Syntheses of pseudoceramines A-D and a new synthesis of spermatinamine, bromotyrosine natural products from marine sponges

Article abstract of DOI:10.1039/c1ob06722b

Herein we report the total syntheses of pseudoceramine A-D (2-5) and spermatinamine (1) isolated from the marine sponge Pseudoceratina sp. Direct acyl substitution of α-hydroxyiminoesters with amine nucleophiles was developed as a key transformation. The synthetic compounds confirm the reported structures and importantly gives access to non-symmetrical spermine based natural products carrying two different bromotyrosine building blocks. Our new synthesis of spermatinamine is two steps shorter and more efficient than the previously reported sequence. The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c1ob06722b

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PAPER
Syntheses of pseudoceramines A–D and a new synthesis of spermatinamine,
bromotyrosine natural products from marine sponges†
a
a
b
¨
J. Mikael Hillgren, Christopher T. Oberg and Mikael Elofsson*
Received 12th October 2011, Accepted 15th November 2011
DOI: 10.1039/c1ob06722b
Herein we report the total syntheses of pseudoceramine A-D (2–5) and spermatinamine (1) isolated
from the marine sponge Pseudoceratina sp. Direct acyl substitution of a-hydroxyiminoesters with
amine nucleophiles was developed as a key transformation. The synthetic compounds confirm the
reported structures and importantly gives access to non-symmetrical spermine based natural products
carrying two different bromotyrosine building blocks. Our new synthesis of spermatinamine is two
steps shorter and more efficient than the previously reported sequence.
Introduction
Marine sponges of the order Verongida are known to produce a
multitude of bromotyrosine derived secondary metabolites, many
with antimicrobial and cytotoxic activities.1 In a recent bioassay-
guided screening of a natural product extract library, the novel bro-
motyrosine alkaloid spermatinamine (1; Fig. 1) was isolated from
an extract of Pseudoceratina sp. as an inhibitor of isoprenylcysteine
carboxyl methyltransferase (Icmt).2 The structure of 1 features
a bromotyrosyl–spermine–bromotyrosyl sequence, constituting a
rare example of a spermine alkaloid from a marine source. More
recently, a bioassay-guided screening for inhibitors of the type III
secretion (T3S) system of the Gram-negative bacterium Yersinia
pseudotuberculosis led to the isolation and characterisation of
four novel bromotyrosine alkaloids, pseudoceramines A–D (2–
5; Fig. 1), from the same natural source.3 The screening assay has
the capacity to identify putative inhibitors of the T3S system as
well as general antibiotics targeting bacterial growth.4 In addition
to pseudoceramines A–D the initial screen also identified 1 as
a putative T3S inhibitor. Further biological assays, however,
indicated that the observed T3S inhibition most likely results from
a general growth inhibition rather than specific interactions with
components of the T3S system. Pseudoceramines A (2) and B
(3) were found to share the bromotyrosyl–spermine–bromotyrosyl
sequence of 1, while pseudoceramine C (4) was the first example
of a bromotyrosine coupled to a spermidine derivative.
Fig. 1 Structures for spermatinamine (1) and pseudoceramines A–D
(2–5).
motyrosine building block and the spermine derivative followed
by carbodiimide-mediated coupling of the two.5 The convergent
synthesis gave 1 in 15.4% yield over five steps (longest sequence)
and offered a short and potentially efficient entry to synthetic
analogues. A more recent approach gave 1 in 30.6% overall yield in
six steps.6 This latter synthesis utilised a protected bromotyrosine,
readily available from 3,4-dibromo-4-hydroxybenzaldehyde, in the
key step. No synthetic analogues of 1 have, to the best of our knowl-
edge, been described using the aforementioned routes and no
syntheses of the pseudoceramines 2–5 have been reported to date.
The total syntheses of the pseudoceramines are needed to
provide proof of their structures, in particular 3, which had its
absolute (S) configuration assigned tentatively during the course of
its isolation. In addition, whilst the structure of spermatinamine is
symmetrical, the pseudoceramines are all non-symmetrical, which
rules out direct application of any of the previous total syntheses
of spermatinamine to the pseudoceramines. This prompted the
development of new, general synthetic routes that could be
used for any natural bromotyrosine–polyamine derivative and
which could easily be branched out in diverted total syntheses
of synthetic symmetrical or non-symmetrical analogues. Finally,
Two total syntheses of spermatinamine (1) have been reported
in the literature. The first featured separate syntheses of the bro-
aUmea˚ Centre for Microbial Research, Department of Chemistry, Umea˚
University, SE-60187, Umea˚, Sweden
bLaboratories for Chemical Biology Umea˚, Umea˚ Centre for Microbial
Research, Laboratories for Molecular Infection Medicine Sweden, Depart-
ment of Chemistry, Umea˚ University, SE-60187, Umea˚, Sweden. E-mail:
mikael.elofsson@chem.umu.se; Tel: +46-90-7869328
† Electronic supplementary information (ESI) available: 1H and 13C NMR
spectra for compounds 2–5, 13, 14, 17–19, 21, 26–33, and 1H NMR
spectrum for compound 34. See DOI: 10.1039/c1ob06722b
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Scheme 1 Synthesis of the bromotyrosine oxime building block 6.
the pseudoceramines together with spermatinamine provide a
structural class with diverse biological profiles and which could
provide interesting templates for the synthesis of new drug-
like synthetic analogues. Here is reported the first syntheses of
pseudoceramines A–D (2–5), as well as a new improved synthesis
of spermatinamine (1).
Results and discussion
All five natural products have in common an O-methyltyrosine
derived fragment that was readily available in the form of acid
6 by emulating the procedure of de Lera et al. (Scheme 1).5 O-
Methyl-L-tyrosine (7) was first brominated and then protected as
the ester before tungstate mediated oxidation delivered the oxime
10. Finally, ester hydrolysis using LiOH produced the acid 6. This
sequence turned out to be gratifyingly robust, and in our hands the
yields leading up to 6 were even higher overall than those reported
previously.5
The natural next step was to attempt the synthesis of the simplest
structure, pseudoceramine D (5), by coupling 6 with diamine
11 in the presence of dicyclohexylcarbodiimide (DCC) and N-
hydroxyphtalimide (NHP), again using the work of de Lera and
co-workers as the standard.5 All our attempts, however, led to
complex mixtures of products. Substituting for a different cou-
pling reagent, benzotriazol-1-yl-oxytripyrrolidinophosphonium
hexafluorophosphate (PyBOP), or pre-forming the relevant acid
chloride or pentafluorophenol (Pfp) ester for direct acyl substi-
tution by the amine did not improve the results. Finally it was
found that direct acyl substitution of the ester 10 with 11 could be
achieved, analogous to the transformation demonstrated by the
group of Spilling (Scheme 2).7 Heating the two components in
methanol in a sealed tube or at reflux for 2–48 h (scaling up the
reaction led to shorter reaction times) not only gave 5 in high (95%)
yield in the final step, but also shortened the synthesis sequence
by one step (69.7% overall, 4 steps).
