Estrano[17,16-e]pyrimidine-peptide conjugates

Article abstract of DOI:10.1016/S0039-128X(03)00145-4

The preparation of a steroidal heterocycle linked to the tripeptide Cys-Gly-Cys is described. Initially, an estrane-derived steroidal heterocycle containing an aminopyrimidine ring fused to the 16,17-position of the steroidal nucleus was synthesized. Thereafter, protected amino acids were coupled iteratively by the DCC method, commencing at the amino group of the aminopyrimidine unit.

Full text of DOI:10.1016/S0039-128X(03)00145-4

Steroids 68 (2003) 751–757
Estrano[17,16-e]pyrimidine-peptide conjugates
Tomohiro Matsumotoa, Masataka Watanabea, Shuntaro Matakab, Thies Thiemannb,∗
a
Interdisciplinary Graduate School of Engineering Sciences, Kyushu University, Kyushu, Japan
b
Institute of Materials Chemistry and Engineering, Kyushu University, 6-1 Kasuga-koh-en Kasuga-shi, Fukuoka 816-8580, Japan
Received 30 May 2003; accepted 3 July 2003
Abstract
The preparation of a steroidal heterocycle linked to the tripeptide Cys-Gly-Cys is described. Initially, an estrane-derived steroidal
heterocycle containing an aminopyrimidine ring fused to the 16,17-position of the steroidal nucleus was synthesized. Thereafter, protected
amino acids were coupled iteratively by the DCC method, commencing at the amino group of the aminopyrimidine unit.
© 2003 Elsevier Inc. All rights reserved.
Keywords: Synthesis; Estranes; Fused steroids; Peptides; Pyrimidines
1. Introduction
conjugates. In the following, the synthesis of an estra-
1,3,5(10),16-tetraeno[17,16-e]pyrimidine is described, and
its transformation to a conjugate with a protected tripeptide
fragment Cys-Gly-Cys, where Cys-Gly-Cys represents a
suitable motif for binding rhenium and technetium.
Peptidyl steroids, where amino acids or peptides are
linked to the steroidal frame, have been reported to play im-
portant roles in the processes in living systems and some of
these, such as cholyl glycine and cholyl taurine, have been
found in the human body. These endogenous bile acid amino
acid conjugates have led to the development of labelled
analogs for clinical purposes, such as for the [14C]-cholyl-
glycine breath test [1], but have also been held possibly
responsible for triggering illnesses such as gastrointestinal
cancer via their nitroso-derivatives [2]. Within this context
[3] and for other purposes [4] a number of cholic acid peptide
conjugates have been prepared. Furthermore, peptide conju-
gates of the androstane series have been reported [5–7]. Less
frequent are studies on peptide derivatives of the estrane
series. Here, the preparation of 17␤peptidylestra-1,3,5(10)-
trien-3-ols [8] as well as of 11␤-estradiol dolastatine-15 and
dolastatine-10 derivatives [9] have to be mentioned. Never-
theless, estrane derivatives play a decisive role in targeting
a number of estrogen-modulated illnesses such as estrogen
receptor positive breast cancer [10]. In this area research
focusses both on the development of estrane-derived radi-
olabelled compounds as diagnostic agents [11] as well as
on estrane-derived drug delivery systems with a good bind-
ing affinity to the estrogen receptor. In their own studies
on radiolabelled estradiol derivatives [12,13], the authors
have become interested to find new connective approaches
to radiolabelled estrane derivatives and to peptide estrane
2. Experimental
Starting materials estrone (Wako Pure Chemical Indus-
tries, Ltd.), N-(butoxycarbonyl)-S-benzyl-l-cysteine (Tokyo
Kasei Kogyo Co, Ltd.) and N-(butoxycarbonyl)glycine
(Wako Pure Chemical Industries, Ltd.) were used as pur-
chased. 3-O-Methylestrone (KOH, MeI, DMSO [14]) and 3-
O-benzylestrone (BnBr, NaH, DMF [15]) were synthesized
analogous to known procedures. N,N-Dimethylformamide
and dichloromethane were dried over CaH2 and toluene
was dried over sodium ketyl. Ethyl formate was distilled
over phosphorous pentoxide. Melting points were measured
on a Yanaco microscopic hotstage and are uncorrected.
