New analogues of the antitumor alkaloid girolline: The 4-deazathiogirolline series

Article abstract of DOI:10.1055/s-2004-834907

Aminothiazole analogues of the antitumor alkaloid girolline have been asymmetrically synthesised using the chiral pool. The key-steps involved the Hantzsch reaction to build the thiazole heterocycle and the functionalisation of the side chain using the tr

Full text of DOI:10.1055/s-2004-834907

PAPER
97
New Analogues of the Antitumor Alkaloid Girolline: The 4-Deazathiogirolline
Series
New
Analogues
o
a
f
the
A
ntit
s
umor
A
lka
t
loid G
i
irollineen Nay, Bruno Schiavi, Alain Ahond, Christiane Poupat,* Pierre Potier
Institut de Chimie des Substances Naturelles du CNRS, Avenue de la Terrasse, BP 1, 91198 Gif-sur-Yvette cedex, France
Fax +33(1)69077247; E-mail: Christiane.Poupat@icsn.cnrs-gif.fr
Received 29 July 2004
protected) were cytotoxic at moderate dilutions (ca. 5
Abstract: Aminothiazole analogues of the antitumor alkaloid gi-
mM); 5-deazathiogirolline itself was inactive.
rolline have been asymmetrically synthesised using the chiral pool.
The key-steps involved the Hantzsch reaction to build the thiazole
heterocycle and the functionalisation of the side chain using the
triphenylphosphane chemistry (i.e. azidation–chlorination of a di-
ol). Biological activity of analogues has been evaluated (cytotoxic-
ity on KB tumor cells and anti-HIV activity).
Herein, the synthesis and activities of the second series,
the ‘4-deazathiogirolline’ (3), is presented. These new
chiral compounds have the side chain of the natural prod-
uct on position 4 of the aminothiazole.
Asymmetric synthesis of 4-deazathiogirolline derivatives
emerged from the chiral pool, starting from D-(+)-arabitol
(4)3 (Scheme 1). Compound 5 was obtained in three steps
and 57% overall yield according to the litterature.4 Dehy-
drohalogenation of 5 in the presence of zinc and in EtOH
resulted in allylic alcohol 6. Its instability observed during
silica gel chromatography forced us to use it crude and
benzylation was done subsequently in the presence of
benzyl bromide and NaH in DMF. Benzylic protection
was preferred to the silyl ether that had been previously
used as in the 5-deazathiogirolline synthesis.2,5 Indeed, in
the present series, benzyl derivatives were stable during
the whole synthesis work while the silyl ethers proved to
be labile especially in weak acidic conditions (acidolysis
of acetonide 11).
Key words: girolline, aminothiazole, chiral pool, antitumor, struc-
ture-activity relationships
Girolline (1, Figure 1) is an original metabolite extracted
in 1988 from the New Caledonian Sponge Cymbastela
cantharella (formerly Pseudaxinyssa cantharella).1 It dis-
played an interesting cytotoxicity in vitro (IC50 = 0.14
mM on KB cells) and a promising antitumor activity in
vivo that afterwards led it to the clinical trials. Unfortu-
nately, the bioassays were stopped at phase I due to unde-
sirable side-effects on the cardiovascular system
including severe hypotension.
OH
NH2, HCl
HN
N
Cl
O
OH
NH2, HCl
a
girolline 1
I
O
HO
b, c
OH
5
O
O
O
OH OH
OH
OH
5
4
5
4
OBn
OR
1
NH2, HCl
NH2, HCl
N
S
d
S
N
Cl
Cl
3
O
3'
O
NH2, HCl
5-deazathiogirolline 2
NH2, HCl
4-deazathiogirolline 3
O
O
8
R = H (6)
R = Bn (7)
OBn
Figure 1 Girolline and targeted analogues.
OBn
5
1'
4
O
S
2
g
Br
O
e, f
Consequently, the synthesis of analogues of girolline has
been undertaken to improve the therapeutic index. Ami-
nothiazole analogues 2 and 3 were especially designated
for that purpose. Depending on the position of the side
chain, two series could be envisaged. The first one was de-
scribed in 2002.2 It consisted of the ‘5-deazathiogirolline’
derivatives 2, bearing the side chain on position 5 of the
aminothiazole. Only some synthetic intermediates (all
N
O
R1 R2
O
BocHN
R1 = H, R2 = OH, (9)
R1,R2 = O (10)
11
OBn
h
OH
S
N
OH
BocHN
12
Scheme 1 Reactions and conditions: (a) Three steps according to
the literature4 (57%); (b) Zn, EtOH, reflux; (c) NaH, BnBr, DMF, 40
°C (83% over 2 steps); (d) mCPBA, NaHCO3, CH2Cl2, r.t. (90%); (e)
Li2NiBr4, THF, r.t. (94%); (f) DMSO, (COCl)2, CH2Cl2, –78 °C then
Et3N (75%); (g) NaHCO3, BocNH(C=S)NH2, DMF, 50 °C (83%); (h)
PPTS, MeOH, reflux (72%).
