Studies on the synthesis of estrogen-GHRPS linkers

Article abstract of DOI:10.1081/SCC-120018924

A series of linkers consisted of estrone, estradiol, and GHRPS (growth hormone releasing peptides) were prepared with carboxyl-methyl or succinyl as the conjugated group. As a protective group in the side chain of Tyr, the Dcb (2,6-dichlorobenzyl) in the intermediates which is unstable to HF can be removed in high yield in the presence of Pd/C (10%), H₂, and 4.4% formic acid in methanol.

Full text of DOI:10.1081/SCC-120018924

Synthetic Communications
An International Journal for Rapid Communication of Synthetic Organic
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ISSN: 0039-7911 (Print) 1532-2432 (Online) Journal homepage: http://www.tandfonline.com/loi/lsyc20
Studies on the Synthesis of Estrogen-GHRPS
Linkers
Chao Wang , Ming Zhao , Weina Cui , Jian Yang & Shiqi Peng
To cite this article: Chao Wang , Ming Zhao , Weina Cui , Jian Yang & Shiqi Peng (2003) Studies
on the Synthesis of Estrogen-GHRPS Linkers, Synthetic Communications, 33:10, 1633-1641,
DOI: 10.1081/SCC-120018924
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SYNTHETIC COMMUNICATIONSÕ
Vol. 33, No. 10, pp. 1633–1641, 2003
Studies on the Synthesis of
Estrogen-GHRPS Linkers
Chao Wang, Ming Zhao, Weina Cui, Jian Yang,
*
and Shiqi Peng
College of Pharmaceutical Sciences,
Peking University, Beijing, P.R. China
ABSTRACT
A series of linkers consisted of estrone, estradiol, and GHRPS
(growth hormone releasing peptides) were prepared with carboxyl-
methyl or succinyl as the conjugated group. As a protective group in
the side chain of Tyr, the Dcb (2,6-dichlorobenzyl) in the intermedi-
ates which is unstable to HF can be removed in high yield in the
presence of Pd/C (10%), H2, and 4.4% formic acid in methanol.
*Correspondence: Shiqi Peng, College of Pharmaceutical Sciences, Peking
University, Beijing 100083, P.R. China.
1633
DOI: 10.1081/SCC-120018924
Copyright & 2003 by Marcel Dekker, Inc.
0039-7911 (Print); 1532-2432 (Online)
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1634
Wang et al.
INTRODUCTION
The phenomenon that the effects of the peptides were enhanced by
steroids through increasing their receptor numbers was named ‘‘permis-
sive action.’’[1–3] Based on this concept in our previous article the oligo-
peptides (for instance urotoxin tripeptides or kyotophin) and steroids
(for instance hydrocortisone or estrone) were combined to give the
corresponding linkers, of which the immunosuppressive or analgesic
effects were higher than that of the steroids or oligopeptides.[4,5] As a
rational spread, the linkers consisting of estrogen and GHRPS (growth
hormone releasing peptides) were synthesized, both of which were known
as the agents preventing osterporosis. In the present article we report
ageneral method, in which the deprotection of Dcb (2,6-dichlorobenzyl)
in Tyr of the intermediates was specially discussed, to prepare the
estrogen-GHRPS linkers.
RESULTS AND DISCUSSION
The protective tetrapeptide (1) was prepared by use of stepwise
syntheses (from C terminal to N terminal) via. solution method, with
DCC (dicyclohexylcarbodiimide) and HOBt (1-hydroxybenzotriazole)
as the coupling agents in the peptide formation, with hydrogen chloride
in ethyl acetate as the deprotection agent for the removal of Boc, in
77–90% yields. At 0ꢀC 1 was treated with 2 M NaOH and hydrochloric
acid successfully to offer the corresponding peptide acid (2) in 96% yield.
Under the normal conditions 2 was coupled with L-Leu-OMe, Leu-NH2,
Gly-OMe, or Gly-NH2 to give 3a–d in 73%, 56%, 75%, and 58% yield,
respectively. After removal of Boc with 4 M of hydrogen chloride in ethyl
acetate 3a–d were converted into 6a–d in excellent yields. After saponifi-
cation and acidification of 3a,c, 7a,c were obtained in 88% and 92% yield
respectively. Similarly 4a,c were converted into 5a,c in 91% and 89%
yield respectively (Sch. 1).
The O-alkylation of estrone with ethyl bromoacetate in the presence
of sodium ethoxide gave 3-O-carbethoxymethylestrone (8) in 83% yield.
After treating 8 with 2 M NaOH and then acidifying with 2 M of hydro-
chloric acid 3-O-carboxymethylestrone (9) was obtained in 94% yield,
and then which was coupled with 6a–d in the presence of DCC and
HOBt to offer Nꢀ-(3-O-carboxymethylestrone-protective pentapeptides
(10a–d) in 64%, 85%, 63% and 60% respectively. Reducing 10a–d
with sodium borohydride Nꢀ-(3-O-carboxymethylestradiol)-protective

