Structures of cribochalines A and B, branched-chain methoxylaminoalkyl pyridines from the micronesian sponge, Cribochalina sp. absolute configuration and enantiomeric purity of related O-methyl oximes

Article abstract of DOI:10.1016/S0040-4020(00)00189-7

Two new 3-alkyl pyridines, cribochalines A (2) and B (3), were isolated from the North Pacific sponge Cribochalina sp. The known related oxime, ikimine A, was shown to be a 2.8:1 mixture of the (S)- and (R)-enantiomers. Cribochaline A exhibited antifungal activity against Candida albicans ATCC and Fluconazole-resistant strains C. albicans 96-489, C. krusei and C. glabrata. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4020(00)00189-7

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 2921±2927
Structures of Cribochalines A and B, Branched-Chain
Methoxylaminoalkyl Pyridines from the Micronesian Sponge,
Cribochalina sp. Absolute Con®guration and Enantiomeric Purity
of Related O-Methyl Oximes
*
Gillian M. Nicholas and Tadeusz F. Molinski
Department of Chemistry, University of California, Davis, CA 95616, USA
Received 14 December 1999; revised 2 March 2000; accepted 3 March 2000
AbstractÐTwo new 3-alkyl pyridines, cribochalines A (2) and B (3), were isolated from the North Paci®c sponge Cribochalina sp. The
known related oxime, ikimine A, was shown to be a 2.8:1 mixture of the (S)- and (R)-enantiomers. Cribochaline A exhibited antifungal
activity against Candida albicans ATCC and Fluconazole-resistant strains C. albicans 96±489, C. krusei and C. glabrata. q 2000 Elsevier
Science Ltd. All rights reserved.
Introduction
Sponges of the family Niphatidae, order Haplosclerida,
produce a diverse array of alkaloids that are structurally
related by the presence of a pyridine ring or its reduced
forms.1±5 The simplest compounds in this class are
30-aminoalkylpyridines in which the substituent at the
aromatic ring is a long-chain (C12±C18) v-aminoalkyl
group. Further variations occur when the side chain contains
unsaturation or is further oxidized at the terminus to a
methoxylamine or oxime, as represented by the structure
of ikimine A (1, C12 chain) from an unidenti®ed sponge
collected in Ant Atoll, Pohnpei.6 Here, we report two new
antifungal compounds, cribochalines A (2) and B (3), from
Cribochalina sp., also from Ant Atoll, which are closely
related to 1. We also re-isolated, from the same sponge,
(1)-16 as an E/Z mixture of oxime geometrical isomers.
Stereochemical analysis reveals that 1 is a non-racemic
mixture of (1)- and (2)-enantiomers. Cribochaline A (2)
was observed to undergo facile autoxidation to 1, suggesting
that 2 is the substrate for a non-enzymic autoxidation and
that 1 and related oximes or their O-Me ethers may be
artifacts. Including the natural product 4, the foregoing
observations unify the three oxidation levels of alkylpyri-
dinesÐamine, alkoxylamine and oximeÐfound in the
Niphatid sponges.
Results and Discussion
The n-hexane-soluble fraction of an MeOH extract of
lyophilized Cribochalina sp. exhibited signi®cant brine
shrimp lethality (BSL, LC100 ,85 ppm) and moderate,
broad-spectrum antifungal activity against Candida
Keywords: alkaloid; oxidation; Porifera.
* Corresponding author. Tel.: 11-530-752-6358; fax: 11-530-752-8995;
e-mail: tfmolinski@ucdavis.edu
albicans ATCC 14503 and Fluconazolew-resistant strains,
0040±4020/00/$ - see front matter q 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(00)00189-7

2922
G. M. Nicholas, T. F. Molinski / Tetrahedron 56 (2000) 2921±2927
Scheme 1. (a) Et3N, DMAP, CH2Cl2; (b) BH3´THF, THF, re¯ux; (c) Pd(OH)2, H2, MeOH; (d) (S)-MTPACl, py, DMAP; (e) NH4OH, aq; (f) BH3´SMe2, THF,
re¯ux.
C. albicans 96±489, C. krusei and C. glabrata. Separation
of the active fraction by silica chromatography gave a
refractory mixture containing homologous ninhydrin-
positive compounds, identi®ed as 3-aminoalkylpyridines
by a combination of HPLC diode-array UV spectra and
1H NMR. The dif®cult separation was ®nally achieved by
use of Et3N-buffered aqueous MeOH on reversed phase
(C18) HPLC to provide pure samples of 1, 2 and 3.
COSY) identical with those of 2. The CH3CHCH2±NH±
spin system is also present; consequently, we assigned
structure 3Ðthe higher homologue of 2Ðto the second
compound.
Neither optical rotation nor the absolute stereochemistry of
1 is reported in the literature.6 Our sample of (1)-1 was
dextrorotatory ([a]Dˆ17.5), but optical rotations of alkyl-
amines are characteristically low in magnitude and
unreliable for determination of absolute con®guration.
Instead, we turned to analysis of circular dichroism (CD)
of a suitable aroyl carboxylate derivative of 1. (1)-Ikimine
A (1) was reduced (LiAlH4, THF, quantitative) to the known
alkaloid (1)-niphatesine C [4, HCl salt, [a]2D3ˆ13.08 (c
0.16, MeOH). lit.2 19.4 (c 0.053, MeOH)], which was
converted to the naphthoamide 5. The b-methyl group of
5 was expected to exert a detectable, albeit weak, Cotton
effect on the primary naphthoamide. Unfortunately, the CD
spectrum of 5 showed no signi®cant Cotton effects (De,0)
at the expected wavelengths, suggesting the weak optical
activity detected in both 1 and 2 is attributable to a partial
racemate with low optical purity. The absolute con®guration
and %ee of the more abundant (1)-enantiomer of 1 were
determined as follows.
