Synthesis of parazoanthine B and analogs

Article abstract of DOI:10.1016/j.tet.2015.04.040

A versatile synthesis of the natural product parazoanthine B (2) and its analogs, parazoanthine C (3) and 18-deoxy-parazoanthine B (4), has been accomplished by a key coupling reaction between a hydantoinic compound and an α-bromo-acetophenone derivative. The synthetic approach is designed to address preparation of a wider group of parazoanthine B analogs characterized by the presence of the 5,6-double bond of Z-configuration.

Full text of DOI:10.1016/j.tet.2015.04.040

Accepted Manuscript
Synthesis of parazoanthine B and analogs
Francesco Tinto, Dario Pagano, Emiliano Manzo
PII:
S0040-4020(15)00538-4
10.1016/j.tet.2015.04.040
TET 26643
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 16 February 2015
Revised Date: 24 March 2015
Accepted Date: 13 April 2015
Please cite this article as: Tinto F, Pagano D, Manzo E, Synthesis of parazoanthine B and analogs,
Tetrahedron (2015), doi: 10.1016/j.tet.2015.04.040.
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ACCEPTED MANUSCRIPT
Synthesis of parazoanthines B and analogs
Francesco Tinto, Dario Pagano and Emiliano Manzo*
O
N
NH
N
H2N
OH
N
NH2
N
H
NH2
NH
O
HN
O
O
R
2: R = OH
3: R = OCH3
4: R = H
HN
R
R=OH, OCH3, H
N
O
H
1

ACCEPTED MANUSCRIPT
Synthesis of parazoanthine B and analogs
*
Francesco Tinto, Dario Pagano and Emiliano Manzo
Istituto di Chimica Biomolecolare, CNR, Via Campi Flegrei 34, I 80078-Pozzuoli (Na),
Italy.
*
Corresponding author.
Tel. + 390818675310; Fax +390818041770. E-mail address: emanzo@icb.cnr.it
2

ACCEPTED MANUSCRIPT
Abstract
A versatile synthesis of the natural product parazoanthine B (2) and its analogs,
parazoanthine C (3) and 18-deoxy-parazoanthine B (4), has been accomplished by a key
coupling reaction between a hydantoinic compound and an α-bromo-acetophenone
derivative. The synthetic approach is designed to address preparation of a wider group of
parazoanthine B analogs characterized by the presence of the 5,6-double bond of Z-
configuration.
Keywords
Parazoanthine B, parazoanthine C, hydantoin, chemical synthesis.
3

ACCEPTED MANUSCRIPT
1.Introduction
Parazoanthines are a group of unique naturally-occurring marine alkaloids derived
from the Mediterranean sea anemone Parazoanthus axinellae.1 The chemical framework
characteristic of these molecules is a 3,5-alkyl disubstituted hydantoin core bearing a
terminal guanidine and an aromatic ring. Hydantoins and derivatives have been widely
used in biomedical studies including novel therapeutic agents of interest as anti-
convulsants2 and antimuscarinics,3 antiulcers and antiarrythmics,4 antivirals, antidiabetics,5
and inhibitors of antagonist of serotonin and fibrinogen receptors,6 inhibitors of glycine
binding site of the NMDA receptor7 and antagonists of leukocyte cell adhesion.8 In the
framework of a SAR program and in consideration of the intriguing structural features of
this rare family of marine alkaloids, we have recently started a synthetic project to prepare
a series of natural and chemical variants of these substances. Within this frame,
parazoanthine A (1) has been obtained by a concise biomimetic approach based on the
coupling reaction of L-arginine methyl ester dihydrochloride with isocyanate derivatives of
p-coumaric acid.9 However, other natural parazoanthines with 5,6 double bond of Z-
configuration, remain inaccessible by this procedure for the impossibility to introduce an
unsaturation on L-arginine moiety, requiring for their preparation an alternative synthetic
procedure.
4

ACCEPTED MANUSCRIPT
O
NH
3N
10
N
H
NH2
6
5
NH
17
15
13
O
HO
1
O
NH
N
H
NH2
N
NH
O
R
2: R = OH
3: R = OCH3
4: R = H
Figure 1. Structure of parazoanthine A (1), parazoanthine B (2), parazoanthine C (3) and
18-deoxy-parazoanthine B (4).
2.Results and discussion
We report a synthetic strategy for the preparation of natural parazoanthine B (2),
parazoanthine C (3) and non-natural 18-deoxy-parazoanthine B (4).
The key step of this procedure is the coupling of a hydantoinic derivative 5a/5b
(stereoisomers mixture in the ratio of 1.5/1) with α-bromo-4’-methoxy-acetophenone (6),
both prepared as illustrated in Schemes 1 and 2, respectively.
In particular, the mixture of Z/E isomers 5a/5b, in the ratio of 1/1.5, was prepared by
Horner-Wadsworth-Emmons reaction of the aldehyde (7), derived from of 3-amino-1-
propanol, with diethyl 2,4-dioxoimidazolidine-5-phosphonate (8), prepared according to
reference 10.
5

