Estrano[17,16-e]pyrimidine-amino acid and estrano[17,16-e]pyrimidinepeptide conjugates

Article abstract of DOI:10.3184/030823409X460722

Estrone has been converted to an aminopyrimidino [17,16-e]-annelated estrane derivative. A number of amino acids dipeptides and tripeptides have been linked to the aminopyrimidino unit of the annelated steroid. The tripeptide motifs may be used for the complexation of metals, such as of technetium as a radio label for the potential detection of estrogen positive breast cancer cells.

Full text of DOI:10.3184/030823409X460722

JOURNAL OF CHEMICAL RESEARCH 2009
JUNE, 391–396
RESEARCH PAPER 391
Estrano[17,16-e]pyrimidine-amino acid and estrano[17,16-e]pyrimidine-
peptide conjugates
Tomohiro Matsumotoa, Kodai Shiinea, Shuntaro Matakab and Thies Thiemanna,c,d
aInterdisciplinary Graduate School of Engineering Sciences and bInstitute of Materials Chemistry and Engineering, Kyushu
University, 6-1, Kasuga-koh-en, Kasuga-shi, Fukuoka 816-8580, Japan
cCIMAGO, University of Coimbra, P-3030 Coimbra, Portugal
dPresent address: College of Science, United Arab Emirates University, Al Ain, United Arab Emirates
Estrone has been converted to an aminopyrimidino [17,16-e]-annelated estrane derivative. A number of amino acids,
dipeptides and tripeptides have been linked to the aminopyrimidino unit of the annelated steroid. The tripeptide
motifs may be used for the complexation of metals, such as of technetium as a radiolabel for the potential detection
of estrogen positive breast cancer cells.
Keywords: steroids, estranes, breast cancer, diagnostic agents
Recently, a number of estradiol based steroidal metal
complexes1-3 have been proposed as potential radiodiagnostic
and therapeutic agents for estrogen receptor positive breast
cancer. In these cases, the estradiol derivative interacts with
the estrogen receptor, which is found in relatively high
concentrations in the cell nuclei of ER-positive breast cancer
cells. The use of technetium [99mTc] as the metal component
has been emphasised by a number of research groups.4,5
Inactive rhenium steroid composites6,7 have been studied as
model compounds for technetium analogues as all isotopes
of technetium itself are radioactive. In our own search for
suitable radioligands for the estrogen receptor,8-11 we have
recently examined one estrano[17,16-e]pyrimidine-tripeptide,
Cys–Gly–Cys–aminopyrimidinoestrane 1 (Fig. 1),12 where
the tripeptide was to be utilised as a ligand to bind rhenium
or technetium.
studied extensively. For this reason, a number of tripeptide
motifs are known to function as very suitable ligands for
[TcO]3+.13-16 These include Cys–Gly–Cys (2), and Me2Gly–
Ser–Cys (3) (Fig. 1). The strategy followed here is to
link a tripeptide motif to an estradiol based steroid via an
aminopyrimidine unit annelated to the estrane skeleton. We
have found previously that D-areno annelated estranes show a
good biodistribution in estrogen receptor-rich tissues.10 While
this in itself is no indication that aminopyrimidinoestranes
will exhibit an equally favourable behaviour in vivo, the
following constitutes an effort to provide a series of estrane-
tripeptide derivatives to be radiolabelled and tested in vivo at
a later stage. The work is a continuation of the preparation
of the aminopyrimidinoestrane–Cys–Gly–Cys motif,
reported previously.12 In the following, the preparations of
an aminopyrimidinoestrane linked to the Ser–Gly–Cys–,
Ser–Gly–Ser, Gly–Ala–Ser, Ser–Ala–Ser motifs are described.
Suitably protected estrones 6 were converted to 16-
hydroxymethylidene-estrones 7 according to a procedure by
Tapolcsanyi et al.17,12 As the reactivity of the 17-keto group in
The synthesis of three further estranopyrimidine-tripeptides
is reported in this paper.
During the last decade, the binding of technetium,
especially of the [TcO]3+ core, to small peptides has been
O
O
O
N
O
HN
O
NH
HN
S
O
N
N
S
NH2
H
N
N
Tc
S
S
Bn
Bn
[TcO (Cys-Gly-Cys)]
BnO
O
1
2
O
O
O
O
O
N
OH
O
O
O
NH
HN
NH
HN
O
N
N
NH2
Tc
O
O
H2N
S
S
Bn
Bn
Bn
[TcO (Me2Gly-Gly-Cys)]
Cys-Ala-Ala (N3S)
Ser-Gly-Ser (N2O2)
3
Figure 1 Protected estratetraeno[17,16-e]-2'-(Cys–Gly–Cys–amino)pyrimidine (1)12 known tripeptide motifs for [TcO]3+, 2 and 3,
and envisaged protected ligands 4 and 5.
* Correspondent. E-mail: thies@cm.kyushu-u.ac.jp

392 JOURNAL OF CHEMICAL RESEARCH 2009
O
O
OH
TFA.