The formula for 2 (C19H34N2O) revealed four degrees of
unsaturation, which is satis®ed by a pyridine ring. The
pyridine ring was established by a UV chromophore con-
sistent with an alkylpyridine [lm1ax 210 nm (e 5630), 262
(4530), 269 (3330)] and by the H NMR spectrum of 2,
which showed the characteristic down®eld signals of a
3-substituted pyridine [d 8.41 (bs, 1H), 7.51 (m, 1H), 7.20
(dd, Jˆ5.1, 7.8 Hz, 1H), 8.41 (m, 1H)]. The methoxylamine
group (NHOMe) was assigned by the presence of a weak IR
1
band (v 3240, NH) together with H and 13C signals corre-
sponding to a deshielded MeO group (dH 3.53, s, 3H; dC
60.1, q). Weak optical activity ([a]2D4ˆ21.08) in compound
2 suggested an alkaloid belonging to the class of chiral
branched alkylpyridines. COSY analysis located the methyl
group (d 0.91, d, Jˆ6.9 Hz) b to the ±NHOMe terminus
1
through coupling to the ±CH±CH2±N H spin system [d
1.61 (m, CH), 2.65 (dd, Jˆ7.5, 12.3 Hz, 1H), 2.85 (dd,
Jˆ6.0, 12.3 Hz, 1H)]. The balance of the 1H and 13C signals
in the side chain were attributed to 9£CH2 groups including
one benzylic CH2 (2.59, t, Jˆ7.5 Hz, 2H, H12) comprising a
straight methylene chain. Thus, the complete structure of 2
has a C12 sidechain attached to C30 (pyridine numbering)
with an Me branch at C2. Comparison of literature for H
NMR data for ikimines6 and related 3-(methoxyamino-
alkyl)pyridines2,4 fully supported this assignment.
(1)-Niphatesine C2 (4), obtained from 1 as described above,
was converted to Mosher's amide 6 ((S)-MTPACl, DMAP,
py, 44%) as shown in Scheme 1. For the purposes of
comparison, (R)-Mosher's amides 7a and 7b were prepared
from (^)-2-methylhexanoyl chloride (8) as follows. Treat-
ment of 8 with (R)-a-methylbenzylamine (Et3N, DMAP,
CH2Cl2) gave a 1:1 mixture of amides that were easily
separated by silica chromatography (1:9 EtOAc/n-hexanes)
to provide the less polar diastereomer (1)-9 ([a]Dˆ11028
(c 2.0, CHCl3) and more polar (1)-10 ([a]Dˆ175.88 (c 2.1,
CHCl3)) in yields of 38 and 39%, respectively. Reduction of
(1)-10 (BH3´THF, THF, re¯ux) gave the corresponding
1
Compound 3 (C20H36N2O) differs from 2 by having one
extra CH2 but otherwise exhibited spectral data (UV, H,
1

G. M. Nicholas, T. F. Molinski / Tetrahedron 56 (2000) 2921±2927
2923
Table 1. Selected 1H NMR chemical d, J's and integrations of 6, (2R)-7a and (2S)-7b (400 MHz, C6D6)
#
(2S)-6a
(2R)-6b
(2R)-7a
(2S)-7b
d
mult, J (Hz), int.
d
mult, J (Hz), int.
d
mult, J (Hz), int.
d
mult, J (Hz), int.
Ome
3.16a
0.68
3.12
2.81
bs, 3H
3.16a
0.67
2.98
±
bs, 3H
3.14a
0.63
2.93
±
bs, 3H
d, 6.7, 3H
m, 2H
±
3.15a
0.64
3.06
2.79
bs, 3H
2-Me
H-1ab
H-1bb
d, 6.8, 2.2H
m, 0.73H
m, 0.73H
d, 6.5, 0.8H
m, 0.54H
±
d, 6.7, 3H
m, 1H
m, 1H
a Broadened by JHF
b Signals for the (S)-7b extracted from spectrum of (R)-Mosher's amides of (^)-7a,b and presented with normalized integrations.
.
benzylamine (1)-11 [58%, [a]Dˆ142.08 (c, 1.7, MeOH)]
that was hydrogenolyzed (Pd(OH)2, H2, MeOH, 72%) to
(2R)-(1)-2-hexylamine 12a ([a]D of HCl salt, 16.18,
(c 0.9, H2O); lit.7 [a]Dˆ22.418 (c 15.384, H2O)) and subse-
quently converted to (R)-Mosher's amide (1)-7a
([a]Dˆ117.08, (c 0.1, CHCl3)). A mixture of the (R)-
Mosher's amides of racemic amine (^)-7a,b was prepared
by the sequential reactions: ammoniolysis of (^)-8 to
primary amide (^)-13 (aqueous NH4OH, 88%), reduction
to amine (^)-12a,b (BH3´SMe2, THF, 80%) and acylation
with (R)-Mosher's acid chloride (py, DMAP, 30%). Inte-
oximes. The product was identi®ed as the E/Z geometrical
isomers of ikimine A (1) by comparison of 1H NMR spectra
with 1 isolated from the same sponge. The literature 1H data
of 16 matched those of the autoxidation product. Since air
oxidation of secondary amines is known to give imines8 it is
conceivable that 1 arises by N-hydroxylation of methoxyl-
amine 2 followed by acid-catalyzed elimination of H2O,
although the ease of autoxidation in this case is surprising.
