), 0.81 (6H, s, ÑÍ -15, 15ꢀ), 0.88 (6H, s, ÑÍ -14, 14ꢀ), 1.21* (2H, m, H-5ꢁ, 5ꢀꢁ), 2.40*
3
3
3
(2H, m, H-9, 9ꢀ), 2.40* (4H, m, H-11a, 11ꢀa, 11b, 11ꢀb), 4.51 (2H, br.s, H-13a, 13ꢀa), 4.80 (2H, br.s, H-13b, 13ꢀb), 8.40 (2H,
13
br.s, 2NH). C NMR spectrum (100 MHz, CDCl , ꢊ, ppm): 14.54 (C-16), 19.30 (C-2), 21.72 (C-15), 24.08 (C-6), 30.32 (C-11),
3
33.34 (C-4), 33.56 (C-14), 37.66 (C-7), 38.98 (C-1), 39.25 (C-10), 42.03 (C-3), 52.00 (C-9), 55.16 (C-5), 106.75 (C-13), 148.92
15
(C-8), 169.52 (C-12). N NMR spectrum (40 MHz, CDCl , ꢊ, ppm): 134 (NH). HR-EI-MS: found 497.4108. Ñ Í N Î .
3
32 52 2 2
Calcd 496.7750.
X-ray Crystal Structure Experiment. A dataset for a crystal of 1 was collected at room temperature (293 K) on an
Oxford Diffraction Super Nova diffractometer using a Nova Cu Kꢁ microfocusing X-ray tube. The dataset was processed
using the CrysAlis PRO program (England, 2012). The structure was solved by direct methods using the SHELX-S program
[12] and refined by anisotropic full-matrix least squares methods for non-hydrogen atoms using SHELXL-2013 [12]. Positions
of H atoms on O atoms were determined in difference Fourier syntheses and refined with the corresponding geometric H-bond
parameters. Positions of other H atoms were calculated geometrically and refined using a rigid-body model with U = 1.2 U
H
eq
or 1.5 U (U correspond to the C atoms).
eq
eq
Table 1 presents the crystal data and refinement parameters.
Positional and thermal parameters for atoms in the structure of 1 were deposited in the Cambridge Crystallographic
Data Centre (No. CCDC 1453861; deposit@ccdc.cam.ac.uk or http://www.ccdc.cam.ac.uk).
Biological Activity. Antifungal and antibacterial activities of 1, 2, 11, and 15–17 were studied using in vitro diffusion
tests on standard nutrient agar in Petri dishes. Solutions of samples of concentrations 0.5%, 1%, and 2% were prepared by
dissolving the appropriate amounts of test compounds in fixed volumes of DMSO. Microorganism suspensions were prepared
using sequential dilutions. A symmetric inhibition zone formed as an ellipse after incubation for 48 h at 37°C. The minimum
inhibiting concentrations (MICs) were determined in units of ꢄg/mL. Results were observed visually using an Olympus SZY
160 microscope (Japan) and photography.
ACKNOWLEDGMENT
The work was supported by a grant of POSCCE-O 2.2.1; SMIS-CSNR 13984-901, No. 257/28.09.2010, and institutional
project 15.817.02.14A.
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1036