1-deamino-1(15)-carba and -dicarba analogues of endothelin-1

Article abstract of DOI:10.1135/cccc20000407

Sulfanyl-methylene and ethylene bridges were inserted into the molecule of endothelin-1 (ET-1) as other possible isosteric replacements of its outer disulfide linkage. The [1-deamino-1-carba]ET-1, [1-deamino-15-carba]ET-1 and [1-deamino-1,15-dicarba]ET-1 were synthesized either by a fragment condensation of protected cyclic pentadecapeptides with carboxy-terminal hexapeptides of the endothelin-1 sequence or by step-wise coupling on polymer support of the entire henicosapeptide sequences from carboxy-terminus. The analogues were devoid of uterotonic activity in comparison with the parent ET-1.

Full text of DOI:10.1135/cccc20000407

Analogues of Endothelin-1
407
1-DEAMINO-1(15)-CARBA AND -DICARBA ANALOGUES
OF ENDOTHELIN-1
1,
2
Jan HLAVÁČEK *, Renáta MARCOVÁ, Miloš BUDĚŠÍNSKÝ
3
and Jiřina SLANINOVÁ
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic,
1
Flemingovo nám. 2, 166 10 Prague 6, Czech Republic; e-mail: honzah@uochb.cas.cz,
2
3
budesinsky@uochb.cas.cz, slanin@uochb.cas.cz
Received Jan uary 14, 2000
Accepted February 25, 2000
Sulfan yl-m eth ylen e an d eth ylen e bridges were in serted in to th e m olecule of en doth elin -1
(ET-1) as oth er possible isosteric replacem en ts of its outer disulfide lin kage. Th e
[1-deam in o-1-carba]ET-1, [1-deam in o-15-carba]ET-1 an d [1-deam in o-1,15-dicarba]ET-1 were
syn th esized eith er by a fragm en t con den sation of protected cyclic pen tadecapeptides with
carboxy-term in al h exapeptides of th e en doth elin -1 sequen ce or by step-wise couplin g on
polym er support of th e en tire h en icosapeptide sequen ces from carboxy-term in us. Th e an a-
logues were devoid of uteroton ic activity in com parison with th e paren t ET-1.
Key w ord s: Peptides; Solid ph ase syn th esis; Step-wise procedure; Fragm en t con den sation ;
En doth elin ; Disulfide isosters; Uteroton ic activity.
En doth elin s are a group of extrem ely poten t vasoactive cyclic peptides
wh ich con sist of 21 am in o acids an d con tain two disulfide bon ds. Th ey ex-
h ibit a wide spectrum of fun ction s in path oph ysiological states of various
organ s in th e body1.
Th eir an alogues with m odified am in o acid residues, disulfide bon ds,
am in o term in al rin gs an d oth ers were syn th esized an d in vestigated to un -
derstan d en doth elin ph ysiology2. Th e cyclic arran gem en t of en doth elin s
stabilized by two disulfide bon ds was foun d to be im portan t for m ain tain -
in g a h igh receptor bin din g activity. For exam ple a reduction of th e outer
disulfide bon d between th e Cys residues in position s 1 an d 15 in terfered
with th e vasocon strictive an d bin din g activity in correspon din g an alogues3
wh ile th e en doth elin -1 (ET-1) an alogue with am ide bon d replacin g th e
outer disulfide lin kage exh ibited an tagon istic activity4.
We foun d in terestin g to in troduce sulfan yl-m eth ylen e or eth ylen e
bridges in to th e m olecule of ET-1 as an oth er possible isosteric replacem en t
of th is disulfide lin kage an d focussed our effort on th e syn th esis of
Collect. Czech. Chem. Commun. (Vol. 65) (2000)

408
Hlaváček, Marcová, Buděšínský, Slaninová:
1-deam in o-1-carba (3a), 1-deam in o-15-carba (3b ) an d 1-deam in o-1,15-
dicarba (3c) an alogues of ET-1. Firstly, we h ave replaced th e outer disulfide
lin kage wh ich is reported to be m ore im portan t th an th e in n er on e in in ter-
action with a correspon din g receptor5,6. A m on ocyclic an alogue of 1-de-
am in o-ET-1 was reported to lack vasocon strictor activity; h owever, it h as
been also described, th at 1-deam in o replacem en t of th e am in o-term in us in
som e peptide h orm on es does n ot in terfere with th eir biological activity7,8
.
Th erefore we h ave also utilized th at replacem en t in th is study to facilitate
th e preparation of th ese an alogues. Wh ile dicarba substitution of th e outer
disulfide bon d h as been already used in th e syn th esis of correspon din g
[Ala3,11]Et-1 an alogue6, th e syn th esis of m on ocarba an alogues was partially
described in our prelim in ary com m un ication s9,10
.
Now, we wan t to report in detail on a m odified syn th esis of both th e
1-carba an d 15-carba an alogues of 1-deam in o-ET-1 an d also on th e first syn -
th esis of th e correspon din g dicarba an alogue with 2-am in osuberic
(2-am in ooctan ediotic) acid im itatin g th e outer disulfide bon d of th e ET-1.
Th e m eth od of “buildin g blocks” applied to creation of isosteric bon ds in
wh ich on e or both sulfur atom s of correspon din g disulfide lin kage are re-
placed by m eth ylen e group h as been developed an d foun d to be useful in a
differen tiation between steric an d fun ction al roles of th e disulfide bon d in
n euroh ypoph yseal h orm on e11 an d calciton in 12 an alogues.
To com pare altern ative syn th etic routes, th e syn th esis of 1-deam in o-
1-carba or 1-deam in o-15-carba an alogues of th e ET-1 (3a an d 3b) was car-
ried out eith er by segm en t con den sation of th e protected cyclic carba
pen tadecapeptides 1a an d 1b or 2a an d 2b with th e carboxy-term in al h exa-
peptides 4a an d 4b (strategy A), or by step-wise assem blin g th e en tire
h en icosapeptide sequen ces from carboxyl term in us on a polym er support
(strategy B). Th e step-wise strategy B was also used in th e syn th esis of
1-deam in o-1,15-dicarba an alogue of th e ET-1 (3c).
Met-Leu-(tBu)Ser-(tBu)Ser-(Trt)Cys-(tBu)Ser-OC-CH2-CH2-X-Y-CH2
Asp(OtBu)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Val-Tyr(tBu)-Ph e-NH-CH-R
1a, X=CH2, Y=S, R=COOH; 1c, X=CH2, Y=S, R=His(Boc)-Leu-Asp(OtBu)-Ile-Ile-Trp-O-WR
1b, X=S, Y=CH2, R=COOH; 1d , X=S, Y=CH2, R=His(Boc)-Leu-Asp(OtBu)-Ile-Ile-Trp-O-WR
Th e lin ear form s of th e cyclic protected 1-deam in o-1-carba- an d 1-de-
am in o-15-carbapen tadecapeptides 1a an d 1b (strategy A) were prepared on
a weak acid labile 2-ch lorotrityl ch loride resin 13,14, usin g Fm oc/tBu protec-
tion of th e correspon din g am in o acids with an exception of th e Cys(Trt)
Collect. Czech. Chem. Commun. (Vol. 65) (2000)

