2-Amino-4-pyrrolidinothieno[2,3-d]pyrimidine-6-carboxylic acid as an N-terminal surrogate in amino acid and peptide analogues

Article abstract of DOI:10.1055/s-2005-918426

2-Amino-4-pyrrolidinothieno[2,3-d]pyrimidine-6-carboxylic acid (4) (ATPC) is an unnatural amino acid with promise in applications as a building block for the synthesis of peptidomimetics. ATPC was obtained from both 3a and 3b thienopyrimidines by hydrolysis and hydrogenolysis, respectively. The synthesis of eleven ATPC-amino acids and two ATPC-peptides is described. ATPC is incorporated as N-terminal moiety in solution or solid-phase peptide synthesis using Boc or Fmoc methodology and without protection of the ATPC amino group. Georg Thieme Verlag Stuttgart.

Full text of DOI:10.1055/s-2005-918426

PAPER
3159
2-Amino-4-pyrrolidinothieno[2,3-d]pyrimidine-6-carboxylic Acid as an
N-Terminal Surrogate in Amino Acid and Peptide Analogues
T
hieno[2,3-
d
]
py
v
rimidine
A
m
a
ino
A
cid
o
n
r
Peptide
D
e
g
rivatives elos E. Bissyris,a Dimitris Belekos,b Vassiliki Magafa,a Petros G. Tsoungas,c George Varvounis,*b
Paul Cordopatis*a
a
Department of Pharmacy, University of Patras, Rio 26504, Greece
Fax +30(2610)997714; E-mail: pacord@upatras.gr
Department of Chemistry, University of Ioannina, Ioannina 45110, Greece
b
Fax +30(26510)98799; E-mail: gvarvoun@cc.uoi.gr
Ministry of Development, Department of Research and Technology, 14–18 Messogion Ave., Athens 11510, Greece
c
Received 6 June 2005
vealed the topological contribution of the individual
amino acid residues of the active AII molecule.6
Abstract: 2-Amino-4-pyrrolidinothieno[2,3-d]pyrimidine-6-car-
boxylic acid (4) (ATPC) is an unnatural amino acid with promise in
applications as a building block for the synthesis of peptidomimet-
ics. ATPC was obtained from both 3a and 3b thienopyrimidines by
hydrolysis and hydrogenolysis, respectively. The synthesis of elev-
en ATPC-amino acids and two ATPC-peptides is described. ATPC
is incorporated as N-terminal moiety in solution or solid-phase pep-
tide synthesis using Boc or Fmoc methodology and without protec-
tion of the ATPC amino group.
Based on our knowledge regarding the chemistry of
thienopyrimidines5a,7 and AII,8 we decided to develop ef-
ficient protocols for generating new heterocyclic peptido-
mimetics of AII using an appropriate thienopyrimidine
derivative as a molecular scaffold. In order to make this
choice, theoretical models were constructed starting from
the AII peptide NMR derived structure9 and replacing one
or two amino acids by a 2-amino-4-(amino or substituted
amino)thieno[2,3-d]pyrimidine-6-carboxylic acid deriva-
tive. Figure 1 represents the most promising hybrid struc-
ture, H-Asp-ArgVal-Tyr-Ile-ATPC-Phe-OH, constructed
theoretically by replacing residues His7-Pro8 by com-
pound 4 (ATPC) using Macromodel software.10 This hy-
brid structure was energy minimized and further subjected
to conformational search using MonteCarlo/LowMode
(MC/LMOD) mixed mode search strategy.11
Key words: heterocycles, amino acids, peptides, thienopyrimidine,
angiotensin II
The field of peptidomimetics has gained enormous popu-
larity in recent years, particularly with the emergence of
unnatural analogues as components of molecules with
therapeutical potential. The need to replace natural amino
acids in peptides with non-proteinogenic counterparts in
order to obtain drug-like target molecules has stimulated
a great deal of innovation on several fronts.1 The use of
natural peptides as pharmaceuticals suffers from several
constraints such as relatively small bioavailability, lack of
transportation, or rapid metabolic degradation.2 The in-
corporation or substitution of a heterocyclic ring into pep-
tides that acts as a conformationally restricted core, has
been recently employed to prepare modified peptides
(heterocyclic-peptide hybrids) with better in vivo efficacy
than the natural peptide.3
In the late 1960s Roth reported on the inhibition of folate
synthesis and function by certain 2,4-diaminothieno[2,3-
d]pyrimidines.4 Since then, the chemistry and biology of
thienopyrimidines has developed remarkably and up to
this day is an area of considerable interest.5 The octapep-
tide angiotensin II (H-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-
OH, AII) is one of the oldest peptide hormones, known for
its multiplicity of biological actions related to endocrine
or connected to the central and peripheral nervous system.
AII is a potent pressor agent, which has a vital role in the
regulation of blood pressure, in the conservation of total
blood volume and salt homeostasis. Structure-activity re-
lationships studies from several laboratories have re-
Figure 1 Hybrid structure of H-Asp-Arg-Val-Tyr-Ile-ATPC-Phe-
OH, where His7-Pro8 in AII is replaced by 4 (ATPC)
The most populated family superimposes quite well on the
initial structure of the AII peptide (Figure 2), suggesting
that compound 4 (Scheme 1) is a good candidate for syn-
thesizing the thienopyrimidine containing peptide H-Asp-
Arg-Val-Tyr-Ile-ATPC-Phe-OH.
It is within this context that we now present the synthesis
of thirteen 2-[(2-amino-4-pyrrolidin-1-ylthieno[2,3-d]-
pyrimidine-6-carbonyl)amino]amino acid and peptide an-
alogues 5–17 (Table 1). Our strategy involves first the
SYNTHESIS 2005, No. 18, pp 3159–3166
x
x.
x
x
.
2
0
0
5
Advanced online publication: 06.10.2005
DOI: 10.1055/s-2005-918426; Art ID: P07505SS
© Georg Thieme Verlag Stuttgart · New York

3160
E. E. Bissyris et al.
PAPER
synthesis of 4 and then the coupling of the carboxylic acid Further replacement of the second halogen in 2 by mer-
C-terminus of 4 with the N-terminus of eleven amino ac- captoacetic acid benzyl ester followed by a Dieckman
ids and two peptides, using both solution and solid phase type condensation afforded 3b in 64% yield. The latter
methodology.
was converted into the target amino acid 4 in 83% yield,
by hydrogenolysis in DMF–MeOH with 10% Pd-C cata-
lyst. Hydrolysis of 3a in DMSO–MeOH with NaOH gave
4 in 91% yield. The thirteen synthesized ATPC-amino
acid and peptide hybrids 5–17 are depicted in Table 1.
The coupling of 4 with the hydrochloric acid salts of Ala-
OMe and Phe-OEt took place with HOBt, TBTU and i-
Pr2NEt in DMF and gave ATPC-Ala-OMe 5 and ATPC-
Phe-OEt 6. The hydrochloric salt of Leu-OMe with HOBt,
DIC and i-Pr2NEt in DMF gave ATPC-Leu-OMe 7.