Infrared spectra were measured with JASCO IR-700 and
1
Nippon Denshi JIR-AQ2OM machines. H and 13C NMR
spectra were recorded with a JEOL EX-270 spectrometer.
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)). Values of opti-
cal rotation were obtained using SEPA-200 high sensitive
polarimeter (Horiba). Column chromatography was carried
out on Wakogel 300. All coupling experiments were purged
with argon at the start and were carried out under an argon
atmosphere.
∗
Corresponding author. Tel.: +81-92-583-7812; fax: +81-92-583-7811.
E-mail address: thies@cm.kyushu-u.ac.jp (T. Thiemann).
0039-128X/$ – see front matter © 2003 Elsevier Inc. All rights reserved.
doi:10.1016/S0039-128X(03)00145-4

752
T. Matsumoto et al. / Steroids 68 (2003) 751–757
2.1. 16-Hydroxymethylidene-3-benzyloxyestrone (4b)
77%) after column chromatography of the crude product on
silica gel (eluant: CH2Cl2–CH2Cl2/EtOAc 5:1–2:1–1:1);
mp 203–204 ◦C; [α]D = +228◦ (chloroform, c = 0.2); IR
(KBr): 2925, 1691, 1606, 1577, 1498, 1362, 1244, 1173,
4b was synthesized analagous to a procedure by Tapolc-
sanyi et al. [16]. The reaction of 3 (6.12 g, 17.0 mmol),
sodium methoxide (1.83 g, 33.9 mmol), ethyl formate
(20.4 ml) in dry toluene (30 ml) gave 4b (6.41 g, 97%). The
spectral data not provided in [16] is given as follows: mp
112–113 ◦C; IR (KBr): 3452, 3028, 2924, 2870, 1736, 1608,
1568, 1496, 1454, 1369, 1281, 1250, 1159, 1103, 1055,
1128, 1018, 914, 756 cm−1; H NMR (270 MHz, CDCl3):
1
δH 0.93 (s, 3H, CH3), 3.51 (s, 3H, NCH3), 5.03 (s, 2H,
4
OCH2), 6.72 (d, 1H, ArH, J 2.5 Hz), 6.77 (dd, 1H, ArH,
3
4J 2.4 Hz, J 7.9 Hz), 7.11 ∼ 7.16 (m, 3H, Ph), 7.19 (d,
3
1H, ArH, J 8.0 Hz), 7.28 ∼ 7.45 (m, 7H, Ph), 7.61 (s,
1H); 13C NMR (67.8 MHz, CDCl3): δH 14.9, 26.1, 26.8,
27.8, 29.7, 31.9, 37.8, 39.6, 44.2, 47.1, 49.5, 70.0, 108.4,
112.3, 114.9, 120.8, 124.3, 126.3, 127.5, 127.9, 128.6,
129.2, 132.8, 137.3, 137.8, 141.3, 146.5, 156.8, 210.0; MS
(FAB, 3-nitrobenzylalcohol): m/z (%) 478 (MH+, 28), 477
(M+, 16); HRMS found: 478.2750 calcd. for C33H36O2N:
478.2746.
