SYNTHESIS OF BICYCLIC PYRIDINE TRIPEPTIDES

Article abstract of DOI:10.1135/cccc19941451

Dinicotinic acid reacted with L-phenylalanine affording N-dinicotinoyl-bis-L-phenylalanine (VII).The same product was also obtained by mild alkaline hydrolysis of the corresponding ester VI.Coupling of VII with L-ornithine or L-ornithine methyl ester gave rise to the formation of the desired chiral bicyclic tripeptides IIIa and IIIb as enniatin analogues.

Full text of DOI:10.1135/cccc19941451

Bicyclic Pyridine Tripeptides
1451
SYNTHESIS OF BICYCLIC PYRIDINE TRIPEPTIDES
Abd El-Hamid ATTIA, Mohamed H. ABO-GHALIA and Osama I. ABD EL-SALAM
National Research Centre,
Dokki, Cairo, Egypt
Received September 2, 1993
Accepted January 20, 1994
Dinicotinic acid reacted with L-phenylalanine affording N-dinicotinoyl-bis-L-phenylalanine (VII). The
same product was also obtained by mild alkaline hydrolysis of the corresponding ester VI. Coupling
of VII with L-ornithine or L-ornithine methyl ester gave rise to the formation of the desired chiral
bicyclic tripeptides IIIa and IIIb as enniatin analogues.
Cyclic peptides are interesting biological compounds of diverse activities1. The cyclic
peptide antibiotic enniatin2 (I) is merely an example. Recently, Talma et al.3, starting
from 3,5-pyridine dicarboxylic acid (dinicotinic acid) and a variety of L-amino acids,
reported the synthesis of chiral bridged bis-coupled amino acid dihydropyridines II.
These compounds, which have an architectural resemblance to enniatin (I), were also
valuable for their chemical activity comparable to that of enzyme cofactors. Thus, they
were sufficiently active to carry out enantioselective reductions of keto groups to corre-
sponding secondary alcohols.
The present study deals with the synthesis of similar chiral cyclic tripeptide systems,
where L-ornithine performs the final ring closure rather than a dihalogeno compound
used by Talma3. The lateral substituents present positions at which other moieties could
be easily linked. Nicotinoylphenylalanine derivatives are reported to possess biological
activities such as antimicrobial4 or glucose and cholesterol level lowering5.
Synthesis of VII was initially attempted by DCCI/HOSu method6–9 (Scheme 1). The
in situ prepared N-hydroxysuccinimide active ester of dinicotinic acid IV was coupled
with L-phenylalanine in alkaline aqueous medium in a poor yield (16%). It was then
found more convenient to get VII by the mild alkaline hydrolysis of its corresponding
ethyl ester VI, which was successfully obtained by a low temperature coupling of a
freshly prepared 3,5-pyridinedicarbonyl chloride (V) with the amino acid ethyl ester in
presence of triethylamine (TEA). Cyclization of VII by the potentiated DCCI/
1-hydroxybenzotriazole (HOBt) method10–13 afforded a pure bis-coupled product IIIa in
42% yield. Characterization of the obtained compound IIIa was based on the qualitative
chromatographic analysis (TLC) of its hydrolyzed products where dinicotinic acid,
phenylalanine and ornithine were detected. The structure of IIIa was confirmed in light
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Attia, Abo-Ghalia, Abd El-Salam:
of its spectroscopic features. Its mass spectrum is devoid of the molecular ion peak
[M+], which may easily split off the carboxylic group. The ion [M − COOH]+ (m/z 512,
2%) was detected which on further fragmentation revealed ion at m/z 459 (10%) after
cleavage of the bridged alkyl group. Other ion peaks were found to be in agreement
1
with the assigned structure (Scheme 2). The H NMR spectrum proved the bridged
methylene protons in the range 1.1 – 2.9 ppm and the methine proton at δ 3.1.
A trial to esterify IIIa by classical Curtius method14 was unsuccessful. Typical hydro-
lyzed products were obtained as identified by TLC qualitative analysis. This may be
attributed to ring instability under the used reaction conditions. However, coupling
L-ornithine methyl ester with VII by the DCCI/HOBt method afforded the desired bis-
coupled ester IIIb which was further purified by preparative TLC (silica gel). The IR
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SCHEME 1
SCHEME 2
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Attia, Abo-Ghalia, Abd El-Salam:
spectrum showed bands at 3 040 cm−1 and 2 950 cm−1 [ν(CH), aliphatic and aromatic],
at 1 660, 1 550, 1 290 cm−1 (amide I, II and III bands) and at 1 780 cm−1 [ν(C=O, ester)].