NaOMe
HCO2Et
MeNHPh
benzene
benzene
or toluene
RO
7a (R = Me, 93%), 7b (R = Bn, 97%)
6a (R = Me), 6b (R = Bn)
NH2
N
O
N
Ph
N
NH
HCl
H2N
NH2
NaOMe
HCl
RO
8a (R = Me, 80%), 8b (R = Bn, 77%)
9a (R = Me, 94%), 9b (R = Bn, 86%)
Scheme 1 Preparation of 2'aminopyrimidinoestrances by the general method of Brederick.18
7 is low due to the hydrogen bonding with the enol moiety at
(b) the use of isobutyl chloroformate (IBCF) and N-
methylmorpholine (NMM),21 and (c) the use of 2,2'-dipyridyl
disulfide and triphenylphosphine.22 The introduction of N-
tert-butoxycarbonylglycine (10a) proved to be especially
troublesome, giving the desired peptidic coupling product,
when using IBCF and NMM, but in poor yield.12 Nevertheless,
for the construction of the peptidoaminopyrimidinoestranes
described in this communication, the authors reverted to using
the DCC method. Thus, initial experiments coupling of 9b
with N-(tert-butoxycarbonyl)-(L)-a-aminobutyric acid led
to the desired product 11b in 53% yield. Also, the reaction
of 9b with N-(tert-butoxycarbonyl)-(L)-a-alanine gave the
coupling product 11c in fair yield. Both coupling products
were subsequently deprotected with trifluoroacetic acid in
dry CH2Cl2 at room temperature. Finally, reaction of N-tert-
butoxycarbonylglycine using the DCC method also gave the
coupling product 11a. However the yield of 25% could not be
improved (Scheme 2).
C-16, compounds 7 were transformed into enamines 8 using
N-methylphenylammonium trifluoroacetate (Scheme 1).12
The enamines were reacted with guandiunium hydrochloride
(3 equiv.) in the presence of a base according to a general
procedure by Bredereck18 to give the corresponding 2'-
aminopyrimidinoestranes 9 in good yield (Scheme 1).12
Previously, we have carried out an X-ray crystal structural
analysis of 9a19 that showed that in the crystal the amino-
function at C-21 forms a hydrogen bond with N-2. Also, AM1
calculations showed that the electron density at N-2 is higher
than at N-1 or at the amino nitrogen. The result indicates
that protic interaction will take place predominately at N-2.
The combination of N-1, N-2 and the amino function at C-
21 gives a high electron density in the vicinity of the amino
function of the aminopyrimidinyl moiety. This is important
for evaluating the outcome of the coupling reaction with the
first amino acid.
Initially, a number of methods were screened for coupling
of N-tert-butoxycarbonyl protected amino acids 10 to 9
(Scheme 2). These included (a) the traditional DCC method,20
UsingtheDCCmethod,serinyl-aminopyrimidinoestrane12c
was reacted further with BOC-glycine and BOC-(L)-alanine
to provide the dipeptidylsteroids 13 and 14 (Scheme 3). The
NH2
O
O
N
HN
HN
N
Y
Z
N
N
N
N
Y-CO2H (10)
CF3CO2H
CH2Cl2
Method A or B
RO
11
12
9a (R = Me), 9b (R = Bn)
9a (R = Me) Method B 11a: Y = BOC-NH-CH2 (25%)
9a (R = Me) Method A 11a: Y = BOC-NH-CH2 (25%)
9b (R = Bn) Method A 11b: Y = BOC-NH(CH)CH2CH3 (53%) 12a: Z = H2N(CH)CH2CH3 (77%)
9b (R = Bn) Method A 11c: Y = BOC-NH(CH)CH3 (42%) 12b: Z = H2N(CH)CH3 (77%)
9b (R = Bn) Method A 11d: Y = BOC-NH(CH)CH2OBn (40%) 12c: Z = H2N(CH)CH2OBn (96%)
Method A: DCC, CH2Cl2; Method B: IBCF, NMM
Scheme 2 Coupling of the first amino acid to the aminopyrimidinoestrane 9.

JOURNAL OF CHEMICAL RESEARCH 2009 393
OBn
O
O
OBn
NH
HN
NHR
N
H2N
HN
N
O
N
N
BOC-NHCH2CO2H (10a)
19a: R = BOC
19b: R = H (82%)
DCC, CH2Cl2
(34%)
BnO
OBn
13
O
12c
O
OBn
NH
HN
NHR
N
HN
H2N
N
O
N
N
BOC-NHCH(CH3)CO2H (10c)
20a: R = BOC
20b: R = H (89%)
DCC, CH2Cl2
(50%)
BnO
14
12c
Scheme 3 Preparation of aminopyrimidinoestrane-dipeptides 13 and 14.
previously synthesised12 cysteinyl-aminopyrimidinoestrane
12e was also converted to dipeptide 15 (Scheme 4). For the
synthesis of the desired steroidal tripeptide conjugates, 13b,
14b, and the previously synthesised12 16 were reacted further.
While for the transformation of 14b the DCC method was
used, 13b and 16 were reacted with the N-hydroxysuccinimide
ester of the corresponding amino acid (Scheme 5). Along
with the 4-nitrophenyl-, the pentafluorophenyl-, and the tert-
butyl esters, N-hydroxysuccinimides belong to the active
ester reagents that circumvent the formation of urea as a by-
product. Also, 15b was reacted with BOC-alanine to tripeptide
21, using the DCC method (Scheme 6).
MHz). The chemical shifts are relative to TMS (solvent CDCl3,
unless otherwise noted). Mass spectra were measured with a JMS-
01-SG-2 spectrometer [electron impact mode (EI), 70 eV or fast atom
bombardment (FAB)]. Melting points were measured on a Yanaco
microscopic hotstage and are uncorrected. Column chromatography
was carried out on Wakogel C-300. All coupling experiments were
purged with argon at the start and were carried out under an argon
atmosphere.
Starting materials estrone (Wako Pure Chemicals Industries, Ltd.),
N-(butoxycarbonyl)-S-benzyl-L-cysteine (Tokyo Kasei Kogyo Co.,
Ltd), N-(butoxycarbonyl)glycine (Wako), N-(butoxycarbonyl)-L-
alanine (TCI), N-(butoxycarbonyl)-L-a-aminobutyric acid (Sigma-
Aldrich), and N-(butoxycarbonyl)-O-benzyl-L-serine (TCI) were
used as purchased. 3-O-Methylestrone (KOH, MeI, DMSO)23 and
3-O-benzylestrone (BnBr, NaH, DMF)24 were synthesised according
to known procedures. 3-Methoxy- and 3-benzyloxyestra-1,3,5(10),
In conclusion, a number of aminopyrimidinoestrane-
tripeptide conjugates have been prepared successfully.