ACCEPTED MANUSCRIPT
O
O
R1
EtOH/NaOEt (1.4
equiv.)
O
O
HN
OEt
OEt
4
2
6
Boc
R2
HN
O
5
NH
P
N
H
H
N
H
8
r.t./1.5h
O
84%
5a/5b=1.5/1
7
NHBoc
5a. R1 = H R2 =
NBoc
5b. R1 =
R2 = H
(Boc)2O (1 equiv.) I2
10%
Dess-Martin reagent
CH2Cl2
Boc
H2N
OH
HN
N
H
OH
7
r.t./2.5h
r.t. overnight
98%
65%
O
O
Triethylphosphite
(1.4 equiv.)
HAc/Br2 (1equiv.)
85 °C/1.5 h
HN
Br
8. 54%
N
N
r.t./1.5 h
O
O
H
H
Scheme 1. Preparation of synthetic precursors 5a/5b.
Differentiation of the Z/E isomers 5a/5b was not easily determinable on the basis of NMR
chemical shifts, but they could be distinguished on the basis of the long-range (3JC-H
)
heteronuclear 13C-1H coupling constants.1,11 In particular for 5a and 5b the different
3
configurations of the exocyclic double bond were evident by measuring JC4-H6 values
3
(δΗ−6 5.73 and 5.56, δC-4 164.2 and 164.4, JC4-H6 4.7 and 9.8 Hz for 5a and 5b
respectively) by non-proton-decoupled J-HMBC experiments.1,11c Compound 5a revealed
Z-geometry with the olefinic H-6 and the carbonyl C-4 in a cis-orientation, while 5b
showed E-geometry with H-6 and C-4 in a trans-orientation.
6

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On the other hand the precursor (6) was prepared by O-methylation and subsequent α-
bromination of the commercially available 4’-hydroxy-acetophenone with iodomethane in
refluxing acetone and bromine in acetic acid, respectively (Scheme 2).
CH3I (9 equiv.)
K2CO3 (5equiv.)
Acetone
O
O
O
HAc/Br2 (1 equiv.)
50°C/overnight
Br
reflux 3h
R
R
HO
4'-OH-acetophenone
6. R = OCH3, 57%
12. R = H, 62%
4'-OMe-acetophenone,
R = OCH3, 95%
acetophenone, R = H
Scheme 2. Preparation of synthetic precursors 6 and 12.
The coupling reaction between the prepared precursors 5a/5b and 6 (scheme 3) consisted
of the use of potassium carbonate and tetrabutylammonium bromide (TBAB) at 50 °C in
anhydrous N,N-dimethylformamide (DMF) addressing the N-imide bromine substitution to
form compound 9. Compound 9 presented the 3,5-alkyl disubstituted hydantoinic moiety
typical of parazoanthines with an exocyclic 5,6-double bond with the desired Z geometry.
The E isomer product was revealed only in trace probably due to the thermal isomerization
of the double bond as indicated by an analytical test in which a small amount of Z/E
mixture 5a/5b was stirred at 50 °C in DMF, showing a significant increase of the
proportion of the Z-isomer (5a). Reduction with sodium borohydride of the coupling
adduct gave compound 10, that was then converted into 11 via dehydration and Boc-
cleavage by two equivalents of p-toluenesulfonic acid at 110 °C in toluene.
Compound 11 displayed a trans-double bond (JH13-H14= 15.4 Hz) between carbon 13 and
14 of the parazoanthine skeleton and was subsequently guanylated by 3,5-dimethyl-1-
pyrazolylformaminidium nitrate12 and N,N-diisopropylethylamine (DIPEA) in anhydrous
DMF to give parazoanthine C (3). This last product showed spectroscopic data identical
7