1
Its H NMR spectrum exhibited the same signals found in that of IIIa with the excep-
tion of the downfield difference of 1.1 ppm for methyl ester protons. In addition,
13C NMR spectrum of IIIb gave signals corresponding to the bridgehead methylene
carbons in the range 39.57 – 33.88 ppm and the methine carbon at δ 56.68.
Results for the biological properties of the obtained products will be published sepa-
rately.
EXPERIMENTAL
All melting points are uncorrected. The infrared spectra (in KBr, given in cm−1) were recorded on
Unicam SP-1000 spectrophotometer. The 1H NMR spectra (given in δ ppm) were recorded on
EM-390 Varian spectrophotometer in (CD3)2SO using tetramethylsilane as an internal standard.
The mass spectrum (ionization by electron impact, EI) was performed by the organic chemistry
laboratory, Bonn University, Germany. Acceleration voltage: 2 kV, electron energy: 70 eV (100 µA)
and the ion source temperature: 150 °C. The spectra were recorded during heating of the sample (150 °C).
The reactions were followed up and the purity of the synthesized compounds was checked by thin-
layer chromatography (Merck, aluminium sheets, silica gel 60, F254) in the proper solvent systems
BuOH–pyridine–AcOH–H2O 120 : 40 : 12 : 48 (S1); acetone–AcOH–H2O 18 : 1 : 2 (S2); S2–light
petroleum 2 : 1 (S3), visualizing the spots in ultraviolet light and/or ninhydrin.
3,5-Pyridinedicarbonyl Dichloride (V)
A mixture of dinicotinic acid (IV) (1.67 g, 10 mmol) and thionyl chloride (5 ml) in dry dioxane
(15 ml) was refluxed for 1 h. Excess solvent was distilled off under reduced pressure and the crude mass
was triturated with dry benzene (3 × 15 ml). The acid chloride was obtained as a dark brown oil
which was immediately used.
Nα-Dinicotinoyl-bis-L-phenylalanine Ethyl Ester (VI)
Triethylamine (5.7 ml; 40 mmol) was added in portions to a well stirred cold solution (−5 °C) of
3,5-pyridinedicarbonyl dichloride (V) (2 g; 10 mmol) and L-phenylalanine ethyl ester hydrochloride
(4 g; 20 mmol) in 450 ml mixture benzene–methylene chloride 4 : 5. The reaction mixture was
stirred for 3 h at the same temperature with simultaneous adjustment of pH ≈ 8. Triethylamine hy-
drochloride was filtered off, and washed with water (3 × 10 ml), 1 M sodium bicarbonate (3 × 10 ml)
and 1 M HCl (3 × 10 ml); then dried over anhydrous sodium sulfate. The solvent was distilled off in
vacuo and the obtained oil was solidified and recrystallized from ethanol–hexane to afford white
crystals of VI (3.45 g; 67% yield); m.p. 122 – 123 °C; RF = 0.52 (S1); [α]3D0 = −77.3 (c = 0.05,
absolute ethanol); IR spectrum (KBr): 3 313 (NH), 1 733 (C=O, ester), 1 647 (amide I), 1 527
(amide II) and 1 293 (amide III). For C29H31N3O6 (517.6) calculated: 67.29% C, 6.04% H, 8.12% N;
found: 67.40% C, 5.99% H, 8.03% N.
Nα-Dinicotinoyl-bis-L-phenylalanine (VII)
Method A: Dry HCl gas was bubbled in a suspension of 3,5-pyridinedicarboxylic acid (IV) (0.84 g;
5 mmol) in glacial acetic acid for 30 min. The solvent was distilled off and the crude mass was left
in dessicator over sodium hydroxide pellets under reduced pressure. To a cold solution (−5 °C) of the
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obtained acid hydrochloride and HOSu (1.27 g; 11 mmol) in 50 ml tetrahydrofurane, DCCI (2.27 g;
11 mmol) was added in portions. A solution of L-Phe (1.65 g; 10 mmol) in 1 M NaHCO3 (11 ml) was
added and the reaction mixture was stirred for 3 h at 0 °C with adjustment of its pH ≈ 8 and left
overnight at room temperature. Dicyclohexylurea was filtered off and THF was distilled off. The cold
( ≈ 0 °C) water solution was acidified to pH ≈ 3 with 1 M HCl. The obtained gelatinous product was
extracted with ether and the ethereal solution was dried over anhydrous sodium sulfate. Solvent was
distilled off under reduced pressure and the obtained product was identified as VII, white crystals
when crystallized from aqueous ethanol (0.37 g; 16% yield); m.p. 223 – 224 °C; RF = 0.49 (S2);
[α]3D0 = −70 (c 0.05, absolute ethanol); IR spectrum (KBr): 3 313 (NH), 2 500 (OH), 1 705 (C=O,
1
acid), 1 674 (amide I), 1 573 (amide II) and 1 293 (amide III). H NMR spectrum ((CD3)2SO): 3.2 d,
4 H (2 CH2); 4.6 m, 2 H (2 CH); 6.7 s, 2 H (2 COOH); 7 – 7.4 m, 10 H (2 C6H5); 8.4 s, 1 H
(pyr-H-4); 8.8 – 9.1 d, 4 H (pyr-H-2,6 + 2 NH). For C25H23N3O6 (461.5) calculated: 65.07% C,
5.02% H, 9.11% N; found: 65.00% C, 5.14% H, 9.14% N.