Investigations concerning their metal complexing behaviour
are currently underway.
16-tetraeno[17,16-e]-2'-aminopyrimidines
9
were synthesised
according to
a
previously published procedure.12 N,N-
Dimethylformamide and dichloromethane were dried over CaH2 and
toluene was dried over sodium ketyl. Ethyl formate was distilled over
phosphorus pentoxide.
3-Methoxyestra-1,3,5(10),16-tetraeno[16,17-e]-2'-(N-tert-butoxy-
carbonyl-L-glycylamino)pyrimidine (11a); general method B:
N-Methyl morpholine (NMM) (0.13 mL, 1.19 mmol) and
subsequently isobutyl chloroformate (IBCF, 0.08 mL, 0.60 mmol)
were added to a solution of 9a (200 mg, 0.60 mmol) and N-(tert-
Experimental
General
IR spectra were measured with JASCO IR-700 and Nippon Denshi
JIR-AQ2OM machines. 1H and 13C NMR spectra were recorded
with a JEOL EX-270 spectrometer (1H at 270 MHz and 13C at 67.8
SBn
O
O
SBn
NH
HN
NHR
N
HN
H2N
N
O
N
N
BOC-NHCH(CH3)CO2H (10c)
15a: R = BOC
15b: R = H (96%)
DCC, CH2Cl2
(56%)
BnO
15
12e [ref. 12]
Scheme 4 Preparation of aminopyrimidinoestrane-dipeptides 15.

394 JOURNAL OF CHEMICAL RESEARCH 2009
XBn
O
O
R1
NH
O
O
NH2
NH
HN
HN
HN
R1
O
NHR
N
N
N
N
BOC-NHCH(CH2OCH2Ph)CO2Succ.
(17) for 18 and 19
O
X
BOC-NHCH(CH2OCH2Ph)CO2H,
Bn
Bn
DCC, CH2Cl2 for 20
18a: R = BOC
18b: R = H (74%)
CH2Cl2
16: X = S, R1 = H [ref. 12]
13b: X = O, R1 = H
14b: X = O, R1 = CH3
18: X = S, R1 = H (56%)
19: X = O, R1 = R = H (43% over 2 steps, incl. TFA, CH2Cl2)
20: X = O, R1 = CH3, R = H (33% over 2 steps, incl. TFA)
Scheme 5 Preparation of aminopyrimidinoestrane-tripeptides 18–20.
butoxycarbonyl)-L-glycine (10a, 157 mg, 0.89 mmol) in dry THF
(10 mL). Both additions were carried out at 0°C, and the resulting
mixture was stirred at 0°C for 1.5 h. CH2Cl2 (40 mL) was added to
the solution, which was then washed with water (20 mL) and aq.
NaHCO3 (30 mL). The organic phase was dried over anhydrous
MgSO4 and concentrated in vacuo. The residue was subjected to
column chromatography on silica gel (CH2Cl2/EtOAc 15:1) to give
11a (74 mg, 25%) as a colourless solid; m.p. 160–161°C. (Found:
MH+, 493.2814. C28H37O4N4 requires MH+, 493.2815). dH 1.00 (3H,
s, CH3), 1.48 (9H, s, But), 3.79 (3H, s, OCH3), 4.51 (2H, brs), 6.66
(1H, d, 4J = 2.7 Hz), 6.72 (1H, dd, 3J = 7.9 Hz, 4J = 2.7 Hz), 7.21 (1H,
d, 3J = 7.9 Hz), 8.27 (1H, brs, NH), 8.34 (1H, s); dC 17.2, 25.0, 26.1,
27.5, 28.4, 29.6, 32.7, 34.0, 37.6, 44.3, 46.5, 55.0, 55.2, 111.6, 114.0,
126.2, 128.8, 132.1, 137.6, 152.6, 155.6, 157.7, 183.6; MS (FAB, 3-
nitrobenzyl alcohol) m/z (%) 493 (1.2) [MH+], 492 (2.0) [M+].
3-Benzyloxy-estra-1,3,5(10),16-tetraeno[17,16-e]-2'-(N-tert-
butoxycarbonyl-L-a-alaninylamino)pyrimidine (11c): 9b (300 mg,
0.73 mmol), N-(tert-butoxycarbonyl)-(L)-a-alanine (310 mg,
1.63 mmol) and DCC (320 mg, 1.55 mmol) in dry CH2Cl2 (15 mL)
were reacted (20 h, 40°C) and subjected to work-up according to
method A. Column chromatography on silica gel (EtOAc/hexane 3:1-
EtOAc) yielded 11c (179 mg, 42%) as a colourless solid; m.p. 124–
125°C.(Found:MH+,583.3284.C35H43O4N4 requiresMH+,583.3285).
dH 1.00 (3H, s, CH3), 1.46 (9H, s, But), 3.49 (1H, brs, NH), 5.05 (2H,
4
3
s, OCH2Ph), 6.76 (1H, d, J = 2.4 Hz), 6.81 (1H, dd, J = 8.6 Hz,
3
4J = 2.6 Hz), 7.22 (1H, d, J = 8.6 Hz), 7.30–7.45 (5H, m, Ph), 8.39
(1H, s); dCꢀ17.1, 18.5, 24.9, 25.6, 26.0, 27.4, 27.5, 28.3, 29.6, 32.7, 33.9,
37.5, 44.3, 46.4, 49.2, 54.9, 69.9, 112.4, 114.9, 126.2, 127.4, 127.9,
128.6, 129.1, 132.4, 137.2, 137.7, 152.7, 155.6, 156.9, 183.5; MS (FAB,
3-nitrobenzylalcohol) m/z (%) 583 (10) [MH+], 582 (2.1) [M+].