The imperfect optical purity of 1 may be a consequence of
the oxidation process itself and not lack of ®delity in the
enzymatic biogenesis of the branched chain (probably
involving methylation by S-adenosyl methionine).
1
gration of the H NMR spectrum of the latter con®rmed
that it was a 1:1 mixture of amides 7a and 7b.
The chemical correlation established in this work (1!4!5)
supports the notion that loss of optical activity occurs after
formation of 2, given that natural (1)-niphatesine C (4)
exhibits higher optical activity ([a]Dˆ19.48) than synthetic
4 ([a]Dˆ138), obtained by reduction of 1. A plausible
biogenic sequence is shown in Scheme 2, starting with
enzymatic oxidation±methylation of 4. Slow racemization
of chiral oxime i in protic media, for example, via the
tautomeric intermediate N-methoxy enamine ii, could
account for loss of optical activity in 1 that derives from
an amine precursor of higher optical purity. Unfortunately,
insuf®cient 2 was left after spontaneous autoxidation to 1,
and natural 4 was unavailable to us to test this hypothesis.9
1
Analysis of the H NMR spectra (CDCl3) of the natural
product derivative 6 showed doubling of several signals
due to the presence of diastereomers (2R)-6a and (2S)-6b.
For example, the C2 Me signals were doubled at d 0.67 (d,
Jˆ6.5 Hz, 0.8H) and 0.68 (d, Jˆ6.8 Hz, 2.2H). The corre-
sponding chemical shifts of (R)-Mosher's amides 7a,b also
1
showed the same ®ne differences in H NMR in chemical
shift for the C2 Me signal in CDCl3, but the diastereomers
were most clearly distinguished in C6D6 by chemical shift
and J coupling patterns of the diastereotopic H1 proton
signals (see Table 1). The spectrum of the model compound
(2R)-(1)-7a displayed a collapsed multiplet (d 2.93, m, 2H)
for the C1 methylene group. Conversely, interpretation of
1
the H NMR spectrum of (^)-7a,b revealed the H1 dia-
Given the ease of autoxidation of 2, it is likely that 2 is the
biosynthetic precursor of 1 and the latter arises by non-
enzymatic oxidation. The examination of H NMR spectra
stereotopic protons of 7b are dispersed between two multi-
plets centered at d 3.06 and 2.79 ppm. In the natural product
derivative 6, both sets of diastereotopic proton multiplets
1
of samples of crude extracts obtained from the sponge did
indeed show evidence of the presence of lesser amounts of
both a- and b-methyl substituted oxime signals (E-isomers
d 7.2 d, or t; Z-isomers, d 6.3±6.6 d or t) together with
ole®nic and propargylic proton signals, although the
majority of the alkylpyridine components of Cribochalina
extract are fully saturated in the alkylamine sidechains.
1
are observed. Careful integration of H signals shows that
the diastereomeric ratio (dr) of 6 is 2.8:1, with a predomi-
nance of the (2S) isomer 6b. Accepting the reasonable
assumption that no kinetic enrichment takes place during
formation of 6, we conclude that our samples of (1)-ikimine
A (1) and (1)-niphatesine C (4) each occur as a 2.8:1
mixture of enantiomers, with an excess of the (2S) enantio-
mer (47% ee).
Biological Activity
Upon prolonged storage in CDCl3 (48C, ,2 weeks),
compound 2 underwent autoxidation (ca. 40±50% conver-
sion), giving a 3:1 mixture of E/Z isomers of O-methyl-
Compound 2 showed moderate antifungal activity against
®ve organisms in the disk-diffusion assay at 300 mg/disk: C.
Scheme 2.

2924
G. M. Nicholas, T. F. Molinski / Tetrahedron 56 (2000) 2921±2927
albicans (ATCC 14503) 14 mm zone of inhibition, C. albi-
cans (96±489) 11 mm, C. albicans (UCD-FR1) 17 mm, C.
krusei 13 mm at 100 mg/disk and C. glabrata 15 mm. (1)-
Ikimine A (1) was inactive and insuf®cient amounts of 3
were available for testing.
the most active was the hexane fraction that showed 100%
mortality after 12 h, compared to the other two that showed
53±86% mortality but only after 48 h. Only the hexane and
chloroform fractions showed antifungal activity at 300 mg/
disk. The hexane fraction (1.6 g) was separated by silica gel
chromatography and eluted with a stepped gradient (hexane
to EtOAc), to give 21 fractions. Fractions 20±21 both
showed activity in the BSL assay (86±100% mortality at
100 ppm) and signi®cant antifungal activity (13 mm,
300 mg/disk) against C. albicans (ATCC). Fraction 20 was
further separated by C18 reversed-phase HPLC (Dynamax
Conclusion
Two new alkaloids, cribochalines A (2) and B (3) have been
isolated and ikimine A (1) is shown to be partially racemic
(2.8:1 S to R). The relationship of cribochaline B to ikimine
A suggests a non-enzymatic biosynthesis of 1 and related
oximes from the corresponding methoxylamines.
Ê
60 A, 21.4£250 mm, 1:9 H2O/MeOH, 0.05% Et3N), to give
cribochaline A (2, 7.8 mg, 0.011% dry wt) with a retention
time of 13.8 min and cribochaline B (3, 1.9 mg, 0.001% dry
wt) with a retention time of 17.8 min. (1)-Ikimine A (1)6
was obtained from separation of the CHCl3-soluble fraction
II by silica ¯ash chromatography (EtOAc/n-hexane
gradient) and HPLC.