Scheme 3 Synthesis of spermatinamine 1.
derivative 15, starting with a sequence of esterification, benzyla-
tion, Boc-group cleavage and tungstate mediated oxidation of a
primary amine to give oxime 19. The final hydrogenolysis step
to give 13 in 70% yield was achieved using a palladium catalyst
that had been poisoned with zinc(II)bromide, thereby avoiding
hydrodebromination of the aryl bromide moieties.8,9 Building
block 14 was available from tyrosine 20 by bromination followed
by esterification.
In order to obtain the non-symmetrical pseudoceramines 2 and
3, it was envisioned that selectively protecting one of the primary
amine moieties in 12 would allow for the acyl substitution of
first 10, then 13 or 14, respectively, after deprotection of the
common intermediate. Thus, spermine 22 was selectively mono-
Boc-protected using the method of Blagbrough and co-workers
(Scheme 5).10 In our hands, the best result achieved was 55% of
the desired intermediate 23 along with 10% of the di-Boc-protected
product 24 (the latter was used to make polyamine 12 according to
previously reported5 procedures). Selective Cbz-protection of the
other primary amine moiety in 23 was achieved in 52% yield using
the procedure of Adamczyk et al.11 From this point onwards,
purification of the intermediates proved difficult, and repeated
attempted purification by chromatography on silica gel generally
resulted in reduced yields. Thus, intermediate 28 could be isolated
in low yields only after preparative HPLC. It was observed that any
attempts to isolate polyamine intermediates in their free base forms
in general led to considerable decomposition; this is in accordance
with observations made by Moya and Blagbrough.12
Although the convergent synthesis of intermediate 28 pro-
vided a branching point for the synthesis of the two remaining
bistyrosine natural products, an improved synthesis was clearly
desirable. Changing directions to a slightly more linear approach,
it was found that acyl substitution of ester 10 with monopro-
tected spermine 23 gave an isolable product 29 in 85% yield
(Scheme 6). This intermediate could be subjected to reductive
amination followed by protecting group cleavage to give the
ammonium salt 30 in 67% over two steps. Attempts to isolate
the intermediate after the first step led to a lowered overall
yield.
Scheme 2 Synthesis of pseudoceramine D (5).
We applied the same method in the synthesis of spermatinamine
(1) by heating 10 with the known ammonium salt 12 5 in methanol
with added triethylamine to liberate the amines (Scheme 3). After
heating for 42 h, 1 was obtained in 72% yield (58.3% longest
sequence, 4 linear steps).
Next, tyrosine derivatives 13 and 14 were prepared. These
compounds are needed as building blocks for targets 2 and 3
(Scheme 4, top and bottom, respectively). Building block 13 was
available in five steps from commercially available bromotyrosine
The final acyl substitution reaction between 30 and 13 to
give pseudoceramine A 2 proceeded in 30% yield (12.5% longest
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Scheme 4 Synthesis of the bromotyrosine building blocks 13 and 14.
Scheme 5 Synthesis of the protected intermediate 28
Scheme 6 Synthesis of pseudoceramines A (2) and B (3).
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Scheme 7 Synthesis of pseudoceramine D (4).
sequence, 7 steps), which was considered fully acceptable given
the extended reaction time and the rich substitution pattern of
the substrates (Scheme 6). Intermediate 14 did not react under
the acyl substitution conditions en route to pseudoceramine B
3. We believe this is mainly due to the a-hydroxyiminoester
being more activated towards nucleophiles than the a-aminoester
and that this result suggests a limitation of the potential scope
of the method. Instead, acid 21 was reacted with 30 under
de Lera’s conditions5 to give pseudoceramine B 3 in 3% yield,
with the major by-product isolated being imide 31. Variations
of coupling agents (HATU, bromotrispyrrolidinophosphonium
hexafluorophosphate, DIC/HOAt) and the preformed Pfp ester
were tested without success. Currently, we speculate that the by-
product arises from neighboring group participation of the a-
methylamine functionality as the intermediate O-acylisourea is
formed, leaving unreacted NHP in the reaction mixture which then
reacts with 30 to form 31. By Boc-protection of 21 and subjecting
the resultant acid 32 to the coupling conditions, imide formation
was averted and protected pseudoceramine 33 was formed in 33%
yield. Protecting group cleavage then furnished pseudoceramine B
3 in 67% yield (9.2% longest sequence, 7 steps). The absolute (S)
stereochemistry was confirmed for 3 by matching (+) sign of the
specific rotation measurement with the isolated natural product.
The prospect of using the newly synthesised pseudoceramine
D (5) in the synthesis of pseudoceramine C (4), together with
the discovery of the robust reductive amination-protecting group
cleavage protocol that furnished intermediate 30, motivated
synthesis of aldehyde 34 from known precursor 35 (Scheme 7).
Although in our hands 34 turned out to be unstable and could
not be isolated, 34 could be used in crude form (approximately
Chemical Co. All experiments were conducted under a nitrogen
atmosphere, in dried glassware, using anhydrous solvents unless
stated otherwise. Reagent grade tetrahydrofuran was distilled
from molten potassium while dichloromethane was distilled from
calcium hydride. Light petroleum (bp 40–60 C) is referred to as
petrol.
◦
Column chromatography was carried out using Merck silica
gel 60 and thin layer chromatography was performed on Merck
aluminium-backed plates, pre-coated with silica gel 60 (F254
)
and visualised by UV light and staining with alkali KMnO4.
Preparative reversed-phase HPLC was performed on a Gilson
GX-271 system fitted with a Machery-Nagel C18 HTEC, 5 mm
particle size column, using a gradient of acetonitrile/water (10–
60% over 20 mins) with added formic acid (0.005%) at a flow of
20 mL min-1 and detection at 210 nm.
Low-resolution mass-spectra were recorded on a Waters Micro-
mass ZG 2000 instrument with an electro spray ion source (ES+
R
and ES-) following liquid chromatography using an XTerraꢀ
MS C18 5 mm particle size, 4.6 ¥ 50 mm column and a wa-
ter/acetonitrile/0.2% formic acid eluent system. High-resolution
mass-spectra were recorded on a Bruker micrOTOF II instrument
1
with an electrospray ion source (ES+). H NMR and 13C NMR
spectrawere recorded at 298K onBruker DRX-360(1H: 360 MHz;
13C: 90 MHz), DRX-400 (1H: 400 MHz; 13C: 100 MHz) or DRX-
500 (1H: 500 MHz; 13C: 125 MHz) spectrometers, with chemical
shift values being reported in ppm relative to residual CHCl3 (dH
7.26 and dC 77.16 ppm) or CD3OH (dH 3.31 and dC 49.00 ppm)
as internal standard. Peak assignments could be established from
complementary HMBC, HMQC and COSY experiments. Infrared
spectra were recorded for neat (oils) or thin film (solids) samples
on NaCl discs using a Bruker Alpha-T spectrophotometer. Optical
rotation was measured on a Perkin Elmer polarimeter 343 and are
reported in 10-1 deg cm2 g-1. Melting points were measured using a
Bu¨chi/Dr Tottoli melting point determination apparatus and are
uncorrected.