3-Benzyloxyestra-1,3,5(10),16-tetraeno[17,16-e]-2'-(N-tert-
butoxycarbonyl-L-a-aminobutyrylamino)pyrimidine (11b); general
methodA: N,N-Dicyclohexylcarbodiimide (DCC; 350 mg, 1.69 mmol)
3-Benzyloxyestra-1,3,5(10),16-tetraeno[17,16-e]-2'-(O-benzyl-N-
tert-butoxycarbonyl-L-serinylamino)pyrimidine (11d): 9b (1.00 g,
2.43 mmol), (N-tert-butoxycarbonyl)-O-benzyl-L-serine (1.44 g,
4.86 mmol) and DCC (1.00 g, 4.86 mmol) in dry CH2Cl2 (20 mL) were
reacted (5 h, r.t.) according to method A. Column chromatography on
silica gel (CH2Cl2/EtOAc 9:1-2:1-1:1) gave 11d (665 mg, 40%) as a
was added to
a solution of 3-benzyloxy-estra-1,3,5(10),16-
tetraeno[17,16-e]-2'-aminopyrimidine (9b, 300 mg, 0.73 mmol)
and N-(tert-butoxycarbonyl)-(L)-a-aminobutyric acid (360 mg,
1.77 mmol) in dry CH2Cl2 (15 mL) and the resultant mixture was
stirred for 15 h at 40°C. The precipitated urea was removed by
filtration, and the filtrate was concentrated in vacuo. The residue was
subjected to column chromatography on silica gel (EtOAc/Hexane
2:1-EtOAc) to give 11b (230 mg, 53%) as a colourless solid, m.p.
185–187°C. (Found: MH+, 597.3437. C36H45O4N4 requires MH+,
597.3441). dH 1.00 (3H, s, CH3), 1.46 (9H, s, But), 5.04 (2H, s,
colourless solid; m.p. 74–76°C. KBr/cm-1
nmax 3734, 3329, 2929, 2852,
2360,2337,1701,1626,1574,1506,1369,1244,1165,669,420;dH (270
MHz) 1.00 (3H, s, CH3), 1.47 (9H, s, But), 5.04 (2H, s, OCH2Ph), 5.05
(2H, s, OCH2Ph), 6.76 (1H, d, 4J = 2.7 Hz), 6.79 (1H, dd, 3J = 8.6 Hz,
4J = 2.7 Hz), 7.21 (1H, d, 3J = 8.6 Hz), 7.30–7.45 (10H, m, 2 Ph), 8.41
(1H, s); dC 13.7, 16.6, 24.4, 25.1, 25.6, 26.9, 27.1, 27.8, 29.1, 32.3,
33.4, 37.1, 43.8, 45.9, 48.8, 54.4, 58.4, 59.9, 62.1, 69.2, 69.5, 73.1,
111.9, 114.4, 125.7, 127.0, 127.1, 127.4, 127.8, 127.9, 128.0, 128.1,
128.8, 131.9, 136.7, 137.2, 152.1, 155.1, 156.4, 182.9; MS (FAB, 3-
nitrobenzyl alcohol) m/z (%) 691 (11.5) [MH+], 690 (24.5) [M+].
3-Benzyloxy-estra-1,3,5(10),16-tetraeno[17,16-e]-2'-(L-a-amino-
butyrylamino)pyrimidine (12a); general Method C: 11b (67.9 mg,
0.114 mmol) was dissolved in dry CH2Cl2 (10 mL). Trifluoroacetic
acid (0.4 mL, 5.18 mmol) was added to the solution, and the reaction
4
3
4
OCH2Ph), 6.76 (1H, d, J = 2.4 Hz), 6.81 (1H, dd, J = 8.6 Hz, J
3
= 2.6 Hz), 7.22 (1H, d, J = 8.6 Hz), 7.30–7.45 (5H, m, Ph), 8.40
(1H, s); dCꢀ10.0, 17.2, 24.9, 25.5, 25.9, 26.0, 27.4, 28.3, 29.5, 27.4,
29.5, 31.6, 32.7, 33.8, 37.5, 44.3, 46.5, 54.9, 69.9, 112.4, 114.9,
126.2, 127.4, 127.9, 128.6, 132.3, 137.2, 137.7, 152.7, 155.6, 156.8,
183.5; MS (FAB, 3-nitrobenzylalcohol) m/z (%) 597 (0.99) [MH+]
596 (0.36) [M+].
SBn
O
O
NH
O
O
NH2
NH
HN
HN
HN
H3C
O
N
N
N
N
H2N
S
BOC-NHCH(CH3)CO2H
DCC
Bn
CH2Cl2
21: (40% over 2 steps, incl. TFA, CH2Cl2)
Scheme 6 Preparation of aminopyrimidinoestrane-tripeptides 21.