Method B: A solution of 1 M KOH (5 ml, 5 mmol) was added dropwise to a stirred cold solution
(−5 °C) of VI (1.04 g; 2 mmol) in methyl alcohol (15 ml). Stirring was maintained at the same tem-
perature for 3 h. Methyl alcohol was distilled off under reduced pressure and water was added to a
volume of 25 ml. The cold aqueous solution (−5 °C) was acidified with 1 M HCl to pH ≈ 3, while
stirring. The obtained gelatinous product was then extracted with ether and the ethereal extract was
dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure and the
resulted residue was found to be VII by TLC when compared with authentic sample, which was re-
crystallized from aqueous ethanol to give white crystals of VII (0.49 g; 53% yield); m.p. 223 – 224 °C.
Cyclo[Nα-dinicotinoyl-bis-L-phenylalanyl-L-ornithine] (IIIa)
To a well stirred solution of VII (115 mg; 0.25 mmol) and HOBt (67.5 mg; 0.5 mmol) in dimethyl-
formamide (3 ml), L-ornithine (51 mg; 25 mmol) was added with equivalent amount of triethylamine
in dimethylformamide (2 ml), at 0 °C. The reaction temperature was kept at about −5 °C. Then,
DCCI (103 mg; 0.5 mmol) was added portionwise to the reaction mixture over 20 min. After the
addition was completed, the mixture was stirred for 5 h at −5 °C, the formed dicyclohexylurea was
filtered off and the reaction mixture was washed with acetonitrile (2 × 5 ml). The combined filtrate
was kept in refrigerator overnight and the newly formed dicyclohexylurea was filtered off. Solvent
was removed and the obtained residue was triturated with water, 1 M HCl and water again; on treat-
ment with ether then solidified. The obtained solid was filtered off, washed thoroughly with ether
and recrystallized from ethanol–ether to give 63 mg (42% yield) of IIIa as a yellowish white powder;
m.p. 154 °C (decomp.); RF = 0.61 (S2); [α]3D0 = −93° (c 0.05 absolute ethanol). IR spectrum: 3 230
1
(NH), 2 500 (OH), 1 725 (C=O), 1 660 (amide I), 1 530 (amide II), and 1 275 (amide III). H NMR
spectrum: 1.1 m, 2 H (CH2); 2.9 m, 8 H (4 CH2); 3.1 d, 3 H (3 CH); 7.1 – 7.4 m, 10 H
(2 C6H5); 8 – 8.5 m, 5 H (pyr-H-4 + 4 NH); 9 d, 3 H (pyr-H-2,6 + COOH). Mass spectrum, m/z: 512
(M+ − COOH), 484 (2), 471 (3), 459 (10), 310 (5), 175 (5), 91 (100).