Experimental
General procedures
(1)-Ikimine A (1).6 3:1 E/Z mixture, [a]2D4ˆ17.58 (c 0.13,
CHCl3). E isomer: 1H NMR (300 MHz, CDCl3) dH 1.05 (d,
Jˆ6.9 Hz, 3H), 1.25±1.30 (m, CH2`s), 1.62 (m, 2H), 2.32
(m, 1H), 2.64 (t, Jˆ6.9 Hz, 2H), 3.80 (s, 3H), 7.19 (d,
Jˆ7.5 Hz, 1H), 7.34 (dd, Jˆ4.8, 7.8 Hz, 2H), 7.64 (br d,
Jˆ7.8 Hz, 2H), 8.45 (m,1H), 8.46 (br s, 1H). HREIMS m/z
304.2509 [M1H]1, Calcd for C19H32N2O 304.2514. As
noted by Carroll et al.,6 the E/Z oxime isomers of ikimine
A were separable on silica HPLC (E-1 elutes ®rst, silica
Microsorb, 10£250 mm, 5:95 EtOAc/n-hexane, 3.0 mL/
min), but re-equilibrated within hours.
1H NMR, COSY and 13C NMR spectra were measured on
General Electric QE-300 (1H NMR 300 and 13C NMR
75 MHz) and Varian Inova 400 (400 and 100 MHz) spectro-
meters. 13C assignments were made from analysis of the
DEPT spectra. IR spectra were measured on a Mattson
Galaxy 3000 FTIR spectrometer and UV spectra were
obtained on a Hewlett Packard 8450A diode array spectro-
photometer. Optical rotations were recorded on a Jasco Dip
digital 370 polarimeter using a 1 dm cell. Measurements of
mass spectra were made at the Regional Mass Spectrometry
Facility at the University of California, Riverside.
Cribochaline A (2). [a]2D4ˆ21.08 (c 0.2, MeOH). UV lmax
(MeOH) 210 nm (e 5630), 262 (4530), 269 (3330). 1H NMR
(300 MHz, CDCl3) dH 8.41 (bs, 1H), 7.51 (m, 1H), 7.20 (dd,
Jˆ5.1, 7.8 Hz, 1H), 8.41 (m, 1H), 2.59 (t, Jˆ7.5 Hz, 2H),
1.60 (m), 3.51 (s, 2H), 2.65 (dd, Jˆ7.5, 12.3 Hz, 1H), 2.85
(dd, Jˆ6.0, 12.3 Hz 1H), 1.62 (m), 1.2±1.3, 0.91 (d,
Jˆ3.3 Hz, 3H). 13C NMR (75 MHz, CDCl3) dC 147.6
(C-2), 147.6 (C-6), 138.1 (C-3), 137.9 (C-4), 123.9 (C-5),
61.5 (OCH3), 58.2 (NCH2), 34.9, 32.9, 31.0, 29.1±29.9,
26.8, 17.9 (CH3). IR (NaCl plate, ®lm) 3240 (br weak),
2926, 2854, 1575, 1477, 1464, 1422 cm21. HRFABMS
m/z 307.2757 [M1H]1 Calcd for C19H35N2O 307.2749.
Collection and bioactivity
The mauve and grey-colored sponge Cribochalina sp. (97±
108) was collected at Ant Atoll #1, Pohnpei, FSM (68
45.6710 N, 1578 59.6190 E) in December 1997 at a depth
of 24 m, and the sponge was kept frozen (2208C) until
required. Voucher samples and spicule mounts are stored
in the Chemistry Department, UC Davis and are available
from the corresponding author. A portion of the sample was
allowed to stand in EtOH (,12 months, 48C) after which the
EtOH extract showed signi®cant activity in the brine shrimp
lethality (BSL) assay (LC50 85 ppm after 24 h) and moder-
ate antifungal activity in the disk diffusion assay (300 mg/
disk) against Candida albicans (ATCC 14503) 9 mm, C.
krusei (8 mm), C. albicans (96±489, 8 mm), C. albicans
(UCD-FR1, 8 mm) and C. glabrata (8 mm).
1
Cribochaline B (3). H NMR (300 MHz, CDCl3) dH 8.45
(m, 1H), 7.60 (m, 1H), 7.31 (dd, Jˆ5.1, 7.8 Hz, 1H), 8.45
(m, 1H), 2.63 (t, Jˆ7.5 Hz, 2H), 1.60 (m), 3.53 (s, 2H), 2.85
(dd, Jˆ6.0, 12.0 Hz 1H), 1.62 (m), 1.18±1.30 (CH2's), 0.89
(d, Jˆ3.3 Hz, 3H). IR (NaCl plate, ®lm) 3240 (br weak),
2926, 2854, 1575, 1477, 1464, 1422, 1026, 790, 713 cm21
.
Extraction and isolation
HRFABMS m/z 321.2907 [M1H]1 Calcd for C20H37N2O
321.2905.
The sponge 97±108 (dry weight 70.2 g) was lyophilized and
extracted with MeOH (4£600 mL) to give a dark green
solution. The water content (v/v) of the MeOH extract
was adjusted, followed by sequential partitioning against
the solvents. Concentration of each sequential organic
extract provided n-hexane (10% v/v, I, 1.68 g) and CHCl3
(40% v/v, II, 3.53 g) soluble fractions. The MeOH was
removed from the aqueous phase under vacuum, and the
remaining liquid partitioned against n-butanol (III, 1.61 g).
The remaining aqueous phase was lyophilized (IV, 13.81 g).
