Synthesis of Nα-Fmoc N,N′-bis-Boc-5-, 6- and 8-guanyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (5-GTIC, 6-GTIC and 8-GTIC)

Article abstract of DOI:10.1055/s-2004-834875

N^α-Fmoc N,N′-bis-Boc-5-, 6- and 8-guanyl-Tic-OH (5-, 6- and 8-GTIC), three conformationally constrained amino acids with basic properties, have been synthesized by microwave irradiation. These amino acids combine the basic features of arginine with the aromatic features of phenylalanine and can be utilized as functional tools in peptide-based structure-activity studies.

Full text of DOI:10.1055/s-2004-834875

PAPER
3011
Synthesis of Na-Fmoc N,N¢-bis-Boc-5-, 6- and 8-guanyl-1,2,3,4-tetrahydroiso-
quinoline-3-carboxylic Acid (5-GTIC, 6-GTIC and 8-GTIC)
Synthesis of
N
-
F
a emoc
N
,
N
¢
-b
a
is-Boc-gua
t
nyl-1, 2
r
,3, 4-tetr
i
ahydro
c
isoquinolin
e
e-3-carboxylic
A
c
S
ids everino,a Vincenzo Santagada,*a Francesco Frecentese,a Elisa Perissutti,a Sara Terracciano,a
Ferdinando Fiorino,a Donatella Cirillo,a Severo Salvadori,b Gianfranco Balboni,b Giuseppe Caliendoa
a
Dipartimento di Chimica Farmaceutica e Tossicologica, Università di Napoli ‘Federico II’, Via D. Montesano 49, 80131 Napoli, Italy
Fax +39(81)678649; E-mail: santagad@unina.it
Dipartimento di Scienze Farmaceutiche, Università di Ferrara, via Fossato di Mortara, 4100 Ferrara, Italy
b
Received 13 July 2004; revised 1 September 2004
In order to refine the evaluation of the guanidine group on
Abstract: Na-Fmoc N,N¢-bis-Boc-5-, 6- and 8-guanyl-Tic-OH (5-,
the aromatic ring of the Tic residue, we report the synthe-
6- and 8-GTIC), three conformationally constrained amino acids
sis of the unknown 5-, 6- and 8-guanidine isomers. The
application of microwave energy to organic compounds
with basic properties, have been synthesized by microwave irradia-
tion. These amino acids combine the basic features of arginine with
the aromatic features of phenylalanine and can be utilized as func- for conducting synthetic reactions at highly accelerated
rates is an emerging technique.9 The synthetic procedure
tional tools in peptide-based structure–activity studies.
for 5- and 6-guanidine isomers 10 and 11 is summarized
in Scheme 1, while the synthetic procedure for 8-guani-
dine isomer 22 is reported in Scheme 2. In several steps,
the synthetic procedure was performed using a microwave
oven (ETHOS 1600, Milestone) especially designed for
organic synthesis with superior results. The key interme-
diates for the synthesis of the two unconventional amino
acids 10 and 11 were compounds 4 and 5, H-Tic(5 and 6-
NH2)-OMe. In particular, H-Tic(5-NH2)-OMe (4) was ob-
tained starting from commercially available L-1,2,3,4-tet-
rahydro-3-isoquinolinecarboxylic acid (H-Tic-OH, 1)
which was submitted to an unconventional nitration with
commercially available nitronium tetrafluoroborate.10
The obtained mixture of 5- and 7-nitro derivatives could
not be separated by column chromatography and was con-
verted into the corresponding N-benzyloxycarbonyl de-
rivatives, and then esterified to the resultant methyl esters
(2) which were separated by silica gel column chromatog-
raphy.10 Hydrogenation of intermediate 2 afforded H-
Tic(5-NH2)-OCH3 (4). H-Tic(6-NH2)-OCH3 (5), instead,
was obtained starting from L-1,2,3,4-tetrahydro-3-iso-
quinolinecarboxylic acid (H-Tic-OH, 1) which was then
treated with fuming nitric acid and concentrated sulfuric
acid at –10 °C to obtain the 6-nitro derivative in a mixture
with the corresponding 7-nitro isomer.8 The mixture of
two regioisomers was converted, by methylation with a
mixture of methanol and sulfuric acid, into the corre-
sponding methyl ester derivatives which were separated