Cyclo[Nα-dinicotinoyl-bis-L-phenylalanyl-L-ornithine Methyl Ester] (IIIb)
L-Ornithine methyl ester (37 mg; 0.25 mmol) was added to a well stirred mixture of VII (115 mg;
0.25 mmol) and HOBt (67.5 mg; 0.5 mmol) in dimethylformamide. The reaction temperature was
kept at −5 °C. Then, DCCI (103 mg; 0.5 mmol) was added in portions to the reaction mixture over
20 min, and stirring was maintained for 5 h at same temperature. The reaction mixture was diluted
with 25 ml acetonitrile and kept overnight in refrigerator. The formed dicyclohexylurea was filtered
off and washed with ethyl acetate (2 × 5 ml). The obtained residue after solvent distillation was dis-
solved in 25 ml ethyl acetate, washed with water, 1 M NaHCO3, water, 1 M HCl, water and dried over
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Attia, Abo-Ghalia, Abd El-Salam:
anhydrous magnesium sulfate. The solvent was distilled off and the obtained oily product was puri-
fied by preparative thin-layer chromatography (silica gel plates). A product of RF = 0.5 (S3) was
separated in methanol to give 54 mg (36%) brownish white crystals identified as IIIb; m.p. 172 °C;
[α]3D0 = 115 (c 0.05, absolute ethanol); IR spectrum: 3 300 (NH), 1 780 (C=O, ester), 1 660 (amide I),
1
1 550 (amide II) and 1 290 (amide III). H NMR spectrum: 1.1 t, 2 H (CH2); 2.8 m, 4 H (2 CH2);
3.1 m, 4 H (2 CH2); 3.6 s, 3 H (CH3); 4.5 m, 2 H (CH2); 7 – 7.4 m, 10 H (2 C6H5); 8.2 s, 4 H
(4 NH); 8.45 s, 1 H (pyr-H-4); 9 s, 2 H (pyr-H-2,6). 13C NMR spectrum: 174.36 d, 1 C (carboxy);
166.26 t, 4 C (C-2, 5, 12, 15); 152.41 d, 2 C (C-16, 20); 140.21 t, 2 C (C-17, 19); 136.12 d, 1 C
(C-1); 130.96 s, 6 C (o, p-Ph); 129.94 s, 4 C (m-Ph); 128.12 s, 2 C (i-Ph); 56.68 m, 3 C (C-4, 7, 13);
53.57 s, 1 C (CH3); 46.95 s, 2 C (CH2-benzyl); 39.57 m, 1 C (C-10); 38.82 m, 1 C (C-8); 33.88 m,
1 C (C-9).
Esterification of IIIa
Dry HCl gas was bubbled in 10 ml cold methanolic solution of IIIa (55 mg; 0.1 mmol) over a period
of 30 min. The resulted clear solution was then left at room temperature overnight. Solvent was dis-
tilled off under reduced pressure and the obtained residue was dissolved in water (10 ml) and neu-
tralized with 1 M NaHCO3. By TLC qualitative analysis, the expected product IIIb was not identified,
but only decomposition products were formed [dinicotinic acid (RF = 0.43), L-phenylalanine (RF
0.05), L-ornithine (RF = 0.1); (S1)].
=
Alkaline Hydrolysis of IIIb
1 M KOH was added to a cold (−5 °C) stirred methanolic solution of IIIb. Stirring was maintained at
the same temperature for 3 h. Methyl alcohol was distilled off under reduced pressure and the water
solution was acidified with 1 M citric acid to pH 3. The solid formed was filtered off and identified
as IIIa on comparison with authentic sample by TLC.
Acidic Hydrolysis of IIIa and IIIb
An acidic solution of IIIa or IIIb (10 mg; 5 ml 6 M HCl) was heated on a steam bath for 5 h. TLC
qualitative analysis of the neutralized reaction mixture indicated the presence of dinicotinic acid,
L-phenylalanine and L-ornithine on comparison with authentic samples.
REFERENCES
1. Schroder H., Lubke K.: The Peptides, Vol. II, p. 405. Academic Press, New York 1966.
2. Schroder H., Lubke K.: The Peptides, Vol. II, p. 112. Academic Press, New York 1966.
3. Talma A. G., Jouin P., De Varies J. G., Troostwijk C. B., Buning G. H. W., Waninge J. K.,
Visscher J., Kellog R. M.: J. Am. Chem. Soc. 107, 2981 (1985).
4. Abo-Ghalia M. H., Salem E. M.: Indian J. Chem., B 19, 135 (1980).
5. Uno H., Funabiki H., Irie A., Yoshimura Y.: Yakugaku Zasshi 87, 1393 (1967); Chem. Abstr. 68,
69294 (1968).
6. Anderson G. W., Zimmerman J. E., Callahan F.: J. Am. Chem. Soc. 86, 1839 (1964).
7. Wunsch E., Dress F.: Chem. Ber. 99, 110 (1966).
8. Wunsch E., Zwick A., Wendlberger G.: Chem. Ber. 100, 173 (1967).
9. Bodanszky M., Bodanszky A.: The Practice of Peptide Synthesis, p. 146. Springer, Heidelberg
1984.
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10. Bodanszky M., Bodanszky A.: The Practice of Peptide Synthesis, p. 145. Springer, Heidelberg
1984.
11. Konig W., Geiger R.: Chem. Ber. 103, 2024 (1970).
12. Windridge G. C., Jorgensen E. C.: J. Am. Chem. Soc. 93, 6318 (1971).
13. Bosshard H. R., Schechter I., Berger A.: Helv. Chim. Acta 56, 717 (1973).
14. Curtius T.: Ber. Dtsch. Chem. Ges. 16, 753 (1883).
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