ATPC-Leu-OH 8 was obtained from the hydrolysis of 7 in
1 M NaOH–MeOH solution. Boc-Asp(OBn)-NH2 was
first converted into its TFA salt and then reacted with
HOBt, TBTU and i-Pr2NEt in DMF to afford ATPC-
Asp(OBn)-NH2 9. ATPC-Leu-Glu(t-Bu)NH2 10 was pre-
pared in a similar manner from the p-toluenesulfonic acid
salt of H-Leu-Glu(Ot-Bu)NH2. The hybrids ATPC-Val-
OH 11, ATPC-Lys(Boc)-OH 12, ATPC-Thr(t-Bu)-OH
13, ATPC-Pro-NH2 14 and ATPC-Leu-Gln-Lys-Thr-Tyr-
OH 15 were synthesized in solid phase utilizing standard
Fmoc/t-Bu methodology. The treatment of side-chain pro-
tected derivatives 12 and 13 with TFA–anisole produced
the compounds 16 and 17, respectively. Evidently, the
amino group of 4 is unreactive under these conditions and
therefore advantageously no protection was required.
Thienopyrimidine 4 was obtained by the sequence delin-
eated in Scheme 1. Thienopyrimidine 3a was synthesized
according to the published procedure.6 This method was
also used to synthesize thienopyrimidine 3b from 2-ami-
no-4,6-dichloropyrimidine-5-carbaldehyde 1. Thus, pyr-
rolidine replaced selectively one of the chlorine atoms of
1 to give pyrrolidinopyrimidine 2 in 85% yield, improved
by 15% with respect to the earlier method.
In conclusion, N-terminal unprotected thienopyrimidine 4
was used to prepare an array of diverse N-terminal peptide
hybrids of acids, amides or esters, side-chain protected or
not, using Boc or Fmoc methodology, in solution or solid
phase peptide synthesis. Among these, hybrid 15 is an an-
Figure 2 Superposition of AII peptide (H-Asp-Arg-Val-Tyr-Ile-
His-Pro-Phe-OH, white) and hybrid (H-Asp-Arg-Val-Tyr-Ile-ATPC-
Phe-OH, cyan)
H2N
N
Cl
H2N
N
Cl
H2N
N
N
SCH2CO2R
CHO
i: 85%
ii
N
N
N
CHO
CHO
Cl
N
1
2
H2N
N
S
R
CO2R
N
a
b
Me
Bn
N
3
H2N
N
S
iii: 91%
iv: 83%
CO2H
3a
3b
N
N
4
Scheme 1 Reagents and conditions: i) HN(CH2)4, Et3N, CHCl3, 0 °C, 12 h; ii) HSCH2CO2CH3 or HSCH2CO2CH2C6H5, Et3N, toluene, reflux,
12 h; iii) NaOH (1 M), MeOH, r.t.,16 h; iv) 1 atm H2, 10% Pd-C, DMF, MeOH, r.t., 6 h
Synthesis 2005, No. 18, 3159–3166 © Thieme Stuttgart · New York

PAPER
Thieno[2,3-d]pyrimidine Amino Acid or Peptide Derivatives
3161
Table 1 Synthesized ATPC-Amino Acid and Peptide Hybrids 5–17
(continued)
alogue of the human band 3 Protein (547–553), in which
4 has replaced the N-terminal dipeptide. Thus, 4 is able to
couple with any amino acid type (aliphatic, aromatic, ba-
sic etc.) and could be a useful tool as an N-terminal surro-
gate in peptide design and synthesis.
Prod- Structure
uct
Reaction
procedure
12
B
NHBoc
H2N
H2N
H2N
N
O
N
S
S
S
Table 1 Synthesized ATPC-Amino Acid and Peptide Hybrids 5–
17
C
H
N
N
N
Prod- Structure
uct
Reaction
procedure
OH
N
N
O
5
A, A
H2N
N
O
N
S
13
14
15
B
B
O
N
t-Bu
C
H
C
H
O
N
OMe
N
N
OH
N
N
O
O
6
7
8
9
A,A
A,B
H2N
H2N
H2N
O
N
S
S
S
O
N
H2N
C
H
C
N
N
N
O
OEt
N
N
N
O
O
O
O
N
See experi-
mental
NH2
C
H
NH2
O
OMe
N
N
O
O
N
H
N
N
H
See experi-
mental
O
N
O
H
H
H
C
H
N
N
H2N
N
N
S
C
N
OH
OH
O
O
N
N
O
HO
See experi-
mental
H2N
O
N
O
S
HO
C
H
N
16
17
C
C
NH2
O
H2N
N
O
N
S
S
NH2
N
C
H
O
N
N
OH
N
N
10
11
See experi-
mental
H2N
N
O
N
H
S
O
C
H
N
H2N
O
N
O
N
C
H
OH
OH
N
O
O
H2N
N
O
O
t-Bu
B
H2N
N
N
O
C
S
N
N
Melting points were determined on an Electro thermal IA 9000 ap-
paratus and are uncorrected. IR spectra were recorded with KBr pel-
H
O
HO
1
lets on a Jasco, FT/IR-5300 spectrophotometer. H NMR spectra
were obtained at 400.13 MHz and 13C NMR at 100.62 MHz on a
Bruker Avance spectrometer. DMSO-d6 and TMS were used as the
1
solvent and the internal standard, respectively. Individual H and
13C NMR assignments, wherever made, are based on homo- and/or
heteronuclear 2D experiments. Low resolution mass spectra were
recorded on a Micromass-Platform LC by ESI or on a JEOL
Synthesis 2005, No. 18, 3159–3166 © Thieme Stuttgart · New York

3162
E. E. Bissyris et al.
PAPER
AX505W spectrometer by EI whereas high-resolution ESI spectra
were measured on a Bruker Apex III spectrometer. HPLC was per-
formed on Amersham Pharmacia, Mod.10 ÄCTA instrument. De-
rivatives were analyzed on a Nucleosil 100 C18 column (5 mm
particle size; 250 × 4.6 mm) using linear gradient from 5–85% B
over 30 min where A = 0.1% TFA in water and B = 0.1% TFA in
MeCN, at a flow rate of 1 mL/min, monitored at 220 nm and 254
nm. Semipreparative column Lichrosorb RP-18 (7 mm particle size;
250 × 8.0 mm) was employed with a linear gradient from 5–85% B
over 30 min and a flow rate of 1.5 mL/min monitored at 220 nm and
354 nm. Flash chromatography was conducted using Merck silica
gel 60 (230–400 mesh) and TLC was run on Merck silica gel 60 F254
plates (0.2 mm). All solvents were dried and/or purified according
to standard procedures.12
standard sample synthesized from 3a by Method A described
above.
ATPC-Amino Acid Esters; General Procedure A
Method A: To a suspension of 4 (0.08 g, 0.30 mmol), the hydrochlo-
ric salt of alanine methyl ester or phenylalanine ethyl ester (0.60
mmol), 1-hydroxybenzotriazole (0.05 g, 0.30 mmol) and 2-(1H-
benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate
(0.10 g, 0.30 mmol) in anhyd DMF (3 mL) was added i-Pr2NEt
(0.31 mL, 1.8 mmol). The mixture was stirred at r.t. for 3 h during
which time the pH was adjusted at 7.5–8.0 with i-Pr2NEt. The sol-
vent was evaporated in vacuo and the residue partitioned between
5% aq citric acid and EtOAc. The aqueous layer was extracted with
EtOAc (3 × 20 mL) and the combined organic layers were washed
with H2O, 5% aq NaHCO3, brine and dried (Na2SO4). The solvent
was evaporated in vacuo and the residue purified by column chro-
matography using EtOAc–MeOH (95:5) as eluent to give ester 5
and EtOAc–hexane (9:1) to give ester 6 or 7.