SYNTHESIS 2005, No. 1, pp 0097–0101
x
x
.
x
x
.2
0
0
4
Advanced online publication: 02.11.2004
DOI: 10.1055/s-2004-834907; Art ID: P08704SS
© Georg Thieme Verlag Stuttgart · New York

98
B. Nay et al.
PAPER
Alkene compound 7 was oxidized to the epoxide 8 in the Hydrogenolysis of benzyl protecting group was not possi-
presence of mCPBA. The reaction was not stereoselective ble no matter what the conditions (0–70 psi, solvents) and
and a mixture (ca. 1:1) of diastereoisomers was obtained. the catalysts (based on Pd or Pt) were. Only the azide
The addition of sodium hydrogen carbonate in the reac- group of intermediate 14 was reduced into an amine (com-
tion mixture prevented the decomposition of the epoxide pound 15) in the presence of Pd/C. The resulting amine
during the course of the reaction. Regioselective opening group was therefore suspected to be responsible for cata-
of the epoxide on the terminal position proceeded in high lyst deactivation.11 Its protection as a tert-butylcarbamate
yield when lithium tetrabromonickelate was used in did not improve benzyl deprotection and we finally attrib-
THF.6 The resulting bromohydrine 9 was then oxidized in uted this failure either to steric hindrance of the secondary
the Swern conditions7 and bromoketone 10 was obtained benzyloxy group, or to catalyst poisoning by the sulfur
in 75% yield after chromatography. Compound 10 was atom of the substrate.
used quickly after purification due to its slight instability.
In the end, benzyl group of intermediate 15 was removed
Condensation of compound 10 with thiourea mono-tert- by boron trichloride (excess) in CH2Cl2.12 At the same
butylcarbamate8 (Hantzsch reaction) yielded the ami- time, tert-butylcarbamate was also cleaved and 4-deaza-
nothiazole intermediate 11. The acetonide was cleaved in thiogirolline analogue 3 was released. Purification was
refluxing MeOH and in the presence of PPTS it gave the successively done by silica gel chromatography and on
diol 12.
Sephadex LH20 column, furnishing the neutral form of
compound 3 in 96% yield. In order to obtain the dihydro-
chloride salt, compound was concentrated in the presence
of 6 N HCl.
Our first attempts to functionalise the diol 12 consisted in
dichlorination. This strategy has already been used suc-
cessfully in the 5-deazathiogirolline series.2 It was not yet
applicable to the present series. The unstable dichloride 4-Deazathiogirolline (3) and its synthetic precursors were
was obtained in only 27% yield and the reaction was not tested in vitro on KB cells (Table 1). Unfortunately, the
reproducible, often giving a complex mixture.
target compound 3 was inactive and none of the interme-
diates gave better results than those of the 5-deazathiogi-
rolline series. It is noteworthy that some of the
intermediates in both series, particularly the azide com-
pounds and the protected derivatives, are more active than
the analogues themselves (IC50 of 14 is 8 mM).
Instead, the replacement of the terminal hydroxyl group of
diol 12 with lithium azide9 was done by use of the Mit-
sunobu method (DEAD/PPh3)10 and in the presence of 12-
crown-4 ether (Scheme 2). The remaining hydroxyl of
compound 13 was then submitted to chlorination with
PPh3/NCS. The azide group did not suffer from reduction
by triphenylphosphane provided that care was taken of the
disappearance of the reagent (reaction of PPh3 with NCS)
before adding 13. For that purpose, PPh3 and excess NCS
were preliminarily refluxed, the reaction being monitored
by TLC until complete disappearance of triphenylphos-
phane was observed.
Table 1 Cytotoxicity Assays of Analogues on KB Tumor Cells
Compound
% killeda
1b
11
12
13
14
52
8
15
3
100
35
18
20
34
18
>10
IC50 (mM)
0.14 >10
>10
>10
>10
a Determined at 10–5 M.
b Girolline as reference
OBn
a
b
Alternatively, compound 3 was also evaluated for anti-
HIV-1 activity. IC50 of compound 3 (concentration of
compound giving 50% of residual reverse transcriptase
activity released in culture medium of infected cells) was
obtained at 0.1 mM (IC50 of AZT as reference = 6 nM) on
virus type HIV-1 Bal in peripheral blood mononuclear
cell (PBMC) cultures (checking cytotoxicity on these
cells gave CC50 >0.3 mM, see experimental part).
N3
S
12
N
OH
13
BocHN
OBn
OBn
N3
NH2
S
S
c
N
N
Cl
Cl
14
BocHN
BocHN
15
This work shows that the aminoimidazole heterocycle is
important for the cytotoxic activity of girolline alkaloid.
Replacement of the aminoimidazole moiety with ami-
nothiazole results in dramatic loss of activity. It is too ear-
ly to bank on the cause of this effect owing to the fact that
the mechanism of action of girolline is still unknown.
However some explanation could be envisaged, e.g. un-
like aromatic properties or different pKa of both heterocy-
cles, that could somewhat influence interactions with
possible biological targets.
OH
d,e
NH2
S
N
Cl
3, 2 HCl
H2N
Scheme 2 Reactions and conditions: (a) PPh3, DEAD, LiN3, 12-
crown-4, CH2Cl2, reflux (94%); (b) PPh3/NCS, THF, reflux (71%);
(c) H2, Pd/C, MeOH, r.t. (68%); (d) BCl3, CH2Cl2, –78 °C to 0 °C then
MeOH (96%); (e) MeOH–H2O (1:1), 6 N HCl.