ACCEPTED MANUSCRIPT
with those of the natural molecule1 (see Experimental section) and was also demethylated
by boron tribromide in dichloromethane, to give parazoanthine B (2) (see Experimental
section).
O
R1
O
DMF/K2CO3(1 equiv.) TBAB
O
Boc
R2
6
12
HN
N
NaBH4 (1,2 equiv.)
EtOH
N
O
NH
R =
H
NH
O
50°C/2h
r.t./1h
R
NHBoc
5a. R1 = H
2
9. R = OCH3, 37%
13. R = H, 33%
5b. R1 = NHBoc R2 = H
5a/5b=1.5/1
DIPEA (1.5 equiv.)
DMF
O
O
3,5-dimethyl-1-
pyrazolylformaminidium
nitrate (1.5 equiv.)
OH
p-TsOH (2 equiv.)
toluene
Boc
NH
NH2
N
N
N
H
NH
NH
110°C/1h
r.t./overnight
O
O
R
R
11. R = OCH3, 49%
15. R = H, 45%
10. R = OCH3, 95%
14. R = H, 96%
O
O
NH
BBr3
CH2Cl2
N
NH2
N
N
NH2
N
H
H
NH
NH
O
O
R
40°C/1h
HO
3. R = OCH3, 63%
4. R = H, 62%
2. 46%
Scheme 3. Synthetic approach to get parazoanthine B (2), parazoanthine C (3) and 18-
deoxy-parazoanthine B (4).
3
For the synthetic parazoanthines B (2) and C (3), the JC4-H6 measures (4.9 and 5.0 Hz
respectively), along with their NMR data, were consistent with the natural Z-configuration.
Using the same synthetic approach of the Scheme 3, 18-deoxy-parazoanthine B (4) was
prepared from α-bromoacetophenone (12) generated by α-bromination of commercial
acetophenone (Scheme 2). As described above, stereochemistry Z of the exocyclic double
bond in the synthetic analog 4, 18-deoxy-parazoanthine B, was proved by 3JC4-H6 value (4.9
Hz).
8

ACCEPTED MANUSCRIPT
3.Conclusion
In summary, we accomplished the first synthesis of parazoanthines with a Z-double bond
between C5 and C6. The present approach opens the way for the preparation of other
natural and no natural analogs, as well as provides a general approach to the preparation of
parazoanthines analogs differing in the alkyl chain length or type and/or in the phenyl
substitution pattern.
4.Experimental Section
4.1.General experimental procedures.
1
All products and synthetic intermediates structure were assigned using H, 13C, COSY,
HSQC, HMBC NMR spectra.
1H, 13C and 2D NMR spectra were acquired by the NMR Service of the Institute of
Biomolecular Chemistry of the National Council of Research (ICB-CNR) and recorded on
a Bruker DRX-600 spectrometer, equipped with a TCI CryoProbe™, fitted with a gradient
along the Z-axis and on Bruker instruments at 400 and/or 300 MHz.
Samples for NMR spectroscopic analysis were dissolved in the appropriate solvent; spectra
in CDCl3 (1H,13C): 7.26, 77.0 ppm; CD3OD (1H,13C): 3.34, 49.0 ppm. HRESIMS were
performed on a Micromass Q-TOF MicroTM coupled with a HPLC Waters Alliance 2695.
TLC plates (Kieselgel 60 F254) and silica gel powder (Kieselgel 60, 0.063-0.200 mm) were
from Merck.
9

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All the reagents and solvents were purchased from Sigma-Aldrich and used without any
further purification.
4.2.Synthetic procedure.
4.2.1.Preparation of N-Boc-aminopropanal (7)
O
O
O
N
H
H
(Boc)2O (0.873 g, 4.0 mmol) and iodine (0.102 g, 0.4 mmol) were added to 3-amino-1-
propanol (313 mL, 4.0 mmol) and the mixture was stirred for 2.5 h at room temperature;
after portioning between sodium thiosulfate (6.0 g) and sodium bicarbonate (10.0 g) water
solution (100 mL) and diethylether (200 mL), the organic phase was evaporated to give N-
tert-butoxycarbonyl-amino-1-propanol (0.644 g, 3.9 mmol, 98%) that was oxidized with
Dess-Martin reagent (2.80 g, 6.6 mmol) in dichloromethane (34 mL) overnight at room
temperature; the mixture was partitioned between sodium thiosulfate (9.0 g) and sodium
biocarbonate (10.0 g) water solution (100 mL) and dichloromethane (100 mL); the water
was extracted with dichloromethane (100 mL) three times; the organic phase was
evaporated and purified by silica gel chromatography using a gradient of n-hexane and
1
ethyl acetate to give 7 (0.433 g, 2.5 mmol, 65%): colorless oil; H-NMR (400 MHz,
CDCl3): δ 9.65 (1H, bs, H-1), 3.27 (2H, bq, J= 6.2 Hz, H2-3), 2.55 (2H, t, J= 5.9 Hz, H2-
2), 1.40-1.22 (9H, s, Boc methyls); HRESIMS m/z 196.0953 [M+Na]+ (calcd for
C8H15NNaO3, 196.0950).13
4.2.2.Preparation of diethyl 2,4-dioxoimidazolidin-5-yl-5-phosphonate (8)
10