by chromatography on silica gel column obtaining H-
Tic(6-NO2)-OCH3 (Scheme 1, 3). Intermediate 3 was then
hydrogenated affording the desired H-Tic(6-NH2)-OCH3
(5). Successively, the Fmoc-protection on the a-amino
group of H-Tic(5- or 6-NH2)-OCH3 (4 and 5) was intro-
duced by treatment with Fmoc-OSu in aqueous sodium
carbonate obtaining derivatives 6 and 7. Under these con-
ditions, the amino group on the ring did not react. The
amino group was successively converted into the corre-
sponding guanidine moiety by reaction with N,N¢-bis-
Boc-S-methyl-isothiourea, HgCl2 and triethylamine in
DMF. The obtained two products 8 and 9 were purified
Key words: unconventional amino acid, microwave, synthesis,
GTIC
There is a continuing need to develop ‘novel’ amino acids
that can provide specific three-dimensional properties to a
peptide. Among the variety of Phe analogues which pro-
vide conformational restriction1 of the peptide backbone
and/or the lateral chain, the most extensively employed
amino acids in structure–activity studies have been the a-
methyl-L-phenylalanine (H-a-Me-Phe-OH) and L-
1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (H-Tic-
OH) residues. Tic and Tic(OH) have been used in medic-
inal chemistry as a phenylalanine replacement in many bi-
ologically active peptides (e.g., opioid,2 substance P,3
FTase inhibitors,4 Bradykinin,5 Kallikrein6). These amino
acids are now commercially available. The continuing
level of interest in Tic is reflected by the fact that several
analogues of Tic have been the targets of many synthetic
studies.7 In a previous paper 6,8 we described a facile syn-
thesis of the Fmoc-N,N¢-bis-Boc-7-guanyl-Tic-OH
(GTIC), a new Tic derivative substituted on the aromatic
ring with a basic guanidine group. This amino acid com-
bines the basic features of arginine with the aromatic fea-
tures of phenylalanine. We synthesized this amino acid to
evaluate if a guanidino function in the 7 position of H-Tic-
OH, either in the delta selective opioid dipeptide antago-
nists, H-Tyr/Dmt-Tic-R (R = OH, NH2) 8 or as fluorogen-
ic substrates for human tissue kallikrein,6 could modify
the receptor affinity and activity. The results of the bind-
ing assay and Ki values (opioid 8 and inhibition of human
tissue kallikrein6), respectively, showed that 7-GTIC in-
stead of Tic drastically modified the receptor affinity and
selectivity.6,8
SYNTHESIS 2004, No. 18, pp 3011–3016
x
x.
x
x
.
2
0
0
4
Advanced online publication: 21.10.2004
DOI: 10.1055/s-2004-834875; Art ID: P07704SS
© Georg Thieme Verlag Stuttgart · New York

3012
B. Severino et al.
PAPER
NH
COOH
(1)
NO2+BF4-, CH3CN
HNO3 / conc. H2SO4
NH
NH
NO2
COOH
COOH
5- and 7-nitro-Tic
NO2
6- and 7-nitro-Tic
a) PhCH2OCOCl, NaHCO3
b) K2CO3, CH3I
c) Chromatography on silica gel
a) CH3OH/H2SO4, MW
b) Chromatography on silica gel
Z
N
NH
O2N
COOCH3
COOCH3
NO2
(3) H-Tic(6-NO2)-OCH3
(2) Z-Tic(5-NO2)-OCH3
Fmoc
N
Fmoc-OSu/Na2CO3
NH
MW
COOCH3
NH2
COOCH3
NH2
(6) Fmoc-Tic(5-NH2)-OCH3
(7) Fmoc-Tic(6-NH2)-OCH3
(4) H-Tic(5-NH2)-OCH3
(5) H-Tic(6-NH2)-OCH3
Fmoc
Fmoc
1 N NaOH/4 °C, 4 h
N
N
N,N'-bis-Boc-S-methyl-isothiourea,
HgCl2 / TEA
MW
COOH
COOCH3
NH
NH
NH-Boc
NH-Boc
Boc-N
Boc-N
(10) Fmoc-Tic(N,N'-bis-Boc-5)-OH
(11) Fmoc-Tic(N,N'-bis-Boc-6)-OH
(8) Fmoc-Tic(N,N'-bis-Boc-5)-OCH3
(9) Fmoc-Tic(N,N'-bis-Boc-6)-OCH3
Scheme 1 Synthetic procedure of 5- and 6-GTIC
using silica gel chromatography and diethyl ether–hexane concentrated hydrochloric acid afforded a mixture of the
as eluents. The final products 10 and 11, Fmoc-Tic(N,N¢- corresponding amines Ac-Tic(7-NH2, 6- or 8-NO2)-OCH3
bis-Boc-5- or 6-)-OH, were obtained by saponification of (16a and 16b), respectively, which were separated by col-