Analogues of Endothelin-1
409
residue, startin g with Fm oc-Ph e-OH. Fm oc-Cys(CH2-CH2-CH2-COOH)-OAll
(5c) an d Fm oc-Hcy(CH2CH2COOH)-O-All (5f) were coupled in th e last step
of th e syn th eses via th eir side-ch ain carboxyl groups. In a cleavage of th e
protected peptides from th e resin by AcOH a m ixture of solven ts con tain -
in g TFE h as been used. Th e TFE, kn own to in terfere with h ydrogen bon ds
in peptide ch ain s, alleviated a low solubility of th e protected pen ta-
decapeptides. To close th e 1-carba an d 15-carba bridges in th e correspon d-
in g pen tadecapeptides, th e TPTU reagen t in th e sam e solven t m ixture was
successfully used as a substitute for th e BOP used in our previous syn th eses.
As a con sequen ce, in creased reaction rates with n early 90% yields of th e
cyclization reaction s were ach ieved. After th e allyl protectin g group was re-
m oved by [Pd(Ph 3P)4] in th e presen ce of m orph olin e th e protected cyclo-
peptides 1a an d 1b were separately coupled to th e side-ch ain protected
h exapeptide boun d to th e Wan g resin 4a an d th e resin boun d protected
peptides 1c an d 1d were obtain ed. After a sim ultan eous rem oval of th e
side-ch ain protectin g groups an d th e 1-deam in o-1-carba- an d 1-deam in o-
15-carbah en icosapeptides from th e resin with TFA, th e in n er disulfide lin k-
age between Cys residues in th e position s 3 an d 11 was closed by air oxida-
tion at pH 8.5 in an am m on ium –trifluoroacetate buffer (procedure A)
givin g an alogues 3a an d 3b.
Met-Leu-(Bzl)Ser-(Bzl)Ser-(MeBzl)Cys-(Bzl)Ser-OC-CH2-CH2-X-Y-CH2
Asp(OcHx)-Lys(ClZ)-Glu(OcHx)-Cys(MeBzl)-Val-Tyr(Bzl)-Ph e-NH-CH-R
2a, X=CH2, Y=S, R=COOH; 2c, X=CH2, Y=S, R=His(Bom )-Leu-Asp(OcHx)-Ile-Ile-Trp(For)-O-MR
2b, X=S, Y=CH2, R=COOH; 2d, X=S, Y=CH2, R=His(Bom )-Leu-Asp(OcHx)-Ile-Ile-Trp(For)-O-MR
2e, X=CH2,Y=CH2, R= His(Bom )-Leu-Asp(OcHx)-Ile-Ile-Trp(For)-O-MR
Th e lin ear form s of th e protected 1-deam in o-1-carba- an d 1-deam in o-
15-carbapen tadecapeptides 2a an d 2b (strategy A) were altern atively pre-
pared on th e Merrifield ch lorom eth yl resin usin g Nα-Fm oc an d th e side-
ch ain protection : Asp(OcHx), Glu(OcHx), Tyr(Bzl), Lys(ClZ), Cys(MeBzl),
Ser(Bzl) with th e exception of am in oterm in al Boc-Ser(Bzl)-OH. Th e Fm oc-
Cys(CH2CH2CH2COOtBu)-OH for 1-carba or Fm oc-Hcy(CH2CH2COOtBu)-OH
for 15-carba lin kages were coupled to th e resin as Cs salts in th e first step.
After th e cleavage of th e Nα-Boc protection group of Ser residue in position 2
an d tBu group in th e side ch ain of th e bridge un its (sim ulated position 1 in
th e n atural en doth elin ) with TFA, th e outer sulfan yl–m eth ylen e bon d,
isosteric with disulfide bridge, was again closed usin g TPTU in solven t m ix-
ture con tain in g TFE. Th e protected cyclic peptides 2a an d 2b were split off
Collect. Czech. Chem. Commun. (Vol. 65) (2000)

410
Hlaváček, Marcová, Buděšínský, Slaninová:
th e resin with 0.1 M NaOH in MeOH-dioxan e15 an d after acidification th eir
carboxylic term in i were separately coupled to th e h exapeptide boun d to
Merrifield resin 4b yieldin g th e protected peptides on resin 2c, 2d . After th e
peptides were deprotected an d cleaved from th e resin sim ultan eously usin g
TFMSA (ref.16), th ey were directly subjected to th e m odified air oxidation
procedure differen t from th at used for in term ediates obtain ed from th e
peptide resin s 1c an d 1d . To alleviate th e low solubility of th ese peptides,
we ch ose air oxidation at h igh con cen tration of DMSO in aqueous solu-
tion 17 (procedure B) to close th e in n er disulfide bon d. Th e degree of oxida-
tion was m on itored by HPLC.
Leu-Ser-Ser-Cys-Ser-OC-CH2-CH2-X-Y-CH2
Met-Asp-Lys-Glu-Cys-Val-Tyr-Ph e-NH-CH-CO-His-Leu-Asp-Ile-Ile-Trp-OH
3a, X=S, Y=CH2; 3b, X=CH2, Y=S; 3c, X=CH2, Y=CH2
Th e step-wise syn th eses (strategy B) of th e protected precursors 2c–2e of
[1-deam in o-1-carba]- (3a), [1-deam in o-15-carba]- (3b) an d [1-deam in o-1,15-
dicarba]en doth elin -1 (3c) from th eir carboxylic term in i were also started
with th e carboxy-term in al h exapeptide built on th e Merrifield resin 4b. Th e
α-am in o an d side ch ain protection , couplin g reagen ts, con dition s, an d th e
rem oval of protectin g groups were essen tial th e sam e as th ose used in th e
preparation of the protected peptides 2c and 2d by the segm ent condensation.
In addition, the Fm oc-Asu(OtBu)-OH (5g) for 1,15-dicarba linkage was utilized.
The closures of the 1-carba, 15-carba and 1,15-dicarba linkages were performed
with the whole henicosapeptide sequences bound to Merrifield resin.
Th e peptides were sim ultan eously deprotected an d detach ed from th e
resin an d th eir in n er disulfide groups closed accordin g to procedure B used
for th e peptides obtain ed from peptide resin s 2c an d 2d , wh ich were pre-
pared by th e segm en t con den sation (strategy A). Th e HPLC elution tim es
for th e an alogs 3a an d 3b with 1-carba an d 15-carba bridges, respectively,
were iden tical. Th e reten tion tim e of th e dicarba an alogue 3c was in creased.
Th e scarcely soluble an alogues 3a–3c were con verted to th eir sodium salts
wh ich could be th en easily h an dled durin g th eir HPLC purification an d th e
followin g bioassay. Uteroton ic activity of com poun ds 3a–3c was assayed
an d com pared with th at of th e n ative ET-1.
H-His(R1)-Leu-Asp(R2)-Ile-Ile-Trp(R3)-O-X
4a, R1 = Boc, R2 = OtBu, R3 = H, X = Wang resin; 4b, R1 = Bom, R2 = OcHx, R3 = For, X = Merrifield resin
Collect. Czech. Chem. Commun. (Vol. 65) (2000)

Analogues of Endothelin-1
411
Th e protected h exapeptide segm en ts boun d to th e Wan g an d Merrifield
resin s 4a an d 4b were prepared accordin g to th e syn th etic protocols for th e
syn th eses of th e pen tadecapeptides 1a, 1b an d 2a, 2b usin g th e correspon d-
in g protected am in o acids an d resin s.
Fm oc-AA(R1)-O-R2
5a, AA = Cys, R1 = CH2CH2CH2COOtBu, R2 = H
5b, AA = Cys, R1 = CH2CH2CH2COOtBu, R2 = All
5c, AA = Cys, R1 = CH2CH2CH2COOH, R2 = All
5d , AA = Hcy, R1 = CH2CH2COOtBu, R2 = H
5e, AA = Hcy, R1 = CH2CH2COOtBu, R2 = All
5f, AA = Hcy, R1 = CH2CH2COOH, R2 = All
5g, AA = Asu, R1 = OtBu, R2 = H
EXPERIMENTAL
Ch lorom eth ylated Merrifield (0.6 m m ol Cl/g), Boc-Trp(For)-Merrifield (0.46 m m ol/g),
2-ch lorotritylch loride (1.3 m m ol/g) an d 4-(ben zyloxy)ben zyl alcoh ol Wan g (0.74 m m ol/g)
resin s were purch ased from Calbioch em –Novabioch em AG (Läufelfin gen , Switzerlan d),
TPTU, TBTU an d HBTU from Sen n Ch em icals In tern ation al (Gen tilly, Fran ce) an d protected
am in o acids were purch ased from Bach em (Buben dorf, Switzerlan d) or were prepared in our
laboratory followin g gen eral protocols18 an d publish ed papers19–27. In th e Fm oc- an d Boc-
am in o acid preparation s, th e prescribed pH of th e reaction m ixtures was m ain tain ed usin g a
pH m eter with an autom atic titrator (Radiom eter, Copen h agen , Den m ark). Th ey were ch ecked
for purity by th in -layer ch rom atograph y (TLC), h igh -perform an ce liquid ch rom atograph y
(HPLC), elem en tal an alysis an d m ass spectroscopy. Th e TLC of th e protected am in o acids
prepared was perform ed on precoated Silica gel 60 F254 plates (Merck, Darm stadt, Germ an y)
in th e followin g system s: butan -2-ol–98% form ic acid–water (75 : 13.5 : 11.5) (S1);
butan -2-ol–25% aqueous am m on ia–water (85 : 7.5 : 7.5) (S2); butan -1-ol–acetic acid–water
(4 : 1 : 1) (S3); butan -1-ol–pyridin el–acetic acid–water (15 : 10 : 3 : 6) (S4). Colum n ch rom a-
tograph y was carried out with Silica gel 60 (Merck, Darm stadt, Germ an y). Th e com poun ds
were visualized directly un der ultraviolet ligh t or by th e n in h ydrin an d ch lorin e-TDM detec-
tion 28. Meltin g poin ts were m easured on a Kofler block an d were n ot corrected. Optical rota-
tion s were m easured on a Perkin –Elm er 141 MCA polarim eter at 22 °C an d [α]D values are
given in 10–1 deg cm 2 g–1. Solven ts were evaporated in vacuo on a rotary evaporator (bath
tem perature 30 °C); DMF was evaporated at 30 °C an d 150 Pa. An average level of th e first
am in o acid substitution on th e correspon din g resin was determ in ed from a weigh t in crease,
spectroscopically by th e m easurem en t of th e diben zofulven e–piperidin e com plex absorption
after th e Fm oc group cleavage by 5% piperidin e in DCM–DMF (1 : 1) for 10 m in an d 20%
piperidin e in DMF for 15 m in , an d by th e quan titative am in o acid an alysis of th e am in o
acid loaded on th e resin . In th e Boc-am in o acid substitution assessm en t, on ly th e weigh t in -
crease an d am in o acid an alysis were used. Progress in peptide syn th esis was followed by th e
Kaiser29 an d Brom oph en ol Blue30 tests. Th e sam ples for am in o acid an alysis were h ydrolyzed
with 6 M HCl con tain in g 3% of ph en ol at 110 °C for 20 h . Th e am in o acid an alyses were
Collect. Czech. Chem. Commun. (Vol. 65) (2000)