The hexane, chloroform and n-butanol fractions all showed
some activity in the brine shrimp lethality (BSL) assay, but
Reduction of (1)-ikimine A
(1)-Niphatesine
C A solution of 1 (5.5 mg,
(4)6
0.018 mmol) in THF (1 mL) was added to a stirred slurry
of LiAlH4 (14 mg, 0.37 mmol) in THF (1 mL). The mixture
was allowed to stir at 258C for 16 h at which time no starting
material was detected by TLC. The mixture was treated with
water (2 drops), followed by aqueous NaOH (2 mL, 4 M),
stirred for 5 min, diluted with EtOAc (15 mL) and the
phases separated. The aqueous phase was extracted with

G. M. Nicholas, T. F. Molinski / Tetrahedron 56 (2000) 2921±2927
2925
EtOAc (3£5 mL), and the combined organic layers were
washed with brine, dried (MgSO4) and concentrated to
give 4 as a colorless oil (5.0 mg, quant., purity .90%).
Puri®cation of the oil by silica chromatography (3:7
MeOH (satd. NH3)/CHCl3) yielded pure 4 as the free base
Jˆ7.4 Hz, 2H), 0.7±1.4 (m,), 0.68 (d, Jˆ6.8 Hz, 2.2H,
(S)-CH3), 0.67 (d, Jˆ6.5 Hz, 0.8H, (R)-CH3). 13C NMR
(75 MHz, CDCl3) dC 165.8, 148.5, 145.5, 138.1, 137.9,
132.5, 129.2, 128.3, 127.5, 123.9, 45.3, 34.3, 33.2, 33.0,
30.9, 29.8, 29.5, 29.5, 29.3, 29.1, 26.8 17.7. IR (NaCl
plate, ®lm,) n 3338 (br), 2922, 2849, 1686 (CvO), 1524,
1464 1268, 1162, 1103 21. HRCIMS (NH3) m/z 493.3021
[M1H]1, Calcd for C28H40N2O2F3 493.3041.
1
(2.5 mg). H NMR (300 MHz, CD3OD) dH 8.36 (m, 1H),
8.33 (m, 1H), 7.68 (m, 1H), 7.34 (dd, Jˆ5.1, 7.5 Hz, 1H),
2.69 (dd, Jˆ5.7, 12.6 Hz, 1H, CH2), 2.64 (t, Jˆ7.8 Hz,
2H), 2.54 (dd, Jˆ7.5, 12.6 Hz, 1H, CH2), 1.61 (m, 2H),
1.28 (m, CH2`s), 0.94 (d, Jˆ7.8 Hz, 3H). HRCIMS
(NH3) m/z 277.2655 [M1H]1, Calcd for C18H33N2
277.2643.
(R)-Mosher's amide of (2R)-1-amino-2-methylhexane
(7a). A solution of (S)-MTPA chloride (50 mg, 0.2 mmol)
and DMAP (catalytic amount) in pyridine (500 mL) was
added to a stirred solution of amine 12a (1.7 mg,
11 mmol) in pyridine (500 mL). After 16 h, the mixture
was stirred vigorously with aqueous NaHCO3 for 30 min
and the two phases separated. The aqueous phase was
extracted with CH2Cl2, and the combined organic phases
were washed with brine, dried (MgSO4) and concentrated
to a colorless oil. Puri®cation of the residue on silica
(gradient from 1:4±1:1 EtOAc/n-hexane) gave 7a (2.1 mg,
52%). [a]2D5ˆ117.08 (c 0.1, CHCl3). UV (MeOH) lmax
206 nm (e 11500), 257 nm (470). 1H NMR (300 MHz,
C6D6) dH 7.71 (d, Jˆ8.2 Hz, 2H), 6.95±7.10 (m, 3H),
6.18 (m, 1H, NH), 3.14 ((m, 3H, OCH3), 2.93 (m, 2H,
(R)-CH2), 1.26 (m, 1H, CH), 1.00±1.20 (m, 6H), 0.83 (t,
Jˆ6.8 Hz, 3H, CH3), 0.63 (d, Jˆ6.7 Hz, 1.5H, (R)-CH3). IR
(NaCl plate, ®lm,) n 3338 (br), 1683 (CvO), 1524, 1455,
1269, 1164, 1106 cm21. HRCIMS m/z 332.1842 [M1H]1
Calcd for C17H25NO2F3 332.1837.