1
1026, 903, 808, 781, 758, 731, 698, 515 cm−1; H NMR
(270 MHz, CDCl3): δH 0.98 (s, 3H, CH3), 1.48 ∼ 1.65 (m,
5H), 1.94 ∼ 2.56 (m, 5H), 2.89 ∼ 2.91 (m, 2H), 5.04 (s,
4
2H, OCH2), 6.73 (d, 1H, ArH, J 2.2 Hz), 6.78 (dd, 1H,
4
3
ArH, J 2.2 Hz, J 7.9 Hz), 7.10 (s, 1H, CH), 7.18 (d, 1H,
3J 7.9 Hz), 7.29 ∼ 7.44 (m, 5H, Ph); 13C NMR (67.8 MHz,
CDCl3): δC 13.8, 14.6, 21.6, 24.7, 25.7, 25.8, 26.5, 26.7,
29.6, 31.5, 31.6, 35.9, 37.7, 38.3, 44.0, 48.0, 50.4, 51.0,
70.0, 112.4, 115.0, 126.2, 127.4, 127.9, 128.5, 132.3, 137.3,
137.7, 156.9, 158.3, 214.9; MS (FAB, 3-nitrobenzylalcohol):
m/z (%) 389 (MH+, 1.43), 388 (M+, 4.66); HRMS found:
388.2036 calcd. for C26H38O3: 388.2038.
2.4. 3-Methoxy-estra-1,3,5(10),16-tetraeno[17,16-e]
2ꢀ-aminopyrimidine (6a)
To a solution of 5a (2.0 g, 4.98 mmol) in n-propanol
(50 ml) guanidine hydrochoride (1.43 g, 14.9 mmol) and
sodium methoxide (805 mg, 14.9 mmol) were added.
Then, the solution was stirred at 80 ∼ 90 ◦C for 70 h.
Dichloromethane (200 ml) and water (150 ml) were added
to the cooled solution. The separated organic phase was
washed with water (2 × 100 ml), dried over anhydrous
MgSO4 and concentrated in vacuo. The residue was sub-
jected to column chromatography on silica gel (eluant:
CH2Cl2/EtOAc 1:1–EtOAc) to give 6a (1.57 g, 94%); mp:
286–289 ◦C; [α]D = +108◦ (chloroform, c = 0.2); IR
(KBr): 3396, 3157, 2922, 1635, 1554, 1498, 1473, 1250,
2.2. 3-Methoxy-16-(N-methyl-N-phenylaminomethylidene)-
estra-1,3,5(10)-triene-17-one (5a)
A solution of 4a (2.20 g, 7.04 mmol) and N-methylphenyl-
ammonium trifluoroacetate (2.34 g, 10.6 mmol) in benzene
(30 ml) was stirred under reflux and the ensuing water was
removed under azeotropic conditions, using a Dean-Stark
condenser. Thereafter, dichloromethane (100 ml) and water
(100 ml) were added to the cooled solution. The separated
organic phase was washed with water (100 ml), dried over
anhydrous MgSO4 and concentrated in vacuo. The crude
product was subjected to column chromatography on silica
gel (eluent: CH2Cl2–CH2Cl2/EtOAc 9:1–7:1–1:1) to give
5a (2.26 g, 80%); mp 204–206 ◦C; [α]D = +263◦ (chloro-
form, c = 0.2); IR (KBr): 2920, 1692, 1605, 1576, 1499,
1245, 1128, 905, 756, 691, 600 cm−1; 1H NMR (270 MHz,
CDCl3): δH 0.93 (s, 1H, CH3), 3.51(s, 3H, NCH3), 3.78 (s,
1107, 1039, 949, 806, 777 cm−1 1H NMR (270 MHz,
;
CDCl3): δH 0.98 (s, 3H, CH3), 3.79 (s, 3H, OCH3), 5.09
4
(br, 2H, NH2), 6.65 (d, 1H, ArH, J 2.5 Hz), 6.71 (dd, 1H,
4
3
3
ArH, J 2.8 Hz, J 8.0 Hz), 7.21 (d, 1H, ArH, J 8.0 Hz),
8.07 (s, 1H, ArH); 13C NMR (67.8 MHz, CDCl3): δH 17.1,
26.2, 27.2, 27.4, 29.6, 32.8, 37.5, 44.3, 46.2, 54.8, 55.2,
111.5, 113.9, 123.4, 126.1, 132.3, 137.7, 152.7, 157.6,
162.1, 183.2; MS (FAB, 3-nitrobenzyl alcohol): m/z (%)
336 (MH+, 17), 335 (M+, 74); HRMS found: 335.1999
calcd. for C21H25ON3: 335.1998.