the methyl ester derivatives with 1 N NaOH. The synthet- umn chromatography on silica gel (Scheme 2, yield 25%
ic route utilized to obtain 5- and 6-isomers (Scheme 1) and 32%, respectively for the 8- and 6-isomers). Deami-
was not suitable to achieve 8-isomer 22. Therefore, in or- nation of Ac-Tic(7-NH2, 8-NO2)-OCH3 (16a) with hypo-
der to obtain the derivative with the guanidine moiety in phosphorous acid gave Ac-Tic(8-NO2)-OCH3 (17), the
8-position, we optimized the following procedure as re- acetamide moiety of which was hydrolysed to give the re-
ported in Scheme 2. In fact, the synthetic procedure for 8- quired H-Tic(8-NO2)-OCH3 (18). Reduction of the nitro
guanidine isomer 22 started from H-Tic(7-NO2)-OCH3 compound to the corresponding amine H-Tic(8-NH2)-
(12) which was synthesized as previously described8 and OCH3 (19) was straightforward (yield 98%), and Fmoc-
converted into the corresponding N-acetyl derivative 13 protection of the a-amino group was carried out with
by treatment with acetic anhydride/sodium acetate. Re- Fmoc-OSu in aqueous sodium carbonate to give Fmoc-
duction of the nitro group to the corresponding amine Ac- Tic(8-NH2)-OCH3 (20). Under these conditions, the ami-
Tic(7-NH2)-OCH3 (14) was accomplished using H2 and no group on the ring did not react. The yield after this pro-
C/Pd (yield 95%), followed by N-protection with trifluo- cedure was modest (55%), presumably because of
roacetic anhydride/trifluoroacetic acid (TFAA/TFA) (15, competition between the protection reaction and break-
yield 85%). An unconventional nitration of Ac-Tic(7- down of the Fmoc group by the secondary amine followed
+
NHTfa)-OCH3 (15) using NO2 BF4 – in sulfolane gave a by trapping of the amine by dibenzofulvene. The 8-amino
mixture of 6- and 8-NO2 isomer derivatives (the anhy- group was converted into the corresponding guanidine
drous environment was necessary to avoid the partial hy- moiety (21) by reaction with N,N¢-bis-Boc-S-methyl-
drolysis of the methyl ester moiety). Subsequent isothiourea, HgCl2 and TEA in DMF. Hydrolysis of the
hydrolysis of the trifluoroacetamide derivatives using methyl ester with 1 N NaOH afforded the final compound
Synthesis 2004, No. 18, 3011–3016 © Thieme Stuttgart · New York

PAPER
Synthesis of Na-Fmoc N,N¢-bis-Boc-guanyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic Acid
3013
NO2
COCH3
NO2
COCH3
NH2
N
(CH3CO)2O/CH3COONa
COOCH3
NH
N
H2, C/Pd
COOCH3
COOCH3
(14) Ac-Tic(7-NH2)-OCH3
(13) Ac-Tic(7-NO2)-OCH3
(12) H-Tic(7-NO2)-OCH3
CF3CONH
N
NH2
COCH3
N
COCH3
a) NO2+BF4- in sulfolane
(CF3CO)2O/CF3COOH
b) conc. HCl/ MW
MW
COOCH3
COOCH3
c) Chromatography on silica gel
NO2
(16a) Ac-Tic(7-NH2, 8-NO2)-OCH3
(15) Ac-Tic(7-NHTfa)-OCH3
(16b) Ac-Tic(7-NH2, 6-NO2)-OCH3
NO2
NO2
NO2
NH2
COCH3
conc. HCl/MW
NH2
COCH3
N
NH
a) HCl / NaNO2
NH
N
H2, C/Pd
b) H3PO2
COOCH3
COOCH3
COOCH3
COOCH3
(16a) Ac-Tic(7-NH2, 8-NO2)-OCH3
(17) Ac-Tic( 8-NO2)-OCH3
(19) H-Tic( 8-NH2)-OCH3
(18) H-Tic( 8-NO2)-OCH3
NH-Boc
NH-Boc
Boc-N
Boc-N
N
NH2
NH
H
Fmoc
Fmoc
N
Fmoc
N,N'-bis-Boc-S-methyl-isothiourea
N
Fmoc-OSu/Na2CO3
MW
N
1N NaOH / 4 °C, 4 h
HgCl2 , TEA, DMF
COOCH3
COOCH3
COOH
MW
(21) Fmoc-Tic( N,N'-bis-Boc-8)-OCH3
(20) Fmoc-Tic( 8-NH2)-OCH3
(22) Fmoc-Tic( N,N'-bis-Boc-8)-OH
Scheme 2 Synthetic procedure of 8-GTIC
Methyl 5-Amino-1,2,3,4-tetrahydroisoquinoline-3-carboxylate
(4)
Fmoc-Tic(N,N¢-bis-Boc-8)-OH (22) which was purified
by column chromatography on silica gel using a mixture
of diethyl ether–hexane as eluent (yield 60%). Under
these conditions the Fmoc group was poorly cleaved. The
final compounds and all intermediates were characterized
by 1H NMR and mass spectroscopy.