1
50% pure by analysis of H NMR integrals). When this crude
material was used in excess in the reductive amination with 5 and
the subsequent cleavage of the Boc-group, pseudoceramine C (4)
was formed in 32% yield.
Conclusions
In summary, the first total syntheses of pseudoceramines A–D
(2–5) have been achieved, utilising a direct acyl substitution
of a-hydroxyiminoesters with amine nucleophiles as the key
transformation. The yields in the key steps ranged from excellent
(pseudoceramine D) to acceptable (pseudoceramines A and
B). In addition, the most efficient synthesis of spermatinamine
(1) to date has been completed. The spectral data for the
synthetic pseudoceramines A–D are in agreement with the
published structures.3 Work is ongoing to expand the scope
of the methodology and generate synthetic analogues of the
pseudoceramines for biological evaluation.
Experimental procedures
(E)-3-(3,5-Dibromo-4-methoxyphenyl)-2-hydroxyimino-N-(3-
(methylamino)propyl)propanamide, pseudoceramine D (5). To a
5 mL Biotage microwave tube was added known ester 10 (191
mg, 0.500 mmol), 3-(methylamino)propylamine 11 (0.157 mL,
1.50 mmol) and methanol (1 mL). The tube was sealed and the
contents heated at 65 ◦C for 22 h, at which point LCMS analysis
showed complete consumption of starting material. After cooling,
the reaction mixture was concentrated and the residue purified
by column chromatography on silica gel (89 : 10 : 1 to 20 : 5 : 1
to 4 : 2 : 1 dichloromethane/methanol/aqueous ammonia) to give
the freebase form of pseudoceramine D 5 (208 mg, 95%) as a
pale yellow glass; nmax/cm-1 (film) 2928, 2863, 1656, 1527, 1471,
1421; dH (400 MHz, CD3OD) 7.48 (2H, s), 3.84 (2H, s), 3.80
(3H, s), 3.35–3.29 (2H, m), 2.57 (2H, t, J = 7.1 Hz), 2.37 (3H, s),
Experimental
General considerations
Tyrosine derivatives were purchased from Bachem GmbH; all
other commercial chemicals were acquired from Sigma Aldrich
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1.72 (2H, tt, J = 7.1, 7.1 Hz); dC (100 MHz, CD3OD) 165.6,
153.8, 152.1, 137.4, 134.5, 118.6, 61.0, 49.4, 37.9, 35.7, 29.7,
28.8; m/z LRMS (ES+) 440.1 (69%, [M(81Br2) + H]+), 438.2
(100, [M(79Br81Br) + H]+), 436.2 (67, [M(79Br2) + H]+), 409.1
(60, [M(81Br2) - CH4N]+), 407.1 (96, [M(79Br81Br) - CH4N]+),
405.1 (60, [M(79Br2) - CH4N]+); HRMS (ES+) found 437.9858,
layer extracted with dichloromethane (2 ¥ 10 mL). The pooled
organic layers were dried (Na2SO4) and concentrated, and the
crude residue purified by column chromatography on silica gel
(190 : 9 : 1 dichloromethane/methanol/aqueous ammonia) to give
the amine 18 (1.48 g, 69%) as a colourless oil; [a]2D1 +5.9 (c 0.8,
dichloromethane); nmax/cm-1 (neat) 3381, 3032, 2949, 1738, 1455;
C14H2079Br81BrN3O3 requires 437.9851.
dH (400 MHz, CDCl3) 7.60 (2H, d, J = 7.4 Hz), 7.43–7.34 (3H, m),
+
7.40 (2H, s), 5.01 (2H, s), 3.73 (3H, s), 3.70 (1H, dd, J = 7.9, 5.1
Hz), 3.00 (1H, dd, J = 13.7, 5.1 Hz), 2.77 (1H, dd, J = 13.7, 7.9 Hz),
1.65 (2H, br s); dC (100 MHz, CDCl3) 175.0, 151.7, 136.5, 136.3,
133.5, 128.54, 128.46, 118.5, 74.7, 55.6, 52.3, 39.7; m/z LRMS
(ES+) 446.2 (70%, [M(81Br2) + H]+), 444.2 (100, [M(79Br81Br) +
H]+), 442.2 (70, [M(79Br2) + H]+), 386.2 (70), 384.2 (100), 382.2
(+)-(S)-Methyl 3-(3,5-dibromo-4-hydroxyphenyl)-2-(2-methyl-
prop-2-yloxycarbamoyl)propanoate (16). Boc-3,5-dibromo-Tyr-
OH 15 (4.00 g, 9.11 mmol) was dissolved in a 1 : 9 mix-
ture of methanol/dichloromethane (50 mL) and (trimethylsi-
lyl)diazomethane (2.80 mL, 5.60 mmol) was added dropwise.
After stirring for 1 h at room temperature any remaining reagent
was quenched by adding two drops of acetic acid. The reaction
mixture was diluted with dichloromethane (100 mL), washed with
saturated aqueous NaHCO3 (3 ¥ 10 mL) and brine (10 mL),
dried (Na2SO4) and concentrated to give the ester 16 (2.49 g,
98%) as a colourless oil which was used in further synthesis with
no purification necessary; [a]2D1 +24.0 (c 0.25, dichloromethane);
(70); HRMS (ES+) found 443.9648, C17H1879Br81BrNO3 requires
+
443.9633.
(E)-Methyl 3-(4-benzyloxy-3,5-dibromophenyl)-2-(hydroxy-
imino)propanoate (19). A solution of sodium tungstate dihydrate
(73.7 mg, 0.223 mmol) and 37% aqueous hydrogen peroxide
(0.250 mL, 2.43 mmol) in water (2 mL) was added to a solution
of amine 18 (99.0 mg, 0.223 mmol) in ethanol (2 mL) at 0 ◦C.