4
3H, OCH3), 6.63 (d, 1H, ArH, J 2.4 Hz), 6.70 (dd, 1H,
4
3
ArH, J 2.5 Hz, J 8.0 Hz), 7.11 ∼ 7.16 (m, 3H), 7.20 (d,
3
1H, ArH, J 8.3 Hz), 7.32 ∼ 7.39 (m, 2H), 7.6 (s, 1H);
13C NMR (67.8 MHz, CDCl3): δH 14.9, 26.1, 26.8, 27.8,
29.7, 31.9, 37.8, 39.6, 44.2, 47.1, 49.5, 55.2, 108.3, 111.4,
113.9, 120.8, 124.3, 126.3, 129.2, 132.5, 137.8, 141.3,
146.4, 157.5; MS (FAB, 3-nitrobenzylalcohol): m/z (%) 402
(MH+, 29), 401 (M+, 100); HRMS found: 401.2353 calcd.
for C37H31O2N: 401.2355.
2.5. 3-Benzyloxy-estra-1,3,5(10),16-tetraeno[17,16-e]-
2ꢀ-aminopyrimidine (6b)
6b was prepared analogously to 6a. The reaction of
5b (7.25 g, 6.80 mmol), guanidine hydrochloride (1.95 g,
20.4 mmol), and sodium methoxide (1.10 g, 20.4 mmol) in
n-propanol (50 ml) gave 6b (2.41 g, 86%); mp 243–245 ◦C;
[α]D = +77◦ (chloroform, c = 0.2); IR (KBr): 3367, 3184,
2931, 2360, 1738, 1643, 1608, 1560, 1498, 1456, 1377,
2.3. 3-Benzyloxy-16-(N-methyl-N-phenylamino-
methylidene)-estra-1,3,5(10)-triene-17-one (5b)
5b was prepared according to a procedure analogous
to the preparation of 5a. The reaction of 4b (4.79 g,
12.3 mmol), N,N-methylphenylammonium trifluoroacetate
(4.08 g, 18.4 mmol) in benzene (30 ml) gave 5b (4.53 g,
1252, 1047, 810, 737, 694 cm−1 1H NMR (270 MHz,
;
CDCl3): δH 0.98 (s, 3H, CH3), 4.97 (br, 2H, NH2), 5.04
(s, 2H, OCH2), 6.74 (d, 1H, ArH, 4J 2.4 Hz), 6.78 (dd,
1H, ArH, 4J 2.4 Hz, 3J 8.0 Hz), 7.21 (d, 1H, ArH, 3J

T. Matsumoto et al. / Steroids 68 (2003) 751–757
753
7.9 Hz), 7.32 ∼ 7.46 (m, 5H, Ph), 8.08 (s, 1H, ArH); 13C
NMR (67.8 MHz, CDCl3): δH 14.2, 17.1, 26.2, 27.2, 27.4,
29.6, 32.8, 37.6, 44.4, 46.2, 54.9, 70.0, 112.4, 114.9, 123.5,
126.2, 127.5, 127.9, 128.6, 132.6, 137.3, 137.8, 152.8,
156.9, 162.2, 183.2; MS (FAB, 3-nitrobenzylalcohol): m/z
(%) 412 (MH+, 13), 411 (M+, 5); HRMS found: 412.2396
calcd. for C27H30ON3: 412.2389.
3-nitrobenzylalcohol): m/z (%) 706 (MH+, 1.17), 705 (M+,
2.41); HRMS found: 705.3469 calcd. for C42H49O4N4S:
705.3475; anal. calcd. for C42H52N4O6S (8·2H2O): C
68.10; H 7.02; N 7.57, found: C 68.39, H 7.18, N 7.56.