Synthesis 2005, No. 1, 97–101 © Thieme Stuttgart · New York

PAPER
New Analogues of the Antitumor Alkaloid Girolline
99
All reactions were carried out under an atmosphere of Ar, using
freshly distilled solvents. CH2Cl2 was distilled from CaH2 and THF
was distilled from sodium/benzophenone. DMF was dried over mo-
lecular sieves 3 Å before use. Reaction temperatures were measured
externally. Analytical TLC was carried out on 0.25 mm precoated
silica gel plates (60F254) from Merck. Preparative flash chromatog-
raphy was performed on silica Chromagel SDS (France) 60 Å (40–
(1-H), 3.06 (m, A + B, 2-H), 3.17 (dd, J = 4.4, 6.6 Hz, B, 3-H), 3.56
(dd, J = 3.7, 5.9 Hz, A, 3-H), 3.88 (m, A + B, 4-H), 4.14 (m, A + B,
5-H), 4.56 (d, J = 11.8 Hz, B) 4.60 (d, J = 11.7 Hz, A), 4.71 (d, J =
11.8 Hz, B), 4.86 (d, J = 11.7 Hz, A, CH2Ph), 7.33 (m, A + B, PhH).
13C NMR (75 MHz, CDCl3): d = 24.8, 24.9, 26.0 [C(CH3)2], 43.0,
43.2 (C-1), 51.1, 52.5 (C-2), 65.8, 66.6 (C-5), 71.8, 73.2 (C-3), 74.9,
75.9 (C-4), 76.6, 80.9 (CH2Ph), 108.9 [C(CH3)2], 127.2, 127.3,
127.4, 127.8 (Ph), 137.7, 137.8 (ipso Ph).
1
60 mm). H and 13C NMR chemical shifts (d) are quoted in ppm
from TMS. NMR spectra were measured on Bruker AC-250 or AC-
300 spectrometers. IR spectra were obtained on a Nicolet FT-IR 205
spectrometer (transmittance mode). Exact masses of compounds
(MeOH solutions) were measured on a Micromass TOF LCT (UK)
spectrometer equipped with an electrospray source (ESI+) and ref-
HRMS (ESI+): m/z [M + Na]+ calcd for C15H20O4Na: 287.1259;
found: 287.1257.
(2R/S,3S,4R)-1-Bromo-3-benzyloxy-4,5-O-isopropylidene-
2,4,5-trihydroxypentane (9)
erenced externally with Leucine Enkephanlin ([M + H]+
=
To a solution of epoxide 8 (1.43 g, 5.41 mmol) in THF (50 mL) was
added a 0.5 M solution of Li2NiBr4 in THF (32.5 mL, 16.24 mmol).
After 16 h of stirring at r.t., Et2O (50 mL) was added and the mixture
was washed with phosphate buffer pH 7 (50 mL). The aqueous layer
was extracted with Et2O (50 mL), the combined organic extracts
were washed with brine (50 mL) and evaporated. Purification of the
crude extract was done by silica gel flash chromatography (CH2Cl2
→ CH2Cl2–MeOH, 98:2 → 95:5), giving bromohydrine 9 (1.75 g,
94%, mixture ca. 1:1 of two stereoisomers) as a colourless oil; Rf
0.35 (CH2Cl2–MeOH, 98:2).
556.2771). UV spectra were measured in MeOH using a Cary Vari-
an 100 spectrometer. A Perkin Elmer 241 MC polarimeter was used
to measured [a]D (589 nm) values.
(+)-(3S,4R)-3-Benzyloxy-4,5-O-isopropylidene-4,5-dihydroxy-
1-pentene (7)
Arabitol derivative 5 (6.7 g, 19.3 mmol) was refluxed in absolute
EtOH (20 mL) for 45 min in the presence of coarse zinc powder (6.3
g, 97 mmol). After cooling, the mixture was filtered over Celite®
that was rinsed with EtOAC (30 mL). Evaporation gave the crude
allylic alcohol 6, which was readily used for benzylation without
further purification (instability observed on silica gel). Crude alco-
hol 6 was deprotonated in DMF (100 mL) by addition of NaH (3.1
g, 77 mmol). Once gas releasing was complete, benzyl bromide (4.6
mL, 38.6 mmol) was added dropwise. The mixture was stirred at 40
°C overnight. After 16 h, the reaction was quenched by dropwise
addition of water (10 mL). It was extracted with Et2O (2 × 50 mL).
The combined organic extracts were washed with water (2 × 50 mL)
before evaporation. The crude product was purified by silica gel
flash chromatography (CH2Cl2–heptane 1:1 → CH2Cl2) to yield the
pure benzyl ether 7 (4 g, 83% over two steps) as a colourless oil; Rf
0.70 (CH2Cl2); [a]D +40 (c = 1.3, CHCl3).
IR (film): 3445 (OH), 3065, 3031, 2986, 2934, 1497, 1215, 1157,
1070, 917, 847 cm–1.
1H NMR (300 MHz, CDCl3): d = 1.36, 1.44, 1.45 [3 × s, C(CH3)2 of
both stereoisomers A and B], 3.41 (m, 3-H, A + B), 3.67 (m, 1-H, A
+ B), 3.90 (m, 2-H, A + B, 5-H, A), 4.07 (m, 5-H, B), 4.21 (dd, J =
6.6, 12.5 Hz, 4-H, A), 4.31 (dd, J = 6.6, 12.5 Hz, 4-H, B), 4.66 (d,
J = 11.8 Hz), 4.72 (s), 4.79 (d, J = 11.8 Hz) (CH2Ph, A + B), 7.34
(m, PhH, A + B).