The resulting mixture was stirred at 0 ◦C for 30 mins, then at room
temperature for 17 h. To the mixture was added saturated aqueous
NH4Cl (5 mL) and brine (2 mL) and the aqueous layer extracted
with ethyl acetate (3 ¥ 10 mL). The combined organic layers were
dried (Na2SO4) and concentrated to give the oxime 19 (101 mg,
99%) as colourless platelets; mp 167–168 ◦C (hexanes); nmax/cm-1
(film) 3061, 3035, 2923, 1729, 1441; dH (400 MHz, CDCl3) 9.47
(1H, br s), 7.59 (2H, d, J = 7.4 Hz), 7.51 (2H, s), 7.43–7.34 (3H, m),
4.99 (2H, s), 3.91 (2H, s), 3.89 (3H, s); dC (100 MHz, CDCl3) 163.6,
151.8, 150.3, 136.4, 134.4, 133.6, 128.6, 128.5, 118.6, 74.8, 53.2,
29.4; m/z LRMS (ES+) 501.1 (50%, [M(81Br2) + C2H4N]+), 499.1
(100, [M(79Br81Br) + C2H4N]+), 497.1 (45, [M(79Br2) + C2H4N]+),
475.2 (60), 458.1 (75, [M(79Br81Br) + H]+); HRMS (ES+) found
n
max/cm-1 (neat) 3388, 2978, 1739, 1699, 1478; dH (400 MHz,
CDCl3) 7.22 (2H, s), 5.81 (1H, s), 5.02 (1H, d, J = 7.0 Hz),
4.51 (1H, m), 3.74 (3H, s), 3.05 (1H, dd, J = 13.9, 5.9 Hz), 2.92
(1H, dd, J = 13.9, 5.9 Hz), 1.44 (9H, s); dC (100 MHz, CDCl3)
171.9, 155.1, 148.6, 132.9, 131.0, 109.9, 80.4, 54.5, 52.6, 37.1,
28.4; m/z LRMS (ES-) 454.2 (42%, [M(81Br2) - H]-), 452.2 (100,
[M(79Br81Br) - H]-), 450.2 (40, [M(79Br2) - H]-); HRMS (ES+)
found 475.9523, C15H1979Br81BrNNaO5 requires 475.9507. The
+
1H NMR data agree with literature values.13
(+) - (S) - Methyl 3 - (4 - benzyloxy - 3, 5 - dibromophenyl) - 2 - (2-
methylprop-2-yloxycarbamoyl)propanoate (17). Benzyl bromide
(0.153 mL, 1.29 mmol) was added to a stirred suspension of ester
16 (389 mg, 0.859 mmol) and K2CO3 (178 mg, 1.29 mmol) in DMF
(9 mL) and stirred at room temperature for 4 h, at which point TLC
analysis showed complete consumption of starting material. The
reaction mixture was diluted with ethyl acetate (20 mL), filtered
and concentrated to give the benzyl ether 17 (430 mg, 92%) as
colourless platelets which was used in further synthesis with no
purification necessary; mp 54–59 ◦C (hexanes); [a]D21 +21.9 (c 1.17,
chloroform); nmax/cm-1 (film) 2977, 1744, 1714, 1499, 1453; dH
(360 MHz, CDCl3) 7.60 (2H, d, J = 7.4 Hz), 7.44–7.34 (3H, m),
7.31 (2H, s), 5.07 (1H, d, J = 7.3 Hz), 5.01 (2H, s), 4.54 (1H, m),
3.76 (3H, s), 3.10 (1H, dd, J = 13.6, 5.9 Hz), 2.95 (1H, dd, J = 13.9,
5.9 Hz), 1.45 (9H, s); dC (90 MHz, CDCl3) 171.9, 155.0, 151.9,
136.4, 135.3, 133.7, 133.6, 128.6, 118.6, 80.4, 74.8, 54.4, 52.7, 37.2,
28.4; m/z LRMS (ES+) 566.1 (10%, [M(79Br81Br) + Na]+), 446.1
(50, [M(81Br2) - C5H8O2]+), 444.1 (100, [M(79Br81Br) - C5H8O2]+),
442.1 (50, [M(79Br2) - C5H8O2]+); HRMS (ES+) found 565.9995,
479.9257, C17H1579Br81BrNNaO4 requires 479.9245.
+
(E) - Methyl 3 - (4 - hydroxy - 3, 5 - dibromophenyl) - 2 - (hydroxy-
imino)propanoate (13). Benzyl ether 19 (229 mg, 0.501 mmol),
zinc(II)bromide (338 mg, 1.50 mmol) and 10% palladium on
charcoal (53.0 mg, 49.8 mmol) were suspended in dry ethyl
acetate (20 mL). The reaction vessel was purged under reduced
pressure and back-filled with hydrogen gas three times, then
stirred vigorously under hydrogen (1 atm) for 30 mins, at which
point TLC analysis showed complete consumption of starting
material. The mixture was dryloaded on silica and subjected to
chromatography on silica gel (1 : 3 ethyl acetate/heptanes) to give
the phenol 13 (128 mg, 70%) as colourless platelets; mp 174–175
◦C (ethyl acetate); nmax/cm-1 (film) 3409, 2949, 1731, 1474; dH
(400 MHz, CD3OD) 7.37 (2H, s), 3.80 (2H, s), 3.79 (3H, s); dC
(100 MHz, CD3OD) 165.7, 151.0, 150.8, 133.8, 132.0, 112.0, 52.9,
29.7; m/z LRMS (ES-) 365.9 (65%, [M(79Br81Br) - H]-), 253.0 (48,
[M(81Br2) - C4H6NO3]-), 251.0 (100, [M(79Br81Br) - C4H6NO3]-),
249.0 (51, [M(79Br2) - C4H6NO3]-); HRMS (ES+) found 389.8792,
+
C22H2579Br81BrNNaO5 requires 565.9977.
(+)-(S)-Methyl 2-amino-3-(4-benzyloxy-3,5-dibromophenyl)-
propanoate (18). Carbamate 17 (2.61 g, 4.81 mmol) was dissolved
in a mixture of dichloromethane (10 mL) and TFA (10 mL)
and stirred at room temperature for 2 h, at which point TLC
analysis indicated complete conversion. The reaction mixture
was diluted with dichloromethane (40 mL) and made basic with
saturated aqueous NaHCO3 (20 mL) and aqueous 10 M NaOH
(10 mL) under vigorous stirring and cooling in an ice-bath.
The resulting biphasic mixture was separated and the aqueous
+
C10H979Br81BrNNaO4 requires 389.8776.
(+)-(S)-3-(3,5-Dibromo-4-methoxyphenyl)-2-methylamino-
propanoic acid hydrochloride (21). Me-Tyr(Me)-OH 20 (500 mg,
2.39 mmol) was dissolved in 2 M HCl (12 mL) and cooled to 0 ◦C.
Bromine (1.00 mL, 19.5 mmol) was added dropwise via addition
funnel, the resulting mixture stirred for 15 mins and then warmed
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to room temperature. Stirring was continued as a stream of air was
passed through the mixture for 22 h. All solvents were evaporated
and the crude residue triturated from diethyl ether and petrol to
[M - C9H19N2O2]+), 143.4 (100); HRMS (ES+) found 465.3445,
C25H45N4O4 requires 465.3441.