2.8. 3-Benzyloxy-estra-1,3,5(10),16-tetraeno[17,16-e]-
2ꢀ-(S-benzyl-l-cysteinylami-no)pyrimidine (9)
2.6. 3-Hydroxy-estra-1,3,5(10),16-tetraeno[17,16-e]-
To a solution of 8 (98.5 mg, 0.140 mmol) in dry
dichloromethane (6 ml) was added trifluoroacetic acid
(0.135 ml, 1.75 mmol) and the reaction mixture was stirred
for 30 min at RT. Then, further trifluoroacetic acid (0.135 ml,
1.75 mmol) was added to the solution, which was stirred
for another 2.5 h at RT. Dichloromethane (30 ml) was added
to the solution, and the mixture was washed with aqueous
sodium hydrogencarbonate (20 ml) and water (2 × 20 ml).
The organic phase was dried over anhydrous Na2SO4
and concentrated in vacuo to give 9 (80.3 mg, 95%); mp
71–72 ◦C (dec.); [α]D = +21◦ (chloroform, c = 0.2); IR
(NaCl): 3750, 3674, 3324, 2924, 2850, 2360, 2352, 2342,
1704, 1625, 1579, 1494, 1459, 1419, 1376, 1342, 1311,
2ꢀ-aminopyrimidine (7)
6b (302 mg, 0.734 mmol) was added to an deaerated mix-
ture of methanol (7.5 ml) and benzene (7.5 ml). Palladium
carbon (60 mg, 10 wt.%) was added to the solution, which
then was saturated with hydrogen. The reaction mixture was
stirred for 20 h under a hydrogen atmosphere at RT. The
palladium carbon was removed by filtration and the filtrate
was concentrated in vacuo to give 7 (234 mg, 99%); mp
284–286 ◦C; [α]D = +99◦ (ethanol, c = 0.2); IR (KBr):
3494, 3336, 3207, 2933, 1624, 1560, 1469, 1358, 1288,
1244, 1184, 1105, 957, 812, 652 cm−1; 1H NMR (270 MHz,
DMSO): δH 1.03 (s, 3H, CH3), 6.52 (br, 2H, NH2), 6.60 (d,
1257, 1240, 1088, 1025, 803, 697, 670 cm−1 1H NMR
;
4
3
4
1H, J 2.2 Hz), 6.64 (dd, 1H, ArH, J 7.6 Hz, J 2.2 Hz),
(270 MHz, CDCl3): δH 0.92 (s, 3H, Me), 3.68 (s, 2H,
3
4
7.19 (d, 1H, ArH, J 8.0 Hz), 7.17 ∼ 7.25 (m, 5H, Ph),
SCH2), 4.97 (s, 2H, OCH2), 6.67 (d, 1H, ArH, J 2.3 Hz),
8.30 (s, 1H, ArH); 13C NMR (67.8 MHz, DMSO): δH 26.2,
26.9, 27.2, 29.3, 33.1, 37.5, 44.1, 46.0, 54.6, 113.1, 115.3,
121.2, 126.1, 130.4, 137.4, 152.3, 155.3, 163.2, 182.2; MS
(FAB, 3-nitrobenzylalcohol): m/z (%) 322 (MH+, 1.4), 321
(M+, 0.8); HRMS found: 322.1912 calcd. for C30H35O2N4:
322.1919.
6.71 (dd, 1H, ArH, 4J 2.3 Hz, 3J 8.6 Hz), 7.13 (d, 1H,
3
ArH, J 8.6 Hz), 7.18 ∼ 7.37 (m, 10H, Ph), 8.36 (s, 1H,
ArH), 9.90 (br, 2H, NH2); 13C NMR (67.8 MHz, CDCl3):
δH 17.1, 24.9, 25.6, 25.8, 26.0, 26.1, 27.4, 27.5, 29.2, 29.4,
29.6, 32.8, 33.4, 33.9, 36.5, 37.0, 37.5, 44.3, 46.5, 49.1,
51.6, 54.8, 54.9, 56.0, 70.0, 112.4, 114.9, 126.2, 127.0,
127.2, 127.5, 127.9, 128.6, 128.8, 129.0, 129.3, 132.4,
137.2, 137.6, 137.7, 138.0, 152.7, 155.7, 156.8, 157.0,
171.6, 183.4; MS (FAB, 3-nitrobenzylalcohol): m/z (%)
606 (MH+, 0.76), 605 (M+, 1.51); HRMS found: 605.2945
calcd. for C37H41O2N4S: 605.2950.