412
Hlaváček, Marcová, Buděšínský, Slaninová:
perform ed on a Bioch rom 20 in strum en t (Ph arm acia, Sweden ). 1H an d 13C NMR spectra
were m easured on FT NMR spectrom eter Varian 500 (1H at 500 MHz, 13C at 125.7 MHz) in
DMSO-d6 with tetram eth ylsilan e as an in tern al referen ce. Molecular weigh ts of th e peptides
were determ in ed usin g m ass spectroscopy with FAB tech n ique (Microm ass, Man ch ester, En g-
lan d). For HPLC, a Spectra Ph ysics in strum en t with an SP 8800 pum p, an SP 4290 in tegrator
an d Th erm o Separation Products Spectra 100 UV detector was used. Peptides 3a–3c were
purified by sem ipreparative HPLC on 250 × 10 m m colum ns: 10 µm Vydac RP-18 (The Separa-
tion s Group, Hesperia CA, U.S.A.) an d 10 µm LiCh roCard, Purosph ere RP-18 (Merck,
Darm stadt, Germ an y), flow rate 3 m l/m in , detection at 200 n m usin g gradien t 50-100% ACN
in 0.05% aqueous TFA with in 60 m in . Th e an alytical HPLC was carried out on th e 250 × 4 m m
colum n : 5 µm LiCh rosph er WP-300 RP-18 (Merck, Darm stadt, Germ an y), flow rate 1 m l/m in ,
detection at 200 n m , 5–100% gradien t of ACN in 0.05% aqueous TFA with in 60 m in .
Strategy A
Protected Cyclopen tadecapeptides 1a an d 1b
Fm oc-Ph e-OH (0.21 g, 0.78 m m ol) was loaded to th e 2-ch lorotritylch loride resin (1 g) with
an average level of th e substitution 0.42 m m ol/g of th e resin . After th e Fm oc protectin g
group was rem oved, th e H-Ph e-resin was th en gradually acylated with 3 equivalen ts (1.3
m m ol) of Fm oc-am in o acids with correspon din g side-ch ain protection [Tyr(tBu), Val,
Cys(Trt), Asp(OtBu), Lys(Boc), Glu(OtBu), Met, Leu, Ser(tBu) twice, Cys(Trt) and Ser(tBu)] using
TBTU (0.49 g, 1.5 m m ol) and DIEA (0.52 m l, 3 m m ol) in DMF (20 m l). Th e Fm oc groups were
rem oved by treatm en t with 20% piperidin e in DMF (2 × 20 m l), 10 an d 30 m in . After th e
last Fm oc deprotection , th e peptide resin was wash ed with MeOH, dried in desiccator an d
divided in to two parts. Both parts were swollen in DMF (10 m l) an d acylated eith er with
Fm oc-Cys(CH2CH2CH2COOH)-O-All (5c; 0.32 g, 0.7 m m ol; for th e com poun d 1a) or with
Fm oc-Hcy(CH2CH2COOH)-O-All (5f; 0.32 g, 0.7 m m ol; for th e com poun d 1b ) via th eir
side-ch ain carboxylic groups as described above. After th e Fm oc deprotection an d fin al
wash in gs with DMF (5 × 10 m l), both peptides were split off th e resin by treatin g with an
AcOH (5 m l)–TFE (5 m l)–DCM (30 m l)–DMF (2 m l) m ixture for 30 m in at room tem perature.
Th e solven ts were evaporated, th e solid residues dissolved in a DCM (30 m l)–TFE (3 m l)–DMF
(30 m l) m ixture an d stirred with TPTU (0.45 g, 1.5 m m ol), HOBt (0.2 g, 1.5 m m ol) an d
DIEA (0.6 m l, 3 m m ol) at room tem perature for about 3 days un til th e Kaiser test in dicatin g
th e presen ce of free α-am in o group was n egative. Both reaction m ixtures were evaporated to
dryn ess an d eth yl acetate was added to th e residues wh ich precipitated. After filtration an d
wash in g with eth yl acetate, th e protected cyclic pen tadecapeptide allyl esters were dried in a
desiccator to dryness, 0.4 g (0.15 m m ol) of each was dissolved under argon in DCM (12 m l)–TFE
(2 m l)–DMF (12 m l) an d [Pd(Ph 3P)4] (0.23 g, 0.2 m m ol) with PPh 3 (0.06 g, 0.2 m m ol) an d
m orph olin e (0.018 m l, 0.2 m m ol) were added to th e solution s. Th e reaction m ixtures were
stirred un der argon for 2 h at room tem perature, solven ts were evaporated, th e crude cyclic
pen tadecapeptide acids 1a an d 1b were wash ed with water, eth yl acetate, dieth yl eth er an d
purified with a DMF–dieth yl eth er m ixture. After dryin g in desiccator, th e yields were 0.23 g
(0.09 m m ol; 43%) an d 0.21 g (0.08 m m ol; 38%) of th e protected cyclopeptides 1a an d 1b,
respectively, wh ich were ch ecked for h om ogen eity by HPLC, FAB MS an d am in o acid an aly-
sis (Table I).
Collect. Czech. Chem. Commun. (Vol. 65) (2000)

Analogues of Endothelin-1
413
TABLE I
An alytical data of protected cyclic pen tadecapeptides 1a, 1b, 2a, 2b an d an alogues 3a–3c
Am in o acid com position a
Com -
poun d
Form ulab
HPLCc
MW/(M+ + 1)
Trp
Ile
Asp Leu
His Ph e Tyr Cys(C3)d
Asu Hcy(C2)e
Val Cys Glu Lys Met Ser
1a
C138H191N15O26S4
42.75
42.54
40.23
40.05
–
–
1.01 0.97
–
1.06 0.91
1.01
–
2 604.4/2 603.3
1.00 1.81 1.05 1.02 0.92 2.60
–
f
1b
–
–
1.05 0.99
–
1.04 0.94
–
1.00 1.84 1.02 1.03 0.87 2.57
–
0.97
2a
C133H168ClN15O26S4
–
–
1.07 1.05
–
1.02 0.96
0.97
–
2 556.6/2 555.1
1.00 1.86 1.01 1.07 0.91 2.68
–
f
2b
–
–
1.02 1.02
–
1.02 0.92
–
1.00 1.81 0.99 1.05 0.86 2.54
–
1.02
3ag
3ah
3bg
3bh
3c
C110H160N24O32S4
22.15 0.74 1.76 2.01 1.82 1.03 1.03 0.98
1.00 1.83 1.05 1.04 0.89 2.62
1.04
–
2 458.9/2 458
–
f
0.76 1.76 1.98 1.85 0.99 1.03 1.01
1.00 1.81 1.04 1.06 0.93 2.63
1.02
–
–
f
f
22.12 0.76 1.72 1.97 1.85 1.01 1.02 0.99
1.00 1.85 1.01 1.03 0.92 2.65
–
–
0.95
0.78 1.75 1.99 1.88 0.99 1.01 1.01
1.00 1.83 1.02 1.01 0.95 2.68
–
–
0.96
C111H162N24O32S3
2 440.9/2 440
22.98 0.81 1.73 1.98 1.84 1.04 1.05 1.01
1.00 1.91 1.02 1.01 0.89 2.57 0.93
–
–
a
b
Am ino acid analyses were perform ed on Biochrom 20 instrum ent (Pharm acia, Sweden). Deter-
c
mined with FAB technique (VG Analytical, England). Retention time in minutes, 25 × 0.4 cm RP-18
colum n , 5 µm (Lich rosph er WP-300, Merck Darm stadt, Germ an y), flow rate 60 m l/h , detec-
tion at 222 n m , gradien t 50–100% of aceton itrile in 0.05% aqueous TFA, 60 m in , Spectra
Ph ysics SP 8800 HPLC pum p, an SP 4290 in tegrator an d Th erm o Separation Products Spectra
d
e
f
100 UV d etector.
H-Cys(CH2CH2CH2COOH)-OH. H-Hcy(CH2CH2COOH)-OH. Th e
g
h
sam e m olecular weigh t was con firm ed by m ass spectroscopy. Prepared by strategy A. Pre-
pared by strategy B.
Collect. Czech. Chem. Commun. (Vol. 65) (2000)