Benzyl 2-Amino-4-pyrrolidinothieno[2,3-d]pyrimidine-6-car-
boxylate (3b)
Mercaptoacetic acid benzyl ester (1.27 g, 7 mmol) and Et3N (1.41
g, 14 mmol) were added dropwise to a stirred suspension of 2-ami-
no-4-chloro-6-(pyrrolidin-1-yl)pyrimidine-5-carbaldehyde (2; 1.58
g, 7 mmol) in toluene (50 mL). The mixture was refluxed for 24 h,
the solvent evaporated under reduced pressure and the residue
stirred with cold water (20 mL) for a few minutes. The product was
filtered off, dried and recrystallized from toluene as pale yellow mi-
crocrystals (2.1 g, 85%); mp 219–221 °C.
Method B: To an ice-cold suspension of 4 (0.08 g, 0.30 mmol) and
1-hydroxybenzotriazole (0.07 g, 0.45 mmol) in anhyd DMF (3 mL),
diisopropylcarbidiimide (0.051 mL, 0.33 mmol) was added. The
mixture was stirred for 10 min at 0 °C and for a further 10 min at r.t.
after which the hydrochloric salt of leucine methyl ester (0.60
mmol) and i-Pr2NEt (0.11 mL, 0.60 mmol) were added. The reac-
tion mixture was stirred at r.t. for 8 h during which time the pH was
adjusted to 7.5–8.0 with i-Pr2NEt. The solvent was evaporated in
vacuo and the residue partitioned between 5% aq citric acid and
EtOAc. The aqueous layer was extracted with EtOAc (3 × 20 mL)
and the combined organic layers were washed with H2O, 5% aq
NaHCO3, brine and dried (Na2SO4). The solvent was evaporated in
vacuo and the residue purified by column chromatography using
EtOAc–hexane (9:1) as eluant to give ester 7.
IR (Nujol): 3450, 3300, 3160, 1700 cm–1.
1H NMR (CDCl3): d = 1.96 [br, s, 4 H, (CH2)2], 3.70 (br s, 4 H,
CH2NCH2), 4.81 (br s, 2 H, NH2), 5.23 (s, 2 H, CH2), 7.27–7.38 (m,
5 H, Ph), 8.04 (s, 1 H, H-5).
13C NMR (CDCl3): d = 26.4 (2), 49.7 (2), 67.11, 110.9, 120.78,
128.57, 128.65, 128.99, 129.94, 136.36, 157.67, 161.21, 163.27,
174.21.
MS (EI, 70 eV): m/z (%) = 354 (100) [M+], 325 (25), 278 (96), 249
(52), 209 (14) 177 (18), 121 (13), 91 (26), 70 (32).
HRMS–ESI: m/z [M+] calcd for C18H18N4O2S: 354.1185; found:
354.1180.
2-[(2-Amino-4-pyrrolidin-1-ylthieno[2,3-d]pyrimidine-6-carbo-
nyl)amino]propionic Acid Methyl Ester (5) (ATPC-Ala-OMe)
Obtained as a colorless solid (Method A); yield: 0.08 g (78%); mp
245 °C (decomp.); Rf 0.28 (EtOAc–MeOH, 95:5), Rf 0.52 (BuOH–
AcOH–H2O, 4:1:1); tR = 16.40 min.
IR (KBr): 3449, 3308, 2968, 2943, 2862, 1741, 1641, 1610 cm–1.
2-Amino-4-pyrrolidinothieno[2,3-d]pyrimidine-6-carboxylic
Acid (4) (ATPC)
1H NMR (DMSO-d6): d = 1.38 (d, J = 7.2 Hz, 3 H, b-CH3 of Ala),
1.95 (br s, 4 H, CH2CH2), 3.63 (s, 3 H, OCH3), 3.68 (br s, 4 H,
CH2NCH2), 4.44–4.48 (m, 1 H, a-H of Ala), 6.30 (s, 2 H, NH2), 8.21
(s, 1 H, H-5), 8.83 (d, J = 7.2 Hz, 1 H, NH).
13C NMR (DMSO-d6): d = 17.82 (b-CH3 of Ala), 25.74 (CH2CH2),
48.97 (a-CH of Ala), 49.43 (CH2NCH2), 52.80 (OCH3), 108.31,
125.21 (C-5), 157.59, 161.63, 162.68, 172.91 (CO), 174.10 (CO).
Method A: To an ice-cold stirred suspension of thienopyrimidine 3a
(0.56 g, 2 mmol) in DMSO (9 mL) and MeOH (3 mL) was added 8
M NaOH (8.6 mL). The reaction mixture was stirred vigorously at
0 °C for 30 min, then at r.t. for 16 h. The resulting solution was
cooled to 0 °C and brought to pH 5 with ice-cold 5% aq citric acid.
The white precipitate was filtered, washed with water and dried in
vacuo to yield 4 (0.48 g; 91%); mp 268 °C (decomp.).
IR (KBr): 3450, 3300, 2982, 2960 2880, 1678 cm–1.
1H NMR (DMSO-d6): d = 1.96 [br s, 4 H, (CH2)2], 3.65 (br s, 4 H,
CH2NCH2), 6.24 (br s, 2 H, NH2), 8.10 (s, 1 H, H-5).
MS (ESI): m/z = 350.12 [M + H]+.
HRMS–ESI: m/z [M + H]+ calcd for C15H20N5O3S: 350.1287;
found: 350.1298.
13C NMR (DMSO-d6): d = 26.4 (2), 49.7 (2), 127.85., 128.86,
2-[(2-Amino-4-pyrrolidin-1-ylthieno[2,3-d]pyrimidine-6-carbo-
nyl)amino]-3-phenylpropionic Acid Ethyl Ester (6) (ATPC-
Phe-OEt)
134.76, 155.92, 162.43, 162.62, 166.84.
MS (EI, 70 eV): m/z (%) = 264 (100) [M+], 235 (91), 220 (23), 209
Obtained as a colorless solid (Method A); yield: 0.10 g (76%); mp
173–175 °C; Rf 0.24 (EtOAc–hexane, 9:1); Rf 0.88 (CH2Cl2–
MeOH, 4:1); tR = 21.73 min.
(35), 191 (26), 165 (9), 148 (6), 121 (11), 70 (25).
HRMS–ESI: m/z [M+] calcd for C11H12N4O2S: 264.0681; found:
264.0686.
IR (KBr): 3346, 3217, 3028, 2976, 2874, 1732, 1658, 1637, 746
cm–1.