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Synthesis of Estrogen-GHRPS Linkers
1635
Scheme 1. I) With DCC, HOBt as the coupling agent; HCl/CH3CO2C2H5 as the
deprotection agent of Boc; II) 2 M NaOH for reaction, and then 4 M HCl, 0ꢀC for
acidification; III) DCC, HOBt; IV) Pd/C (10%) and H2; or Pd/C (10%) and 4.4%
HCOOH in CH3OH; or Pd/C (10%), H2 and 4.4% HCOOH in CH3OH; V) 2 M
NaOH, 0ꢀC; VI) 4M of hydrochloride in ethyl acetate; VII) 2 M NaOH, 0ꢀC;
wherein a: AA¼Leu, R¼OMe; b: AA¼Leu, R¼NH2; c: AA¼Gly, R¼OMe;
d: AA¼Gly, R¼NH2.
pentapeptides (11a–d) in 67%, 69%, 70%, and 75% yield, respectively
(Sch. 2).
Acylation of the 17-hydroxyl in the estradiol with succinic anhydride
in the presence of DMAP (4-dimethylaminopyridine) and estradiol-17-O-
hemisuccinate (16) was obtained in 66% yield. Treating 16 with 6b,d
and DCC, Nꢀ-(17-O-succinyl-estradiol)-protective pentapeptide amides
(17b,d) were provided in 70% and 72% yield, respectively (Sch. 3).
In the presence of DCC and DMAP estradiol was acylated by 7a,c
and the corresponding protective pentapeptidyl-17-O-estradiols (19a,c)
were obtained in 67% and 69% yields, respectively (Sch. 4).
The reactions for removal of Dcb from the side chain of Tyr in all the
protective intermediates (3a–d, 10a–d, 11a–d, 17b,d and 19a,c) by catal-
ytic hydrogenation, catalytic transfer hydrogenation, and the combina-
tion of catalytic hydrogenation with catalytic transfer hydrogenation
were carried out in our experiments. 3c,d can be converted into 4c,d in
27% and 20% yield respectively in the presence of Pd/C (5%) and H2,
but the hydrogenation of 3a,b, 10a–d, 11a–d, 17b,d and 19a,c gave no
desired products. In the presence of Pd/C (10%) and 4.4% formic acid in
methanol, or Pd/C (10%) and 70% formic acid in methanol, the yields of
4c,d were increased to 84% and 88% respectively, however the hydro-
genation of 3a,b under this experimental condition the desired products
were not obtained either. On the other hand, if the deprotection of 3a–d

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1636
Wang et al.
Scheme 2. I) NaOEt, C2H5OH, BrCH2CO2C2H5; II) 2 M NaOH; III) DCC,
HOBt, NMM, 3a–f; IV) NaBH4; V) Pd/C (10%), H2 and 4% HCOOH in
CH3OH; VI) 2 M NaOH, 0ꢀC; VII) Pd/C (10%), H2 and 4.4% HCOOH in
CH3OH; VIII) 2 M NaOH; wherein AA¼Leu or Gly, R¼NH2 or OCH3.
was carried out in the presence of Pd/C (10%), H2 and 4.4% formic acid
in methanol the yields of 4a–d were increased to 91–94%. Under the same
condition 10a–d, 11a–d, 17b,d and 19a,c were converted into the desired
products 14a–d, 12a–d, 18b,d and 20a,c in 90–92% yields smoothly. The
results suggested that the benzyl type protective groups in the intermedi-
ates which is unstable to HF can be removed in the presence of Pd/C
(10%), H2 with 4.4% formic acid in methanol successfully and this
method may be generally convenient for the same situation.