13C NMR (75 MHz, CDCl3): d = 25.1, 26.4, 26.5 [C(CH3)2], 33.7,
36.7 (C-1), 66.0 (C-5), 71.7, 71.9, 74.2, 74.8, 75.7, 76.4, 77.9, 79.6
(CH2Ph, C-2, C-3, C-4), 108.8, 109.3 [C(CH3)2], 127.9, 128.1,
128.4, 128.5 (Ph), 137.4, 137.6 (ipso Ph).
IR (film): 3065, 3031, 2986, 2874, 1497, 1251, 1212, 1158, 1074,
930, 850 cm–1.
HRMS (ESI+): m/z [M + Na]+ calcd for C15H2179BrO4Na: 367.0521;
found: 367.0502.
1H NMR (250 MHz, CDCl3): d = 1.35, 1.41 [2 × s, 2 × 3 H,
C(CH3)2], 3.74 (dd, J = 6.3, 7.3 Hz, 1 H, 5-Ha), 3.87 (dd, 4.9, 7.3
Hz, 1 H, 5-Hb), 4.06, 4.12 (2 × m, 2 × 1 H, 3-H, 4-H), 4.40, 4.60 (2
× d, J = 11.7 Hz, 2 × 1 H, PhCH2), 5.30–5.42 (m, 2 H, 1-H), 5.83
(ddd, J = 7.3, 10.2, 17.1 Hz, 1 H, 2-H), 7.28–7.35 (m, 5 H, PhH).
13C NMR (63 MHz, CDCl3): d = 25.3, 25.6 [C(CH3)2], 66.8 (C-5),
70.5, 77.6 (C-3, C-4), 81.0 (CH2Ph-3), 109.5 [C(CH3)2], 119.7 (C-
1), 127.6, 127.8, 128.3 (Ph), 135.2 (C-2), 138.1 (ipso Ph).
(3S,4R)-1-Bromo-3-benzyloxy-4,5-O-isopropylidene-4,5-dihy-
droxypentane-2-one (10)
To a solution of anhyd DMSO (720 mL, 10.14 mmol) in CH2Cl2 (17
mL) was added dropwise and at –78 °C (CO2–EtOH) a solution of
oxalyl chloride (652 mL, 7.6 mmol) in CH2Cl2 (17 mL). After 10
min, a solution of compound 9 (1.75 g, 5.07 mmol) in CH2Cl2 (17
mL) was added dropwise. The mixture was then stirred at –78 °C for
15 min before adding Et3N (4.3 mL) at the same temperature. The
reaction was warmed to r.t. and stirred for further 90 min. The reac-
tion was quenched by the addition of water (50 mL) and the organic
extract was washed with water (50 mL) and brine (50 mL). After
evaporation of solvent, filtration on a short silica gel column
(CH2Cl2) furnished bromoketone 10 (1.3 g, 75% yield) as a slightly
brown oil (unstable); Rf 0.80 (CH2Cl2–MeOH, 99:1); [a]D +22 (c =
0.89, CHCl3).
HRMS (ESI+): m/z [M + Na]+ calcd for C15H20O3Na: 271.1310;
found: 271.1302.
(2R/S,3S,4R)-3-Benzyloxy-1,2-epoxy-4,5-O-isopropylidene-4,5-
dihydroxypentane (8)
To a solution of compound 7 (1.5 g, 6.05 mmol,) in CH2Cl2 (60 mL)
were successively added NaHCO3 (1.52 g, 18.14 mmol,) and mCP-
BA (3.13 g, 18.14 mmol,). After 24 h at r.t., the reaction was
quenched by the addition of sat. aq Na2S2O3 solution (40 mL). The
organic extract was then washed with sat. aq NaHCO3 solution (60
mL) and brine (60 mL). After evaporation, silica gel flash chroma-
tography (CH2Cl2 → CH2Cl2–MeOH, 98:2) furnished pure epoxide
8 (1.43 g, 90%) as a mixture (ca. 1:1) of two stereoisomers A and
B; colourless oil; Rf 0.60 (CH2Cl2–CH3OH, 98:2).
IR (film): 3065, 3032, 2988, 2935, 1738 (C=O), 1497, 1258, 1218,
1075, 915, 844 cm–1.
1H NMR (250 MHz, CDCl3): d = 1.32, 1.40 [2 × s, 2 × 3 H,
C(CH3)]2, 3.88 (dd, J = 5.9, 8.8 Hz, 1 H, 5-Ha), 4.06 (dd, J = 5.9,
8.8 Hz, 1 H, 5-Hb), 4.08 (d, J = 5.9 Hz, 1 H, 3-H), 4.30 (q, J = 5.9
Hz, 1 H, 4-H), 4.35 (s, 2 H, 1-H), 4.56 (d, J = 11.2 Hz, 1 H, CH2Ph),
4.63 (d, J = 11.2 Hz, 1 H, CH2Ph), 7.34 (m, 5 H, PhH).