414
Hlaváček, Marcová, Buděšínský, Slaninová:
Segm en t Syn th esis of th e Protected 1-Deam in o-1-carba- (1c) an d 1-Deam in o-15-carba- (1d )
h en icosapeptides on Resin
Th e protected cyclic peptides 1a an d 1b (0.13 g, 0.05 m m ol of each ) were separately agitated
with th e side-ch ain protected h exapeptide 4a lin ked to Wan g resin (0.2 g, 0.4 m m ol/g for
each of th e cyclic peptides) in th e m ixture of solven ts DCM (15 m l)–DMF (7.5)–TFE (3 m l)
with HBTU (0.11 g, 0.3 m m ol) in th e presen ce of DIEA (0.1 m l) at 40 °C for 3 days in ultra-
son ic bath . Th e peptide resin s 1c an d 1d were separated by filtration , wash ed with th e sam e
solven t m ixture (3 × 15 m l), with DCM (3 × 15 m l), MeOH (3 × 15 m l) an d dried in desicca-
tor.
An alogues 3a an d 3b by Oxidative Procedure A
Peptide-resin s 1c an d 1d were treated with 50% TFA in DCM (3 × 10 m l) in th e presen ce of
EDT (0.5 m l) and anisole (1 m l). The resins were filtered off, washed with 50% AcOH (3 × 10 m l),
th e filtrates evaporated to on e th ird of th eir volum es an d diluted with 100 m l of water. Th e
solution s were wash ed with eth yl acetate an d dieth yl eth er to rem ove th e EDT an d an isole
an d were freeze-dried (both about 0.11 g). Both products were oxidized by air in an
AcOH–TFA (1 : 1; 10 m l) solution at pH 8.5, wh ich was adjusted an d m ain tain ed by 50%
am m on ium h ydroxide. Th e peptide solution s (about 400 m l of each ) were stirred at room
tem perature for 24 h , th en acidified with AcOH to pH 4.5, evaporated to on e th ird of th eir
volum es an d fin ally freeze-dried to obtain th e crude an alogues 3a an d 3b wh ich were puri-
fied by preparative HPLC (Table I).
Protected Cyclopen tadecapeptides 2a an d 2b
Th e Cs salts of th e Fm oc-Cys(CH2CH2CH2COOtBu)-OH (5a ; 0.45 g, 0.65 m m ol) or
Fm oc-Hcy(CH2CH2COOtBu)-OH (5d ; 0.45 g, 0.65 m m ol) were separately loaded as th e first
am in o acid residues to ch lorom eth ylated Merrifield resin (0.5 g) in DMF (15 m l) at 50 °C for
30 h in th e presen ce of 18-crown -6 (0.24 g, 0.65 m m ol). Th e average substitution on th e
resin was 0.25 m m ol/g. Fm oc group in th is an d all th e followin g steps was rem oved by treat-
m en t with 20% piperidin e in DMF (2 × 10 m l), 15 m in . Th e am in o acid-resin was wash ed
with DMF (5 × 10 m l) an d th en gradually acylated with 3 equivalen ts (0.37 m m ol) of
Fm oc-am in o acids with correspon din g side-ch ain protection [Ph e, Tyr(BrZ), Val, Cys(MeBzl),
Asp(OcHx), Lys(ClZ), Glu(OcHx), Met, Leu, twice Ser(Bzl), Cys(MeBzl)] usin g TBTU (0.12 g,
0.4 m m ol) an d DIEA (0.13 m l, 0.8 m m ol) in DMF (10 m l). Th e last couplin g was carried out
usin g Boc-Ser(Bzl)-OH. After cleavage of th e Boc an d tBu ester groups with 50% TFA/DCM
(2 × 10 m l), 1 h , a closure of th e 15-carba bridge was carried out on th e resin usin g TPTU
(0.23 g, 0.74 m m ol), HOBt (0.11g, 0.74 m m ol) an d DIEA (0.24 m l, 1.48 m m ol) in DMF (10 m l)
at 50 °C for 3 days in an ultrason ic bath . Each of th e full-sequen ce-peptide resin s was
treated at room tem perature with a m ixture of MeOH (5 m l)–dioxan e (13 m l)–4 M NaOH
(0.5 m l) in ultrason ic bath for 3 m in at room tem perature an d th en AcOH (0.5 m l) was added.
Th e resin s were filtered off an d wash ed with a DCM–DMF–TFE m ixture (3 : 3 : 1; 3 × 10 m l).
Th is cleavage was repeated twice an d fin ally all filtrates were collected an d solution s evapo-
rated to dryn ess. Th e residues were triturated with eth yl acetate, separated by filtration an d
purified from a DMF–dieth yl eth er m ixture. Th e yields were 0.13 g (0.05 m m ol; 40%) an d
0.12 g (0.047 m m ol; 38%) of th e protected cyclopeptides 2a an d 2b, respectively, wh ich
were ch ecked for h om ogen eity by HPLC, FAB MS an d am in o acid an alyses (Table I).
Collect. Czech. Chem. Commun. (Vol. 65) (2000)

Analogues of Endothelin-1
415
Segm en t Syn th esis of th e Protected 1-Deam in o-1-carba- (2c) an d 1-Deam in o-15-carba- (2d )
h en icosapeptides on Resin
Solution s of protected cyclic pen tadecapeptides 2a or 2b (0.08 g, 0.03 m m ol), HBTU (0.06 g,
0.15 m m ol), HOBt (0.04 g, 0.3 m m ol) an d DIEA (0.1 m l, 0.6 m m ol) in a DMF (3 m l)–DCM
(3 m l)–TFE (1 m l) m ixture were stirred at room tem perature for 30 m in an d th en added
separately to side-ch ain protected h exapeptide 4b (0.11 g, 0.46 m m ol/g) lin ked to Merrifield
resin . Th e con den sation was allowed to proceed in ultrason ic bath at 50 °C for 3 days. Th e
peptide-resin s were separated by filtration , wash ed with a DCM–DMF–TFE m ixture (3 : 1;
3 × 10 m l), MeOH (3 × 10 m l) an d dried in desiccator.
Strategy B
Step-Wise Syn th esis of th e Protected 1-Deam in o-1-carba- (2c), 1-Deam in o-15-carba- (2d )
an d 1-Deam in o-1,15-dicarba- (2e) h en icosapeptides on Resin
Each of th e Fm oc-Cys(CH2CH2CH2COOtBu)-OH (5a ; 0.24 g, 0.5 m m ol), Fm oc-
Hcy(CH2CH2COOtBu)-OH (5d; 0.24 g, 0.5 mmol) and Fmoc-Asu(OtBu)-OH (5 g; 0.23 g, 0.5 m m ol)
in DMF solution (20 m l) was activated with HBTU (0.6 g, 1.5 m m ol), HOBt (0.2 g, 1.5
m m ol) in th e presen ce of DIEA (0.51 m l, 3 m m ol) an d coupled to th e h exapeptide (0.36 g,
0.46 m m ol/g) on th e Merrifield resin 4b with yieldin g th e correspon din g carboxy-term in al
resin boun d h eptapeptides. Th e Fm oc protectin g group in th is an d all th e followin g steps
was rem oved by treatm en t with 20% piperidin e in DMF (2 × 10 m l), 30 m in . However, after
th is couplin g (wh ich was repeated twice with th e five fold excess of 5a, 5d an d 5g), th e
am in o acid substitution was foun d to be 0.15 m m ol/g on ly. Th erefore each of th e h epta-
peptide ch ain s boun d to resin was capped by treatm en t with an acetic acid an h ydride–DIEA
m ixture (2 : 1; 20 m l) for 2 h , wash ed with DCM (3 × 20 m l), DMF (5 × 20 m l) an d gradu-
ally acylated with 3 equivalen ts (0.16 m m ol) of side-ch ain protected Fm oc-am in o acids [Ph e,
Tyr(BrZ), Val, Cys(MeBzl), Asp(OcHx), Lys(ClZ), Glu(OcHx), Met, Leu, twice Ser(Bzl),
Cys(MeBzl)] usin g TBTU (0.05 g, 0.16 m m ol) an d DIEA (0.08 m l, 0.48 m m ol) in DMF (10 m l).
Th e last couplin g was carried out usin g Boc-Ser(Bzl)-OH. After cleavage of th e Boc an d tBu
ester groups with 50% TFA in DCM (2 × 10 m l), 1 h , th e peptide-resin s were treated with
TPTU (0.08 g, 0.25 m m ol), HOBt (0.06 g, 0.49 m m ol) an d DIEA (0.12 m l, 0.73 m m ol) in
DMF (15 m l) at 50 °C for 3 days in ultrason ic bath . Th e cyclized peptides boun d to th e resin
2c, 2d an d 2e were separated from th e reaction m ixture by filtration an d were wash ed with
3 × 20 m l portion s of DMF, isopropyl alcoh ol, MeOH an d dried in desiccator.
Com poun ds 3a, 3b an d 3c by Oxidative Procedure B
Each of th e protected peptide resin s 2c, 2d an d 2e was stirred with an EDT (0.2 m l)–m-cresol
(0.8 m l)–DMS (3 m l)–TFA (5 m l)–TFMSA (1 m l) m ixture at –5 to 0 °C for 30 m in an d th en it
was filtered, wash ed with cold dieth yl eth er an d dried to rem ove residual eth er. Th e pep-
tide-resin s were furth er treated with a th ioan isole–EDT 2 : 1 m ixture (1.5 m l) for 10 m in at
room tem perature an d after coolin g in an ice bath , TFA (10 m l) was added un der stirrin g.
After 10 m in , TFMSA (1 m l) was added dropwise an d th e reaction was stirred for an oth er 1.5 h
at room tem perature. Th e peptides were precipitated un der argon stream with dieth yl eth er
an d togeth er with th e resin filtered off th e solven t con tain in g EDT, m-cresol an d
th ioan isole. After wash in g with dieth yl eth er (3 × 50 m l), th e peptides were dissolved with
Collect. Czech. Chem. Commun. (Vol. 65) (2000)