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Synthesis of Estrogen-GHRPS Linkers
1637
Scheme 3. I) Succinic anhydride, DMAP; II) DCC, HOBt, NMM, 6b,d; III)
Pd/C (10%), H2 and 4.4% HCOOH in CH3OH; wherein AA¼Leu or Gly.
Scheme 4. I) DCC, DMAP and 7a, c; II) Pd/C (10%), H2 and 4.4% HCOOH in
CH3OH; wherein AA¼Leu or Gly.

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1638
Wang et al.
EXPERIMENTAL
The protected amino acids used were of L-configuration. The purity
of the intermediates and the products was confirmed by TLC (Merck
Silicagel pltaes of type 60 F 254, 0.25 mm layer thickness) or HPLC
(Waters, C18 column 3.9 Â 150 mm). FAB-MS was determined by a
VG-ZAB-MS and a HPES-5989x instrument. Optical rotations were
determined at 20ꢀC on aSchmidt þ Haensch Polartronic D instrument.
Boc-Tyr(Dcb)-Gly-Gly-Phe-Phe-OMe
(1):
M.p.
127–130ꢀC;
20
½ꢀꢁD ¼ 22:008 (c ¼ 1 in CHCl3/CH3OH, 15/1); Boc-Tyr(Dcb)-Gly-Gly-
Phe-Leu-OMe (3a): M.p. 126–128ꢀC; ½ꢀꢁD ¼ 1:678 (c ¼ 1.2 in CHCl3/
20
CH3OH, 15/1); FAB-MS (m/e): 828[M þ H]þ; Boc-Tyr(Dcb)-Gly-Gly-
20
Phe-Leu-NH2 (3b): M.p. 138–142ꢀC; ½ꢀꢁD ¼ ꢂ16:408 (c ¼ 1.1 in CHCl3/
CH3OH, 15/1); TOF-MS (m/e): 836[M þ Na]þ; Boc-Tyr(Dcb)-Gly-Gly-
Phe-Gly-OMe (3c): M.p. 124–126ꢀC; ½ꢀꢁD ¼ 0:838 (c ¼ 2.4 in
20
CHCl3/CH3OH, 15/1); FAB-MS (m/e): 772 [M þ H]þ; Boc-Tyr(Dcb)-
Gly-Gly-Phe-Gly-NH2 (3d): M.p. 149–152ꢀC; ½ꢀꢁD ¼ ꢂ12:008 (c ¼ 0.5
20
in CHCl3/CH3OH, 15/1); FAB-MS (m/e): 757[M þ Na]þ. Estrone-3-O-
20
CH2CO-Tyr(Dcb)-Gly-Gly-Phe-Leu-OMe (10a): M.p. 112–115ꢀC;
½ꢀꢁD ¼ 25:008 (c ¼ 0.4 in CHCl3/CH3OH, 15/1); TOF-MS (m/e): 1039
[M þ H]þ; Estrone-3-O-CH2CO-Tyr(Dcb)-Gly-Gly-Phe-Leu-NH2 (10b):
20
M.p. 136–139ꢀC; ½ꢀꢁD ¼ 38:008 (c ¼ 0.4 in CHCl3/CH3OH, 15/1); TOF-
MS (m/e): 1046 [M þ H]þ; Estrone-3-O-CH2CO-Tyr(Dcb)-Gly-Gly-Phe-
20
Gly-OMe (10c): M.p. 101–104ꢀC; ½ꢀꢁD ¼ 12:008 (c ¼ 0.5 in CHCl3/
CH3OH, 15/1); TOF-MS (m/e): 984 [M þ H]þ; Estrone-3-O-CH2CO-
Tyr(Dcb)-Gly-Gly-Phe-Gly-NH2(10d): M.p. 153–156ꢀC; ½ꢀꢁD ¼ 28:008
20
(c ¼ 0.1 in CHCl3/CH3OH, 15/1); TOF-MS (m/e): 990 [M þ H]þ. Boc-
20
Tyr(Dcb)-Gly-Gly-Phe-Leu-17b-O-estradiol (19a): M.p. 138–141ꢀC;
½ꢀꢁD ¼ 11:408 (c ¼ 0.7 in CHCl3/CH3OH, 15/1); TOF-MS (m/e): 1090
[M þ Na]þ; Boc-Tyr(Dcb)-Gly-Gly-Phe-Gly-17b-O-estradiol (19c): M.p.
20
140–142ꢀC; ½ꢀꢁD ¼ 19:008 (c ¼ 0.7 in CHCl3/CH3OH, 15/1); TOF-MS
(m/e): 1011 [M þ Na]þ.
General Procedure for Removal of Boc
Zero point two zero millimolar of Boc protected compound was
stirred in 2 mL of 4 M of hydrochloride in ethyl acetate at room tempera-
ture for 3 h. The reaction mixture was evaporated to remove the solvent.
The residue was dissolved in 10 mL of ethyl acetate and the solution was
evaporated to driness. The resulted solid of 6a–d was used for coupling
reaction directly.