2.7. 3-Benzyloxy-estra-1,3,5(10),16-tetraeno[17,16-e]-
2ꢀ-(S-benzyl-N-tert-butoxycarbo-nyl-l-cysteinylamino)
pyrimidine (8)
To a solution of 6b (300 mg, 0.729 mmol) and N-(tert-
butoxycarbonyl)-S-benzyl-l-cysteine (454.8 mg, 1.46 mmol)
in dry dichloromethane (15 ml) was added Nꢀ,Nꢀ-dicyclo-
hexylcarbodiimide (DCC; 300.8 mg, 1.46 mmol) and the
resulting reaction mixture was stirred for 7 h at RT. The
precipitated urea was removed by filtration, and the fil-
trate was concentrated in vacuo. The crude product was
subjected to column chromatography on silica gel (eluant:
CH2Cl2/EtOAc 12:1–6:1–3:1) to give 8 (220.5 mg, 43%);
mp 85–87 ◦C (dec.); IR (KBr): 3538, 2928, 2850, 2350,
2286, 1710, 1631, 1569, 1498, 1455, 1369, 1250, 1163,
2.9. 3-Benzyloxy-estra-1,3,5(10),16-tetraeno[17,16-e]-
2ꢀ-(N-tert-butoxycarbonyl-l-glycyl-S-benzyl-l-
cysteinylamino)pyrimidine (10)
To a solution of 9 (354.2 mg, 0.586 mmol) and N-(tert-
butoxycarbonyl)-l-glycine (205.2 mg, 1.17 mmol) in dry
dichloromethane (15 ml) was added Nꢀ,Nꢀ-dicyclohexyl-
carbodiimide (241.4 mg, 1.17 mmol) and the reaction mix-
ture was stirred for 4 h at RT. Precipitated urea was removed
by filtration, and the filtrate was concentrated in vacuo. The
crude product was subjected to column chromatography on
silica gel (CH2Cl2/EtOAc 3:1–1:1) to give 10 (272.2 mg,
61%); mp 74–76 ◦C (dec.); IR (NaCl): 3760, 3656, 3290,
3032, 2972, 2924, 2858, 2244, 1710, 1690, 1657, 1602,
1571, 1500, 1455, 1419, 1401, 1368, 1281, 1244, 1167,
1103, 1047, 1026, 911, 864, 809, 731, 698 cm−1; 1H NMR
(270 MHz, CDCl3): δH 1.00 (s, 3H, CH3), 1.45 (s, 9H,
t-Bu), 3.78 (s, 2H, SCH2), 3.88 (br, 2H), 5.05 (s, 2H,
1
1048, 1024, 809, 696 cm−1; H NMR (270 MHz, CDCl3):
δH 1.00 (s, 3H, CH3), 1.43 (s, 2H), 1.47 (s, 9H, t-Bu), 3.79
4
(s, 2H, SCH2), 5.05 (s, 2H, OCH2), 6.75 (d, 1H, ArH, J
2.4 Hz), 6.79 (dd, 1H, ArH, 3J 7.9 Hz, 4J 2.4 Hz), 7.21
(d, 1H, ArH, 3J 7.9 Hz), 7.26∼7.45 (m, 10H, Ph), 8.38
(s, 1H, ArH), 8.55 (br, 1H, NH); 13C NMR (67.8 MHz,
CDCl3): δH 17.2, 25.0, 25.6, 27.5, 28.3, 29.6, 32.7, 33.7,
34.0, 36.7, 37.6, 44.3, 46.5, 49.2, 55.0, 70.0, 112.4, 115.0,
126.2, 127.2, 127.5, 127.9, 128.6, 129.1, 129.4, 132.4,
137.3, 137.7, 137.9, 152.6, 155.4, 156.9, 183.6; MS (FAB,
4
OCH2), 6.76 (d, 1H, ArH, J 2.3 Hz), 6.79 (dd, 1H, ArH,