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O
O
P
HN
OEt
OEt
N
O
H
Hydantoin (2.000 g, 20.0 mmol), was dissolved in glacial acetic acid (8 mL) and heated at
85 °C; a small amount of bromine (0.120 mL) was added and after 1 h and when the red
color disappeared more bromine (1.08 mL) was added dropwise. After 30 min the mixture
was cooled to 0 °C and triethyl phosphite (3.300 g, 28.0 mmol) was added; the mixture
was heated at room temperature, stirred for 1.5 h and evaporated. The residue was
crystallized in diethylether to give 8 (2.55 g, 10.8 mmol, 54%): white solid; NMR data
was identical to those reported in the literature.10
4.2.3.Preparation of N-Boc-(Z/E)-3-(2’,4’-dioxoimidazolidin-5’-ylidene)-propanamine
(5a/5b)
O
O
N
H
O
HN
O
NH
5a
O
HN
O
O
HN
5b
N
H
O
Compound 8 (0.212 g, 0.9 mmol) was dissolved in an ethanolic solution (10 mL) of
sodium ethoxide (74 mg, 1.08 mmol); compound 7 (0.130 g, 0.75 mmol) was added and
the reaction was stirred at room temperature for 1.5 h; the mixture was evaporated and
purified by silica gel chromatography using a chloroform/methanol gradient to give
compound 5a/5b as Z/E isomers mixture in the ratio 1.5/1.0 (0.161 g, 0.63 mmol, 84%):
pale yellow oil; Rf (chloroform/methanol 8/2) = 0.72; IR (film) νmax 3311, 2921, 2901,
11

ACCEPTED MANUSCRIPT
1
2845, 1680, 1620 cm-1; H-NMR (400 MHz, CDCl3): δ 5.73 (1H, t, J= 8.4 Hz, H-3, Z
form), 5.56 (1H, t, J= 8.1 Hz, H-3, E form), 3.23-3.14 (2H, m, H2-1, Z and E forms), 2.79
13
(2H, m, H2-2, E form), 2.35 (2H, m, H2-2, Z form), 1.52-1.23 (9H, s, Boc methyls); C
NMR (CDCl3): δ 164.4 (C, C-4’, E form), 164.2 (C, C-4’, Z form), 156.4 (C, Boc), 131.4
(C, C-5’, Z form), 129.8 (C, C-5’, E form), 116.7 (CH, C-3, E form), 110.5 (CH, C-3, Z
form), 79.7 (C, Boc), 39.7 (CH2, C-1, Z and E form), 28.4 (CH3, Boc), 27.2 (CH2, C-2, Z
form), 26.5 (CH2, C-2, E form); HRESIMS m/z 278.1112 [M+Na]+ (calcd for
C11H17N3NaO4, 278.1117).
4.2.4.Preparation of 4’-OMe-acetophenone
O
H3CO
4’-OH-acetophenone (1 g, 0.0074 mol) was dissolved in acetone (750 mL) with potassium
carbonate (5.1 g, 0.037 mol); the mixture was refluxed for 15 min and iodomethane (9.6 g,
0.066 mol) was added; the reaction was refluxed for 3 h, filtered, evaporated and purified
by silica gel chromatography using a gradient of petroleum ether and diethylether to give
1
4’-OMe-acetophenone (1.6 g, 0.007 mmol, 95%):colorless oil; H-NMR (400 MHz,
CDCl3): δ 7.94 (2H, d, J= 8.1 Hz, H-2’, H-6’), 6.94 (2H, d, J= 8.1 Hz, H-3’, H-5’), 3.86
13
(3H, OMe), 2.54 (3H, s, H3-1); C NMR (CDCl3): δ 198.4 (C), 165.0 (C), 129.4 (2 CH),
127.4 (C), 112.3 (2 CH), 55.9 (OCH3), 23.4 (CH3).
4.2.5.Preparation of 2-bromo-1-(4’-methoxyphenyl)-ethanone (6)
12

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O
Br
H3CO
4’-OMe-acetophenone (1.04 g, 0.0066 mol) was dissolved in glacial acetic acid (7 mL); the
temperature was increased at 50 °C and bromine (0.344 mL, 0.0066 mol) was added; the
reaction was stirred overnight at 50 °C, evaporated and partitioned between a saturated
sodium bicarbonate water solution (100 mL) and diethylether (100 mL); the organic phase
was purified by silica gel chromatography using a gradient of petroleum ether and
1
diethylether to give 6 (0.866 g, 0.0038 mol, 57%): colorless oil; H-NMR (400 MHz,
CDCl3): δ 7.95 (2H, d, J = 8.7 Hz, H-2’, H-6’), 6.94 (2H, d, J = 8.7 Hz, H-3’, H-5’), 4.38
(2H, bs, H2-1), 3.86 (3H, OMe).
4.2.6.Preparation of compound 9
O
O
O
N
O
N
H
NH
O
H3CO
Compound 5a/5b (61 mg, 0.239 mmol) was dissolved in anhydrous DMF (2.5 mL);
potassium carbonate (34 mg, 0.239 mmol) and TBAB (2 mg) were added; the mixture was
heated at 50 °C under argon atmosphere and compound 6 (65 mg, 0.287 mmol) was added
after 15-30 min; after 2 h the reaction was evaporated and purified by silica gel
chromatography using a gradient of n-hexane and dichloromethane to give compound 9
(35 mg, 0.088 mmol, 37%): pale yellow oil; Rf (chloroform/methanol 95/5) = 0.35; IR
1
(film) νmax 3315, 2921, 2901, 2845, 1719, 1626 cm-1; H-NMR (400 MHz, CDCl3): δ 7.95
(2H, d, J= 8.7 Hz, H-16, H-20), 6.96 (2H, d, J= 8.7 Hz, H-17, H-19), 5.87 (1H, bt, J= 8.0
13