Methyl
5-nitro-2-(benzyloxy)-1,2,3,4-tetrahydroisoquinoline-3-
carboxylate (2, 0.70 g, 1.9 mmol) was dissolved in MeOH (100
mL). The solution was degassed with N2 and 10% Pd/C (0.2 g) was
added. The reaction mixture was maintained under H2 atmosphere
for 15 min at r.t. The mixture was then filtered to remove the Pd/C
and, finally, the solvent was removed to give 4 as a white solid (0.36
g, 93%).
In conclusion, the Fmoc-N,N¢-bis-Boc-5-, 6- and 8-gua-
nyl-Tic-OH (GTIC), three conformationally constrained
amino acids, will serve as useful tools in peptide-based
structure–activity studies. Furthermore, the application of
microwave irradiation significantly reduces reaction
times and improves the yields compared with convention-
al procedures.
Mp 124–126 °C; Rf 0.40 (Et2O–EtOH, 9:1).
1H NMR (500 MHz, CDCl3): d = 2.54–2.60 (m, 2 H), 2.76–2.80 (m,
1 H), 3.73–3.75 (dd, 1 H, NH), 3.77 (s, 3 H), 4.04–4.07 (d, 2 H),
6.47–6.49 (d, 1 H), 6.52–6.54 (d, 1 H), 6.95–6.98 (t, 1 H).
ESI-MS: m/z (%) = 207 (MH+).
Methyl 6-Amino-1,2,3,4-tetrahydroisoquinoline-3-carboxylate
(5)
Microwave Equipment and Conditions
(3S)-1,2,3,4-Tetrahydroisoquinoline-3-carboxylic acid
1
(2.5
The synthetic steps performed by microwave irradiation were car-
ried out using a microwave oven (ETHOS 1600, Milestone) espe-
cially designed for organic synthesis. The experimental conditions
used during microwave application were similar, with the same
concentration of starting material and volume of solvent, to those
used by conventional heating. All reactions performed by micro-
wave heating were conducted in standard Pyrex glassware and were
performed by a microwave program which was composed of appro-
priate ramping and holding steps. The temperature of the stirred re-
action mixture was monitored directly by a microwave-transparent
fluoroptic probe inserted into the solution.
g,14.1 mmol) was added slowly to conc. H2SO4 (7.5 mL) main-
tained at a temperature of –10 °C. Conc. HNO3 (1.8 mL) was then
added drop-wise. The reaction mixture was stirred at this tempera-
ture for 3.5 h and then poured into ice water (75 mL). The product
was precipitated by neutralization with NH4OH, filtered, washed
with H2O, then dried to give a brown solid. The mixture of 6- and
7-regioisomers was dissolved in MeOH (50 mL), conc. H2SO4 (1.1
mL) was added and the mixture was stirred under microwave irra-
diation at reflux for 30 min, followed by evaporation. The following
extraction with CH2Cl2 and washing with 5% NaHCO3 and brine
gave a mixture of 6- and 7-nitro-Tic-OMe which was separated by
silica gel column chromatography (Et2O–EtOH, 9:1) to give methyl
6-nitro-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (3, 1 g, 30%).
Mp 82–84 °C; Rf 0.60 (Et2O–EtOH, 9:1).
Synthesis 2004, No. 18, 3011–3016 © Thieme Stuttgart · New York

3014
B. Severino et al.
PAPER
1H NMR (500 MHz, CDCl3): d = 3.07–3.09 (m, 2 H), 3.18–3.22 (m,
1 H), 3.77 (s, 3 H), 4.11–4.25 (d, 2 H), 7.18–7.19 (d, 1 H), 7.99–8.00
(d, 1 H), 8.01(s, 1 H).
9
Mp 113–115 °C; Rf 0.40 (Et2O–hexane, 6:4).
1H NMR (500 MHz, CDCl3): d =1.43 (s, 9 H), 1.50 (s, 9 H), 3.18–
3.25 (m, 2 H), 3.61 and 3.68 (2 s, 3 H, OCH3, rotamers), 4.32–4.65
(m, 6 H), 7.17–7.88 (m, 11 H, Ar).
ESI-MS: m/z (%) = 237 (MH+).
The obtained product 3 (1 g, 4.2 mmol) was dissolved in MeOH (50
mL). The solution was degassed with N2 and 10% Pd/C (0.44 g) was
added. The reaction mixture was maintained under H2 atmosphere
for 1 h at r.t. The mixture was then filtered to remove the Pd/C and,
finally, the solvent was removed to give 5 a white solid (0.8 g,
93%).
ESI-MS: m/z (%) = 671 (MH+).
N-Fmoc N,N¢-Bis-Boc-5-guanyl-1,2,3,4- tetrahydroisoquino-
line-3-carboxylic Acid and N-Fmoc- N,N¢-Bis-Boc-6-guanyl-
1,2,3,4-tetrahydroisoquinoline-3-carboxylic Acid (10 and 11)
Methyl ester groups were removed by treating amino acids 8 or 9 (1
mmol) in MeOH (8 mL) with 1 N NaOH (1.2 equiv) for 4 h at 4 °C.