416
Hlaváček, Marcová, Buděšínský, Slaninová:
TFA (5 m l each ), filtered in to cold dieth yl eth er (200 m l) to precipitate an d th e solids were
dried in desiccator. Each of th e h en isocapeptides con tain in g th e free sulfan yl groups in th e
Cys3, Cys11 residues was oxidized in aqueous 80% DMSO solution (500 m l) at pH m ain -
tain ed at 7–8 by aqueous am m on ia for 4 h . After acidification of th e m ixtures to pH 5, th e
solution s were freeze-dried an d th e crude an alogues 3a–3c subjected to HPLC purification in
th e gradien t of ACN (50–100%) in 0.05% TFA with in 60 m in . A LiCh roCard colum n with
th e Purosph ere RP-18 station ary ph ase recom m en ded for h ydroph obic peptides was used.
Th e purified an alogues 3a an d 3b exh ibited th e HPLC, FAB MS an d am in o acid an alyses
data iden tical to th ose obtain ed for th e sam e an alogues prepared from peptides 1c an d 1d
boun d to th e Wan g resin (Table I).
H-His(Boc)-Leu-Asp(OtBu)-Ile-Ile-Trp-O-WR (4a)
Fm oc-Trp-OH (0.52 g, 1.2 m m ol) was coupled to 4-ben zyloxyalcoh ol resin (WR; 0.5 g) usin g
a Sieber m eth od31 with substitution of 0.4 m m ol/g, th en deprotected with 20% piperidin e
in DMF an d gradually acylated with 3 equivalen ts (0.6 m m ol) of side ch ain protected
Fm oc-am in o acids [Ile twice, Asp(OtBu), Leu an d His(Boc)] usin g TBTU (0.22 g, 0.7 m m ol)
an d DIEA (0.25 m l, 1.4 m m ol) in DMF (20 m l). After th e fin al Fm oc deprotection an d wash -
in g with DMF, propan -2-ol an d MeOH, th e partially protected h exapeptide resin 4a was
dried in desiccator (0.62 g) an d divided in to two parts wh ich were used for th e segm en t con -
den sation with th e cyclic pen tadecapeptides 1a an d 1b.
H-His(Bom )-Leu-Asp(OcHx)-Ile-Ile-Trp(For)-O-MR (4b)
After th e Boc deprotection with 50% TFA in DCM an d n eutralization with 5% DIEA in
DCM, th e H-Trp(For)-O-MR (2.0 g, 0.46 m m ol/g) was gradually acylated with 3 equivalen ts
(2.76 m m ol) of side-ch ain protected Boc-am in o acids [Ile twice, Asp(OcHx), Leu an d
His(Bom )] usin g DIC (0.43 m l, 2.76 m m ol) an d HOBt (0.37 g, 2.76 m m ol) in DMF (30 m l).
After th e fin al Boc deprotection , n eutralization an d wash in g with DCM, propan -2-ol an d
MeOH, th e side-ch ain protected h exapeptide-resin 4b was dried in desiccator (2.6 g) an d di-
vided in to five parts wh ich were used eith er for th e segm en t con den sation with th e cyclic
pen tadecapeptides 2a an d 2b or for th e step-wise syn th esis of th e h en icosapeptides 2c–2e.
Fm oc-Cys(CH2CH2CH2COOtBu)-OH (5a)
A solution of H-Cys(CH2CH2CH2COOtBu)-OH (6.6 g, 25 m m ol) in water (500 m l) was stirred
at pH 9.3 (adjusted an d m ain tain ed with 10% Na2CO3) with Fm oc-OSu (8.5 g, 25.2 m m ol)
in aceton e (150 m l) for 3 h . Aceton e was evaporated, th e alkalin e solution was wash ed with
dieth yl eth er (4 × 100 m l) an d acidified with aqueous HCl to pH 3. Th e separated oil was ex-
tracted with eth yl acetate (3 × 100 m l), th e organ ic solution was wash ed with saturated
KHSO4 an d Na2SO4 an d evaporated to dryn ess. Th e residue was purified on a colum n of Silica
gel 60 in th e gradien t of solven ts EtOAc–PE yieldin g 8.7 g (17.9 m m ol; 72%) of pure product.
M.p. 89–90 °C; [α]D –27.9 (c 0.59, DMF), [ref.22 m .p. 142–143 °C for dicycloh exylam m on ium
salt; [α]D +2.8 (c 0.36, EtOH)]. Th e HPLC reten tion tim e was 19.6 m in (60–100% MeOH in
0.05% TFA, 30 m in ). TLC: RF 0.78 (S1), 0.30 (S2), 0.84 (S3), 0.49 (S4), 0.33 (40% EtOAc in PE).
1H NMR: 12.88 b, 1 H (carboxyl); 7.75 d, 1 H (carbam ate); 7.33 dt, 7.42 bt, 7.73 bd, 7.89 bd,
8 H (fluoren e, arom .); 4.28 dd, 2 H an d 4.31 dd, 2 H (2 × CH2O); 4.23 bt, 1 H (fluoren e,
alif.); 4.12 ddd, 1 H (α-CH); 2.74 dd an d 2.90 dd, 2 H (β-CH2); 2.52 m , 2 H (δ-CH2); 2.27 t, 2 H
Collect. Czech. Chem. Commun. (Vol. 65) (2000)