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Hz, H-6), 4.95 (2H, bs, H2-13), 3.90 (3H, s, OCH3), 3.22 (2H, bq, J= 6.6 Hz, H2-8), 2.44
(2H, m, H2-7), 1.57-1.36 (9H, s, Boc methyls); 13C NMR (CDCl3): δ 189.0 (C, C-14), 162.4
(C, C-4), 164.1 (C, C-18), 156.2 (C, Boc), 153.8 (C, C-2), 131.2 (C, C-5), 130.0 (CH, C-16
and C-20), 129.7 (C, C-15), 115.2 (CH, C-17 and C-19), 111.0 (CH, C-6), 80.2 (C, Boc),
54.9 (CH3, OMe), 43.4 (CH, C-13), 39.9 (CH2, C-8), 29.4, 29.0, 28.6 (CH3, Boc methyls),
29.1 (CH2, C-7); HRESIMS m/z 426.1644 [M+Na]+ (calcd for C20H25N3NaO6, 426.1641).
4.2.7.Preparation of compound 10
O
O
OH
N
O
N
H
NH
O
H3CO
Compound 9 (35 mg, 0.088 mmol) was dissolved in ethanol (3 mL) and sodium
borohydride (4 mg, 0.105 mmol) was added; the mixture was stirred for 1 h at room
temperature and after addition of some drops of water was partitioned between water (20
mL) and ethylacetate (20 mL); the organic phase was evaporated to get 10 (34 mg, 0.084
mmol, 95%): pale yellow oil; Rf (chloroform/methanol 95/5) = 0.30; IR (film) νmax 3331,
2920, 2915, 2855, 1715, 1611 cm-1; 1H-NMR (400 MHz, CDCl3): δ 7.34 (2H, d, J= 7.7 Hz,
H-16, H-20), 6.88 (2H, d, J= 7.7 Hz, H-17, H-19), 5.85 (1H, t, J= 7.3 Hz, H-6), 4.97 (1H,
bd, J= 6.5 Hz, H-14), 3.82 (2H, m, H2-13), 3.81 (3H, s, OCH3), 3.20 (2H, m, H2-8), 2.42
(2H, m, H2-7), 1.47-1.34 (9H, s, Boc methyls); 13C NMR (CDCl3): δ 162.9 (C, C-4), 159.1
(C, C-18), 156.3 (C, Boc), 156.1 (C, C-2), 132.8 (C, C-15), 130.1 (C, C-5), 127.5 (CH, C-
16 and C-20), 115.2 (CH, C-17 and C-19), 111.8 (CH, C-6), 80.1 (C, Boc), 73.4 (CH, C-
14), 54.9 (CH3, OMe), 44.5 (CH, C-13), 39.8 (CH2, C-8), 29.8, 29.2, 28.6 (CH3, Boc
14

ACCEPTED MANUSCRIPT
methyls), 29.0 (CH2, C-7); HRESIMS m/z 428.1794 [M+Na]+ (calcd for C20H27N3NaO6,
428.1798).
4.2.8.Preparation of compound 11
O
NH2
N
NH
O
H3CO
Compound 10 (34 mg, 0.084 mmol) was dissolved in toluene (3 mL) with monohydrated
p-toluensulfonic acid (32 mg, 0.168 mmol); the mixture was heated at 110 °C for 1 h,
evaporated and purified twice by silica gel chromatography using a gradient of chloroform
and methanol to give 11 (11.7 mg, 0.041 mmol, 49%): pale yellow oil; Rf
(chloroform/methanol 6/4) = 0.50; IR (film) νmax 3319, 2955, 2928, 2881, 1719, 1626 cm-1;
1H-NMR (400 MHz, CD3OD): δ 7.50 (1H, d, J= 15.4 Hz, H-14), 7.39 (2H, bd, J= 8.4 Hz,
H-16, H-20), 7.08 (1H, d, J= 15.4 Hz, H-13), 6.93 (2H, d, J= 8.4 Hz, H-17, H-19), 5.86
(1H, t, J= 7.6 Hz, H-6), 3.86 (3H, s, OCH3), 2.95 (2H, bt, J= 7.3 Hz, H2-8), 2.50 (2H, bq,
J= 7.6 Hz, H2-7); 13C NMR (CD3OD): δ 160.6 (C, C-4), 159.2 (CH, C-18), 153.6 (C, C-2),
131.7 (C, C-5), 127.7 (CH, C-16 and C-20), 126.4 (C, C-15), 120.4 (CH, C-14), 116.0
(CH, C-13), 114.4 (CH, C-17, C-19), 110.5 (CH, C-6), 54.9 (CH3, OMe), 40.2 (CH2, C-8),
28.3 (CH2, C-7); HRESIMS m/z 287.1275 [M+H]+ (calcd for C15H18N3O3, 287.1270).
4.2.9.Preparation of compound 12
O
Br
15