+
◦
N-(12-Amino-4,9-diaza-4,9-dimethyldodecyl)-benzyloxycarba-
mate trishydrochloride (27). A 100/458 mg aliquot of crude
spermine derivative 26 (100 mg) was dissolved in a mixture of
aqueous 4 M HCl (2 mL) and 1,4-dioxane (2 mL) and stirred
at room temperature for 20 h. Solvents were evaporated under
reduced pressure and the residue triturated from diethyl ether to
give 27 (105 mg) as a colourless gum, approximately 70–80% pure
give 21 (864 mg, 90%) as tan platelets; mp 174–175 C (petrol);
[a]2D1 +16.3 (c 0.95, methanol); nmax/cm-1 (film) 3406, 2932, 1737,
1472, 1423; dH (400 MHz, CD3OD) 7.56 (2H, s), 4.29 (1H, dd, J =
6.7, 5.6 Hz), 3.85 (3H, s), 3.32 (1H, dd, J = 14.6, 5.6 Hz), 3.20 (1H,
dd, J = 14.6, 6.7 Hz), 2.76 (3H, s); dC (100 MHz, CD3OD) 170.0,
155.2, 135.0, 134.3, 119.4, 62.6, 61.1, 34.8, 32.9; m/z LRMS (ES-)
368.0 (44%, [M(81Br2) - H]-), 366.0 (100, [M(79Br81Br) - H]-), 364.0
(44, [M(79Br2) - H]-), 335.0 (52, [M(79Br81Br) - CH4N]-); HRMS
1
by analysis of H NMR integrals, used in the next step without
further purification; nmax/cm-1 (film) 3405, 2964, 1705, 1630, 1529,
1474; dH (400 MHz, CD3OD) 7.43–7.31 (5H, m), 5.09 (2H, s),
3.41–3.06 (12H, m), 2.92 (3H, s), 2.91 (3H, s), 2.22–2.14 (4H, m),
2.02–1.83 (4H, m); dC (100 MHz, CD3OD) 159.3, 138.4, 129.7,
129.2, 129.0, 67.8, 61.7, 56.8, 56.7, 55.3, 54.3, 40.8, 40.7, 38.8,
38.1, 26.2, 23.6, 22.5; m/z LRMS (ES-) 365.5 (15%, [M + H]+),
+
(ES+) found 367.9328, C11H1479Br81BrNO3 requires 367.9320.
(+)-(S)-Methyl 3-(3,5-dibromo-4-methoxyphenyl)-2-methylami-
nopropanoate (14). Acid 21 (40 mg, 0.10 mmol) was dissolved
in methanol (5 mL) and thionyl chloride (0.14 mL, 2.0 mmol)
was added dropwise. The resulting mixture was refluxed for
22 h, at which point LCMS analysis indicated consumption of
starting material. Solvents were removed under reduced pres-
sure and the residue dissolved in dichloromethane (5 mL) and
washed with saturated aqueous NaHCO3 (5 mL). The aqueous
layer was back-extracted with dichloromethane (2 ¥ 5 mL),
the pooled organic layers dried (Na2SO4) and concentrated
under reduced pressure and the residue purified by chromatog-
raphy on silica gel (1 : 3 ethyl acetate/heptanes, then 89 : 10 : 1
dichloromethane/methanol/aqueous ammonia) to give 14 (25
mg, 66%) as a pale brown film; [a]2D1 +9.1 (c 1.0, dichloromethane);
+
143.4 (100); HRMS (ES+) found 365.2921, C20H37N4O2 requires
365.2917.
(E)-3-(3,5-Dibromo-4-methoxyphenyl)-2-hydroxyimino-N-(12-
benzyloxycarbamoyl-4,9-diazadodecyl)propanamide
bisformate
(28). Acid (37 mg, 0.10 mmol), N,N¢-dicyclohexyl-
6
carbodiimide (23 mg, 0.11 mmol), N-hydroxyphtalimide
(18 mg, 0.11 mmol) and 1,4-dioxane (0.5 mL) were combined
in a 5 mL Biotage microwave tube and the resulting mixture
stirred at room temperature for 24 h. To the mixture was added
a solution of a 52/105 mg aliquot of crude spermine derivative
27 (52 mg) and triethylamine (31 mL, 0.22 mmol) in methanol
(0.5 mL) and the resulting mixture was stirred for 14 h. Solvents
were evaporated under reduced pressure and the residue purified
by column chromatography on silica gel (9 : 1 : 0 to 4 : 2 : 1
dichloromethane/methanol/aqueous ammonia) and preparative
HPLC to give the bisformate salt of 28 (9.0 mg, 8% from 25) as
a colourless, hygroscopic powder; nmax/cm-1 (film) 3300, 2946,
1709, 1661, 1588; dH (400 MHz, CD3OD) 8.47 (2H, s), 7.49 (2H,
s), 7.36–7.28 (5H, m), 5.08 (2H, s), 3.85 (2H, s), 3.81 (3H, s), 3.33
(2H, t, J = 6.3 Hz), 3.21 (2H, t, J = 6.5 Hz), 3.00–2.90 (8H, m), 2.69
(3H, s), 2.67 (3H, s), 1.92–1.83 (4H, m), 1.74–1.69 (4H, m); dC (100
MHz, CD3OD) 169.4, 166.0, 159.1, 153.9, 152.0, 138.3, 137.5,
134.5, 129.5, 129.1, 128.8, 118.6, 67.6, 61.1, 55.2, 40.6, 39.0, 37.5,
28.8, 26.4, 26.1, 23.2; m/z LRMS (ES+) 716.4 (35%, [M(81Br2) +
H]+), 714.4 (70, [M(79Br81Br) + H]+), 712.4 (38, [M(79Br2) + H]+),
494.2 (40, [M(81Br2) - C12H17N2O2]+), 492.2 (83, [M(79Br81Br) -
C12H17N2O2]+), 490.2 (42, [M(79Br2) - C12H17N2O2]+), 409.1
(50, [M(81Br2) - C17H28N3O2]+), 407.1 (100, [M(79Br81Br) -
C17H28N3O2]+), 405.1 (50, [M(79Br2) - C17H28N3O2]+), 277.4 (75);
n
max/cm-1 (film) 2949, 2930, 1736, 1473, 1423; dH (400 MHz,
CDCl3) 7.32 (2H, s), 3.86 (3H, s), 3.70 (3H, s), 3.40 (1H, t,
J = 6.7 Hz), 2.88 (1H, dd, J = 13.3, 6.7 Hz), 2.88 (1H, dd, J =
13.3, 6.7 Hz), 2.38 (3H, s); dC (100 MHz, CDCl3) 174.3, 153.0,
136.2, 133.4, 118.1, 64.2, 60.8, 52.0, 38.0, 34.8; m/z LRMS (ES+)
382.1 (20%, [M(79Br81Br) + H]+), 324.1 (48, [M(81Br2) - C2H3O2]+),
322.1 (100, [M(79Br81Br) - C2H3O2]+), 320.1 (50, [M(79Br2) -
C2H3O2]+); HRMS (ES+) found 381.9489, C12H1679Br81BrNO3
+
requires 381.9476.
Benzyloxy-N-(12-(2-methylprop-2-oxy)carbamoyl-4,9-diaza-4,
9-dimethyldodecyl)carbamate (26). N1-Cbz-N14-Boc-spermine
25 (578 mg, 1.32 mmol), 37% aqueous formaldehyde (2.20 mL,
29.4 mmol), acetic acid (2.50 mL, 43.7 mmol) and NaCNBH3
(665 mg, 10.6 mmol) were added to ethanol (15 mL) at room
temperature and the resulting mixture stirred for 15 h, at which
point LCMS analysis showed completion. The mixture was diluted
with dichloromethane (50 mL) and 10 M aqueous NaOH (10 mL)
and stirred 10 mins. The biphasic mixture was extracted with
dichloromethane (2 ¥ 50 mL), washed with brine (10 mL), dried
(Na2SO4) and concentrated and the residue purified by column
chromatography on silica gel (1 : 1 ethyl acetate/heptanes, then
40 : 5 : 1 dichloromethane/methanol/aqueous ammonia) to give
26 (458 mg) as a colourless syrup, approximately 80–90% pure
HRMS (ES+) found 714.1703, C30H4479Br81BrN5O5 requires
+
714.1689.