Method B: A solution of thienopyrimidine 3b (0.71 g, 2 mmol) in
DMF (27 mL) and MeOH (3 mL) was hydrogenated at r.t. for 6 h at
1 atm pressure in the presence of 10% Pd-C (0.45 g). The catalyst
was removed by filtration and the filtrate was evaporated to dryness
in vacuo. The oily residue was triturated with Et2O, the solid fil-
tered, washed well with Et2O, and dried in vacuo to yield 4 (0.44 g,
83%); mp 268 °C (decomp.). This compound was identical to the
1H NMR (DMSO-d6): d = 1.11 (t, J = 7.04, 7.07 Hz, 3 H,
OCH2CH3), 1.99 (br s, 4 H, CH2CH2), 3.07–3.16 (m, 2 H, b-H2 of
Phe), 3.71 (br s, 4 H, CH2NCH2), 4.07 (dd, J = 7.1, 7.0 Hz, 2 H,
OCH2CH3), 4.61–4.64 (m, 1 H, a-H of Phe), 6.31 (s, 2 H, NH2),
7.20–7.28 (m, 5 H, C6H5), 8.16 (s, 1 H, H-5), 8.87 (d, J = 7.71 Hz,
1 H, NH).
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Thieno[2,3-d]pyrimidine Amino Acid or Peptide Derivatives
3163
13C NMR (DMSO-d6): d = 13.87 (OCH2CH3), 24.79 (CH2CH2),
36.63 (b-CH2 of Phe), 48.48 (CH2NCH2), 54.06 (a-CH of Phe),
60.51 (OCH2CH3), 108.98, 124.30 (C-5), 124.51, 128.17, 128.99,
137.27, 153.20, 156.65, 160.67, 161.81, 171.66 (CO), 171.94 (CO).
MS (ESI): m/z (%) = 440.17 [M + H]+.
HRMS–ESI: m/z [M + H]+ calcd for C22H26N5O3S: 440.1756;
washed with anhyd Et2O and dried under vacuum to give TFA-H-
Asp(OBn)-NH2 (0.19 g, 0.60 mmol). To a suspension of compound
4 (0.08 g, 0.30 mmol) in DMF (4 mL) were added TFA-H-
Asp(OBz)-NH2 (0.19 g, 0.60 mmol), HOBt (0.05 g, 0.30 mmol),
TBTU (0.10 g, 0.30 mmol) and i-Pr2NEt (0.33 mL, 1.80 mmol).The
mixture was stirred at r.t. for 3 h during which time the pH was ad-
justed to 7.5–8.0 with i-Pr2NEt. The solvent was evaporated in vac-
uo and the residue partitioned between 5% aq citric acid (40 mL)
and EtOAc (40 mL). The aqueous layer was extracted with EtOAc
(2 × 40 mL) and the combined organic layers washed with H2O, 5%
aq NaHCO3, brine, and dried (Na2SO4). Evaporation in vacuo gave
a solid, which was recrystallized from EtOAc–Et2O to give com-
pound 9 (0.11 g, 78%) as a colorless solid; mp 153–156 °C; Rf 0.17,
(EtOAc–MeOH, 95:5), Rf 0.55 (BuOH–HOAc–H2O, 4:1:1); tR =
18.29 min.
found: 440.1776.
2-[(2-Amino-4-pyrrolidin-1-ylthieno[2,3-d]pyrimidine-6-carbo-
nyl)amino]-4-methylpentanoic Acid Methyl Ester (7) (ATPC-
Leu-OMe)
Obtained as a colorless solid (Method A); yield: 0.095 g (81%);
Method B, yield: 0.04 g (35%); mp 216–217 °C; Rf 0.25 (EtOAc–
hexane, 9:1); Rf 0.76 (CH2Cl2–MeOH, 4:1); tR = 20.92 min.
IR (KBr): 3449, 3296, 2957, 2868, 1741, 1678, 1610 cm–1.
IR (KBr): 3418, 3333, 3038, 2976, 2957, 2883, 1732, 1674, 1635,
740 cm–1.
1H NMR (DMSO-d6): d = 0.88 (d, J = 5.95 Hz, 3 H, d-CH3 of Leu),
0.93 (d, J = 6.02 Hz, 3 H, d-CH3 of Leu), 1.60–1.68 (m, 1 H, g-H of
Leu), 1.70–1.75 (m, 2 H, b-H2 of Leu), 1.99 (br s, 4 H, CH2CH2),
3.65 (br s, 3 H, OCH3), 3.71 (s, 4 H, CH2NCH2), 4.50–4.54 (m, 1 H,
a-H of Leu), 6.30 (s, 2 H, NH2), 8.18 (s, 1 H, H-5), 8.73 (d, J = 7.89
Hz, 1 H, NH).
13C NMR (DMSO-d6): d = 21.36 (d-CH3 of Leu), 22.80 (d-CH3 of
Leu), 24.44 (g-CH of Leu), 24.91 (CH2CH2), 41.05 (b-CH2 of Leu),
48.63 (CH2NCH2), 50.77 (OCH3), 51.98 (a-CH of Leu), 109.19,
124.35 (C-5), 124.51, 153.42, 160.28, 162.07, 172.14 (CO), 173.21
(CO).
1H NMR (DMSO-d6): d = 1.97 (br s, 4 H, CH2CH2), 2.91 (d, J = 5.97
Hz, 1 H, b-H of Asp), 2.95 (d, J = 5.99 Hz, 1 H, b-H of Asp), 3.66
(s, 4 H, CH2NCH2), 4.81–4.87 (m, 1 H, a-H of Asp), 5.09 (s, 2 H,
CH2Ph), 6.37 (s, 2 H, NH2), 7.17 (s, 1 H, CONH2), 7.27–7.37 (m, 5
H, Ph), 7.43 (s, 1 H, CONH2), 8.12 (s, 1 H, H-5), 8.75 (d, J = 8.27
Hz, 1 H, NH of Asp).
13C NMR (DMSO-d6): d = 24.75 (CH2CH2), 36.35 (b-CH2 of Asp),
48.62 (CH2NCH2), 49.74 (a-CH of Asp), 65.62 (CH2Ph), 109.063,
124.54 (C-5), 127.37, 127.72, 127.87, 128.31, 128.41, 142.85,
156.75, 160.54, 161.77, 170.28 (CO), 174.59 (CO).
MS (ESI): m/z = 392.17 [M + H]+.
HRMS–ESI: m/z [M + H]+ calcd for C18H26N5O3S: 392.1756;
MS (ESI): m/z = 469.14 [M + H]+.
HRMS–ESI: m/z [M + Na]+ calcd for C22H24N6O4SNa: 491.1474;
found: 392.1768.
found: 491.1453.
2-[(2-Amino-4-pyrrolidin-1-ylthieno[2,3-d]pyrimidine-6-carbo-
nyl)amino]-4-methylpentanoic Acid (8) (ATPC-Leu-OH)
To an ice-cold solution of compound 7 (0.059 g, 0.15 mmol) in
MeOH (1 mL), was added dropwise 1 M NaOH (0.17 mL). The
mixture was stirred at r.t. for 4 h, cooled to 0 °C and then additional
1 M NaOH (0.17 mL) was added. Stirring was continued at r.t. for
12 h. The solvent was evaporated in vacuo and the residue dissolved
in H2O (15 mL). The solution was cooled with ice and brought to
pH 2 with 1 M HCl. The colorless precipitate was extracted with
EtOAc (3 × 15 mL), the combined organic layers were washed with
brine, dried (Na2SO4) and evaporated to afford 8 (0.05 g, 88%); mp
285 °C (decomp.); Rf 0.20 (CH2Cl2–MeOH, 4:1), Rf 0.56 (BuOH–
HOAc–H2O, 4:1:1); tR = 18.72 min.