13C NMR (63 MHz, CDCl3): d = 25.0, 26.4 [C(CH3)2], 47.6 (C-1),
66.4 (C-5), 73.5, 75.9 (C-4, C-3), 82.6 (CH2Ph), 110.2 (C(CH3)2),
128.3, 128.4, 128.7 (Ph), 136.5 (ipso Ph), 201.0 (C-2).
IR (film): 3064, 3032, 2987, 2934, 2882, 1371, 1257, 1211, 1156,
1073, 959, 917, 847 cm–1.
1H NMR (300 MHz, CDCl3): d = 1.34 (A), 1.36 (B), 1.37 (A), 1.44
(B) [4 × s, C(CH3)2], 2.64 (dd, J = 2.2, 4.4 Hz, A), 2.74 (dd, J = 3.6,
5.2 Hz, B), 2.79 (dd, J = 2.9, 5.2 Hz, B), 2.85 (ps t, J = 4.4 Hz, A)
Synthesis 2005, No. 1, 97–101 © Thieme Stuttgart · New York

100
B. Nay et al.
PAPER
2-Amino-4-[(1¢S,2¢R)-1¢-benzyloxy-2¢,3¢-O-isopropylidene-2¢,3¢-
dihydroxypropyl)thiazole N2-tert-Butylcarbamate (11)
yield) as a colourless and transparent resin; Rf 0.60 (CH2Cl2–
MeOH, 95:5); [a]D +70 (c = 1.5, CHCl3).
A mixture of bromoketone 10 (1.55 g, 4.53 mmol) and thiourea N-
t-butylcarbamate6 (956 mg, 5.44 mmol) in DMF (45 mL) was heat-
ed at 50 °C in the presence of NaHCO3 (761 mg, 9 mmol) and
stirred for 16 h. After the reaction was complete, Et2O (100 mL) was
added. The mixture was washed with water (2 × 100 mL) and brine
(100 mL) before evaporation of the solvent. Purification by silica
gel chromatography (CH2Cl2 → CH2Cl2–MeOH, 98:2) gave thiaz-
ole derivative 11 (1.58 g, 83% yield) as an amorphous colourless
solid; Rf 0.20 (CH2Cl2–MeOH, 99:1); [a]D +60 (c = 1.0, CHCl3).
IR (film): 3600–2700 (very br), 2102 (N3), 1720 (C=O), 1594,
1551, 1302, 1243, 1157, 1084 cm–1.
1H NMR (300 MHz, CDCl3): d = 1.54 [s, 9 H, OC(CH3)3], 3.42 (m,
2 H, 3¢-H), 4.13 (m, 1 H, 2¢-H), 4.41 (d, J = 11.0 Hz, 1 H, CH2Ph),
4.46 (d, J = 5.9 Hz, 1 H, 1¢-H), 4.60 (d, J = 11.0 Hz, 1 H, CH2Ph),
6.85 (s, 1 H, 5-H), 7.32 (m, 5 H, PhH), 8.95 (br s, 1 H, NH).
13C NMR (63 MHz, CDCl3): d = 28.2 [OC(CH3)3], 53.2 (C-3¢), 71.6,
72.9 (C-2¢, CH2Ph), 77.5 (C-1¢), 83.0 [OC(CH3)3], 111.1 (C-5),
128.0, 128.5 (Ph), 148.2 (ipso Ph), 150.3 (C-4), 152.0 (C-2), 160.6
(C=O).
IR (film): 3226 (br), 2983, 2934, 1722 (C=O), 1556, 1288, 1242,
1158, 1073, 848 cm–1.
HRMS (ESI+): m/z [M + Na]+ calcd for C18H23N5O4SNa: 428.1368;
found: 428.1375.
1H NMR (250 MHz, CDCl3): d = 1.33, 1.40 [2 × s, 2 × 3 H,
C(CH3)2], 1.54 [s, 9 H, OC(CH3)3], 4.03 (m, 2 H, 3¢-H), 4.42 (d, J =
11.7 Hz, 1 H, CH2Ph), 4.52 (m, 2 H, 1¢-H, 2¢-H), 4.57 (d, J = 11.7
Hz, 1 H, CH2Ph), 6.84 (s, 1 H, 5-H), 7.28 (m, 5 H, PhH), 8.84 (br s,
1 H, NH).
13C NMR (63 MHz, CDCl3): d = 25.4, 26.6 [C(CH3)2], 28.2
[OC(CH3)3], 66.2 (C-3¢), 71.3 (C-2¢), 77.4, 77.5 (C-1¢, CH2Ph), 82.6
[OC(CH3)3], 109.5 [C(CH3)2], 110.8 (C-5), 127.6, 127.8, 128.3
(Ph), 138.0 (ipso Ph), 149.1 (C-4), 152.2 (C-2), 159.9 (C=O).
UV (MeOH): lmax = 259 nm.
2-Amino-4-[(1¢S,2¢R)-3¢-azido-1¢-benzyloxy-2¢-chloropro-
pyl)thiazole N2-tert-Butylcarbamate (14)
A mixture of triphenylphosphane (171 mg, 0.65 mmol) and NCS
(109 mg, 0.82 mmol) in THF (2 mL) were heated at reflux for 30
min, giving a dirty purple thick mixture. Complete disappearance of
PPh3 was needed and was controlled by TLC. A solution of hy-
droxyazide 13 (66 mg, 0.163 mmol) in THF (1 mL) was then added
and the mixture was refluxed for 20 min before quenching with wa-
ter (5 mL). Extraction with Et2O (10 mL) was followed by washing
of the extract with water (10 mL) and brine (10 mL). Evaporation
of volatiles gave a residue that was purified by preparative TLC
(CH2Cl2–MeOH, 98:2). Chloroazide 14 (49 mg, 72% yield) was ob-
tained as a colourless and transparent resin; Rf 0.95 (CH2Cl2–
MeOH, 95:5); [a]D +58 (c = 0.5, CHCl3).