The solution was then diluted with H2O, concentrated in vacuo to
remove the MeOH, and washed with EtOAc. After cooling to 0 °C,
the aqueous solution was acidified with citric acid (10%) and the
product extracted with EtOAc. The organic layer was washed with
H2O, dried (Na2SO4) and evaporated. The residue was purified by
silica gel column chromatography (Et2O–hexane, 10:1) (10: 0.41g,
63%; 11: 0.43 g, 65%).
Mp 121–123 °C; Rf 0.40 (Et2O–EtOH, 9:1).
1H NMR (500 MHz, CDCl3): d = 2.84–2.9 (m, 2 H), 2.95–2.99 (m,
1 H), 3.69–3.72 (dd, 1 H, NH), 3.77 (s, 3 H), 3.99–4.03 (d, 2 H),
6.44 (s, 1 H), 6.50–6.52 (d, 1 H), 6.81–6.83 (d, 1 H).
ESI-MS: m/z (%) = 207 (MH+).
N-Fmoc 3-Methyl 5-Amino-1,2,3,4-tetrahydroisoquinoline-3-
carboxylate and N-Fmoc 3-Methyl 6-Amino-1,2,3,4-tetrahy-
droisoquinoline-3-carboxylate (6 and 7)
10
5- or 6-Amino-Tic-OMe (4 or 5, 0.9 g, 4.4 mmol) was suspended in
9% Na2CO3 (9.8 mL) and cooled in ice water. A solution of Fmoc-
OSu (1.18 g, 3.5 mmol) in dioxane (10.5 mL) was then added drop-
wise and the mixture was stirred and heated by microwave at 40 °C
for 20 min. The solvent was evaporated, EtOAc was then added and
the phases were separated. The organic phase was evaporated and
the residue, loaded onto a silica gel column, was purified (Et2O–
hexane, 8:2). Product-containing fractions were pooled and concen-
trated to obtain a white solid (6: 1.67 g, 90%; 7, 1.73 g, 92%).
Mp 196–198 °C; Rf 0.50 (Et2O–hexane, 10:1).
1H NMR (500 MHz, CDCl3): d = 1.45 (s, 9 H), 1.58 (s, 9 H), 3.20–
3.30 (m, 2 H), 4.30–4.50 (m, 6 H), 7.17& ndash;7.80 (m, 11 H, ar-
omatic).
ESI-MS: m/z (%) = 657 (MH+).
11
Mp 185–187 °C; Rf 0.50 (Et2O–hexane, 10:1).
6
1H NMR (500 MHz, CDCl3): d = 1.43 (s, 9 H), 1.50 (s, 9 H), 3.18–
3.25 (m, 2 H), 4.32–4.65 (m, 6 H), 7.17–7.88 (m, 11 H, aromatic).
Mp 77–79 °C; Rf 0.50 (Et2O–hexane, 8:2).
1H NMR (500 MHz, CDCl3): d = 3.11–3.19 (m, 2 H), 3.65 and 3.68
(2 s, 3 H, OCH3, rotamers), 4.22–4.70 (m, 6 H), 6.58–7.85 (m, 11
H, Ar).
ESI-MS: m/z (%) = 657 (MH+).
Methyl 2-Acetyl-7-nitro-1,2,3,4-tetrahydroisoquinoline-3-car-
boxylate (13)
ESI-MS: m/z (%) = 428 (MH+).
Ester 12 (4.2 g, 17.8 mmol) was dissolved in EtOH (20 mL) and
conc. HCl (2 mL) was added. The precipitated hydrochloride salts
(4.7 g), anhyd NaOAc (0.92 g) and acetic anhydride (9.2 mL) were
heated (50–60 °C) on a steam bath for 1 h and the reaction mixture
was poured into water. The product was extracted with CHCl3
(3 × 25 mL) and chromatographed over silica gel (EtOAc–MeOH,
8:2). The resulting acetyl derivative 13 was obtained as a brown oil
(4.6 g, 96%).
7
Mp 80–82 °C; Rf 0.50 (Et2O–hexane, 8:2).
1H NMR (500 MHz, CDCl3): d = 3.19–3.25 (m, 2 H), 3.66 and 3.69
(2 s, 3 H, OCH3, rotamers), 4.22–4.70 (m, 6 H), 6.61–7.84 (m, 11
H, Ar).
ESI-MS: m/z (%) = 428 (MH+).
Rf 0.50 (Et2O).