A solution of primary amine 4 in hydrochloric acid (1 M,
100 mL) was evaporated and dried overnight under vacuum
to give the corresponding hydrochloride salt. [a]2D3ˆ13.08
(c 0.16, MeOH). lit.2 19.4 (c 0.053, MeOH). The 1H NMR
data for the 4´HCl were consistent with that reported for
(1)-niphatesine C (4).2 1H NMR 300 MHz (CD3OD) dH
8.36 (m, 1H), 8.33 (m, 1H), 7.68 (m, 1H), 7.36 (dd,
Jˆ5.1, 7.5 Hz, 1H), 2.84 (dd, Jˆ5.7, 12.3 Hz, 1H, CH2),
2.69 (dd, Jˆ7.5, 12.3 Hz, 1H, CH2), 2.65 (t, Jˆ
7.8 Hz, 2H), 1.61 (m, 2H), 1.28 (m, CH2`s), 0.99 (d, Jˆ
6.6 Hz, 3H).
6-Methoxynaphthoamide 5. A solution of (1)-niphatesine
C (4) (5.0 mg, 10.9 mmol), prepared from (1)-1), and
DMAP (1 crystal) in dry CH2Cl2 (200 mL) was treated
with 6-methoxynaphthoyl chloride (131 mmol, prepared
by reaction of 6-methoxynaphthoic acid and excess
SOCl2, cat. DMF) in CH2Cl2 (800 mL) in the presence of
Et3N (10 mL, 79 mmol). After 16 h, the mixture was
vigorously stirred with aqueous NaHCO3 for 30 min. The
aqueous phase was extracted with CH2Cl2, and the
combined organic phases washed with NaHCO3, dried
(MgSO4), then concentrated to give a colorless oil, which
was separated on silica (1£15 cm, gradient of 1:1 EtOAc /
n-hexane) to give the non-polar 6-methoxynaphthoamide 5
(2.6 mg, 33%). [a]2D3ˆ11.38 (c 0.22, MeOH). CD (MeOH),
no signal. 1H NMR (CDCl3) 7.19 (dd, Jˆ8.7, 2.4 Hz,
1H), 7.15 (m), 6.29 (m, 1H), 3.94 (s, 3H), 3.44 (m, 1H),
3.31 (m, 1H), 2.65 (t, Jˆ7.5 Hz, 2H), 1.78 (m), 1.62
(m), 1.26 (m), 0.99 (d, Jˆ6.6 Hz, 3H). HRDCIMS
(NH3) m/z 461.3164 [M1H]1, Calcd for C30H41N2O2
461.3168.
(R)-Mosher's amides of (^)-1-amino-2-methylhexane
(7a,b). To a solution of hydrochloride salt (^)-12a,b
(601 mg, 39 mmol) in pyridine (500 mL) was added (S)-
MTPACl (40 mg, 0.16 mmol) in pyridine (1 mL) and
DMAP (catalytic amount). After 18 h, the reaction mixture
was stirred with aqueous NaHCO3 for 30 min and the two
phases separated. The aqueous phase was washed with
CH2Cl2 and the combined organic phases were washed
with brine, dried (MgSO4) and concentrated to colorless
oil. Separation of the residue on silica (20% EtOAc/Hexane)
gave a non-polar UV active fraction identi®ed as the 1:1
mixture 7a,b (4.0 mg, 30%). UV (MeOH) lmax 204 nm (e
1
13200), 257 nm (460), 261 nm (450). H NMR 400 MHz
(C6D6) dH 7.71 (d, Jˆ7.6 Hz, 2H), 7.00±7.10 (m, 3H), 6.20
(m, 1H, NH), 3.15 (m, 3H, OCH3), 3.06 (m, 0.5H (S)-CH2),
2.93 (m, 1H, (R)-CH2), 2.79 (m, 0.5H(S)-CH2), 1.30 (m, 1H,
CH), 1.00±1.16 (m, 6H), 0.83 (t, Jˆ6.8 Hz, 1.5H, (R)-CH3),
0.82 (t, Jˆ6.8 Hz, 1.5H, (S)-CH3), 0.64 (d, Jˆ6.7 Hz, 1.5H,
(S)-CH3), 0.63 (d, Jˆ6.7 Hz, 1.5H, (R)-CH3). 13C NMR
75 MHz (CDCl3) dC 166.0, 132.5, 129.2, 128.4, 127.5,
55.0, 45.3, 34.0, 33.2, 29.7, 29.0, 22.9, 17.7, 14.1. IR
(NaCl plate, ®lm,) n 3338 (br) 2956, 2928, 2858, 1662,
1523, 1455, 1270, 1164, 1106 cm21. HRCIMS m/z
332.1832 [M1H]1 Calcd C17H25NO2F3 332.1837.
(R)-Mosher's amide of (1)-4. (S)-MTPA chloride (40 mg,
0.16 mmol) in pyridine (500 mL) was added to a solution of
(1)-amine 4 (2.5 mg, 9.0 mmol) and DMAP (catalytic
amount) in dry pyridine (500 mL). After 15 h, the reaction
mixture was stirred with aqueous NaHCO3 for 30 min and
the two phases separated. The aqueous phase was extracted
with CH2Cl2 and the combined organic phases washed with
brine, dried (MgSO4) and concentrated to colorless oil
(5.1 mg). Separation of the residue on silica (1:4 EtOAc/
n-hexane to 1:1) gave the mixed diastereomers of 6a,b
(2.0 mg, 44%). UV (MeOH) lmax 205 nm (e 20 900),
257 nm (e 3700), 262 nm (e 4200), 268 nm (e 3000),
(^)-2-Methylhexanoyl chloride (8). A solution of (^)-2-
methyl hexanoic acid (1.0 mL, 7.0 mmol) in CH2Cl2 (1 mL)
was treated with oxalyl chloride (6.1 mL, 70 mmol) and
DMF (3 drops). A steady ¯ow of gas evolved for the ®rst
30 min, to give a pale yellow±green solution. The reaction
was allowed to stir at 258C for another hour and the solvent
carefully removed to leave volatile (^)-8, which was used
without further puri®cation.