HRMS (ESI+): m/z [M + Na]+ calcd for C21H28N2O5SNa: 443.1617;
found: 443.1647.
UV (MeOH): lmax = 259 nm.
2-Amino-4-((1¢S,2¢R)-1¢-benzyloxy-2¢,3¢-dihydroxypropyl)thia-
zole N2-tert-Butylcarbamate (12)
A solution of the acetonide 11 (1.21 g, 2.89 mmol) in MeOH (30
mL) was heated at reflux for 16 h in the presence of pyridinium p-
toluenesulfonate (725 mg, 2.89 mmol). EtOAc (100 mL) was then
added and the mixture was washed with aq 32% NH4OH (50 mL),
water (50 mL) and brine (50 mL). The organic extract was evapo-
rated and purified by silica gel chromatography (CH2Cl2–MeOH,
98:2 → 95:5), yielding pure diol 12 (790 mg, 72% yield) as a co-
lourless resin; Rf 0.20 (CH2Cl2–MeOH, 95:5); [a]D +71 (c = 1.0,
CHCl3).
IR (film): 3500–2700 (br), 2105 (N3), 1723 (C=O), 1548, 1301,
1242, 1156, 1075, 1028, 868, 801, 751, 698 cm–1.
1H NMR (300 MHz, CDCl3): d = 1.55 [s, 9 H, OC(CH3)3], 3.44 (dd,
J = 6.6, 13.2 Hz, 1 H, 3¢-Ha), 3.62 (dd, J = 4.4, 13.2 Hz, 1 H, 3¢-Hb),
4.28 (m, 1 H, 2¢-H), 4.41 (d, J = 11.8 Hz, 1 H, CH2Ph), 4.64 (d, J =
5.9 Hz, 1 H, 1¢-H), 4.67 (d, J = 11.8 Hz, 1 H, CH2Ph), 6.90 (s, 1 H,
5-H), 7.34 (m, 5 H, PhH), 8.04 (br s, 1 H, NH).
13C NMR (63 MHz, CDCl3): d = 28.2 [OC(CH3)3], 54.0 (C-3¢), 62.2
(C-2¢), 71.2 (C-1¢), 77.4 (CH2Ph), 83.1 [OC(CH3)3], 111.8 (C-5),
128.1, 128.5 (Ph), 137.3 (ipso Ph), 147.5 (C-4), 152.0 (C-2), 159.9
(C=O).
IR (film): 3368 (very br), 2979, 1721 (C = O), 1557, 1292, 1245,
1158, 1077, 872 cm–1.
1H NMR (300 MHz, CDCl3): d = 1.51 [s, 9 H, OC(CH3)3], 3.50 (br
s, 1 H, OH), 3.69 (m, 2 H, 3¢-H), 4.04 (m, 1 H, 2¢-H), 4.30 (br s, 1
H, OH), 4.38 (d, J = 11.8 Hz, 1 H, CH2Ph), 4.49 (d, J = 5.9 Hz, 1 H,
1¢-H), 4.56 (d, J = 11.8 Hz, 1 H, CH2Ph), 6.81 (s, 1 H, 5-H), 7.28
(m, 5 H, PhH), 9.50 (br s, 1 H, NH).
HRMS (ESI+): m/z [M + Na]+ calcd for C18H2235ClN5O3SNa:
446.1030; found: 446.1006.
13C NMR (63 MHz, CDCl3): d = 28.2 [OC(CH3)3], 63.4 (C-3¢), 71.6
(C-2¢), 73.7 (CH2Ph), 78.0 (C-1¢), 82.9 [OC(CH3)3], 110.7 (C-5),
127.9, 128.5 (Ph), 137.7 (ipso Ph), 148.7 (C-4), 152.2 (C-2), 160.8
(C=O).
UV (MeOH): lmax = 258 nm.
2-Amino-4-[(1¢S,2¢R)-3¢-amino-1¢-benzyloxy-2¢-chloropro-
pyl)thiazole N2-tert-Butylcarbamate (15)
Palladium 10% on charcoal (20 mg) was added to a solution of chlo-
roazide 14 (77 mg, 0.182 mmol) in MeOH (2 mL). The mixture was
vigorously stirred under a H2 atmosphere (balloon). After 1 h, it was
diluted in EtOAc (5 mL), filtered through Celite® and evaporated.
Purification on a silica gel column (CH2Cl2–MeOH, 95:5, flash
chromatography) gave the amine 15 (49 mg 68% yield) as a colour-
less resin; Rf 0.20 (CH2Cl2–MeOH, 95:5); [a]D +70 (c = 0.4,
MeOH).
HRMS (ESI+): m/z [M + Na]+ calcd for C18H24N2O5SNa: 403.1304;
found: 403.1288.
UV (MeOH): lmax = 260 nm.