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Acetophenone ( 0.466 mL, 0.004 mol) was dissolved in glacial acetic acid (4 mL) and
heated at 50 °C; a small amount of bromine (0.040 mL) was added and after 1 h the rest of
bromine (0.166 mL) was added; after overnight of stirring the mixture was evaporated and
partitioned between a saturated sodium bicarbonate water solution (20 mL) and
diethylether (20 mL) that was evaporated and purified by silica gel chromatography using
a gradient of light petroleum ether/diethylether to give compound 12 (0.494 g, 2.48 mmol,
62%): pale yellow oil; 1H-NMR (400 MHz, CDCl3): δ 7.97 (2H, d, J= 8.1 Hz, H-2’, H-6’),
7.59 (1H, bt, J= 7.6 Hz, H-4’), 7.48 (2H, t, J= 8.1 Hz, H-3’, H-5’), 4.45 (2H, bs, H2-1).
4.2.10.Preparation of compound 13
O
O
O
N
O
N
H
NH
O
Compound 5a/5b (61 mg, 0.239 mmol) was dissolved in anhydrous DMF (2.5 mL),
potassium carbonate (34 mg, 0.239 mmol) and TBAB (2 mg) were added; the mixture was
heated at 50 °C and compound 12 (58 mg, 0.287 mmol) was added; after 2h the reaction
was evaporated and purified by silica gel chromatography using a gradient of n-hexane and
dichloromethane to give compound 13 (30 mg, 0.079 mmol, 33%): pale yellow oil; Rf
(chloroform/methanol 95/5) = 0.80; IR (film) νmax 3350, 2918, 2939, 2840, 1728, 1620 cm-
1; 1H-NMR (400 MHz, CDCl3): δ 7.97 (2H, d, J= 7.5 Hz, H-16, H-20), 7.62 (1H, bt, J =
7.7 Hz, H-18), 7.50 (2H, t, J = 7.5 Hz, H-17, H-19), 5.88 (1H, t, J = 7.9 Hz, H-6), 4.99
(2H, bs, H2-13), 3.22 (2H, bq, J = 6.4 Hz, H2-8), 2.44 (2H, m, H2-7), 1.53-1.35 (9H, s, Boc
methyls); 13C NMR (CDCl3): δ 190.5 (C, C-14), 162.8 (C, C-4), 156.3 (C, Boc), 153.4 (C,
C-2), 131.1 (C, C-5), 134.1, 128.7, 127.8 (CH, aromatic), 110.3 (CH, C-6), 80.4 (C, Boc),
16