N-Fmoc 3-Methyl N,N¢-Bis-Boc-5-guanyl-1,2,3,4-tetrahy-
droisoquinoline-3-carboxylate and N-Fmoc 3-Methyl N,N¢-Bis-
Boc-6-guanyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (8
and 9)
Fmoc-5- or 6-amino-Tic-OMe (6 or 7, 1.1 g 2.7 mmol) was dis-
solved in DMF (40 mL).The mixture was cooled to 0 °C and then
added N,N¢-bis-Boc-S-methyl-isothiourea (0.9 g 3.0 mmol), HgCl2
(1.36 g 5.0 mmol) and after 10 min Et3N (1.0 mL). The reaction
mixture was warmed to r.t., heated by microwave to 40 °C for 20
min, and then was filtered. The organic phase was concentrated and
purified on by silica gel column chromatography (Et2O–hexane,
6:4) (8: 1.23 g, 68%; 9: 1.27 g, 70%).
1H NMR (500 MHz, CDCl3) showed a mixture of rotamers dou-
bling most signals: d = 2.19 and 2.27 (2 s, 3 H, COCH3, rotamers),
3.40 (m, 2 H), 3.46 (m, 1 H), 3.62 and 3.64 (2 s, 3 H, OCH3, rota-
mers), 4.79 and 4.80 (2 d, J = 6.1 Hz, 2 H, rotamers), 7.35 (d,
J = 8.2 Hz, 1 H), 8.02 (s, 1 H), 8.05 (d, J = 8.2 Hz, 1 H).
ESI-MS: m/z (%) = 279 (MH+).
Methyl 2-Acetyl-7-amino-1,2,3,4-tetrahydroisoquinoline-3-car-
boxylate (14)
Derivative 13 (4.6 g, 16.5 mmol), dissolved in MeOH (50 mL), was
hydrogenated at 1 atm at r.t. for 30 min in the presence of 10% Pd/
C (460 mg). The filtered solution was evaporated in vacuo to give
an oil, 14, which was crystallized from Et2O (3.9 g, 95%).
8
Mp 120–122 °C;) Rf 0.40 (Et2O–hexane, 6:4).
Mp 78–80 °C; Rf 0.60 (Et2O–EtOH, 9:1).
1H NMR (500 MHz, CDCl3): d = 1.45 (s, 9 H), 1.58 (s, 9 H), 3.20–
3.30 (m, 2 H), 3.55 and 3.65 (2 s, 3 H, OCH3, rotamers), 4.30–4.50
(m, 6 H), 7.17–7.80 (m, 11 H, Ar).
1H NMR (500 MHz, CDCl3) showed a mixture of rotamers dou-
bling most signals: d = 2.13 and 2.23 (2 s, 3 H, COCH3, rotamers),
3.04 (m, 2 H), 3.10–3.14 (m, 1 H), 3.60 and 3.61 (2 s, 3 H, OCH3,
rotamers), 4.58 (d, J = 6.1 Hz, 2 H, rotamers), 6.43 (s, 1 H), 6.52 (d,
J = 8.1 Hz, 1 H), 6.93 (d, J = 8.1 Hz, 1 H).
ESI-MS: m/z (%) = 671 (MH+);
Synthesis 2004, No. 18, 3011–3016 © Thieme Stuttgart · New York

PAPER
Synthesis of Na-Fmoc N,N¢-bis-Boc-guanyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic Acid
3015
ESI-MS: m/z (%) = 249 (MH+).
1H NMR (500 MHz, CDCl3) showed a mixture of rotamers dou-
bling most signals: d = 2.19 and 2.29 (2 s, 3 H, rotamers), 3.18 (m,
2 H), 3.38 (m, 1 H), 3.65 and 3.68 (2 s, 3 H, OCH3, rotamers), 4.93
and 5.18 (dd, J = 6.2 Hz, 2 H, rotamers), 7.40 (t, J = 8.1 Hz, 1 H),
7.48 (d, J = 7.8 Hz, 1 H), 8.02 (d, J = 7.8 Hz, 1 H).
Methyl 2-Acetyl-7-[(trifluoroacetyl)amino]-1,2,3,4-tetrahy-
droisoquinoline-3-carboxylate (15)
A mixture of 14 (3.9 g, 15.7 mmol), trifluoroacetic anhydride (3.7
mL, 26.4 mmol) and trifluoroacetic acid (4.45 mL, 26.4 mmol) in
CH2Cl2 (60 mL) was stirred and heated at reflux under microwave
conditions for 1 min, then cooled and evaporated. The residue was
dissolved in CH2Cl2 and washed with brine. The organic phase was
dried (Na2SO4) and evaporated to dryness. The filtered solution was
evaporated in vacuo to give an oil, 15, which was crystallized from
Et2O (5.15 g, 95%).
ESI-MS: m/z (%) = 279 (MH+).
Methyl 8-Amino-1,2,3,4-tetrahydroisoquinoline-3-carboxylate
(19)
Hydrolysis of 17 (0.59 g, 2.1 mmol) in MeOH (15 mL) and conc.