1
330 nm (e 100). H NMR 400 MHz (C6D6) dH 8.44 (m,
1H), 8.35 (m, 1H), 7.72 (d, Jˆ7.6 Hz, 2H), 7.05±7.10 (m,
3H), 6.91 (d, Jˆ7.6 Hz, 1H), 6.63 (m, 1H), 6.32 (m, 1H,
NH), 3.16 (m, 3H, OCH3), 3.12 (m, 0.73H, (S)-CH2), 2.98
(m, 0.54H, (R)-CH2), 2.81 (m, 0.73H, (S)-CH2), 2.16 (t,

2926
G. M. Nicholas, T. F. Molinski / Tetrahedron 56 (2000) 2921±2927
(1)-(2S)-N-((10R)-1-Phenethyl)-2-methylhexanamide (9)
and (1)-(2R)-N-((10R)-1-phenethyl)-2-methylhexanamide
(10). (R)-1-Phenylethylamine (42 mg, 0.35 mmol) was
added dropwise to a solution of acid chloride (^)-8, triethyl-
amine (177 mg, 1.75 mmol) and DMAP (ca. 1 mg, 8 mmol)
in CH2Cl2 (2 mL). The mixture was stirred for 4 h at which
point TLC indicated the reaction to be complete. The
mixture was diluted with CH2Cl2 (5 mL) and dilute aqueous
NaHCO3 (5 mL) and rapidly stirred for 30 min. The layers
were separated and the aqueous phase extracted with
CH2Cl2 (2£20 mL). The combined organic phases were
washed with brine, dried (MgSO4) and the solvent removed
to give the crude product. Puri®cation by silica chroma-
tography (10% EtOAc/n-hexanes) gave pure (2S,10R)-9
(31.2 mg, 38%), followed by pure (2R,10R)-10 (31.8 mg,
1.35 (d, Jˆ6.9 Hz, 3H), 1.00±1.35 (m, 5H), 0.85 (d,
Jˆ6.6 Hz, 3H). 13C NMR 75 MHz (CDCl3) dC 146.3 (s),
129.5 (d), 128.1 (d), 127.8 (d), 59.7 (d), 55.1 (t), 35.7 (t),
34.1 (d), 30.2 (t), 24.0 (t), 23.8 (q), 18.6 (q), 14.4 (q). IR
(NaCl plate, ®lm) n 2957, 1456, 1213 cm21. HRCIMS
(NH3) m/z 219.1989 M1H]1 Calcd for C15H25N 219.1987.
(1)-(2R)-1-Amino-2-methylhexane hydrochloride (12a).7
Pd(OH)2 (3 mg) was added to a solution of amine 11
(29 mg, 0.13 mmol) in MeOH (2 mL) and stirred vigorously
under an atmosphere of H2 at 258C for 15 h. Removal of
catalyst by ®ltration, acidi®cation of the ®ltrate (5 M HCl,
5 drops) and evaporation of the solvent gave the hydro-
chloride salt of 12a (13.8 mg, 72%). [a]2D3ˆ16.18 (c0.9,
1
H2O); lit. 12b7 [a]Dˆ22.418 (c 15.384, H2O). H NMR
1
39%). Con®gurations were assigned by comparison of H
(300 MHz, D2O) dH 2.71 (dd, Jˆ6.3, 12.6 Hz, 1H), 2.61
(dd, Jˆ7.8, 12.6 Hz, 1H), 1.56 (m, 1H), 0.73 (d,
Jˆ6.6 Hz, 3H), 0.63 (t, Jˆ6.6 Hz, 3H). 1H NMR
300 MHz (CD3OD) dH 2.82 (dd, Jˆ6.3, 12.6 Hz, 1H),
2.66 (dd, Jˆ7.8, 12.6 Hz, 1H), 1.73 (m, 1H), 0.95 (d,
Jˆ6.6 Hz, 3H), 0.87 (t, Jˆ6.6 Hz, 3H). 13C NMR 75 MHz
(CD3OD) dC 46.5, 34.7, 32.8, 29.8, 23.7, 17.3, 14.2.
NMR spectra with those of the known enantiomers (2)- 9
and (2)-1010 and subsequent conversion of (1)-10 to (1)-
12a.
Less polar (2S,10R) diastereomer 9. [a]2D4ˆ11028 (c 2.0,
CHCl3). UV (MeOH) lmax 213 nm (e 5083), 251 (150),
257 (189), 263 (144). 1H NMR (300 MHz, CDCl3) dH
7.24±7.38 (m, 5H), 5.77 (br d, Jˆ5.1 Hz, 1H, NH), 5.14
(m, 1H), 2.14 (m, 1H), 1.63 (m, 1H), 1.48 (d, Jˆ6.9 Hz,
3H), 1.15±1.40 (m, 5H), 1.10 (d, Jˆ6.9 Hz, 3H), 0.88 (t,
Jˆ6.6 Hz, 3H). 13C NMR (75 MHz, CDCl3) dC 175.6 (s),
143.4 (s), 128.4 (d), 127.1 (d), 126.0 (d), 48.3 (d), 41.4 (d),
34.0 (t), 29.5 (t), 22.5 (t), 21.6 (q), 17.7 (q), 13.8 (q). IR
(^)-1-Amino-2-methylhexane hydrochloride (12a,b).
BH3´SMe2 (40 mL of a 10 M solution in THF) was added
to a solution of amide (^)-13 in THF (2 mL). The reaction
was heated at re¯ux for 5 h. Aqueous sodium hydroxide
(1.5 mL, 4 M) was added to the reaction, which was allowed
to stir for a further 30 min. The reaction was diluted with
EtOAc (10 mL) and the layers separated. The aqueous layer
was washed with EtOAc. The combined organics were acidi-
®ed with hydrochloric acid (1 M, 10 mL) and extracted with
H2O. Evaporation of the aqueous layer gave a residue that
was dried overnight under vacuum to give the hydrochloride
salt of (^)-12a,b (36.2 mg, 80%). 1H NMR 300 MHz
(CDCl3) dH 2.75 (dd, Jˆ6.6, 12.3 Hz, 1H), 2.60 (dd,
Jˆ7.5, 12.3 Hz, 1H), 1.61 (m, 1H), 1.08 (m, 6H), 0.77 (d,
Jˆ6.6 Hz, 3H), 0.67 (t, Jˆ6.6 Hz, 3H). 13C NMR 75 MHz
(CDCl3) dC 46.4, 34.7, 32.8, 29.8, 23.7, 17.3, 14.3. IR (NaCl
plate, ®lm) n 2956 (br), 1610, 1508 cm21. HREIMS m/z
116.1436 [M1H]1 Calcd C7H18N requires 116.1439.