(E)-3-(3,5-Dibromo-4-methoxyphenyl)-2-hydroxyimino-N-(12-
(2-methylprop-2-oxy)carbamoyl-4,9-diazadodecyl)propanamide
(29). Ester 10 (216 mg, 0.567 mmol) and N1-Boc-spermine
23 (343 mg, 1.13 mmol) were dissolved in methanol (6 mL)
and the resulting mixture refluxed for 14 h, at which point
LCMS analysis showed completion. After cooling, the reac-
tion mixture was concentrated and the residue purified by
column chromatography on silica gel (89 : 10 : 1 to 20 : 5 : 1
dichloromethane/methanol/aqueous ammonia) to give 29 (312
mg, 85%) as a pale yellow glass; nmax/cm-1 (film) 3290, 2971, 2932,
1
by analysis of H NMR integrals, used in the next step without
further purification; nmax/cm-1 (film) 3337, 3033, 2795, 1703, 1526;
dH (400 MHz, CDCl3) 7.35–7.30 (5H, m), 5.09 (2H, s), 3.30–3.23
(2H, m), 3.18–3.14 (2H, m), 2.43–2.33 (8H, m), 2.20 (3H, s), 2.18
(3H, s), 1.70–1.59 (4H, m), 1.49–1.46 (4H, m), 1.43 (9H, s); dC
(90 MHz, CDCl3) 156.7, 156.3, 137.0, 128.6, 128.3, 128.2, 79.0,
66.5, 57.8, 57.7, 56.5, 56.2, 54.0, 52.0, 42.0, 39.9, 28.6, 26.9, 26.5,
25.04, 24.98; m/z LRMS (ES-) 465.4 (20%, [M + H]+), 277.4 (35,
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2864, 1693, 1659, 1525; dH (400 MHz, CD3OD) 7.48 (2H, s), 3.84
(2H, s), 3.81 (3H, s), 3.35–3.32 (2H, m), 3.09 (2H, t, J = 6.7
Hz), 2.63–2.53 (8H, m), 1.74–1.64 (4H, m), 1.60–1.50 (4H, m),
1.43 (9H, s); dC (100 MHz, CD3OD) 165.6, 158.6, 153.8, 152.0,
137.5, 134.5, 118.6, 79.9, 61.0, 50.4, 48.2, 47.7, 39.1, 38.5, 30.6,
29.9, 28.8, 28.3, 28.1; m/z LRMS (ES-) 652.3 (48%, [M(81Br2) -
H]-), 650.4 (100, [M(79Br81Br) - H]-), 648.4 (45, [M(79Br2) - H]-),
576.2 (52, [M(79Br81Br) - C4H9]-); HRMS (ES+) found 652.1562,
21 (270 mg, 0.669 mmol) was dissolved in water (2 mL), cooled
to 0 ◦C and freebased by careful addition of solid NaHCO3 (141
mg, 1.67 mmol). To the vortexed solution was added dropwise a
solution of di-tert-butyl dicarbonate (175 mg, 0.802 mmol) in THF
(2 mL). After warming to room temperature and stirring for 17 h
the solution was again cooled to 0 ◦C and another portion of di-
tert-butyl dicarbonate (115 mg, 0.527 mmol) in THF (1 mL) was
added, followed by warming to room temperature and stirring for
26 h. Deemed complete by LCMS analysis, the reaction mixture
was diluted with water (5 mL), acidified to pH 2 with aqueous
1 M HCl, extracted with dichloromethane (3 ¥ 10 mL) and the
combined organic layers dried (Na2SO4) and concentrated under
reduced pressure. The crude residue was purified by chromatog-
raphy on silica gel (1 : 3 ethyl acetate/heptanes) to give 32 (200
C25H4279Br81BrN5O5 requires 652.1532.
+
(E)-N-(12-Amino-4,9-dimethyl-4,9-diazadodecyl)-3-(3,5-dibro-
mo-4-methoxyphenyl)-2-(hydroxyimino)propanamide trishydrochl-
oride (30). Boc-protected polyamine 29 (16 mg, 25 mmol), 37%
aqueous formaldehyde (75 mL, 1.0 mmol), acetic acid (0.11 mL, 2.0
mmol) and NaCNBH3 (31 mg, 0.50 mmol) were added to ethanol
(1 mL) at room temperature and the resulting mixture stirred
for 1 h, at which point LCMS analysis showed completion. The
mixture was diluted with dichloromethane (6 mL) and aqueous
10 M NaOH (3 mL) and stirred for 10 mins. The biphasic mixture
was extracted with dichloromethane (3 ¥ 10 mL), washed with
brine (5 mL), dried (Na2SO4) and concentrated to 5 mL total
volume. To the concentrate was added n-dodecanethiol (0.5 mL)
and TFA (0.25 mL) and the resulting solution stirred at room
temperature for 16 h. All volatile solvents were removed by
evaporation under reduced pressure and the residue extracted into
a 1 : 1 mixture of methanol/aqueous 1 M HCl (2 mL) and purified
by preparative HPLC to yield 30 (11.5 mg, 67%) as a colourless
film; nmax/cm-1 (film) 3385, 2965, 1666, 1530, 1471; dH (400 MHz,
CD3OD) 7.50 (2H, s), 3.86 (2H, s), 3.82 (3H, s), 3.39–3.35 (2H,
m), 3.30–3.05 (10H, m), 2.91 (3H, s), 2.87 (3H, s), 2.23–2.13 (2H,
m), 2.02–1.94 (2H, m), 1.90–1.80 (4H, m); dC (100 MHz, CD3OD)
166.1, 153.9, 151.9, 137.5, 134.5, 118.6, 61.1, 56.7, 56.6, 55.2, 54.2,
40.6, 40.5, 37.9, 37.1, 28.9, 25.7, 23.5, 22.35, 22.33; m/z LRMS
(ES+) 580.4 (50%, [M(81Br2) + H]+), 578.4 (100, [M(79Br81Br) +
H]+), 576.4 (48, [M(79Br2) + H]+); HRMS (ES+) found 580.1348,
◦
mg, 64%) as colourless needles; mp 51–52 C (hexanes); [a]D21
-40.7 (c 2.48, dichloromethane); nmax/cm-1 (film) 2976, 2932, 1735,
1690, 1474; dH (400 MHz, CD3OD) obtained as a 5 : 4 mixture of
atropisomers, 7.47 (1.1H, s), 7.45 (0.9H, s), 4.82–4.72 (1H, m), 3.83
(1.6H, s), 3.82 (1.4H, s), 3.24 (1H, dd, J = 14.6, 4.9 Hz), 3.03 (0.4H,
dd, J = 14.6, 10.5 Hz), 2.98 (0.6H, dd, J = 14.6, 10.5 Hz), 2.73
(1.6H, s), 2.72 (1.4H, s), 1.39 (4H, s), 1.34 (5H, s); dC (100 MHz,
CD3OD) 173.7 (Major), 173.5 (minor), 157.5 (m), 157.0 (M), 154.1
(M), 154.0 (m), 138.9 (M), 138.6 (m), 134.6 (M), 134.5 (m), 118.8
(M), 118.6 (m), 81.9 (M), 81.5 (m), 62.0, 61.0 (M), 60.8 (m), 34.9
(M), 34.5 (m), 32.8 (m), 32.4 (M), 28.6 (m), 28.5 (M); m/z LRMS
(ES-) 468.0 (50%, [M(81Br2) - H]-), 466.0 (100, [M(79Br81Br) - H]-),
464.0 (48, [M(79Br2) - H]-), 335.0 (38, [M(79Br81Br) - C6H13NO2]-);
HRMS (ES+) found 489.9682, C16H2179Br81BrNNaO5 requires
+
489.9664.