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Synthesis of Estrogen-GHRPS Linkers
1639
General Procedure for Saponification of the Methyl Esters
A solution of 0.20 mmol of the methyl ester, 15 mL of methanol and
3 mL of 2 M NaOH in methanol was stirred at 0ꢀC for 5 h. The reaction
mixture was neutralized with 2 M of hydrochloric acid to pH 7.0 and
evaporated to remove methanol. The residue was acidified with 2 M of
hydrochloric acid to pH 1.0. The precipitates were collected by filtration
to offer 2, 5a,c, 7a,c, 13a,c and 15a,c.
20
Boc-Tyr(Dcb)-Gly-Gly-Phe-Leu-OH (7a): M.p. 136–138ꢀC; ½ꢀꢁD
¼
ꢂ20:008 (c ¼ 0.4 in CHCl3/CH3OH, 15/1); FAB-MS (m/e) ¼ 723
[M þ Na]þ; H-Tyr-Gly-Gly-Phe-Leu-OH (5a): M.p. 130–133ꢀC; ½ꢀꢁD
¼
20
5:008 (c ¼ 0.4 in H2O); TOF-MS (m/e): 556 [M þ H]þ; H-Tyr-Gly-Gly-
Phe-Gly-OH (5c): M.p. 170–174ꢀC; ½ꢀꢁD ¼ 10:008 (c ¼ 0.4 in H2O); TOF-
20
MS (m/e): 522 [M þ Na]þ; Boc-Tyr(Dcb)-Gly-Gly-Phe-Gly-OH (7c):
20
M.p. 136–140ꢀC; ½ꢀꢁD ¼ 1:508 (c ¼ 1.3 in CHCl3/CH3OH, 15/1); TOF-
MS (m/e) ¼ 780 [M þ Na]þ; Estradiol-3-O-CH2CO-Tyr-(Dcb)-Gly-Gly-
Phe-Leu-OH (13a): M.p. 151–153ꢀC; ½ꢀꢁD ¼ 13:308 (c ¼ 0.75 in
20
CHCl3/CH3OH, 15/1); TOF-MS (m/e) ¼ 1040 [M þ H]þ; Estradiol-3-O-
CH2CO-Tyr-(Dcb)-Gly-Gly-Phe-Gly-OH (13c): M.p. 110–112ꢀC; ½ꢀꢁD
¼
20
24:008 (c ¼ 0.5 in CHCl3/CH3OH, 15/1); TOF-MS (m/e): ¼ 992
[M þ Na]þ; Estradiol-3-O-CH2CO-Tyr-Gly-Gly-Phe-Leu-OH (15a):
20
M.p. 122–125ꢀC; ½ꢀꢁD ¼ 40:008 (c ¼ 0.15 in CHCl3/CH3OH, 15/1);
TOF-MS (m/e): 868 [M þ H]þ; Estradiol-3-O-CH2CO-Tyr-Gly-Gly-
Phe-Gly-OH (15c): M.p. 140–142ꢀC; ½ꢀꢁD ¼ 15:008 (c ¼ 0.4 in
20
CHCl3/CH3OH, 15/1); TOF-MS (m/e) ¼ 813 [M þ H]þ.
General Procedure for Removal of Dcb
A suspension of 0.20 mmol of Dcb protected compound, 5 mg of
Pd/C (5%) and 15 mL of methanol were agitated with hydrogen at
room temperature for 24 h. The reaction mixture was filtrated. The
filtrate was evaporated to offer the goal products.
A suspension of 0.20 mmol of Dcb protected compound, 5 mg of
Pd/C (5%) and 8 mL of 4.4% formic acid in methanol were agitated
with hydrogen at room temperature for 24 h. The reaction mixture was
filtrated. The filtrate was evaporated to offer the goal products.
20
H-Tyr-Gly-Gly-Phe-Leu-OH (5a): M.p. 130–133ꢀC; ½ꢀꢁD ¼ 5:008
(c ¼ 0.4 in H2O); TOF-MS (m/e) ¼ 556 [M þ H]þ; H-Tyr-Gly-Gly-Phe-
20
Leu-NH2 (5b): M.p. 123–126ꢀC; ½ꢀꢁD ¼ 12:008 (c ¼ 1.0 in H2O); TOF-
MS (m/e): 592 [M þ K]þ; H-Tyr-Gly-Gly-Phe-Gly-OH (5c): M.p.