2-Amino-4-[(1¢S,2¢R)-3¢-azido-1¢-benzyloxy-2¢-hydroxypro-
pyl)thiazole N2-tert-Butylcarbamate (13)
To a mixture of diol 12 (130 mg, 0.34 mmol), triphenylphosphane
(269 mg, 1.02 mmol), lithium azide9 (167 mg, 3.4 mmol), 12-
crown-4 ether (166 mL, 1.02 mmol,) and molecular sieves 4 Å (100
mg) in CH2Cl2 (4 mL), was dripped DEAD (162 mL, 1.02 mmol)
while stirring. The reaction mixture was refluxed for 4 h and then
CH2Cl2 (10 mL) was added for treatment. Washing with water (2 ×
20 mL) and brine (20 mL) furnished a salt-free organic extract that
was evaporated. Purification of the residue on preparative TLC
plates (CH2Cl2–MeOH, 96:4) yielded pure azide 13 (129 mg, 94%
IR (film): 3500–2700 (br), 1715 (C=O), 1552, 1368, 1296, 1248,
1159, 1076, 872 cm–1.
1H NMR (300 MHz, CDCl3): d = 1.53 [s, 9 H, OC(CH3)3], 2.81 (dd,
J = 4.9, 14.7 Hz, 1 H, 3¢-Ha], 2.96 (dd, J = 3.9, 14.7 Hz, 1 H, 3¢-Hb),
4.17 (m, 1 H, 2¢-H), 4.41 (d, J = 11.7 Hz, 1 H, CH2Ph), 4.64 (d, J =
5.9 Hz, 1 H, 1¢-H), 4.65 (d, J = 11.7 Hz, 1 H, CH2Ph), 6.89 (s, 1 H,
5-H), 7.34 (m, 5 H, PhH).
Synthesis 2005, No. 1, 97–101 © Thieme Stuttgart · New York

PAPER
New Analogues of the Antitumor Alkaloid Girolline
101
13C NMR (63 MHz, CDCl3): d = 28.3 [OC(CH3)3], 44.9 (C-3¢), 66.9
(C-2¢), 71.7 (C-1¢), 79.1 (CH2Ph), 82.3 [OC(CH3)3], 110.4 (C-5),
127.9, 128.1, 128.5 (Ph), 137.5 (ipso Ph), 148.0 (C-4), 152.8 (C-2),
161.0 (C=O).
HRMS (ESI+): m/z [M + H]+ calcd for C18H2535ClN3O3S: 398.1305;
found: 398.1288.
the reverse transcriptase activity associated with the virus particles
released in the supernatant after 5 d of cell culture in the presence
of different concentrations of compounds. The 50% inhibitory con-
centration of virus multiplication (IC50) was derived from the com-
puter-generated median effect plot of the dose-effect data.15 In
parallel experiments, cytotoxicity of the molecules for uninfected
cells was measured after an incubation of 5 d in their presence using
a colorimetric assay (MTT test).16 The 50% cytotoxic concentration
(CC50) is the concentration which give half the OD540 of the ref-
erence and was calculated using the abovementioned program.
UV (MeOH): lmax = 259 nm.
2-Amino-4-[(1¢S,2¢R)-3¢-amino-2¢-chloro-1¢-hydroxypropyl)-
thiazole Dihydrochloride [4-Deazathiogirolline (3)]
To a solution of amine 15 (44 mg, 0.111 mmol) in CH2Cl2 (1 mL),
was added dropwise and at –78 °C a 1 M solution of BCl3 in CH2Cl2
(2.2 mL, 2.2 mmol). The reaction mixture was allowed to warm
slowly to 0 °C during ca. 4 h and then was cooled back to –25 °C
before adding MeOH (2 mL). The solution was warmed to r.t. and
stirred for 1 h. The solvent was removed and the residue was further
concentrated in the presence of MeOH (3 × 2 mL). The crude prod-
uct was purified on preparative TLC plates (butanone–EtOAc–
HOAc–H2O, 3:5:1:1) and then on Sephadex® LH20 column, yield-
ing the final product 3 ( 22 mg,96%). The dihydrochloride was ob-
tained in quantitative yield (31 mg) as a yellow resin by
concentrating the product with MeOH–35% aq HCl (1:1, 2 mL); Rf
0.15 (butanone–EtOAc–HOAc–H2O, 3:5:1:1); [a]D +21 (neutral
form, c = 0.4, MeOH); [a]D +11 (dihydrochloride, c = 1.1, MeOH).
Aknowledgements
We gratefully thank R. Nicolaÿ for technical aid, Mrs. C. Gaspard
from the ICSN (Gif-sur-Yvette) for the cytotoxicity tests, and Dr.
A.M. Aubertin from the Institut de Virologie INSERM U544/ULP
(Strasbourg) for the anti-HIV tests.
References
(1) (a) Ahond, A.; Bedoya Zurita, M.; Colin, M.; Fizames, C.;
Laboute, P.; Lavelle, F.; Laurent, D.; Poupat, C.; Pusset, J.;
Pusset, M.; Thoison, O.; Potier, P. C. R. Acad. Sci. Paris
1988, 307, 145. (b) Chiaroni, A.; Riche, C.; Ahond, A.;
Poupat, C.; Pusset, M.; Potier, P. C. R. Acad. Sci. Paris 1991,
312, 49.