4-{2-[(2-Amino-4-pyrrolidin-1-ylthieno[2,3-d]pyrimidine-6-
carbonyl)amino]-4-methyl-pentanoylamino}-4-carbamoylbu-
tyric Acid tert-Butyl Ester (10) [ATPC-Leu-Glu(t-Bu)-NH2]
14
Z-Leu-Glu(t-Bu)-NH2 (0.36 g, 0.8 mmol) was hydrogenated in
MeOH (4.5 mL) and H2O (0.5 mL) over 10% Pd-C catalyst (0.13 g)
at r.t. for 2 h. The catalyst was filtered off and the filtrate was evap-
orated to dryness in vacuo. The oily residue was dissolved in Et2O
(30 mL) and p-toluenesulfonic acid (PTSA) (1.62 g, 0.8 mmol) was
added. The precipitate was filtered, washed with anhyd Et2O and
dried under vacuum to give PTSA.H-Leu-Glu(Ot-Bu)-NH2. To a
suspension of compound 4 (0.08 g, 0.30 mmol) in anhyd DMF (4
mL) were added PTSA-H-Leu-Glu(Ot-Bu)-NH2 (0.30, 0.60 mmol),
HOBt (0.05 g, 0.30 mmol), TBTU (0.10 g, 0.30 mmol) and i-Pr2NEt
(0.31 mL, 1.80 mmol). The mixture was stirred at r.t. for 3 h during
which time the pH was adjusted at 7.5–8.0 with i-Pr2NEt. The sol-
vent was evaporated in vacuo, sat. NaHCO3 (30 mL) was added and
the precipitate was filtered off and washed with H2O, 10% citric ac-
id, H2O, and dried under vacuum. Final purification was achieved
by preparative HPLC (Pharmacia LKB-2250) on reversed-phase
support C-18 (Lichrosorb RP) with a linear gradient from 20% to
100% MeCN (0.1% TFA) for 30 min and 100–20% MeCN (0.1%
TFA) for 5 min at a flow rate 1.5 mL/min and UV detection at 220
nm and 254 nm. Pure fractions were collected and lyophilized to
give compound 10 (0.11 g, 65%) as a colorless solid; mp 156–157
°C; Rf 0.86 (CH2Cl2–MeOH, 4:1), Rf 0.56 (MeCN–H2O, 7:1); tR =
20.25 min.
IR (KBr): 3438, 3364, 3092, 2959, 2874, 1716, 1664, 1635 cm–1.
1H NMR (DMSO-d6): d = 0.85 (d, J = 6.0 Hz, 3 H, d-CH3 of Leu),
0.90 (d, J = 6.0 Hz, 3 H, d-CH3 of Leu), 1.55–1.60 (m, 1 H, g-H of
Leu), 1.61–1.71 (m, 2 H, b-H2 of Leu), 1.96 (br s, 4 H, CH2CH2),
3.69 (br s, 4 H, CH2NCH2), 4.38–4.44 (m, 1 H, a-H of Leu), 6.29 (s,
2 H, NH2), 8.15 (s, 1 H, H-5), 8.61 (d, J = 8.4 Hz, 1 H, NH).
13C NMR (DMSO-d6): d = 22.15 (d-CH3 of Leu), 23.75 (d-CH3 of
Leu), 25.35 (g-CH of Leu), 25.55 (CH2CH2), 43.71 (b-CH2 of Leu),
49.46 (CH2NCH2), 51.51 (a-CH of Leu), 110.02, 124.89 (C-5),
157.64, 161.57, 162.81, 172.78 (CO), 175.08 (CO).
MS (ESI): m/z = 378.16 [M + H]+.
HRMS–ESI: m/z [M + H]+ calcd for C17H24N5O3S: 378.1600;
IR (KBr): 3422, 3325, 2962, 2928, 2872, 1672, 1641 cm–1.
found: 378.1617.
1H NMR (DMSO-d6): d = 0.88 (d, J = 6.26 Hz, 3 H, d-CH3 of Leu),
0.92 (d, J = 6.20 Hz, 3 H, d-CH3 of Leu), 1.37 [s, 9 H, C(CH3)3],
1.55–1.63 (m, 1 H, g-H of Leu), 1.66–1.70 (m, 2 H, b-H2 of Leu),
1.71–1.82 (m, 1 H, b-H of Glu), 1.91 (m, 1 H, b-H of Glu), 2.01 (br
s, 4 H, CH2CH2), 2.17–2.25 (m, 1 H, g-H2 of Glu), 3.78 (s, 4 H,
CH2NCH2), 4.17–4.23 (m, 1 H, a-H of Glu), 4.50–4.55 (m, 1 H, a-
H of Leu), 7.03 (s, 2 H, NH2), 7.22 (s, 2 H, CONH2), 8.04 (d, J =
3-[(2-Amino-4-pyrrolidin-1-ylthieno[2,3-d]pyrimidine-6-carbo-
nyl)amino]succinamic Acid Benzyl Ester (9) [ATPC-Asp(OBn)-
NH2]
Boc-Asp(OBn)-NH213 (0.32 g, 1 mmol) was treated with TFA–ani-
sole (10:1) for 1 h at r.t. The solvent was evaporated in vacuo and
the oily residue was triturated with anhyd Et2O, the solid filtered,
Synthesis 2005, No. 18, 3159–3166 © Thieme Stuttgart · New York

3164
E. E. Bissyris et al.
PAPER
2-{(2-Amino-4-pyrrolidin-1-ylthieno[2,3-d]pyrimidine-6-car-
7.99 Hz, 1 H, NH of Glu), 8.29 (s, 1 H, H-5), 8.74 (d, J = 7.89 Hz,
1 H, NH of Leu).
bonyl)amino}-3-methylbutyric Acid (11) (ATPC-Val-OH)
Obtained as colorless solid; yield: 0.05 g (83%); mp 262–264 °C; Rf
0.15 (CH2Cl2–MeOH, 4:1), Rf 0.50 (BuOH–HOAc–H2O, 4:1:1);
tR = 16.64 min.
13C NMR (DMSO-d6): d = 21.54 (d-CH3 of Leu), 23.09 (d-CH3 of
Leu), 24.21 (g-CH of Leu), 24.37 (CH2CH2), 27.31 (b-CH2 of Glu),
27.77 [(CH3)3], 31.31 (g-CH2 of Glu), 40.32 (b-CH2 of Leu), 49.33
(CH2NCH2), 51.73 (a-CH of Leu), 51.77 (a-CH of Glu), 79.71
[C(CH3)3], 110.26, 123.90 (C-5), 128.19, 156.94, 158.27, 161.28,
171.72 (CO), 171.96 (CO), 172.96 (CO).
MS (ESI): m/z (%) = 562.45 [M + H]+.
HRMS–ESI: m/z [M + Na]+ calcd for C26H39N7O5SNa: 584.2631;
IR (KBr): 3445, 3366, 2966, 2921, 2876, 1715, 1660, 1639 cm–1.