15b

JOURNAL OF CHEMICAL RESEARCH 2009 395
mixture was stirred for 17 h at r.t. CH2Cl2 (10 mL) was added to the
solution, and thereafter the mixture was washed with aq. Na2CO3
(2 ¥ 20 mL) and water (2 ¥ 20 mL). The organic phase was dried
over anhydrous MgSO4 and concentrated in vacuo to give 12a (43.7
mg, 77%) as a slowly crystallising, colourless solid. (Found: MH+,
497.2917. C31H37O2N4 requires MH+, 497.2917). dH 1.00 (3H, s, CH3),
27.4, 27.5, 28.3, 29.6, 32.8, 37.5, 44.3, 46.5, 53.3, 54.9, 69.2, 70.0,
73.6, 112.4, 114.9, 126.2, 127.5, 127.9, 128.5, 128.6, 129.4, 132.4,
137.2, 137.3, 137.7, 152.6, 155.5, 156.9, 172.7, 183.4; MS (FAB,
3-nitrobenzyl alcohol) m/z (%) 761 (2.1) [MH+], 760 (3.4) [M+].
3-Benzyloxy-estra-1,3,5(10),16-tetraeno[17,16-e]-2'-(L-alanyl-O-
benzyl-L-serinylamino)pyrimidine (14b): 14a (79 mg, 0.10 mmol)
and trifluoroacetic acid (2ꢀ ¥ꢀ [0.1 mL, 1.3 mmol]) in dry CH2Cl2
(5 mL) were reacted (1h, r.t., then 1h, r.t.) and subjected to work-
up according to method C to give 14b (60.8 mg, 89%) as a slowly
solidifying colourless oil. KBr/cm-1 nmax 3430, 2926, 2360, 1668,
1500, 1456, 1255, 1105, 809, 732; dH 0.98 (3H, s, CH3), 5.05 (2H,
4
1.46 (9H, s, But), 5.04 (2H, s, OCH2Ph), 6.76 (1H, d, J = 2.4 Hz),
3
4
3
6.81 (1H, dd, J = 8.6 Hz, J = 2.6 Hz), 7.22 (1H, d, J = 8.6 Hz),
7.30–7.45 (5H, m, Ph), 8.46 (1H, s); dCꢀ10.3, 17.1, 24.9, 24.9, 25.6,
26.0, 27.4, 27.5, 29.6, 32.7, 33.9, 37.5, 44.3, 46.4, 54.8, 69.9, 112.3,
114.8, 126.1, 127.4, 127.9, 128.5, 132.4, 137.2, 137.7, 152.7, 155.9,
156.8, 183.3; MS (FAB, 3-nitrobenzylalcohol) m/z (%) 497 (1.3)
[MH+], 496 (0.41) [M+].
4
3
s, OCH2), 6.76 (1H, d, J = 2.7 Hz), 6.79 (1H, dd, J = 8.6 Hz,
3
4J = 2.7 Hz), 7.22 (1H, d, J = 8.6 Hz), 7.30–7.45 (10H, m), 8.40
3-Benzyloxy-estra-1,3,5(10),16-tetraeno[17,16-e]-2'-(L-a-
alaninylamino)pyrimidine (12b): 11c (60 mg, 0.124 mmol) and
trifluoroacetic acid (0.6 mL, 7.8 mmol) in dry CH2Cl2 (7 mL) were
reacted (13 h, r.t.) and subjected to work-up according to method C
to give 12b (21 mg, 77%) as a slowly crystallising, colourless solid.
(Found: MH+, 483.2758. C30H35O2N4 requires MH+, 483.2760).
dH 1.00 (3H, s, CH3), 1.25–2.03 (m, 7H) 1.47 (3H, d, CH3(ala),
3J=6.8Hz), 2.29–2.58(4H, m), 2.81–2.97(3H, m), 3.81(1H, brs, NH),
5.05(2H,s,OCH2Ph),6.76(1H,d,4J=2.4Hz),6.82(1H,dd,3J=8.6Hz
4J = 2.6 Hz), 7.22 (1H, d, 3J = 8.6 Hz), 7.30–7.45 (5H, m, Ph), 8.45
(1H, s), 10.0 (2H, brs, NH2); 13C NMR (67.8 MHz, CDCl3) dCꢀ17.1,
21.4, 26.0, 27.4, 27.5, 29.5, 32.7, 37.5, 44.3, 46.4, 51.5, 54.8, 69.9,
112.3, 114.8, 126.1, 127.4, 127.9, 128.5, 129.0, 132.4, 137.2, 137.7,
152.7, 155.8, 156.8, 183.3; MS (FAB, 3-nitrobenzylalcohol): m/z (%)
483 (MH+, 12.69), 482 (M+, 1.45).
(1H, s); MS (FAB, 3-nitrobenzyl alcohol) m/z (%) 660 (7.2) [MH+],
659 (6.5) [M+].
3-Benzyloxy-estra-1,3,5(10),16-tetraeno[17,16-e]-2'-(N-tert-
butoxycarbonyl-L-alanyl-S-benzyl-L-cysteinylamino)pyrimidine
(15a): 12e (94 mg, 0.16 mmol), N-tert-butoxycarbonyl)-L-alanine
(59 mg, 0.31 mmol) and DCC (64 mg, 0.31 mmol) in dry CH2Cl2
(10 mL) were reacted (18 h, r.t.) and subjected to work-up according
to method B. Column chromatography on silica gel (CH2Cl2/EtOAc
3:1–1:1) gave 15a (67 mg, 56%) as a colourless solid, m.p. 134–
136°C. KBr/cm-1 nmax 3423, 2925, 2852, 2360, 1703, 1651, 1506,
1456, 1419, 1373, 1246, 1167, 1024, 696, 418; dH 1.00 (3H, s, CH3),
3
1.37 (3H, d, J = 6.8 Hz, CH3[Ala]), 1.45 (9H, s, But), 3.78 (2H, s,
SCH2), 5.05 (2H, s, OCH2), 6.74 (1H, d, 4J = 2.3 Hz), 6.79 (1H, dd,
3J = 8.6 Hz, 4J = 2.3 Hz), 7.21 (1H, d, 3J = 8.6 Hz), 7.28–7.45 (10H,
m), 8.39 (1H, s); dC 17.1, 18.4, 24.9, 25.6, 26.1, 27.4, 27.5, 28.3, 29.6,
32.7, 33.5, 34.0, 36.7, 37.5, 44.3, 46.5, 49.2, 50.3, 52.7, 54.9, 70.0,
112.4, 114.9, 126.2, 127.2, 127.5, 127.9, 128.6, 129.1, 129.5, 132.4,
137.2, 137.7, 138.0, 152.6, 155.4, 156.9, 172.8, 183.6; MS (FAB,
3-nitrobenzyl alcohol) m/z (%) 777 (2.9) [MH+], 776 (3.8) [M+].