Analogues of Endothelin-1
417
(ζ-CH2); 1.72 t, 2 H (ε-CH2); 1.38 s, 9 H (C(CH3)3). 13C NMR: 172.49, 1 C (COOH); 171.96, 1 C
(CO, ester); 156.19, 1 C (CO, carbam ate); 143.98, 140.91, 127.82, 127.24, 125.49, 120.30, 12 C
(fluoren e, arom .); 79.79, 1 C (C(CH3)3); 65.91, 1 C (CH2O); 54.24, 1 C (α-CH2); 46.80, 1 C
(fluoren e, alif.); 33.78, 1 C (ζ-CH2); 32.71, 1 C (δ-CH2); 30.82, 1 C (β-CH2); 27.91, 3 C
(C(CH3)3); 24.70, 1 C (ε-CH2). For C26H31NO6S (485.6) calculated: 64.31% C, 6.43% H,
2.88% N, 6.60% S; foun d: 64.25% C, 6.44% H, 3.16% N, 6.39% S. FAB MS, m/z: 486.2 (M + 1).
Fm oc-Cys(CH2CH2CH2COOtBu)-O-All (5b)
A m ixture of Fm oc-Cys(CH2CH2CH2COOtBu)-OH (5a; 9.6 g, 20 m m ol) an d NaHCO3 (1.8 g,
21 m m ol) in water (32.4 m l), Aliquat 336 (9.6 m l) an d allyl brom ide (2.5 g, 21 m m ol) in
DCM (31.9 m l) was stirred at room tem perature for 30 h . Allyl brom ide (2.5 g, 21 m m ol)
an d 0.5 M NaHCO3 (5.3 m l) were added an d th e m ixture was stirred for an oth er 24 h . Th e
product was extracted from aqueous layer with DCM (3 × 100 m l), th e DCM solution was
dried with Na2SO4, evaporated to dryn ess an d th e diester was purified on a Silica gel 60 col-
um n in a EtOAc–PE gradien t yieldin g pure com poun d 5b (5.2 g, 49.5%). Th e HPLC reten -
tion tim e was 25.18 m in (60–100% MeOH in 0.05% TFA, 30 m in ). TLC: RF 0.85 (40% EtOAc
in PE) an d 0.69 (30% EtOAc in PE). For C29H35NO6S (525.7) calculated: 66.26% C, 6.71% H,
2.66% N, 6.10% S; foun d: 66.61% C, 6.48% H, 2.39% N, 6.51% S. FAB MS, m/z: 526.4 (M + 1).
Fm oc-Cys(CH2CH2CH2COOH)-O-All (5c)
Th e diester 5b (5.3 g, 10 m m ol) was treated with TFA (6 m l) in th e presen ce of dieth yl sul-
fide (1.1 m l, 10 m m ol) at room tem perature for 2 h . TFA was evaporated an d th e residue
evaporated th ree tim es with toluen e. Th e oily product (4 g) was purified on a Silica gel 60
colum n usin g 50% EtOAc in PE yieldin g 3.2 g (6.8 m m ol; 68%) of th e pure acid 5c, m .p.
94–96 °C; [α]D –28.4 (c 0.47, DMF). Th e HPLC reten tion tim e was 16.43 m in (60–100%
MeOH in 0.05% TFA, 30 m in ). TLC: RF 0.16 (30% EtOAc in PE). 1H NMR: 12.29 b, 1 H (car-
boxyl); 7.75 d, 1 H (NH, carbam ate); 7.89 bd, 7.73 bd, 7.42 bt, 7.33 dt, 8 H (fluoren e,
arom .); 5.89 m , 1 H (β-allyl ester); 5.29 dq an d 5.20 dq, 2 H (2 × γ-allyl ester); 4.58 m , 2 H
(α-allyl ester); 4.31 dd an d 4.28 dd, 2 H (CH2O); 4.23 bt, 1 H (fluoren e, alif.); 4.12 m , 1 H
(α-CH); 2.52 m , 2 H (δ-CH2); 2.27 t, 2 H (ζ-CH2); 2.91 dd an d 2.75 dd, 2 H (β-CH2); 1.72 t, 2 H
(ε-CH2). 13C NMR: 173.15, 1
C (COOH); 171.46, 1 C (CO, ester); 156.32, 1 C (CO,
carbam ate); 143.98, 140.93, 127.83, 127.25, 125.38, 120.32, 12 C (fluoren e, arom .); 117.92, 1 C
(γ-allyl ester); 132.56, 1 C (β-allyl ester); 65.83, 1 C (CH2O); 65.18, 1 C (α-allyl ester); 54.14,
1 C (α-CH); 46.81, 1 C (fluoren e, alif.); 33.58, 1 C (ζ-CH2); 30.82, 1 C (β-CH2); 32.71, 1 C
(δ-CH2); 24.72, 1 C (ε-CH2). For C25H27NO6S (469.6) calculated: 63.95% C, 5.80% H, 2.98% N,
6.83% S; foun d: 63.22% C, 5.67% H, 2.89% N, 6.23% S. FAB MS, m/z: 470.1 (M + 1).
Fm oc-Hcy(CH2CH2COOtBu)-OH (5d )
Th e sam e reaction an d purification con dition s as described for com poun d 5a were used in
th e reaction of H-Hcy(CH2CH2COOtBu)-OH (5.3 g, 20 m m ol) with Fm oc-OSu (7.1 g, 21 m m ol).
Th e yield of pure 5d was 6.7 g (69%). Th e data of TLC, HPLC, elem en tal an d FAB MS an aly-
ses were close to th ose obtain ed for th e Cys derivative 5a. M.p. 86–89 °C; [α]D –21.3 (c 0.52,
DMF), [ref.23 m .p. 87–89 °C; [α]D –17.8 (c 1.8, DMF)]. 1H NMR: 12.87 b, 1 H (carboxyl); 7.85 d,
1 H (NH, carbam ate); 7.89 bd, 7.71 bd, 7.42 bt, 7.33 dt, 8 H (fluoren e, arom .); 4.32 m , 2 H
(CH2O); 4.23 bt, 1 H (fluoren e, alif.); 4.22 m , 1 H (α-CH); 2.67 t, 2 H (ε-CH2); 2.57 m , 2 H
Collect. Czech. Chem. Commun. (Vol. 65) (2000)

418
Hlaváček, Marcová, Buděšínský, Slaninová:
(γ-CH2); 2.51 m , 2 H (ζ-CH2); 1.92 m , 2 H (β-CH2); 1.38 s, 9 H (C(CH3)3). 13C NMR: 173.19,
1 C (COOH); 171.45, 1 C (CO, ester); 156.34, 1 C (CO, carbam ate); 143.98, 140.93, 127.83,
127.25, 125.38, 120.32, 12 C (fluoren e, arom .); 79.98, 1 C (C(CH3)3); 65.98, 1 C (CH2O);
54.64, 1 C (α-CH); 46.80, 1 C (fluoren e, alif.); 32.73, 1 C (ζ-CH2); 31.02, 1 C (β-CH2); 27.89,
3 C (C(CH3)3); 27.65, 1 C (γ-CH2); 24.04, 1 C (ε-CH2).
Fm oc-Hcy(CH2CH2COOtBu)-OAll (5e)
Usin g th e reaction an d purification con dition s described for th e syn th esis of th e Cys deriva-
tive 5b, Fm oc-Hcy(CH2CH2COOtBu)-OH (5d ; 3.8 g, 7.9 m m ol), NaHCO3 (1.34 g) in water
(15.8 m l) an d Aliquat 336 (3.6 m l) were treated with allyl brom ide (1.96 g, 16.2 m m ol) in
DCM (12 m l) to afford 2.1 g of pure 5e (50%) with an alytical HPLC, TLC, elem en tal an d
FAB MS an alysis data close to th ose obtain ed for th e Cys derivative 5b.
Fm oc-Hcy(CH2CH2COOH)-OAll (5f)
Th e sam e reaction an d purification con dition s described in preparation of Cys derivative 5c
were used in treatm en t of diester 5e (2.65 g, 5.0 m m ol) with TFA (3 m l) in th e presen ce of
dieth yl sulfide (0.55 m l). Pure product 5f was obtain ed in a yield of 1.6 g (68%), m .p. 93–95 °C;
[α]D –22.3 (c 0.42, DMF) an d exh ibited an alytical HPLC, TLC, elem en tal an d FAB MS an aly-
sis data close to th ose obtain ed for th e Cys derivative 5c. 1H NMR: 12.29 b, 1 H (carboxyl);
7.85 d, 1 H (NH, carbamate); 7.89 bd, 7.71 bd, 7.42 bt, 7.33 dt, 8 H (fluorene, arom.); 5.89 m, 1 H
(OCH2CH=CH2); 5.29 dq an d 5.20 dq, 2 H (2 × OCH2CH=CH2); 4.58 m , 2 H (OCH2CH=CH2);
4.32 m , 2 H (CH2O); 4.23 bt, 1 H (fluoren e, alif.); 4.22 m , 1 H (α-CH); 2.67 t, 2 H (ε-CH2);
2.57 m , 2 H (γ-CH2); 2.51 m , 2 H (ζ-CH2); 1.92 m , 2 H (β-CH2). 13C NMR: 173.19, 1 C (COOH);
171.96, 1 C (CO, ester); 156.34, 1 C (CO, carbam ate); 143.98, 140.93, 127.83, 127.25,
125.38, 120.32, 12
C (fluoren e, arom .); 117.91, 1 C (OCH2CH=CH2); 132.54, 1 C
(OCH2CH=CH2); 65.83, 1 C (CH2O); 65.08, 1 C (OCH2CH=CH2); 53.04, 1 C (α-CH); 46.82, 1
C (fluoren e, alif.); 34.66, 1 C (ζ-CH2); 31.00, 1 C (β-CH2); 27.64, 1 C (γ-CH2); 26.30, 1 C
(ε-CH2).
Z-Asu-OBzl
Z-Asu-OH (refs24,26) (6.0 g, 18.6 m m ol) was stirred with TFE (2.6 m l, 18.6 m m ol) an d ben zyl
brom ide (3.5 g, 2.4 m l, 20.4 m m ol) in DMF (4 m l) at room tem perature for 20 h . Th e reac-
tion m ixture was poured in to a water with ice (40 m l) an d th e organ ic layer was taken up
with EtOAc. Th is solution was wash ed with 5% NaHCO3 an d water, dried with Na2SO4 an d
evaporated to dryn ess yieldin g an oily product (6.4 g, 15.5 m m ol). Th e purity of 88% was
assessed by HPLC in th e 0–100% gradien t of ACN in 0.05% TFA, 60 m in , 220 n m , elution
tim e 35 m in (con ten t of th e ben zyl ester in th is product was 5.6 g (13.6 m m ol)).
H-Asu(OtBu)-OH
Usin g gen eral procedure18, Z-Asu-OBzl (4.1 g, 10 m m ol) was con verted to th e tert-butyl ester
oily product (4.6 g) con tain in g 92% of Z-Asu(OtBu)-OBzl (by HPLC as described above), elu-
tion tim e 44.8 m in . Th e diester was sapon ified in aceton e (40 m l) solution with 1 M NaOH
(10 m l) at 0–5 °C for 5 h . After acidification with 1 M HCl, th e separated oil was extracted
with EtOAc an d th is solution was wash ed with water, dried with Na2SO4 an d evaporated to
Collect. Czech. Chem. Commun. (Vol. 65) (2000)