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1640
Wang et al.
170–174ꢀC; ½ꢀꢁD ¼ 10:008 (c ¼ 0.4 in H2O); TOF-MS (m/e) ¼ 522
20
[M þ Na]þ; H-Tyr-Gly-Gly-Phe-Gly-NH2 (5d): M.p. 128–130ꢀC;
20
½ꢀꢁD ¼ 60:008 (c ¼ 0.3 in H2O); TOF-MS (m/e) ¼ 499 [M þ K]þ;
20
Estrone-3-O-CH2COTyr-Gly-Gly-Phe-Leu-OMe (14a): M.p. 128–131ꢀC;
½ꢀꢁD ¼ 12:908 (c ¼ 0.46 in CHCl3/CH3OH, 15/1); TOF-MS (m/e): 889
[M þ Na]þ; Estrone-3-O-CH2CO-Tyr-Gly-Gly-Phe-Gly-NH2 (14b): M.p.
20
153–156ꢀC; ½ꢀꢁD ¼ 28:008 (c ¼ 0.1 in CHCl3/CH3OH, 15/1); TOF-MS
(m/e): 990 [M þ Na]þ; Estradiol-3-O-CH2CO-Tyr-Gly-Gly-Phe-Leu-
20
NH2 (12b): M.p. 122–125ꢀC; ½ꢀꢁD ¼ 57:208 (c ¼ 0.22 in CHCl3/CH3OH,
15/1); TOF-MS (m/e): 888 [M þ Na]þ; Estradiol-3-O-CH2CO-Tyr-Gly-
20
Gly-Phe-Gly-NH2 (12d): M.p. 132–136ꢀC; ½ꢀꢁD ¼ ꢂ80:008 (c ¼ 0.30 in
CHCl3/CH3OH, 15/1); TOF-MS (m/e): 854 [M þ H]þ; Estradiol-17-b-
20
O-COCH2CH2CO-Tyr-Gly-Gly-Phe-Leu-NH2 (18b): M.p. 130–132ꢀC;
½ꢀꢁD ¼ 16:008 (c ¼ 0.25 in CHCl3/CH3OH, 15/1); TOF-MS (m/e):
932 [M þ Na]þ; Estradiol-17-b-O-COCH2CH2CO-Tyr-Gly-Gly-Phe-
20
Gly-NH2 (18d): M.p. 132–136ꢀC; ½ꢀꢁD ¼ ꢂ80:008 (c ¼ 0.30 in
CHCl3/CH3OH, 15/1); TOF-MS (m/e): 857 [M þ Na]þ; H-Tyr-Gly-Gly-
20
Phe-Leu-17b-O-diestradiol (20a): M.p. 163–165ꢀC; ½ꢀꢁD ¼ 66:708
(c ¼ 0.30 in CHCl3/CH3OH, 15/1); TOF-MS (m/e): 810 [M þ H]þ; H-
20
Tyr-Gly-Gly-Phe-Gly-17b-O-diestradiol (20c): M.p. 120–122ꢀC; ½ꢀꢁD
¼
23:608 (c ¼ 1.10 in CHCl3/CH3OH, 15/1); TOF-MS (m/e): 877
[M þ Na]þ.
ACKNOWLEDGMENT
The author Shiqi Peng wishes to thank the Key Basic Research
Project (G1998051111) of China for financial support.
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Synthesis of Estrogen-GHRPS Linkers
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Received in Japan June 16, 2002

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