(-)-(S)-(E)-3-(3,5-Dibromo-4-methoxyphenyl)-N-(12-(3-(3,5-
dibromo-4-methoxyphenyl)-2-(N-methyl-(2-methylprop-2-yloxy)-
carbamoyl)propanamido)-4,9-dimethyl-4,9-diazadodecyl)-2-(hyd-
roxyimino)propanamide bishydrochloride (33). Acid 32 (13 mg,
28 mmol), DCC (6.3 mg, 31 mmol) and N-hydroxyphthalimide
(5.0 mg, 31 mmol) were dissolved in 1,4-dioxane (0.5 mL) and
stirred at room temperature for 2 h, at which point DCC (0.60
mg, 3.1 mmol) and N-hydroxyphthalimide (0.50 mg, 3.1 mmol)
were added, followed by further stirring for 18 h. A solution of
ammonium salt 30 (21 mg, 31 mmol) and NEt3 (13 mL, 92 mmol)
in methanol (0.5 mL) was then added and stirring continued
for 7 h. The reaction mixture was concentrated and the crude
residue dissolved in 1 : 1 methanol/aqueous 1 M HCl (2 mL) and
purified by preparative HPLC to give the hydrochloride salt 33
(10 mg, 33%) as a colourless powder; [a]2D1 -19.6 (c 1.4, methanol);
C22H3879Br81BrN5O3 requires 580.1321.
+
(E,E)-N -(12-(3-(3,5-Dibromo-4-hydroxyphenyl)-2-(hydroxy-
imino)propanamido)-4,9-dimethyl-4,9-diazadodecyl)-3-(3,5-dibro-
mo-4-methoxyphenyl)-2-(hydroxyimino)propanamide bishydrochl-
oride, pseudoceramine A (2). To a 5 mL Biotage microwave tube
was added 13 (8.1 mg, 22 mmol), 30 (23 mg, 33 mmol), NEt3
(14 mL, 100 mmol) and methanol (0.5 mL). The tube was sealed
and the contents heated at 65 ◦C for 68 h. After cooling, the
reaction mixture was diluted with aqueous 1 M HCl (0.5 mL)
and purified by preparative HPLC to give the hydrochloride salt
of pseudoceramine A 2 (6.5 mg, 30%) as a pale yellow powder;
n
max/cm-1 (film) 3385, 2926, 1687, 1656, 1594, 1469; dH (500 MHz,
n
max/cm-1 (film) 3300, 3050, 2942, 1658, 1587, 1529, 1471; dH (400
CD3OD) obtained as a 5 : 4 mixture of atropisomers 8.50 (1H,
br s), 7.49 (2H, s), 7.46 (2H, s), 4.80–4.70 (1H, m), 3.86 (2H,
s), 3.82 (3H, s), 3.81 (3H, s), 3.35–3.21 (6H, m), 2.95–2.89 (8H,
m), 2.78 (1.4H, s), 2.73 (1.6H, s), 2.67 (6H, s), 1.92–1.83 (4H,
m), 1.77–1.71 (4H, m), 1.40 (5H, s), 1.31 (4H, s); dC (125 MHz,
CD3OD) 173.1 (Major), 172.6 (minor), 169.5, 166.0, 157.5 (M),
156.8 (m), 154.1 (m), 153.9 (M), 152.1, 138.8 (m), 138.5 (M), 137.5,
134.7, 134.5, 118.9 (m), 118.7 (M), 118.6, 82.1 (m), 81.8 (M), 62.4
(m), 61.7 (M), 61.1, 55.3 (M), 55.2 (m), 40.8, 38.1 (m), 37.6 (M),
34.5 (m), 34.2 (M), 33.0 (M), 31.5 (m), 28.9, 28.6 (M), 28.5 (m),
26.2, 23.6; m/z LRMS (ES-) 1029.8 (75%, [M(79Br81Br3) - H]-),
1027.9 (100, [M(79Br281Br2) - H]-), 1025.8 (60, [M(79Br381Br) - H]-);
MHz, CD3OD) 8.51 (1H, br s), 7.50 (2H, s), 7.35 (2H, s), 3.86
(2H, s), 3.81 (3H, s), 3.79 (2H, s), 3.35–3.32 (4H, m), 2.85–2.67
(8H, m), 2.57 (3H, s), 2.56 (3H, s), 1.91–1.78 (4H, m), 1.63–1.56
(4H, m); dC (100 MHz, CD3OD) 166.2, 165.8, 153.9, 152.8, 152.6,
152.1, 137.5, 134.5, 133.7, 133.6, 130.7, 118.6, 113.0, 61.1, 57.6,
57.4, 55.3, 55.2, 40.7, 37.8, 37.4, 28.8, 28.5, 26.42, 26.39, 24.0, 23.7;
m/z LRMS (ES+) 915.3 (35%, [M(79Br281Br2) + H]+), 409.1 (50),
407.1 (100), 405.1 (48), 393.1 (76, [C12H1379Br81BrN2O3]+); HRMS
+
(ES+) found 914.9912, C31H4379Br281Br2N6O6 requires 914.9937.
(-)-(S)-3-(3,5-Dibromo-4-methoxyphenyl)-2-(N-methyl-(2-me-
thylprop-2-yloxycarbamoyl)propanoic acid (32). Ammonium salt
1252 | Org. Biomol. Chem., 2012, 10, 1246–1254
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+
HRMS (ES+) found 1029.0957, C38H5779Br281Br2N6O7 requires
1029.0981.