Analogues of Endothelin-1
419
dryn ess. Th e oily residue (3.3 g), free of th e ben zyl ester as assessed by HPLC usin g th e
0–100% gradien t of ACN in 0.05% TFA, 60 m in , elution tim e 37.6 m in , was h ydrogen ated in
propan -2-ol (100 m l) solution on Pd black (prepared by gen eral procedure from 9 m l of 10%
PdCl2) for 2 h at room tem perature. Th e catalyst was filtered off an d propan -2-ol eas evapo-
rated yieldin g 2.1 g (86%) of H-Asu(OtBu)-OH (HPLC elution tim e 20.1 m in in th e gradien t
described above).
Fm oc-Asu(OtBu)-OH (5g)
Th e sam e reaction an d purification con dition s as described with com poun d 5a were used in
th e reaction of H-Asu(OtBu)-OH (3.2 g, 13 m m ol) with Fm oc-OSu (4.7 g, 13.9 m m ol). Th e
crude com poun d was purified on a Silica gel 60 colum n in th e gradien t of solven ts
EtOAc–PE yieldin g 4.3 g (71%) of pure derivative 5g, m .p. 89–91 °C; [α]D –16.9 (c 0.51,
DMF). HPLC reten tion tim e was 41.6 m in (0–100% ACN in 0.05% TFA, 60 m in ). TLC: RF
0.78 (S1), 0.32 (S2), 0.85 (S3), 0.54 (S4), 0.39 (40% EtOAc in PE). 1H NMR: 12.88 b, 1 H (car-
boxyl); 7.64 d, 1 H (NH, carbam ate); 7.33 dt, 7.42 bt, 7.73 bd, 7.89 bd, 8 H (fluoren e,
arom .); 4.26 dd an d 4.29 dd, 2 H (2 × CH2O); 4.22 bt, 1 H (fluoren e, alif.); 3.92 ddd, 1 H
(α-CH); 2.17 bt, 2 H (ζ-CH2); 1.67 m an d 1.59 m , 2 H (β-CH2); 1.47 m , 2 H (ε-CH2); 1.38 s, 9 H
(C(CH3)3); 1.26 m , 2 H (δ-CH2). 13C NMR: 174.17, 1 C (COOH); 172.44, 1 C (CO, ester);
156.34, 1 C (CO, carbam ate); 144.05, 140.90, 127.81, 127.24, 125.48, 120.30, 12 C (fluoren e,
arom .); 79.55, 1 C (C(CH3)3); 65.74, 1 C (CH2O); 53.88, 1 C (α-CH); 46.85, 1 C (fluoren e,
alif.); 34.85, 1 C (ζ-CH2); 30.75, 1 C (β-CH2); 28.07, 1 C (δ-CH2); 27.94, 3 C (C(CH3)3);
24.58, 1 C (γ-CH2); 25.38, 1 C (ε-CH2). For C27H33NO6 (467.6) calculated: 69.36% C, 7.11% H,
3.00% N; foun d: 68.96% C, 7.18% H, 3.13% N. FAB MS, m/z: 467.2 (M + 1).
Biological Assay
Uteroton ic Activity Assessm en t
Com poun d 3a, 3b or 3c (0.3 m g) was dissolved in 1 M NaOH (0.5 m l) an d 1 M HCl was
added dropwise un til pH 6 of th e solution was reach ed. Peptides were tested for uteroton ic
activity in vitro in th e previously described rat uteroton ic test for oxytocin accordin g to
Holton 32 in th e Mun sick33 solution eith er in th e absen ce of Mg2+ or in th e presen ce of 1 m M
Mg2+. Syn th etic en doth elin was used as a stan dard for determ in ation of both th e agon istic
an d an tagon istic activities. Fem ale rats were estrogen ized 24–48 h before th e experim en t.
Cum ulative dose respon se curves were con structed usin g data from experim en ts in wh ich
doses were added successively in double concentrations to the bath with an uterus strip in 1 m in
in tervals with out ch an gin g th e fluid un til a m axim al respon se was obtain ed. Th e activity
was determ in ed by com parin g th e th resh old doses of th e stan dard an d th e respective an alog
(IU/m g or EC50). In th e case of an tagon istic activity, th e dose of th e an tagon ists was applied
to th e bath with th e uterus strip, 1 m in prior to th e stan dard dose of en doth elin . Th e an tag-
on istic activity was expressed as EC50 or pA2, i.e. th e con cen tration of th e an alogue, wh ich
reduced th e effect of th e double dose of agon ist, in our case en doth elin , to th e effect of dose x,
an d th e n egative decadic logarith m of th e EC50, respectively. Each an alog was tested on
uteri from 3–5 differen t Wistar rats.
Collect. Czech. Chem. Commun. (Vol. 65) (2000)

420
Hlaváček, Marcová, Buděšínský, Slaninová:
RESULTS AND DISCUSSION
In gen eral, a com bin ation of th e solid ph ase an d solution approach es h as
been chosen in the synthesis of ET-1 analogues 3a–3c using strategies A and B.
Th e strategy A h as followed a con vergen t approach con sistin g in segm en t
con den sation of th e form erly prepared protected cyclic pen tadecapeptide
acids 1a, 1b an d 2a, 2b with correspon din g side-ch ain protected h exa-
peptides boun d to resin s 4a an d 4b usin g th e sam e couplin g reagen ts an d
con dition s.
However, th e preparation of 1a, 1b was differen t from th at of 2a, 2b (an d
con sequen tly, th e preparation of 4a differen t from th at of 4b) as regards
th e peptide ch ain assem bly an d th e side-ch ain protection . Com poun ds 1a,
1b an d 4a were built up on th e m ild-acid-labile resin s with tBu an d Trt
side-ch ain protection an d th e Fm oc-Cys-OAll (5c) or Fm oc-Hcy-OAll (5f)
con tain in g 1-carba(m eth ylen e-sulfan yl) or 15-carba(sulfan yl-m eth ylen e)
lin kages were in serted in to correspon din g peptide ch ain s in th e last
solid-ph ase step. Th e followin g cyclization an d th e carboxy group
deprotection were carried out in solution .
On th e con trary, in preparation of 2a an d 2b th e peptide ch ain s were
built up on th e stron g acid or base-labile resin with a ben zyl-type
side-ch ain protection . Th e 1-carba or 15-carba lin kages were in serted in to
peptide ch ain s in th e first couplin g step wh en th e correspon din g com poun ds
Fm oc-Cys(CH2CH2CH2COOtBu)-OH (5a) or Fm oc-Hcy(CH2CH2COOtBu)-OH
(5d ) Cs salts were coupled to th e Merrifield resin . Th e fin al cyclization was
perform ed on th e resin an d th e C-term in al carboxy group deprotection
was, in fact, iden tical with th e cleavage of th e protected cyclized peptides
2a, 2b from th e resin .
Th e strategy B con sisted in th e step-wise con struction of th e wh ole peptide
sequen ce from th e C-term in al Trp residue boun d to th e Merrifield resin .
The Fmoc-Cys(CH2CH2CH2COOtBu)-OH (5a) or Fmoc-Hcy(CH2CH2COOtBu)-OH
(5d ), an d also Fm oc-Asu(OtBu)-OH (5g) bearin g th e eth ylen e (1,15-dicarba)
lin kage, were coupled to th e growin g peptide ch ain in th e seven th step.
Th is syn th etic procedure was sim ilar to th at utilized for peptides 2a, 2b an d
4b due to th e sam e reagen ts an d th e sam e am in o acid side-ch ain protection
used. Th e on ly differen ce is th e closure of carba bridges, perform ed in th e
last step of th e syn th eses on th e polym er, just before th e deprotection an d
detach m en t of th e h en icosapeptides from th e resin .
Th e strategy B seem s th e m ost straigh tforward an d th e sim plest on e due
to th e overall sh orter syn th etic route. However, we observed a large de-
crease in substitution of growin g peptide on th e Merrifield resin after cou-
Collect. Czech. Chem. Commun. (Vol. 65) (2000)