3-Benzyloxy-estra-1,3,5(10),16-tetraeno[17,16-e]-2'-(L-alanyl-S-
benzyl-L-cysteinylamino)pyrimidine (15b): 15a (62 mg, 0.08 mmol)
and trifluoroacetic acid [2 ¥ (0.1 mL, 1.3 mmol) in dry CH2Cl2 (5 mL)
were reacted (3 h, r.t., then 3 h, r.t.) and subjected to work-up according
to method C to give 15b (51.6 mg, 96%) as a colourless solid; m.p.
160–162°C (dec.). (Found: MH+, 676.3325. C40H46O3N5S requires
3-Benzyloxy-estra-1,3,5(10),16-tetraeno[17,16-e]-2'-(O-benzyl-
L-serinylamino)pyrimidine (12c): 11d (174 mg, 0.25 mmol) and
trifluoroacetic acid (2ꢀ ¥ꢀ [0.24 mL, 3.12 mmol]) in dry CH2Cl2
(10 mL) were reacted (1 h, r.t.; then 3 h, r.t.) and subjected to work-up
according to method C to give 12c (143 mg, 96%) as a colourless solid;
m.p. 84–86°C (dec.). (Found: MH+, 589.3175. C37H41O3N4 requires
MH+, 589.3179). KBr/cm-1
nmax 3327, 2927, 2852, 2360, 1711, 1626,
1576, 1497, 1419, 1311, 1244, 1090, 808, 735, 696, 644; dH 1.00 (3H,
s, CH3), 4.57 (2H, s, OCH2Ph), 5.05 (2H, s, OCH2Ph), 6.74 (1H, d, 4J =
2.6 Hz), 6.78 (1H, dd, 3J = 8.6 Hz, 4J = 2.6 Hz), 7.21 (1H, d, 3J = 8.6 Hz),
7.25–7.46 (10H, m), 8.45 (1H, s); dC 17.1, 24.9, 25.6, 26.1, 27.4, 27.6,
29.6, 32.8, 34.0, 37.5, 44.3, 46.4, 49.1, 54.9, 70.0, 71.9, 73.4, 112.4,
114.9, 126.1, 127.5, 127.7, 127.9, 128.5, 128.6, 129.2, 132.5, 137.2,
137.7, 152.7, 156.7, 156.9, 183.4; MS (FAB, 3-nitrobenzyl alcohol)
m/z (%) 590 (12.6) [MH+], 589 (29.9) [M+].
MH+, 676.3321). KBr/cm-1
nmax 3329, 2927, 2852, 1668, 1626, 1574,
1498, 1454, 1419, 1313, 1244, 1103, 1024, 806, 698; dH 0.98 (3H, s,
4
CH3), 5.04 (2H, s, OCH2), 6.75 (1H, d, J = 2.7 Hz), 6.78 (1H, dd,
3J = 8.6 Hz, 4J = 2.7 Hz), 7.21 (1H, d, 3J = 8.6 Hz), 7.29–7.45 (10H,
m), 8.07 (1H, s); dC 15.3, 17.1, 20.0, 24.9, 25.6, 26.2, 27.2, 27.4, 27.5,
29.6, 29.7, 32.8, 33.9, 35.3, 36.7, 37.5, 44.3, 46.2, 46.4, 49.2, 50.9,
53.9, 54.8, 65.9, 70.0, 112.4, 114.9, 123.3, 126.2, 127.5, 127.9, 128.6,
128.8, 128.9, 129.1, 132.6, 137.3, 137.4, 137.7, 137.8, 152.6, 156.9,
162.2, 166.8, 168.3, 183.3; MS (FAB, 3-nitrobenzyl alcohol) m/z (%)
676 (33.8) [MH+].
3-Benzyloxy-estra-1,3,5(10),16-tetraeno[17,16-e]-2'-(N-tert-
butoxycarbonyl-L-glycyl-O-benzyl-L-serinylamino)pyrimidine (13a):
12c (380 mg, 0.65 mmol), N-(tert-butoxycarbonyl)-L-glycine (10a,
226 mg, 1.29 mmol) and DCC (267 mg, 1.29 mmol) in dry CH2Cl2
(25 mL) were reacted (5 h, r.t.) according to method B. Column
chromatography on silica gel (CH2Cl2/EtOAc 3:1–1:1) gave 13a
(165 mg, 34%) as a slowly crystallising colourless oil. (Found: MH+,
746.3919. C44H52O6N5 requires MH+, 746.3918). KBr/cm-1 nmax
3427, 2924, 2856, 2360, 1664, 1500, 1458, 1375, 1248, 1167, 735,
696; dH 1.00 (3H, s, CH3), 1.45 (9H, s, But), 5.05 (2H, s, OCH2), 6.76
3-Benzyloxy-estra-1,3,5(10),16-tetraeno[17,16-e]-2'-(O-benzyl-
N-tert-butoxycarbonyl-L-serinyl-L-glycyl-S-benzyl-L-cysteinyl
amino)pyrimidine (18a): A solution of 16 (60 mg, 0.09 mmol) and
(N-tert-butoxycarbonyl-O-benzyl-L-serylsuccinimide (17, 53 mg,
0.14 mmol) in dry CH2Cl2 (5 mL) was stirred for 23 h at r.t.