resulting mixture stirred for 20 h. Saturated aqueous ammonium
chloride (5 mL) was then added and the resulting biphasic mixture
was extracted with diethyl ether (2 ¥ 10 mL), the combined organic
layers dried (Na2SO4) and concentrated and the residue dissolved
in acetic acid (4 mL) and water (2 mL) and stirred for 18 h. After
evaporation of solvents, purification of the residue was attempted
by chromatography on silica gel (acetone) to give the unstable
(+)-(S)-(E)-3-(3,5-Dibromo-4-methoxyphenyl)-N-(12-(3-(3,5-
dibromo-4-methoxyphenyl)-2-methylaminopropanamido)-4,9-dim-
ethyl-4,9-diazadodecyl)-2-(hydroxyimino)propanamide tristrifluo-
roacetate, pseudoceramine B (3). Protected pseudoceramine 33
(8.6 mg, 7.8 mmol), n-dodecanethiol (11 mg, 39 mmol) and TFA
(0.5 mL) were dissolved in dichloromethane (0.5 mL) and stirred at
room temperature for 2 h, at which point LCMS analysis showed
complete consumption of starting materials. The reaction mixture
was concentrated under reduced pressure and the crude residue
filtered through a Supelco fritted C18 silica plug, eluting with
a gradient of methanol-water (0–70%) to give the trifluoroacetate
salt of pseudoceramine B 3 (6.6 mg, 67%) as a colourless film; [a]D21
+34.3 (c 0.07, methanol) (lit.,3 +8◦ (c 0.08, methanol)); nmax/cm-1
(film) 3357, 3224, 2961, 2706, 1666, 1545, 1471; dH (500 MHz,
CD3OD) 7.52 (2H, s), 7.50 (2H, s), 4.00 (1H, dd, J = 8.5, 6.0 Hz),
3.86 (2H, s), 3.85 (3H, s), 3.82 (3H, s), 3.45–3.00 (14H, m), 2.86
(3H, s), 2.85 (3H, s), 2.70 (3H, s), 1.99–1.79 (8H, m); dC (125 MHz,
CD3OD) 168.2, 166.3, 155.1, 153.9, 151.9, 137.5, 135.1, 134.6,
134.5, 119.3, 118.6, 63.5, 61.2, 61.1, 56.6, 55.2, 55.0, 40.4, 37.6,
37.1, 35.9, 32.4, 28.9, 25.7, 25.1, 22.29, 22.28; m/z LRMS (ES-)
929.8 (57%, [M(79Br81Br3) - H]-), 927.8 (100, [M(79Br281Br2) - H]-),
925.8 (60, [M(79Br381Br) - H]-); HRMS (ES+) found 1029.0957,
1
aldehyde 34 as a colourless oil, approximately 50% pure by H
NMR analysis of the crude material (68 mg, 18%), which was
used immediately in the next step without further purification; dH
(400 MHz, CDCl3) obtained as a 1 : 1 mixture of atropisomers
9.78 (1H, m), 3.27–3.14 (2H, m), 2.84 (1.5H, s), 2.83 (1.5H, s),
2.00–1.61 (4H, m), 1.45 (4.5H, s), 1.45 (4.5H, s).
(E)-3-(3,5-Dibromo-4-methoxyphenyl)-2-hydroxyimino-N-(8-
(methylamino)-4-methyl-4-azaoctyl)propanamide bishydrochloride,
pseudoceramine C (4). To a solution of 5 (16.1 mg, 36.8 mmol) in
ethanol (1 mL) was added 34 (50% pure, 74.0 mg, 0.184 mmol),
acetic acid (22.1 mg, 0.369 mmol) and NaCNBH3 (11.5 mg, 0.184
mmol). The resulting mixture was stirred at room temperature
for 16 h, then diluted with dichloromethane (5 mL) and aqueous
10 M NaOH (0.5 mL), stirred 5 mins and washed with brine
(3 mL). The aqueous phase was extracted with dichloromethane
(2 ¥ 5 mL) and the combined organic layers dried (Na2SO4) and
concentrated to 2 mL total volume. n-Dodecanethiol (0.25 mL)
and TFA (1 mL) were added and the resulting mixture stirred for
15 h. All volatiles were evaporated under reduced pressure, the
crude residue extracted with a 1 : 1 mixture of methanol/aqueous
1 M HCl (2 mL) and the extract purified by preparative HPLC
to give the hydrochloride salt of pseudoceramine C 4 (6.9 mg,
32%) as a colourless film; nmax/cm-1 (film) 3357, 2974, 2927, 2896,
1720, 1664; dH (500 MHz, CD3OD) 7.50 (2H, s), 3.86 (2H, s),
3.82 (3H, s), 3.41–3.35 (2H, m), 3.22–3.03 (6H, m), 2.85 (3H,
s), 2.72 (3H, s), 1.98–1.93 (2H, m), 1.83–1.72 (4H, m); dC (125
MHz, CD3OD) 166.3, 153.9, 152.0, 137.4, 134.5, 118.6, 61.1,
56.6, 55.2, 40.4, 37.1, 33.6, 28.9, 25.8, 24.1, 22.3; m/z LRMS
(ES+) 523.2 (51%, [M(81Br2) + H]+), 521.3 (100, [M(79Br81Br) +
H]+), 519.2 (47, [M(79Br2) + H]+); HRMS (ES+) found 523.0760,
+
C38H5779Br281Br2N6O7 requires 1029.0981.
(E)-2-(12-(3-(3,5-Dibromo-4-methoxyphenyl)-2-(hydroxy-
imino)propanamido)-4,9-dimethyl-4,9-diazadodecyl)-1,3-dioxoiso-
indole bishydrochloride (31). Acid 21 (9.1 mg, 23 mmol), DCC
(5.1 mg, 25 mmol) and N-hydroxyphthalimide (4.0 mg, 25 mmol)
were dissolved in 1,4-dioxane (0.5 mL) and stirred at room
temperature for 4 h, at which point DCC (0.50 mg, 2.5 mmol) and
N-hydroxyphthalimide (0.40 mg, 2.5 mmol) were added, followed
by further stirring for 19 h. A solution of ammonium salt 30
(17 mg, 25 mmol) and NEt3 (14 mL, 99 mmol) in methanol
(0.5 mL) was then added and stirring continued for 23 h. The
reaction mixture was concentrated and the crude residue dissolved
in 1 : 1 methanol/aqueous 1 M HCl (2 mL) and purified by
preparative HPLC to give, along with the hydrochloride salt of
pseudoceramine B 3 (0.7 mg, 3%), the hydrochloride salt 31 (8.0
mg, 45%) as a colourless film; nmax/cm-1 (film) 3386, 2959, 1711,
1660, 1589, 1471; dH (400 MHz, CD3OD) 8.34 (1H, br s), 7.88–7.85
(2H, m), 7.83–7.80 (2H, m), 7.48 (2H, s), 3.85 (2H, s), 3.81 (3H, s),
3.80 (2H, t, J = 6.6 Hz), 3.35 (2H, t, J = 6.6 Hz), 3.17–3.04 (8H, m),
2.80 (3H, s), 2.79 (3H, s), 2.15–2.08 (2H, m), 1.98–1.91 (2H, m),
1.80–1.76 (4H, m); dC (100 MHz, CD3OD) 169.8, 166.1, 153.9,
152.0, 137.5, 135.5, 134.5, 133.3, 124.2, 118.6, 61.1, 56.7, 55.3,
55.1, 40.6, 40.5, 37.2, 36.0, 28.8, 25.8, 25.1, 22.8, 22.7; m/z LRMS
(ES-) 710.8 (50%, [M(81Br2) - H]-), 708.7 (100, [M(79Br81Br) -
H]-), 706.5 (55, [M(79Br2) - H]-); HRMS (ES+) found 710.1393,
C19H3179Br81BrN4O5 requires 523.0742.
+
Acknowledgements
We thank the Swedish Research Council, the Kempe foundation
and the Carl Trygger foundation for financial support. We would
also like to thank Dao Hoang Phuc for initial studies.
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