3J 8.3 Hz, 4J 2.3 Hz), 7.21 (d, 1H, ArH, 3J 8.3 Hz),

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T. Matsumoto et al. / Steroids 68 (2003) 751–757
7.28 ∼ 7.45 (m, 10H, Ph), 8.41 (s, 1H, ArH); 13C NMR
(67.8 MHz, CDCl3): δH 15.3, 17.1, 26.1, 27.4, 27.6, 28.3,
29.6, 32.7, 33.4, 36.7, 37.5, 44.3, 46.6, 52.8, 55.0, 65.9,
70.0, 112.5, 115.0, 126.2, 127.2, 127.5, 127.9, 128.6, 129.1,
129.6, 132.3, 137.3, 137.7, 137.9, 152.6, 155.3, 156.9,
169.6, 183.6; MS (FAB, 3-nitrobenzylalcohol): m/z (%)
763 (MH+), 762 (M+); HRMS found: 762.3690 calcd. for
C44H52O5N5S: 762.3690; anal. calcd. for C44H54O6N5S
(10·H2O ): C 67.75, H 6.85, N 8.98, found: C 67.74, H
6.87, N 8.95.
for 1.5 h at RT. Then, further trifluoroacetic acid (0.2 ml,
2.59 mmol) was added and the mixture was stirred for an-
other 1.5 h at RT. Dichloromethane (20 ml) was added to the
solution which thereafter was washed with aqueous sodium
hydrogen carbonate (30 ml) and water (2 × 30 ml). The
organic phase was dried over Na2SO4 and concentrated in
vacuo to give 11 (114.0 mg, 89%); mp 158–160 ◦C (dec.);
[α]D = +35◦ (chloroform, c = 0.22); IR (KBr): 3433,
2925, 2856, 2360, 1730, 1670, 1462, 1122, 1072, 740 cm−1
;
1H NMR (270 MHz, CDCl3): δH 0.98 (s, 3H, Me), 5.04 (br
4
and s, 4H, NH2 and OCH2), 6.75 (d, 1H, ArH, J 2.6 Hz),
6.78 (dd, 1H, ArH, 4J 2.6 Hz, 3J 8.6 Hz), 7.21 (d, 1H, ArH,
3J 8.6 Hz), 7.30 ∼ 7.45 (m, 10H, Ph), 8.07 (s, 1H, ArH);
13C NMR (67.8 MHz, CDCl3): δH 17.1, 26.2, 27.2, 27.4,
29.6, 32.8, 35.9, 36.8, 37.5, 44.3, 45.1, 46.2, 53.9, 54.8,
70.0, 112.4, 114.9, 126.2, 127.5, 127.9, 128.6, 128.8, 128.9,
132.6, 152.6, 156.9; MS (FAB, 3-nitrobenzylalcohol): m/z
2.10. 3-Benzyloxy-estra-1,3,5(10),16-tetraeno[17,16-e]-
2ꢀ-(l-glycyl-S-benzyl-l-cysteinylamino)pyrimidine (11)
To a solution of 10 (147.4 mg, 0.193 mmol) in dry
dichloromethane (8 ml) was added trifluoroacetic acid
(0.20 ml, 2.59 mmol) and the reaction mixture was stirred
O
O
O
NaH
KOH, MeI
DMSO
PhCH2Br
DMF
MeO
HO
BnO
O
2
1
3
O
NaOMe
OH
HCO2Et
benzene
RO
RO
2 : R = Me
3 : R = Bn
4a : R = Me
4b : R = Bn
NH2
N
O
NH
NH2
N
HCl
N(Me)Ph
H2N
NaOMe, nPrOH
TFA-MeNHPh
benzene
RO
RO
5a : R = Me
5b : R = Bn
6a : R = Me
6b : R = Bn
NH2
N
N
H2
Pd - C
6b
MeOH / benzene
( 1 / 1 )
HO
7
Scheme 1. Synthetic route to estrano[17,16-e]aminopyrimidines.