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44.3 (CH, C-13), 39.8 (CH2, C-8), 28.7, 28.2, 27.9 (CH3, Boc methyls), 27.1 (CH2, C-7);
HRESIMS m/z 396.1531 [M+Na]+ (calcd for C19H23N3NaO5, 396.1535).
4.2.11.Preparation of compound 14
O
O
OH
N
O
N
H
NH
O
Compound 13 (30 mg, 0.079 mmol) was dissolved in ethanol (3 mL) and sodium
borohydride (3.6 mg, 0.095 mmol) was added; the mixture was stirred for 1 h at room
temperature and after addition of some drops of water was partitioned between water (20
mL) and ethylacetate (20 mL); the organic phase was evaporated to give 14 (28 mg, 0.076
mmol, 96%): pale yellow oil; Rf (chloroform/methanol 95/5) = 0.45; IR (film) νmax 3350,
1
2929, 2909, 2863, 1735, 1618 cm-1; H-NMR (400 MHz, CDCl3): δ 7.42 (2H, bd, J = 7.8
Hz, H-16, H-20), 7.35 (2H, bt, J = 7.8 Hz, H-17, H-19), 7.30 (1H, bt, J = 7.6 Hz, H-18),
5.85 (1H, t, J= 8.4 Hz, H-6), 5.01 (1H, dd, J = 2.7, 7.8 Hz, H-14), 3.86 (2H, m, H2-13),
3.20 (2H, bq, J = 6.1 Hz, H2-8), 2.41 (2H, bq, J = 6.9 Hz, H2-7), 1.50-1.38 (9H, s, Boc
13
methyls); C NMR (CDCl3): δ 162.8 (C, C-4), 156.8 (C, Boc), 156.2 (C, C-2), 140.8 (C,
C-15), 130.3 (C, C-5), 128.2, 127.5, 127.1 (CH, aromatic), 110.6 (CH, C-6), 80.5 (C, Boc),
73.3 (CH, C-14), 47.2 (CH, C-13), 40.4 (CH2, C-8), 28.7, 28.4, 27.5 (CH3, Boc methyls),
27.9 (CH2, C-7); HRESIMS m/z 398.1696 [M+Na]+ (calcd for C19H25N3NaO5, 398.1692).
4.2.12.Preparation of compound 15
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O
NH2
N
NH
O
Compound 14 (28 mg, 0.076 mmol) was dissolved in toluene (3 mL) with monohydrated
p-toluensulfonic acid (26 mg, 0.152 mmol); the mixture was heated at 110 °C for 1 h,
evaporated and purified twice by silica gel chromatography using a gradient of
dichloromethane and methanol to give 15 (8.7 mg, 0.034 mmol, 45%): pale yellow oil; Rf
(chloroform/methanol 7/3) = 0.35; IR (film) νmax 3320, 2935, 2909, 2872, 1716, 1620 cm-1;
1H-NMR (400 MHz, CD3OD): δ 7.67 (1H, d, J= 15.1 Hz, H-14), 7.50 (2H, bd, J = 7.5 Hz,
H-16, H-20), 7.38 (2H, bt, J = 7.5 Hz, H-17, H-19), 7.27 (1H, bt, J = 7.6 Hz, H-18), 7.23
(1H, d, J= 15.1 Hz, H-13), 5.87 (1H, t, J = 7.6 Hz, H-6), 3.11 (2H, bt, J = 7.3 Hz, H2-8),
2.58 (2H, bq, J = 7.3 Hz, H2-7); 13C NMR (CD3OD): δ 162.4 (C, C-4), 153.3 (C, C-2),
137.1 (C, C-15), 131.8 (C, C-5), 128.5, 128.0, 127.3 (CH, aromatic), 120.7 (CH, C-14),
118.4 (CH, C-13), 109.8 (CH, C-6), 40.1 (CH2, C-8), 26.3 (CH2, C-7); HRESIMS m/z
257.1205 [M+H]+ (calcd for C14H16N3O2, 257.1200); HRESIMS m/z 280.2773 [M+Na]+
(calcd for C14H15N3NaO2, 280.2776).
4.2.13.Preparation of compound 4
O
NH
N
NH2
N
H
NH
O
Compound 15 (8.7 mg, 0.034 mmol) was dissolved in anhydrous DMF (0.5 mL) with
DIPEA (0.051 mmol) and 3,5-dimethyl-1-pyrazolylformaminidium nitrate (0.010 g, 0.051
mmol); the mixture was stirred overnight at room temperature under argon atmosphere,
18