1H NMR (DMSO-d6): d = 0.90–0.95 (m, 6 H, g-CH3 of Val), 1.96
(br s, 4 H, CH2CH2), 2.05–2.17 (m, 1 H, b-H of Val), 3.70 (br s, 4
H, CH2NCH2), 4.25–4.29 (m, 1 H, a-H of Val), 6.28 (s, 2 H, NH2),
8.22 (s, 1 H, H-5), 8.55 (d, J = 8.40 Hz, 1 H, NH).
13C NMR (DMSO-d6): d = 20.00 (g-CH3 of Val), 20.23 (g-CH3 of
Val), 25.75 (CH2CH2), 30.75 (b-CH of Val), 49.42 (CH2NCH2),
59.05 (a-CH of Val), 110.03, 125.00 (C-5), 157.68, 161.59, 162.96
172.86 (CO), 174.06 (CO).
found: 584.2625.
ATPC-Amino Acid and Peptide Derivatives by Solid-Phase
Synthesis; General Procedure B
Compounds 11–14 were synthesized by Fmoc solid-phase method-
ology utilizing a 2-chlorotrityl-chloride resin15 as solid support.
Compound 14 was prepared utilizing a 2-chlorotrityl chloride resin
bearing a Rink–Bernatowitz linker to provide the peptide amide16
Glassware manual apparatus was used. The efficiency of the cou-
pling reactions was monitored using the ninhydrin17 or chloranil18
tests.
MS (ESI): m/z = 364.16 [M + H]+.
HRMS–ESI: m/z [M + 3 H]+ calcd for C16H24N5O3S: 366.1599;
found: 366.1592.
2-{(2-Amino-4-pyrrolidin-1-ylthieno[2,3-d]pyrimidine-6-car-
bonyl)amino}-6-tert-butoxycarbonylamino Hexanoic Acid (12)
[ATPC-Lys(Boc)-OH]
Obtained as a colorless solid; yield: 0.07 g (86%); mp 252 °C (de-
comp.); Rf 0.17 (CH2Cl2–MeOH, 4:1), Rf 0.53 (BuOH–AcOH–H2O,
4:1:1); tR = 19.28 min.
Cleavage of the Fmoc Group
The Fmoc-protective group was removed at r.t. with 20% piperidine
in DMF for 5 min followed by a second treatment with 20% piperi-
dine in DMF for 25 min as reported by Barlos et al.15 The resin was
washed three times with DMF, twice with i-PrOH and once with
Et2O. Complete Fmoc-cleavage was checked by running TLC after
releasing the derivative from an aliquot of resin with the cleavage
mixture AcOH–TFE–CH2Cl2 (1:2:7).
IR (KBr): 3440, 3364, 3080, 2974, 2920, 2874, 1720, 1690, 1668,
1631 cm–1.
1H NMR (DMSO-d6): d = 1.27–1.37 [m, 13 H, C(CH3)3, g-CH2, d-
CH2 of Lys], 1.64–1.70 (m, 1 H, b-H of Lys), 1.73–1.79 (m, 1 H, b-
H of Lys), 1.95 (br s, 4 H, CH2CH2), 2.80–2.89 (m, 2 H, e-CH2 of
Lys), 3.69 (br s, 4 H, CH2NCH2), 4.28–4.33 (m, 1 H, a-H of Lys),
6.27 (br s, 2 H, NH2), 6.74 (t, J = 5.20, 5.46 Hz, 1 H, e-NH of Lys),
8.15 (s, 1 H, H-5), 8.59 (d, J = 7.60 Hz, 1 H, NH).
13C NMR (DMSO-d6): d = 21.94 (g-CH2 of Lys), 24.40 (CH2CH2),
29.13 [(CH3)3], 29.99 (b-CH2 of Lys), 31.63 (d-CH2 of Lys), 42.39
(e-CH2 of Lys), 49.43 (CH2NCH2), 53.35 (a-CH of Lys), 78.19
[C(CH3)3], 110.03, 124.87 (C-5), 157.68, 161.59, 162.96, 172.85
(CO), 174.73 (CO).
Activation and Coupling
Fmoc-amino acid (0.50 mmol, three-fold molar excess of attached
to resin amino acid) and HOBt (0.75 mmol) were dissolved in DMF
(3 mL) and cooled to 0 °C. Diisopropylcarbodiimide (DIC, 0.55
mmol) was then added and the resulting solution was stirred for 10
min at 0 °C and for a further 10 min at 20 °C. The mixture contain-
ing the benzotriazolylester of the Fmoc-amino acid was transferred
to the solid phase reactor. After shaking for 2 h, the peptide-resin
was washed three times with DMF, twice with i-PrOH and once
with Et2O.
MS (ESI): m/z = 493.22 [M + H]+.
HRMS–ESI: m/z [M + H]+ calcd for C22H33N6O5S: 493.2233;
found: 493.2245.
Coupling of 4 with Amino Acids and Peptides
To a mixture of compound 4 (0.13 g, 0.50 mmol, three-fold excess
of the resin substitution), HOBt (0.77 g, 0.50 mmol) and TBTU
(0.16g, 0.50 mmol) in anhyd DMF (3 mL) was added i-Pr2NEt (0.35
mL, 2 mmol) and the resulting reaction mixture was transferred to
the solid phase reactor. After shaking for 6 h, the peptide-resin was
washed three times with DMF, twice with i-PrOH and once with
Et2O.
2-[(2-Amino-4-pyrrolidin-1-ylthieno[2,3-d]pyrimidine-6-carbo-
nyl)amino}-3-tert-butoxybutyric Acid (13) [ATPC-Thr(t-Bu)-
OH]
Obtained as colorless solid; yield: 0.062 g (83%); mp 174–178 °C;
Rf 0.33 (CH2Cl2–MeOH, 4:1), Rf 0.62 (BuOH–AcOH–pyridine–
H2O, 4:1:1:2); tR = 18.43 min.
IR (KBr): 3385, 2976, 2925, 2876, 1718, 1651, 1635 cm–1.
Cleavage of the ATPC-Amino Acid and Peptide Derivatives
from 2-Chlorotrityl Resin
1H NMR (DMSO-d6): d = 1.09 (d, J = 5.84 Hz, 3 H, g-CH3 of Thr),
1.12 [br s, 9 H, (CH3)3], 1.96 (br s, 4 H, CH2CH2), 3.69 (br s, 4 H,
CH2NCH2), 4.03–4.06 (m, 1 H, b-H of Thr), 4.29–4.32 (m, 1 H, a-
H of Val), 6.28 (s, 2 H, NH2), 8.12 (br s, 1 H, NH), 8.14 (s, 1 H, H-
5).
13C NMR (DMSO-d6): d = 20.83 (g-CH3 of Thr), 24.56 (CH2CH2),
28.46 [(CH3)3], 48.54 (CH2NCH2), 59.32 (a-CH of Thr), 67.86 (a-
CH of Thr), 73.21 [C(CH3)3], 123.95 (C-5), 156.81, 160.76, 161.59,
171.84 (CO), 172.55 (CO).