HCl (7.5 mL) under microwave irradiation at reflux for 20 min gave
methyl 8-nitro-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (18,
0.49 g, 99%) which was dissolved in MeOH (50 mL). The solution
was degassed with N2 and 10% Pd/C (0.2 g) was added. The reac-
tion mixture was maintained under a H2 atmosphere for 30 min at
r.t. The mixture was then filtered to remove the Pd/C and, finally,
the solvent was removed to give 19 as a white solid (0.38 g, 98%).
Mp 129–131 °C; Rf 0.75 (Et2O–EtOH, 9:1).
1H NMR (500 MHz, CDCl3) showed a mixture of rotamers dou-
bling most signals: d = 2.18 and 2.25 (2 s, 3 H, COCH3, rotamers),
3.08 (m, 2 H), 3.25 (m, 1 H), 3.60 and 3.64 (2 s, 3 H, OCH3, rota-
mers), 4.70 (d, J = 6.2 Hz, 2 H, rotamers), 7.18 (d, J = 8.1 Hz, 1 H,),
7.26 (d, J = 8.1 Hz, 1 H,) 7.44 (s, 1 H).
Mp 124–126 °C; Rf 0.40 (Et2O–EtOH, 7:3).
ESI-MS: m/z (%) = 345 (MH+).
1H NMR (500 MHz, CDCl3): d = 2.84 (m, 2 H), 2.97 (m, 1 H), 3.69
(dd, J = 6.2 Hz, 1 H), 3.77 (s, 3 H), 3.99 (d, J = 6.1 Hz, 2 H), 6.20
(d, J = 7.8 Hz 1 H), 6.35 (d, J = 8.2 Hz, 1 H), 6.80 (t, J = 8.2 Hz, 1
H).
Methyl 2-Acetyl-7-amino-8-nitro-1,2,3,4-tetrahydroisoquino-
line-3-carboxylate (16a)
To an ice-cold solution of 15 (4.6 g, 13.4 mmol) in tetramethylene
ESI-MS: m/z (%) = 207 (MH+).
sulfone (5 mL) was added drop-wise nitronium tetrafluoroborate
+
–
(5.2 g of 85% NO2 BF4 , 33.5 mmol) dissolved in tetramethylene
sulfone (10 mL). After the addition was completed, the reaction
mixture was stirred at r.t. for another 10 min, then the solution was
diluted with H2O and extracted with Et2O. The organic phase was
dried (Na2SO4) and evaporated to dryness. This product contained a
mixture of 6-NO2 and 8-NO2 derivatives. Hydrolysis of the trifluo-
roacetamides in MeOH (50 mL) and conc. HCl (15 mL) under mi-
crowave heating at reflux for 6 min, followed by evaporation,
extraction with CH2Cl2 and washing with H2O gave a mixture of the
nitroamines which were chromatographed on silica gel (Et2O–
EtOH, 9:1). The elution firstly gave methyl 2-acetyl-7-amino-8-ni-
tro-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (16a, 1 g, 32%).
Na-Fmoc 3-Methyl 8-Amino-1,2,3,4-tetrahydroisoquinoline-3-
carboxylate (20)
Intermediate 19 (0.90 g, 4.4 mmol) was suspended in 9% Na2CO3
(9.8 mL) and cooled in ice water. A solution of Fmoc-OSu (1.18 g,
3.5 mmol) in dioxane (10.5 mL) was then added drop-wise and the
mixture was heated under microwave irradiation to 40 °C for 20
min. The solvent was evaporated, EtOAc was then added and the
phases were separated. The organic phase was evaporated and the
residue, loaded onto a silica gel column, was chromatographically
purified (Et2O–hexane, 8:2). Product-containing fractions were
pooled and concentrated to give 20 as a white solid (1.23g, 68%).
Mp 80–82 °C; Rf 0.5 (Et2O–hexane, 8:2).
1H NMR (500 MHz, CDCl3): d = 3.10 (m, 2 H), 3.60 and 3.61 (2 s,
3 H, OCH3, rotamers), 4.19 (m, 6 H), 6.56 (m, 11 H).
Mp 180–183 °C; Rf 0.70 (Et2O–EtOH, 9:1).
1H NMR (500 MHz, CDCl3) showed a mixture of rotamers dou-
bling most signals: d = 2.10 and 2.20 (2 s, 3 H, rotamers), 3.08 (m,
2 H), 3.24–3.28 (m, 1 H), 3.60 and 3.64 (2 s, 3 H, OCH3, rotamers),
4.65 (d, J = 6.1 Hz, 2 H, rotamers), 6.12 (d, J = 7.8 Hz, 1 H), 6.68
(d, J = 8.1 Hz, 1 H).
ESI-MS: m/z (%) = 428 (MH+).
Na-Fmoc 3-Methyl N,N¢-Bis-Boc-8-guanyl-1,2,3,4-tetrahy-
droisoquinoline-3-carboxylate (21)
ESI-MS: m/z (%) = 294 (MH+).