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evaporated and fractioned by silica gel chromatography using a gradient of chloroform and
methanol and further purified by HPLC RP-phase (H2O-TFA 0.1%/MeOH, from 60/40 to
50/50 in 20 minutes, RP-amide analytical column, flow rate 1 mL/min) to give 4 (6.3 mg,
0.021 mmol, 62%): pale yellow oil; Rf (chloroform/methanol 6/4) = 0.30; IR (film) νmax
3340, 2948, 2929, 2852, 1723, 1648 cm-1; 1H-NMR (400 MHz, CD3OD): δ 7.58 (1H, d, J=
15.0 Hz, H-14), 7.46 (2H, d, J = 7.4 Hz, H-16, H-20), 7.37 (2H, bt, J = 7.5 Hz, H-17, H-
19), 7.28 (1H, bt, J = 7.4 Hz, H-18), 7.22 (1H, d, J = 15.0 Hz, H-13), 5.85 (1H, bt, J = 7.7
Hz, H-6), 3.41 (2H, bt, J = 7.0 Hz, H2-8), 2.58 (2H, bq, J =7.0, H2-7); 13C NMR (CD3OD):
δ 162.4 (C, C-4), 158.7 (C, C-10), 153.9 (C, C-2), 137.2 (C, C-15), 131.6 (C, C-5), 129.5
(CH, C-17 and C-19), 129.0 (CH, C-18), 127.2 (CH, C-16 and C-20), 120.8 (CH, C-14),
119.0 (CH, C-13), 110.8 (CH, C-6), 40.9 (CH2, C-8), 26.4 (CH2, C-7); HRESIMS m/z
300.1465 [M+H]+ (calcd for C15H18N5O2, 300.1460).
4.2.14.Preparation of parazoanthine C (3)
O
NH
N
NH2
N
H
NH
O
H3CO
Compound 11 (11.7 mg, 0.041 mmol) was dissolved in anhydrous DMF (0.5 mL) with
DIPEA (0.062 mmol) and 3,5-dimethyl-1-pyrazolylformaminidium nitrate (0.012 g, 0.062
mmol); the mixture was stirred overnight at room temperature under argon atmosphere,
evaporated, fractioned by silica gel chromatography using a gradient of chloroform and
methanol and further purified by HPLC RP-phase (H2O-TFA 0.1%/MeOH, from 60/40 to
50/50 in 20 minutes, RP-amide analytical column, flow rate 1 mL/min) to give
parazoanthine C (3) (8.6 mg, 0.026 mmol, 63%): pale yellow oil; Rf (chloroform/methanol
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6/4) = 0.25; IR (film) νmax 3368, 2951, 2925, 2852, 1720, 1656 cm-1; 1H-NMR (400 MHz,
CD3OD): δ 7.48 (1H, d, J = 14.8 Hz, H-14), 7.36 (2H, d, J = 8.3 Hz, H-16, H-20), 7.06
(1H, d, J = 14.8 Hz, H-13), 6.91 (2H, d, J = 8.3 Hz, H-17, H-19), 5.83 (1H, bt, J = 7.8 Hz,
13
H-6), 3.80 (3H, s, OMe), 3.38 (2H, bt, J = 7.0 Hz, H2-8), 2.54 (2H, bq, J =7.0, H2-7); C
NMR (CD3OD): δ 163.0 (C, C-4), 161.0 (C, C-18), 158.9 (C, C-10), 154.6 (C, C-2), 131.3
(C, C-5), 129.6 (C, C-15), 128.5 (CH, C-16 and C-20), 121.5 (CH, C-14), 117.0 (CH, C-
13), 115.4 (CH, C-17 and C-19), 110.2 (CH, C-6), 55.8 (CH3, OMe), 41.3 (CH2, C-8), 27.5
(CH2, C-7); HRESIMS m/z 330.1571 [M+H]+ (calcd for C16H20N5O3, 330.1566).
4.2.15.Preparation of parazoanthine B (2)
O
NH
N
NH2
N
H
NH
O
HO
Parazoanthine C (3) (8.6 mg, 0.026 mmol) was dissolved in 1 mL of anhydrous
dichloromethane boron tribromide solution (0.3 M), prepared by diluting a more
concentrated dichloromethane boron tribromide solution (1M); the mixture was stirred for
1 h at 40 °C under argon atmosphere, evaporated, stirred with methanol/water solution
(1/1, 1.5 mL) for ten minutes, dried and purified twice by silica gel chromatography using
a gradient of dichloromethane and methanol to give 2 (3.8 mg, 0.012 mmol, 46 %): pale
1
yellow oil; IR (film) νmax 3347, 2956, 2928, 2857, 1716, 1671 cm-1; H-NMR (400 MHz,
CD3OD): δ 7.43 (1H, d, J = 14.7 Hz, H-14), 7.27 (2H, d, J = 8.2 Hz, H-16, H-20), 6.98
(1H, d, J = 14.7 Hz, H-13), 6.78 (2H, d, J = 8.2 Hz, H-17, H-19), 5.80 (1H, bt, J = 7.8 Hz,
13
H-6), 3.35 (2H, bt, J = 7.1 Hz, H2-8), 2.54 (2H, bq, J = 7.0, H2-7); C NMR (CD3OD): δ
162.9 (C, C-4), 158.9 (C, C-10), 157.7 (C, C-18), 154.5 (C, C-2), 131.3 (C, C-5), 128.5
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(CH, C-16 and C-20), 128.4 (C, C-15), 121.9 (CH, C-14), 116.7 (CH, C-17 and C-19),
116.2 (CH, C-13), 110.2 (CH, C-6), 41.1 (CH2, C-8), 27.4 (CH2, C-7); HRESIMS m/z
316.1405 [M+H]+ (calcd for C15H18N5O3, 316.1410).
Supporting Data
NMR data of synthetic compounds are included.
Acknowledgement
The authors thank Dr. Alessandro Esposito and Dr. Dominique Melk of ICB-NMR service,
M. Zampa of “Servizio di Spettrometria di Massa” for ESIMS.
References
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(6)
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6909–6919.
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S1
Supporting Information
Synthesis of parazoanthine B and analogs
Francesco Tinto, Dario Pagano and Emiliano Manzo*
Consiglio Nazionale delle Ricerche- Istituto di Chimica Biomolecolare, Via Campi Flegrei,
34 -80078-I Napoli- Italy
emanzo@icb.cnr.it
Table of contents:
NMR data of synthetic compounds
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