Analogues of Endothelin-1
421
plin g of th e carba bridge con tain in g com poun ds 5a, 5d an d 5g,
respectively, in th e seven th step. Th e decreased yields of th e couplin g were
also observed wh en th e com poun ds 5a an d 5d as Cs salts were lin ked to th e
sam e type of th e resin at th e begin n in g of th e syn th eses of protected
pen tadecapeptides 2a an d 2b wh en followin g th e strategy A.
Th is disadvan tage could be excluded by couplin g of th e 1-carba or
15-carba lin kage con tain in g com poun ds 5c or 5f via th eir side-ch ain
carboxylic groups in th e syn th esis of protected cyclic pen tadecapeptides 1a
an d 1b (strategy A). Th e reaction route was gen erally m ore com plicated;
h owever, it afforded better yields of solid-ph ase couplin gs an d possibility to
purify in dividual in term ediates in solution after th e pen tadecapeptide se-
quen ces of th e 1a an d 1b were detach ed from th e resin . A sligh t excess of
h exapeptide boun d resin 4a an d a larger excess of reagen ts was n ecessary
for its satisfactory con den sation with th e protected acids 1a an d 1b. In
com parison with th e strategy B an d th e strategy A wh ere th e cyclic pro-
tected pen tadecapeptides 2a, 2b an d h exapeptide boun d resin 4b were elab-
orated, th e em ploym en t of th e AcOH-labile 2-ch lorotrityl resin (1a an d 1b)
an d th e TFA-labile Wan g resin (4a) allowed th e usage of m ilder con dition s
for side-ch ain deprotection an d detach m en t of th e wh ole h en icosapeptide
sequen ces from th e resin (1c, 1d ) with out an y dam age to th e peptide m ole-
cules.
For th e oxidative closure of th e in n er disulfide bridge between Cys3 an d
Cys11 residues, basically two approach es were utilized. Th e first on e (proce-
dure A) con sisted in th e oxidation by air oxygen in a h igh ly diluted
am m on ium acetate–trifluoroacetate solution at pH 8, th e secon d on e (pro-
cedure B) used DMSO as a solvent and an oxidation reagent, as well. Even the
second approach was found to be more efficient due to a shorter time of the
oxidation, we have encountered well known problems with evaporation of
DMSO and with a measurement of exact pH values in aqueous 80% DMSO.
Com poun ds 3a–3c were subjected to a bioassay of a sm ooth m uscle con -
traction an d th e results were com pared with th at obtain ed in application of
ET-1. Non e of th e com poun ds used, at an y con cen tration , produced con -
traction of th e rat uterus “in vitro”. Nor was an tagon istic activity detected
for th e th ree com poun ds tested. Th e results of th e bioassay h ave con firm ed
a suggestion th at α-am in o group of ET-1 is a structural elem en t essen tial for
bin din g of th e peptide to a correspon din g receptor an d for a tran sduction
of biological activity. Th e deletion of th is group in 1-deam in o an alogues
3a–3c h as caused th e drop in th e ET vasocon strictory activity described al-
ready by Japan ese auth ors6 for [Ala3,11,Asu15]ET-1.
Collect. Czech. Chem. Commun. (Vol. 65) (2000)

422
Hlaváček, Marcová, Buděšínský, Slaninová:
On th e oth er h an d, th e substitution of th e outer disulfide bridge by th e
sulfan yl-m eth ylen e (carba-1, carba-15) or eth ylen e (1,15-dicarba) bridges
could be h ardly respon sible for such dram atic extin ction of biological activ-
ity. Th e cyclic arran gem en t, suggested to be im portan t for ach ievin g a h igh
receptor bin din g activity of ET-1, was also preserved in th ese carba an a-
logues. It was even reported th at th e two ET-1 an alogues, in wh ich Cys resi-
dues of eith er on e of th e disulfide bon ds h ave been replaced by Ala th us
givin g rise to a m on ocyclic an alogue, still possess about 2 an d 10% of th e
activity of ET-1 (refs5,34). Th ese an alogues readily bin d to th e rat cerebral
m em bran e an d are full agon ists wh en tested with th e rat aorta35,36. Cleav-
age of th e Cys3-Cys11 disulfide bon d, an d protection as a Cys(Acm ) or, al-
tern atively, reduction of both disulfides with subsequen t carboxam ido-
m eth ylation give rise to a weak partial agon ist (with respect to ET-1) judg-
in g from th e con striction test with porcin e aorta37. Hen ce th e decrease in
biological activity seem s to be rath er due to th e ch aracter of th e protectin g
groups th an to th e reduction of disulfide bon ds an d th e disulfide bon d can -
n ot be a n ecessary prerequisite for th e activation an d recogn ition of th e re-
ceptor.
Th erefore, th e syn th esis an d bioassay of th e ET-1 an alogues with th e
cystath ion in e buildin g block con tain in g th e carba lin kage an d bearin g also
th e α-am in o group, are n ecessary for fin din g th e true effect of th e carba
substitution on th e ET-1 con form ation an d for recogn ition of its receptors.
SYMBOLS
ACN, aceton itrile; AcOH, acetic acid; OAll, allyl ester; Asu, 2-am in osuberic acid; Boc,
tert-butyloxycarbon yl; BOP, [(ben zotriazol-1-yl)oxy]tris(dim eth ylam in o)ph osph on ium h exa-
fluoroph osph ate; BrZ, [(2-brom oben zyl)oxy]carbon yl; Bom , ben zyloxym eth yl; Bz, Ben zyl;
cHx, cycloh exyl; ClZ, [(2-ch loroben zyl)oxy]carbon yl; DCM, dich lorom eth an e; DIC,
N,N-diisopropylcarbodiim ide; DIEA, N,N-diisopropyleth ylam in e; DMF, N,N-dim eth yl-
form am ide; DMS, dim eth yl sulfide; DMSO, dim eth yl sulfoxide; EC50, effective con cen tra-
tion ; EDT, eth an e-1,2-dith iol; ET, en doth elin ; EtOAc, eth yl acetate; Fm oc, (fluoren -
1-yl-m eth oxy)carbon yl; For, form yl; HBTU, O-(ben zotriazol-1-yl)-1,1,3,3-tetram eth yluron ium
h exafluoroph osph ate; Hcy, h om ocystein e; HOBt, 1-h ydroxyben zotriazole; MeBzl,
4-m eth ylben zyl; MR, Merrifield resin ; OSu, succin im ide ester; PE, ligh t petroleum ; tBu,
tert-butyl; TBTU, O-(ben zotriazol-1-yl)-1,1,3,3-tetram eth yluron ium tetrafluoroborate; TDM,
4,4′-bis(dim eth ylam in o)diph en ylm eth an e; TFE, 2,2,2-trifluoroeth an -1-ol; TFMSA, trifluoro-
m eth an esulfon ic acid; TFA, trifluoroacetic acid; TPTU, O-[2-(1H)-oxopyridin -1-yl]-
1,1,3,3-tetram ethyluronium tetrafluoroborate; Trt, triphenylm ethyl; WR, Wang resin. The nom en-
clature and sym bols of am ino acids follow published recom m endations of the IUPAC/IUB Joint
Com m ission on Biochem ical Nom enclature (Eur. J. Biochem. 1984, 138, 9).
Collect. Czech. Chem. Commun. (Vol. 65) (2000)

Analogues of Endothelin-1
423
This paper was supported by the Grant Agency of the Czech Republic (grants No. 203/97/0155 and
No. 203/98/1330).
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