Intermediate 20 (1.1 g, 0.0027 mol) was dissolved in DMF (40 mL).
The mixture was cooled to 0 °C, then N,N¢-bis-Boc-S-methyl-
isothiourea (0.9 g, 3.0 mmol), HgCl2 (1.36 g, 5.0 mmol) were added
followed by Et3N (1.0 mL) after 10 min. The reaction was heated
under microwave irradiation at 40 °C for 20 min. Then the mixture
was filtered, the organic phase was concentrated and purified by sil-
ica gel column chromatography (Et2O–hexane, 6:4) to give 21 (1.19
g, 66%).
Continued elution gave methyl 2-acetyl-7-amino-6-nitro-1,2,3,4-
tetrahydroisoquinoline-3-carboxylate (16a, 1.57 g, 43%).
Mp 182–184 °C; Rf 0.40 (Et2O–EtOH, 9:1).
1H NMR (500 MHz, CDCl3) showed a mixture of rotamers dou-
bling most signals: d = 2.07 and 2.08 (2 s, 3 H, rotamers), 3.08 (m,
2 H), 3.25 (m, 1 H), 3.60 and 3.64 (2 s, 3 H, OCH3, rotamers), 4.55
(d, J = 6.1 Hz, 2 H, rotamers), 6.81 (s, 1 H), 7.81 (s, 1 H).
Mp 121–123 °C; Rf 0.40 (Et2O–hexane, 6:4).
ESI-MS: m/z (%) = 294 (MH+).
1H NMR (500 MHz, CDCl3): d = 1.34 (s, 9 H), 1.46 (s, 9 H), 3.24
(m, 2 H), 3.60 and 3.66 (s, 3 H, rotamers), 4.45 (m, 6 H), 7.62 (m,
11 H).
Methyl 2-Acetyl-8-nitro-1,2,3,4-tetrahydroisoquinoline-3-car-
boxylate (17)
A stirred solution of 16a (0.78 g, 2.66 mmol) in HCl (6 N, 8 mL)
was treated drop-wise with a solution of NaNO2 (0.22 g, 3.2 mmol)
in H2O (1 mL) at 0 °C. After 100 min at 0 °C, hypophosphorous
acid (50% aq soln, 2.7 mL) was added drop-wise and the mixture
was kept at 4 °C for 18 h, then poured into H2O and extracted with
CH2Cl2. The organic phase was dried (Na2SO4) and evaporated to
dryness giving methyl 2-acetyl-8-nitro-1,2,3,4-tetrahydroisoquino-
line-3-carboxylate (17, 0.59 g, 80%).
ESI-MS: m/z (%) = 671 (MH+).
Na-Fmoc N,N¢-Bis-Boc-8-guanyl-1,2,3,4-tetrahydroisoquino-
line-3-carboxylic Acid (22)
The methyl ester group was removed by treating intermediate 21 (1
mmol) in MeOH (8 mL) with 1 N NaOH (1.2 equiv) for 4 h at 4 °C.
The solution was then diluted with H2O, concentrated in vacuo to
remove the MeOH, and washed with EtOAc. After cooling to 0 °C,
the aqueous solution was acidified with citric acid (10%) and the
Mp 138–140 °C; Rf 0.60 (Et2O–EtOH, 9:1).
Synthesis 2004, No. 18, 3011–3016 © Thieme Stuttgart · New York

3016
B. Severino et al.
PAPER
product extracted with EtOAc. The organic layer was washed with
H2O, dried (Na2SO4) and evaporated. The residue was purified by
column chromatography on silica gel (Et2O–hexane, 10:1) to give
compound 22 (0.39 g, 60%).
(4) Caliendo, G.; Fiorino, F.; Greco, P.; Perissutti, E.; De Luca,
S.; Giuliano, A.; Santelli, G.; Severino, B.; Santagada, V. Il
Farmaco 1999, 54, 785.
(5) Guba, W.; Haessner, R.; Breiphol, G.; Henke, S.; Knolle, J.;
Santagada, V.; Kessler, H. J. Am. Chem. Soc. 1994, 116,
7532.
(6) Pimenta, D. C.; Melo, L. R.; Caliendo, G.; Santagada, V.;
Fiorino, F.; Severino, B.; De Nucci, G.; Juliano, L.; Juliano,
M. Biol. Chem. 2002, 383 (5), 853.
Mp 182–184 °C; Rf 0.50 (Et2O–hexane, 10:1).
1H NMR (500 MHz, CDCl3): d = 1.34 (s, 9 H), 1.46 (s, 9 H), 3.22–
3.26 (m, 2 H), 4.34–4.70 (m, 6 H), 7.65 (m, 11 H).
ESI-MS: m/z (%) = 657 (MH+).
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Synthesis 2004, No. 18, 3011–3016 © Thieme Stuttgart · New York