(NaCl plate, ®lm) n 3295, 2966, 1643, 1640, 1545 cm21
.
HRCIMS m/z 233.1771 [M1H]1 Calcd for C15H22NO
233.1779.
More polar (2R,10R) diastereomer 10. [a]2D4ˆ175.88 (c 2.1,
CHCl3). UV (MeOH) lmax 214 nm (e 4980), 251 (e 144),
257 (e 190), 263 (e 146). H NMR (300 MHz, CDCl3) dH
1
7.24±7.38 (m, 5H), 5.84 (br d, Jˆ6.9 Hz, 1H, NH), 5.13 (m,
1H), 2.16 (m, 1H), 1.63 (m, 1H), 1.48 (d, Jˆ6.9 Hz, 3H),
1.37±1.18 (m, 5H), 1.13 (d, Jˆ6.9 Hz, 3H), 0.83 (t,
Jˆ6.6 Hz, 3H). 13C NMR 75 MHz (CDCl3) dC 175.6 (s),
143.4 (s), 128.4 (d), 127.1 (d), 126.0 (d), 48.3 (d), 41.4 (d),
34.0 (t), 29.5 (t), 22.6 (t), 21.6 (q), 17.7 (q), 13.9 (q). IR
(NaCl plate, ®lm) n 3312, 2934, 1640, 1537 cm21
.
(^)-2-Methylhexanamide (13). Concentrated aqueous
ammonium hydroxide (1 mL, 8.2 mmol) was added to
acid chloride 8 (110 mg, 0.74 mmol) and the mixture stirred
vigorously for 30 min. The mixture was partitioned against
EtOAc (2£5 mL) and the organic layers washed with brine
(5 mL), dried and concentrated to give primary amide (^)-
13 (86.3 mg, 88%). 1H NMR 300 MHz (CDCl3) dH 5.46 (br
m, 2H, NH2), 2.25 (m, 1H), 1.64 (m, 1H), 1.40 (m, 1H), 1.30
(m, 4H), 1.15 (d, Jˆ6.9 Hz, 3H), 0.89 (t, Jˆ6.6 Hz, 3H). 13C
NMR 75 MHz (CDCl3) dC 182.6 (s), 41.5 (d), 35.0 (t), 30.8
(t), 23.7 (t), 18.4 (q), 14.3 (q). IR (NaCl plate, ®lm) n 3355
(br) 3195 (br), 2933, 1562, 1415 cm21. HRCIMS (NH3) m/z
130.1229 [M1H]1 Calcd. C7H16NO 130.1231.
HRCIMS m/z 233.1787 [M1H]1 Calcd for C15H22NO
233.1779.
((1R)-1-(N-((20R)-2-Methylhexyl)amino)ethyl)benzene (11).
BH3´THF (1.0 M, 420 mL, 0.42 mmol) was added dropwise
to a solution of amide 10 (48 mg, 0.21 mmol) in THF
(200 mL) at 08C. The mixture was then heated at re¯ux
for 2 h before cooling to 08C and quenching by dropwise
addition of aqueous NaOH (2 mL, 5 M). The biphasic
system was then heated to 508C, stirred for 45 min and
cooled to 258C before dilution with CH2Cl2 (5 mL). The
layers were separated and the aqueous phase was extracted
with CH2Cl2 (2£5 mL). The combined organic phases were
dried (MgSO4), ®ltered and the solvent removed to give the
crude product, which was puri®ed by silica chromatography
(1:9 EtOAc/n-hexane) to provide pure benzylamine 11
(26 mg, 58%). UV (MeOH) lmax 209 nm (e 3748), 251
(131), 257 (151), 263 (129). [a]2D4ˆ142.08 (c 1.7, MeOH).
1H NMR 300 MHz (CDCl3) dH 7.19±7.30 (m, 5H), 3.69 (q,
Jˆ6.6 Hz, 1H), 2.24 (m, 1H), 2.22 (m, 1H), 1.56 (m, 1H),
Acknowledgements
We thank Sarah D'Souza (ACS Project SEED) for valuable
assistance with brine shrimp lethality assays, Mary Kay
Harper (Scripps Institution of Oceanography) for identi®-
cation of the sponge, Roger Mulder for assistance with 2D

G. M. Nicholas, T. F. Molinski / Tetrahedron 56 (2000) 2921±2927
2927
5. Stierle, D. B.; Faulkner, D. J. J. Nat. Prod. 1991, 54, 1134±
1136.
NMR experiments and Rich Kondrat (UC Riverside) for MS
measurements. This work was funded by the Public Health
Service, National Institutes of Health (AI 39987). The
400 MHz NMR spectrometer was purchased with assistance
from the NSF (CHE-9808183).
6. Carroll, A. R.; Scheuer, P. J. Tetrahedron 1990, 46, 6637±6644.
7. Levene, P. A.; Mikeska, L. A. J. Biol. Chem. 1924, 84, 571±
599.
8. Rosenblatt, D. H.; Burrows, E. P. Oxidation of Amines. In: The
Chemistry of Functional Groups. Supplement F, part 2; Patai, S.,
Ed.; Wiley: New York, 1982, pp 1137±1140.
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Á
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