(2) Schiavi, B.; Ahond, A.; Al-Mourabit, A.; Poupat, C.;
Chiaroni, A.; Gaspard, C.; Potier, P. Tetrahedron 2002, 58,
4201.
(3) D-Arabitol was prepared from D-arabinose by NaBH4
reduction in water–HOAc. Purification proceeded more
easily through its pentaacetylated derivative (Ac2O) that was
then submitted to methanolysis (CH3ONa), providing the
arabitol in 86% yield. Changing the initial sugar could lead
to other stereochemistries: Holý, A. Coll. Czech. Chem.
Comm. 1982, 47, 2786.
IR (film): 3500–2700 (br), 1626 (s), 1283, 1150, 1021, 982, 825,
767 cm–1.
1H NMR (300 MHz, CD3OD): d = 3.35 (m, 1 H, 3¢-Ha), 3.56 (d,
J = 12.5 Hz, 1 H, 3¢-Hb), 4.58 (d, J = 7.4 Hz, 1 H, 2¢-H), 5.16 (s, 1
H, 1¢-H), 6.87 (s, 1 H, 5-H).
13C NMR (63 MHz, CD3OD): d = 45.1 (C-3¢), 62.7 (C-2¢), 69.9 (C-
1¢), 106.4 (C-5), 141.3 (C-4), 172.8 (C-2).
HRMS (ESI+): m/z [M + H]+ calcd for C6H1135ClN3OS: 208.0311;
found: 208.0349.
UV (MeOH): lmax = 257 nm.
(4) (a) Nakagawa, T.; Tokuoka, H.; Shinoto, K.; Yoshimura, J.;
Sato, T. Bull. Chem. Soc. Jpn. 1967, 40, 2150. (b) Mulzer,
J.; Greifenberg, S.; Becksett, A.; Gotwald, M. Liebigs Ann.
Chem. 1992, 1131. (c) Garegg, P. J.; Samuelsson, B. J.
Chem. Soc., Perkin Trans. 1 1980, 2866.
Cytotoxicity Tests
The KB cells were serially cultured in MEM (minimum essential
medium, with Earle’s salt solution), purchased from Seromed and
containing 10% fetal calf serum, L-glutamine (2 mM), penicillin G
(60 mg/mL), streptomycin sulphate (60 mg/mL) and gentamycin (40
mg/mL). For the test, KB cells were grown as monolayers in 24-well
plastic plates (25000 cells seeded per well in 1 mL medium). Serial
dilutions of the stock EtOH solutions of the compounds were made
in the medium and added to the cell cultures under a volume of 10
mL per well, immediately after plating the cells. All cultures were
incubated at 37 °C in a 95% air–5% CO2 humidified incubator. Af-
ter 3 d, cell viability was determined by addition of 100 mL/well of
a 0.02% solution in medium of the vital dye neutral red, followed
by further 8–16 h incubation. The cell monolayers were washed
with phosphate-buffered saline, submitted to lysis with a 1% sodi-
um lauryldodecyl sulphate solution and to photometric quantifica-
tion of the extracted dye at 540 nm, using a ELX 800 microplate
reader (BIO-TEK Instruments, Inc.) as originally described by
Borenfreund and Puerner.13
(5) Schiavi, B. PhD Thesis; Université de Paris-Sud, Orsay:
France, 2000.
(6) (a) Bonini, C.; Roghi, G. Synthesis 1994, 225. (b) Dawe, R.
D.; Molinski, T. F.; Turner, J. V. Tetrahedron Lett. 1984, 25,
2061.
(7) Omura, K.; Swern, D. Tetrahedron 1978, 34, 1651.
(8) Schiavi, B.; Ahond, A.; Poupat, C.; Potier, P. Synth.
Commun. 2002, 32, 1671.
(9) Hofman-Bang, N. Acta Chem. Scand. 1957, 11, 581.
(10) Mitsunobu, O. Synthesis 1981, 1.
(11) Czech, B. P.; Bartsch, R. A. J. Org. Chem. 1984, 49, 4076.
(12) (a) Ludek, O. R.; Meier, C. Synthesis 2003, 2101. (b) For a
review on cleavage of ether, see: Bhatt, M. V.; Kulkarni, S.
U. Synthesis 1983, 249.
(13) Borenfreund, E.; Puerner, J. A. Toxicol. Lett. 1985, 24, 119.
(14) Moog, C.; Wick, A.; Le Ber, P.; Kirn, A.; Aubertin, A. M.
Antiviral Res. 1994, 24, 275.
Evaluation of Antiviral Activity
The effects of the compounds on the replication of HIV-1 were
evaluated, as previously described, in CEM-SS cells acutely infect-
ed with HIV-1 LAI or in PBMC infected with HIV-1 Bal or in MT4
cells infected with HIV-1 IIIB.14 The compounds were solubilised
in DMSO at an initial concentration of 10–2 M. The 1% dilution in
culture medium as well as serial dilutions were used in the antiviral
assays. The production of virus was measured by quantification of
(15) Chou, J.; Chou, T. C. In Computer Software for Apple II
Series and IBM-PC and Instruction Manual; Elsevier-
Biosoft, Elsevier Science Publisher: Cambridge U.K., 1985,
19.
(16) Mosmann, T. J. Immunol. Methods 1983, 65, 55.
Synthesis 2005, No. 1, 97–101 © Thieme Stuttgart · New York