The solution was concentrated in vacuo and the residue was subjected
to column chromatography on silica gel (CH2Cl2/EtOAc 1:1) to give
18a (48 mg, 56%) as a slowly crystallising colourless oil. (Found:
MH+, 939.4484. C54H63O7N6S requires MH+, 939.4479). KBr/cm-1
4
3
4
(1H, d, J = 2.7 Hz), 6.79 (1H, dd, J = 8.6 Hz, J = 2.7 Hz), 7.22
3
(1H, d, J = 8.6 Hz), 7.30–7.45 (10H, m), 8.40 (1H, s); MS (FAB,
3-nitrobenzyl alcohol) m/z (%) 746 (27.5) [MH+].
3-Benzyloxy-estra-1,3,5(10),16-tetraeno[17,16-e]-2'-(L-glycyl-O-
benzyl-L-serinylamino)pyrimidine (13b): 13a (157 mg, 0.21 mmol)
and trifluoroacetic acid [2ꢀ¥ꢀ(0.21 mL, 2.73 mmol)] in dry CH2Cl2
(9 mL) were reacted (1h, r.t., then 1h, r.t.) and subjected to work-
up according to method C to give 13b (111 mg, 82%) as a slowly
solidifying colourless oil. KBr/cm-1 nmax 3429, 2924, 2360, 1668,
1498, 1456, 1252, 1105, 806, 735, 696; dH 0.98 (3H, s, CH3), 5.04
(4H, brs and s, NH2, OCH2), 6.75 (1H, d, 4J = 2.7 Hz), 6.79 (1H, dd,
3J = 8.6 Hz, 4J = 2.7 Hz), 7.21 (1H, d, 3J = 8.6 Hz), 7.30–7.45 (10H,
m), 8.07 (1H, s); MS (FAB, 3-nitrobenzyl alcohol) m/z (%) 646 (9.8)
[MH+], 645 (7.8) [M+].
n
max 3430, 2930, 1668, 807; dH 0.90 (3H, s, CH3), 1.34 (9H, s, But),
5.00 (2H, s, OCH2), 6.67 (1H, d, 4J = 2.3 Hz), 6.70 (1H, dd, 3J = 8.6 Hz,
4J = 2.3 Hz), 7.12 (1H, d, 3J = 8.6 Hz), 7.18–7.37 (15H, m), 8.30 (1H,
s); MS (FAB, 3-nitrobenzyl alcohol) m/z (%) 939 (51.3) [MH+].
3-Benzyloxy-estra-1,3,5(10),16-tetraeno[17,16-e]-2'-(O-benzyl-
L-serinyl-L-glycyl-S-benzyl-L-cysteinylamino)pyrimidine
(18b):
Trifluoroacetic acid (0.06 mL, 0.78 mmol) was added to a solution
of 18a (43 mg, 0.046 mmol) in dry CH2Cl2 (3 mL) and the resulting
reaction mixture was stirred for 1 h at r.t. Then, further trifluoroacetic
acid (0.06 mL, 0.78 mmol) was added to the solution, which was
stirred for another hour at r.t. CH2Cl2 (30 mL) was added, and
the solution was washed with aq. NaHCO3 (20 mL) and water
(2 ¥ 20 mL). The organic layer was dried over anhydrous Na2SO4
and concentrated in vacuo to give 18b (28.7 mg, 74%) as a slowly
solidifying colourless oil. (Found: MH+, 839.3951. C49H55O5N6S
requires MH+, 839.3955). KBr/cm-1 nmaxꢀ 3345,ꢀ 2928,ꢀ 2359,ꢀ 1640,ꢀ
809,ꢀ737;ꢀdH 0.98 (3H, s, CH3), 5.04 (2H, s, OCH2), 6.75 (1H, d,
3-Benzyloxy-estra-1,3,5(10),16-tetraeno[17,16-e]-2'-(N-tert-
butoxycarbonyl-L-alanyl-O-benzyl-L-serinylamino)pyrimidine (14a):
12c (123 mg, 0.21 mmol), N-(tert-butoxycarbonyl)-L-alanine (16c,
79 mg, 0.42 mmol), and DCC (86.5 mg, 0.42 mmol) in dry CH2Cl2
(10 mL) were reacted (19 h, r.t.) and subjected to work-up according
to method B. Column chromatography on silica gel (CH2Cl2/EtOAc
3:1–1:1) gave 14a (79 mg, 50%) as a slowly crystallising colourless
oil. KBr/cm-1
n
max 3425, 2927, 2360, 1653, 1502, 1454, 1369, 1245,
4J = 2.7 Hz), 6.79 (1H, dd, J = 8.6 Hz, J = 2.7 Hz), 7.21 (1H, d,
3J = 8.6 Hz), 7.29–7.45 (10H, m), 8.36 (1H, s); MS (FAB, 3-nitro-
benzyl alcohol) m/z (%) 839 (3.6) [MH+].