The peptide-resin ester was treated with a mixture of CH2Cl2–tri-
fluoroethanol–HOAc (7:2:1, 15 mL/g peptide resin) for 2 h at r.t. In
the case of compound 14 cleavage of peptide-linker bond was ac-
complished in 2 h at r.t. using a solution of TFA–CH2Cl2–anisole–
water (80:15:2.5:2.5, 15 mL/g peptide resin). The resin was separat-
ed by filtration and washed three times with the splitting mixture.
The combined filtrates were concentrated in vacuo and the crude
peptide was precipitated by addition of Et2O, collected by filtration,
washed with Et2O and dried under vacuum. Crystallization or pre-
parative HPLC provided pure products.
MS (ESI): m/z = 422.18 [M + H]+.
HRMS–ESI: m/z [M + H]+ calcd for C19H27N5O4S: 422.1862;
found: 422.1854.
Synthesis 2005, No. 18, 3159–3166 © Thieme Stuttgart · New York

PAPER
Thieno[2,3-d]pyrimidine Amino Acid or Peptide Derivatives
3165
1-(2-Amino-4-pyrrolidin-1-ylthieno[2,3-d]pyrimidine-6-carbo-
nyl)pyrrolidine-2-carboxylic Acid Amide (14) (ATPC-Pro-NH2)
Obtained as a colorless solid; yield: 0.047 g (78%); mp 235–238 °C;
Rf 0.67 (CH2Cl2–MeOH, 4:1), Rf 0.23 (BuOH–AcOH–H2O, 4:1:1);
tR = 13.47 min.
1H NMR (DMSO-d6): d = 0.87 (d, J = 6.23 Hz, 3 H, d-CH3 of Leu),
0.92 (d, J = 6.23 Hz, 3 H, d-CH3 of Leu), 1.02 (d, J = 6.31 Hz, 3 H,
g-CH3 of Thr), 1.24–1.29 (m, 2 H, g-CH2 of Lys), 1.48–1.51 (m, 2
H, d-CH2 of Lys), 1.53–1.59 (m, 1 H, g-H of Leu), 1.63–1.66 (m, 2
H, b-H2 of Leu), 1.67–1.73 (m, 2 H, b-H2 of Lys), 1.75–1.80 (m, 1
H, b-H of Gln), 1.88–1.93 (m, 1 H, b-H of Gln), 2.00 (br s, 4 H,
CH2CH2), 2.10–2.13 (m, 2 H, g-CH2 of Gln), 2.70–2.76 (m, 2 H, e-
CH2 of Lys), 2.82 (dd, J = 7.08, 7.48 Hz, 1 H, b-H of Tyr), 2.90 (dd,
J = 5.74, 5.63 Hz, 1 H, b-H Tyr), 3.77 (br s, 4 H, CH2NCH2), 3.90–
3.95 (m, 1 H, b-H of Thr), 4.20–4.22 (m, 1 H, a-H of Thr), 4.23–
4.25 (m, 1 H, a-H Gln), 4.30–4.35 (m, 1 H, a-H of Lys), 4.37–4.40
(m, 1 H, a-H Tyr), 4.53–4.56 (m, 1 H, a-H of Leu), 6.64 (d, J = 8.45
Hz, 2 H, H-3, H-5 of Tyr), 6.77 (br s, 1 H, CONH2 of Gln), 6.99 (d,
J = 8.45 Hz, 2 H, H-2, H-6 of Tyr), 7.22 (br s, 1 H, CONH2 of Gln),
7.68 (br s, 4 H, NH2, e-NH2 of Lys), 7.78 (d, J = 8.37 Hz, 1 H, NH
of Thr), 7.90 (d, J = 7.63 Hz, 1 H, NH of Lys), 7.95 (d, J = 7.97 Hz,
1 H, NH of Tyr), 8.23 (d, J = 7.37 Hz, 1 H, NH of Gln), 8.35 (s, 1
H, H-5), 8.75 (d, J = 8.08 Hz, 1 H, NH of Leu).
IR (KBr): 3450, 3375, 2982, 2878, 1680, 1614 cm–1.
1H NMR (DMSO-d6): d = 1.88 (br s, 2 H, g-H2 of Pro), 1.93 (br s, 4
H, CH2CH2), 2.06 (br s, 2 H, b-H2 of Pro), 3.43–3.46 (m, 2 H, d-H2
of Pro), 3.68 (br s, 4 H, CH2NCH2), 4.32–4.40 (m, 1 H, a-H of Pro),
6.30 (s, 2 H, NH2), 6.90 (s, 1 H, CONH2), 7.35 (s, 1 H, CONH2),
7.80 (s, 1 H, H-5).
13C NMR (DMSO-d6): d = 25.03 (g-CH2 of Pro), 25.84 (CH2CH2),
29.94 (b-CH2 of Pro), 49.06 (d-CH2 of Pro), 49.31 (CH2NCH2),
61.10 (a-CH of Pro), 125.21 (C-5), 157.59 (C-4), 161.67 (C-2),
172.91 (CO), 174.78 (CO).
MS (ESI): m/z = 361.14 [M + H]+.
HRMS–ESI: m/z [M + H]+ calcd for C16H21N6O5S: 361.1446;
13C NMR (DMSO-d6): d = 19.40 (g-CH3 of Thr), 21.53 (d-CH3 of
Leu), 22.09 (g-CH2 of Lys), 23.11 (d-CH3 Leu), 24.38 (g-CH of
Leu), 24.40 (CH2CH2), 26.63 (b-CH2 of Gln), 30.73 (b-CH2 of Lys),
31.38 (d-CH2 of Lys), 31.50 (g-CH2 of Gln), 36.19 (b-CH2 of Tyr),
38.78 (e-CH2 of Lys), 40.52 (b-CH2 of Leu), 49.28 (CH2NCH2),
51.71 (a-CH of Leu), 52.22 (a-CH of Lys), 52.40 (a-CH of Gln),
53.71 (a-CH of Tyr), 57.84 (a-CH of Thr), 66.73 (b-CH Thr),
124.35 (C-5), 110.18, 115.08, 127.10, 130.16, 156.03, 158.34,
161.67, 169.78 (CO), 173.90 (CO).
found: 361.1453.
2-{(2-Amino-4-pyrrolidin-1-ylthieno[2,3-d]pyrimidine-6-car-
bonyl)amino}-Leu-Gln-Lys-Thr-Tyr-OH (15) (ATPC-Leu-
Gln-Lys-Thr-Tyr-OH)
Compound 15 was synthesized by Fmoc solid phase methodology
utilizing a 2-chlorotrityl-chloride resin as the solid support. The first
amino acid was attached to the resin and the peptide chain was elon-
gated using Fmoc-Thr(t-Bu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-
Gln(Trt)-OH and Fmoc-Leu-OH. To an ice-cold suspension of the
suitable Fmoc-amino acid [0.50 mmol, three-fold molar excess at-
tached to resin Tyr(t-Bu)] and HOBt (0.77 g, 0.50 mmol) in DMF
(3 mL) diisopropylcarbodiimide (0.085 mL, 0.55 mmol) was added.