T. Matsumoto et al. / Steroids 68 (2003) 751–757
755
(%) 663 (MH+, 2.31), 662 (M+, 4.80); HRMS found:
662.3164 calcd. for C39H44O3N5S: 662.3165.
anal. calcd. for C54H62O6N6S2: C 67.90, H 6.54, N 8.80,
found: C 68.18, H 6.73, N 8.78.
2.11. 3-Benzyloxy-estra-1,3,5(10),16-tetraeno[17,16-e]-
2ꢀ-(S-benzyl-N-tert-butoxycarbonyl-l-cysteinyl-l-glycyl-
S-benzyl-l-cysteinylamino)pyrimidine (12)
3. Results and discussion
Recently, various steroidal derivatives with fused-ring
heterocycles have been the synthetic targets of a number
of groups. Thus, the preparation of steroidal derivatives
fused to heterocycles such as pyrazole [17–23;16–24],
isoxazole [16–28], pyrazoline [24,29] or pyrimidine
[23,25,26,30–33], in the 2,3- and/or 16,17-positions of
the steroidal frame have been forwarded, where the com-
pounds exhibit a variety of biological activities. Importantly,
many of these syntheses rely on additions of heteronucle-
ophiles to 16-hydroxymethylene-estrones, which represent
1,3-dicarbonyl compounds and can be easily prepared by
hydroxymethylenation (␣-formylation) of suitably protected
estrone.
To a solution of 11 (105.3 mg, 0.159 mmol) and N-(tert-
butoxycarbonyl)-S-benzyl-l-cysteine (99.1 mg, 0.318 mmol)
in dry dichloromethane (10 ml) was added Nꢀ,Nꢀ-dicyclo-
hexylcarbodiimide (65.6 mg, 0.318 mmol) and the reaction
mixture was stirred for 15 h at RT. The precipitated urea
was removed by filtration, and the filtrate was concen-
trated in vacuo. The crude residue was subjected to column
chromatography on silica gel to give 12 (51.8 mg, 34%);
mp 130–132 ◦C (dec.); IR (KBr): 3396, 2925, 2360, 1651,
1500, 1245, 1163, 698 cm−1; 1H NMR (270 MHz, CDCl3):
δH 1.00 (s, 3H, Me), 1.43 (s, 9H, t-Bu), 3.74, 3.76 (s, 2H,
4
SCH2), 5.05 (s, 2H, OCH2), 6.76 (d, 1H, ArH, J 2.6 Hz),
Thus, for the synthesis of estratetraeno[17,16-e]-2ꢀ-
aminopyrimidines 6a, 6b and 7 the hydroxyl group was
6.79 (dd, 1H, ArH, 3J 8.3 Hz, 4J 2.6 Hz), 7.24 (d, 1H, ArH,
3J 8.3 Hz), 7.29 ∼ 7.42 (m, 15H, Ph), 8.37 (s, 1H, ArH);
Scheme 2. Preparation of estrano[17,16-e]pyrimidine peptide conjugates.

756
T. Matsumoto et al. / Steroids 68 (2003) 751–757
protected by either methylation [14] or benzylation [15].
Thereafter 3-O-protected 6a and 6b were treated with
ethylformate to give the ketoenol 4a and 4b, respectively.
4a and 4b were converted to the corresponding enamines
5a and 5b. Condensation of 5a and 5b with guanidine
hydrochloride gave the aminopyrimidines 6a and 6b. 6b
was readily deprotected to 7 by reductive O-debenzylation
(Pd/C, H2). Rather than to continue the synthesis with
O-methylated aminopyrimidine 6a, which would necessi-
tate a more difficult deprotective demethylation step, the
authors opted for carrying out subsequent steps with 6b
(Scheme 1).
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Acknowledgements
The authors thank Ms Yasuko Tanaka for MS and HRMS
(EI and FAB) measurements. The elemental analyses were
performed at the Center for Instrumental Analysis, Hakozaki
Campus, Kyushu University.

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