3
4
1167, 1105, 735, 698; dH 1.00 (3H, s, CH3), 1.38 (3H, d, 3J = 6.8 Hz,
CH3[Ala]), 1.44 (9H, s, But), 5.05 (2H, s, OCH2), 6.76 (1H, d,
4J = 2.7 Hz), 6.79 (1H, dd, J = 8.6 Hz, J = 2.7 Hz), 7.22 (1H, d,
3-Benzyloxy-estra-1,3,5(10),16-tetraeno[17,16-e]-2'-(O-benzyl-L-
serinyl-L-glycyl-O-benzyl-L-serinylamino)pyrimidine (19):Asolution
3
4
3J = 8.6 Hz), 7.27–7.45 (10H, m), 8.40 (1H, s); dC 17.1, 18.7, 26.1,

396 JOURNAL OF CHEMICAL RESEARCH 2009
of 13b (64 mg, 0.10 mmol) and N-(tert-butoxycarbonyl)-O-
benzyl-L-serylsuccinimide (17, 58 mg, 0.15 mmol) in dry CH2Cl2
(5 mL) was stirred for 17 h at r.t. The solution was concentrated in
vacuo and the residue was subjected to column chromatography
on silica gel (CH2Cl2/EtOAc 1:1 – 1:2) to give BOC-protected 19
(45.8 mg, 50%). BOC-protected 19 (45.8 mg, 0.05 mmol) was
dissolved in dry CH2Cl2 (3 mL) and trifluoroacetic acid (0.06 mL,
0.78 mmol) was added. The resultant solution was stirred for 1 h at r.t.
Then, additional trifluoroacetic acid (0.06 mL, 0.78 mmol) was added
and the solution stirred for 2 h at r.t. CH2Cl2 (30 mL) was added to
the solution, which was washed with aq. NaHCO3 (20 mL) and water
(2 ¥ 20 mL). The organic phase was dried over anhydrous Na2SO4
and concentrated in vacuo to give 19 (35 mg, 86%) as a slowly
solidifying oil. KBr/cm-1 nmaxꢀ3341,ꢀ2926,ꢀ2360,ꢀ1636,ꢀ1508,ꢀ1455,ꢀ
1418,ꢀ1241,ꢀ1103,ꢀ1027,ꢀ809,ꢀ737,ꢀ697;ꢀdHꢀ0.92 (3H, s, CH3), 4.98
(2H, s, OCH2), 6.68 (1H, d, 4J = 2.6 Hz), 6.71 (1H, dd, 3J = 8.6 Hz,
4J = 2.6 Hz), 7.14 (1H, d, 3J = 8.6 Hz), 7.20–7.38 (10H, m), 8.00 (1H,
s); MS (FAB, 3-nitrobenzyl alcohol) m/z (%) 823 (65.7) [MH+].
3-Benzyloxyestra-1,3,5(10),16-tetraeno[17,16-e]-2'-(L-serinyl-
L-alanyl-O-benzyl-L-serinylamino)pyrimidine (20): DCC (38 mg,
0.18 mmol), was added to a solution of 14b (61 mg, 0.09 mmol) and
N-(tert-butoxycarbonyl)-O-benzyl-L-serine (17, 54 mg, 0.18 mmol)
in dry CH2Cl2 (7 mL) and the resulting reaction was stirred for 5 h
at r.t. The precipitated urea was removed by filtration, and the filtrate
was concentrated in vacuo. The residue was subjected to column
chromatography on silica gel (CH2Cl2/EtOAc 2:1-1:1) to give the
BOC-protected Ala-Ala-Cys-aminopyrimidinoestrane (31 mg, 36%).
The compound was taken up in CH2Cl2 (3 mL), trifluoroacetic acid
(0.05 mL, 0.65 mmol) was added to the solution, and the mixture
was stirred for 1 h at r.t. Then, further trifluoroacetic acid (0.05 mL,
0.65 mmol) was added to the solution, which was stirred for an
additional hour at r.t. CH2Cl2 (30 mL) was added to the mixture,
which was washed with aq. NaHCO3 (20 mL) and water (2 ¥ 20 mL).
The organic phase was dried over anhydrous MgSO4 and concentrated
in vacuo to give 20 (24 mg, 91%) as a slowly crystallising solid.
(Found: MH+, 837.4335. C50H57O6N6 requires MH+, 837.4340).
KBr/cm-1 nmaxꢀ 3337, 2927, 2856, 2360, 1636, 1604, 1499, 1454,
1419, 1375, 1240, 1103, 1027, 809, 735, 697; dH 0.98 (3H, s, CH3),
1.39 (3H, d, 3J = 6.8 Hz, Me[Ala]), 5.05 (2H, s, OCH2), 6.75 (1H,
d, 4J = 2.7 Hz), 6.79 (1H, dd, 3J = 8.6 Hz, 4J = 2.7 Hz), 7.21 (1H, d,
3J = 8.6 Hz), 7.29–7.45 (10H, m), 8.40 (1H, s); MS (FAB,
3-nitrobenzyl alcohol) m/z (%) 837 (8.2) [MH+].
(2 ¥ 20 mL). The organic phase was dried over anhydrous MgSO4
and concentrated in vacuo to give 21 (22.8 mg, 95%) as a slowly
solidifying solid. KBr/cm-1 nmaxꢀ3329, 2927, 2853, 2359, 1648, 1605,
1560, 1498, 1455, 1424, 1377, 1258, 1105, 1027, 808, 736, 696;
d
H 0.98 (3H, s, CH3), 5.04 (2H, s, OCH2), 6.75 (1H, d, 4J = 2.6 Hz),
3
4
3
6.78 (1H, dd, J = 8.6 Hz, J = 2.6 Hz), 7.21 (1H, d, J = 8.6 Hz),
7.29–7.45 (10H, m), 8.07 (1H, s); MS (FAB, 3-nitrobenzyl alcohol)
m/z (%) 747 (48.3) [MH+].
Received 5 February 2009; accepted 14 April 2009
Paper 09/0435 doi: 10.3184/030823409X460722
Published online: 30 June 2009
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42%). It was taken up in dry CH2Cl2 (3 mL), trifluoroacetic acid
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