The mixture was stirred for 10 min at 0 °C and then for a further 10
min at r.t. The mixture containing the benzotriazolyl ester of the
Fmoc-amino acid was transferred to the solid phase reactor and
shaken for 2 h. The peptide-resin was washed three times with
DMF, twice with i-PrOH and once with Et2O. The efficiency of the
coupling reactions was monitored using the ninhydrin test. The
Fmoc-protective group was removed at r.t. with 20% piperidine in
DMF for 5 min followed by a second treatment with 20% piperidine
in DMF for 25 min. The resin was washed three times with DMF,
twice with i-PrOH and once with Et2O. Complete Fmoc-cleavage
was checked by running TLC after releasing the derivative from an
aliquot of resin with the cleavage mixture AcOH–TFE–CH2Cl2
(1:2:7). After the synthesis of Leu-Gln(Trt)-Lys(Boc)-Thr(t-Bu)-
Tyr(t-Bu)-resin was completed, a mixture of compound 4 (0.13 g,
0.50 mmol, three-fold excess of the resin substitution), HOBt (0.77
g, 0.50 mmol) and TBTU (0.16g, 0.50 mmol) in anhyd DMF (3 mL)
in which was added i-Pr2NEt (0.35 mL, 2 mmol) was transferred to
the solid phase reactor. After shaking for 6 h, the peptide-resin was
washed three times with DMF, twice with i-PrOH and once with
Et2O. Cleavage of peptide-resin bond and removal of the protecting
groups were accomplished in 4 h at r.t. using a solution of TFA–
CH2Cl2–1,2-ethanedithiol–anisole–H2O (80:10:5:3:2) (15 mL/g
peptide resin). The peptide was precipitated upon concentration of
solvent and addition of Et2O, collected by filtration, washed with
Et2O on the filter and dried in vacuo. The peptide was desalted by
gel filtration chromatography on a Sephadex G-15 column eluted
with aqueous AcOH (15%) at a flow rate of 6 mL/h. Final purifica-
tion was achieved by semi-preparative HPLC. Pure peptide frac-
tions were collected and lyophilized. The product gave a single spot
on TLC. Analytical HPLC produced single peaks with at least 98%
of the total peptide peak integrals. Yield: 0.084 g (56%); mp 132–
133 °C; Rf 0.31 (BuOH–AcOH–H2O, 4:1:1), Rf 0.52 (BuOH–
AcOH–pyridine–H2O, 4:1:1:2); tR = 16.12 min.
MS (ESI): m/z = 899.08 [M + H]+.
HRMS–ESI: m/z [M + Na]+ calcd for C41H59N11O10SNa: 920.4065;
found: 920.4059.
Side-Chain Deprotection; General Procedure C
The side-chain protected derivatives 12 or 13 (0.1 mmol) were treat-
ed with TFA–anisole (10:1, 2 mL) for 1 h at r.t. The solvent was
evaporated in vacuo and the oily residue triturated with anhyd Et2O.
The solid was filtered, washed with anhyd Et2O and dried under
vacuum to give pure 16 or 17.
6-Amino-2-{(2-amino-4-pyrrolidin-1-ylthieno[2,3-d]-pyrimi-
dine-6-carbonyl)amino}hexanoic Acid (16) (ATPC-Lys-OH)
Obtained as a colorless solid; yield: 0.027 g (96%); mp 169–171 °C
(decomp); Rf 0.05 (BuOH–HOAc–H2O, 4:1:1), Rf 0.35 (BuOH–
AcOH–pyridine–H2O, 4:1:1:2); tR = 12.21 min.
IR (KBr): 3425, 3319, 3119, 2953, 2879, 1724, 1682, 1643 cm–1.
1H NMR (DMSO-d6): d = 1.38–1.41 (m, 2 H, g-CH2 of Lys), 1.53–
1.57 (m, 2 H, d-CH2 of Lys), 1.71–1.74 (m, 1 H, b-H of Lys), 1.83–
1.85 (m, 1 H, b-H of Lys), 1.99 (br s, 4 H, CH2CH2), 2.74–2.79 (m,
2 H, e-CH2 of Lys), 3.77 (br s, 4 H, CH2NCH2), 4.34–4.40 (m, 1 H,
a-H of Lys), 7.26 (br s, 2 H, NH2), 7.72 (br s, 3 H, e-NH3 of Lys),
8.27 (s, 1 H, H-5), 8.86 (d, J = 8.0 Hz, 1 H, NH).
13C NMR (DMSO-d6): d = 23.61 (g-CH2 of Lys), 25.56 (CH2CH2),
27.41 (b-CH2 of Lys), 31.46 (d-CH2 of Lys), 41.19 (e-CH2 of Lys),
49.99 (CH2NCH2), 53.07 (a-CH of Lys), 110.90, 124.81 (C-5),
156.99, 158.31, 162.38, 174.23 (CO), 175.44 (CO).
MS (ESI): m/z = 393.17 [M + H]+.
HRMS–ESI: m/z [M + H]+ calcd for C17H25N6O5S: 393.1709;
found: 393.1721.
2-{(2-Amino-4-pyrrolidin-1-ylthieno[2,3-d]pyrimidine-6-car-
bonyl)amino}-3-hydroxybutyric Acid (17) (ATPC-Thr-OH)
Obtained as a colorless solid; yield: 0.025 g (91%); mp 187–190 °C
(decomp.); Rf 0.25 (MeCN–H2O, 7:1), Rf 0.46 (EtOAc–MeOH,
95:5); tR = 13.11 min.
IR (KBr): 3427, 3327, 2959, 2932, 2870, 1734, 1684, 1670, 1647
cm–1.
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E. E. Bissyris et al.
PAPER
IR (KBr): 3412, 3339, 3229, 2978, 2930, 2878, 1718, 1689, 1657
cm–1.
1H NMR (DMSO-d6): d = 1.10 (d, J = 5.20 Hz, 3 H, g-CH3 of Thr),
1.95 (br s, 4 H, CH2CH2), 3.76 (br s, 4 H, CH2NCH2), 4.19–4.21 (m,
1 H, b-H of Thr), 4.41–4.44 (m, 1 H, a-H of Thr), 6.97 (s, 2 H, NH2),
8.35 (s, 1 H, H-5), 8.61 (d, J = 8.00 Hz, 1 H, NH).
13C NMR (DMSO-d6): d = 21.41 (g-CH3 Thr), 24.77 (CH2CH2),
50.00 (CH2NCH2), 59.46 (a-CH of Thr), 67.18 (b-CH of Thr),
110.87, 125.25 (C-5), 157.59, 162.68, 162.82, 172.91 (CO), 172.93
(CO).
C.; Hillerman, S. M.; Soderstrom, C. I.; Knauth, E. A.; Marx,
M. A.; Rossi, A. M. K.; Sobolov, S. B.; Sun, J. Bioorg. Med.
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MS (ESI): m/z = 366.41 [M + H]+.
HRMS–ESI: m/z [M + H]+calcd for C15H19N6O4S: 366.4153; found:
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Acknowledgment
The authors thank the University of Patras for a K. Karatheodoris
Research Grant (to V.M.), Professor E. Mikros, Department of
Pharmacy, University of Athens for molecular modelling work, and
A. Cakebread and R. Tye, King’s College London, for mass spectra
obtained on machines funded by the University of London.
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Synthesis 2005, No. 18, 3159–3166 © Thieme Stuttgart · New York