Ala-PQ (4). Removal of Boc group of the derivative 4 gave the desired
Phe-Ala-PQ.TFA (5). Compound 6 [Boc-Arg(Tos)-PQ] was obtained
by coupling PQ diphosphate with Boc-Arg(Tos)-OH. Treatment of 6
with HF gave Arg-PQ.HF (7). The derivative 6 was Boc deprotected
to give 8 [Arg(Tos)-PQ.TFA]. Boc-Lys(Cl-Z)-Arg(Tos)-PQ (9) was
obtained through the coupling of 8 with Boc-Lys(Cl-Z)-OH. HF
treatment of 9 yielded Lys-Arg-PQ (10). The coupling of the derivative
8 to Boc-Phe-OH gave Boc-Phe-Arg(Tos)-PQ (11), which was fully
deprotected using HF to yield the dipeptide derivative Phe-Arg-PQ.HF
(12). All the reactions were monitored by TLC with appropriate
solvent systems. The deprotected aminoacyl and dipeptide PQ
derivatives were purified by RP-HPLC. The final compounds were
characterized by RP-HPLC, 1H and 13C NMR, mass spectrometry and
amino acid analyses.
Syn th esessBoc-Ala-PQ (2)sOne millimole of PQ diphosphate (1)
was dissolved in dimethylformamide (DMF) and triethylamine (TEA)
in an acetone-dried ice bath. Boc-Ala-OH (1.2 mmol) was added. The
pH of the solution was adjusted to 7-8 with N-methylmorpholine
(NMM). HOBt (1.2 mmol) and DIC (1.2 mmol) were added and the
pH was again adjusted to 7-8. The reaction mixture was stirred
overnight at room temperature and monitored by TLC (chloroform:
methanol:acetic acid, 95:5:3, v/v/v). The mixture was diluted with
0.2 M Na2CO3′ (pH 11), and the product was extracted with ethyl
ether and washed with 0.2 M citric acid solution (pH 2). After drying
over Na2SO4, the solvents were eliminated and the crude compound
was dissolved in water and lyophilized.
ized: yield, 72%; mp, 162-164 °C; Rf (A) ) 0.49; Rf (B) ) 0.48; mass
spectrometry: M+ ) 414.2, M+1 ) 415.2, m/ e (intensity): 59 (17.2),
70 (100), 87 (56.98), 175 (19.35), 201 (53.76), 259 (8.38); 1H NMR
(DMSO-d6, d): 8.53-8.51 (d, 1H, Het-H), 8.46 (ml, 1H, NH), 8.16 (ml,
2H, NH2), 8.09-8.05 (d, 1H, Het-H), 8.84 (ml, 1H, NH), 7.84 (ml, 1H,
NH), 7.45-7.38 (q, 1H, Het-H), 6.47 (d, 1H, Het-H), 6.26 (d, 1H, Het-
H), 3.80(s, 3H, OCH3), 3.67 (m, 1H, CHCH3), 3.14-3.10 (ml, 2H, CH2-
NH2), 1.64-1.53(ml, CH2), and 1.20-1.17 (d, 3H, CH3); 13C NMR
(DMSO-d6, ppm): 168.06 (C17), 156.86 (C22), 158.98 (C6), 144.57 (C8),
144.25 (C2), 134.89 (C9), 129.61 (C4), 122.14 (C3), 96.19 (C5), 91.78
(C7), 54.98 (C11), 51.96 (C18), 46.95 (C15, C12), 33.41 (C13), 15.78 (C20),
24.23 (C14), and 20.20 (C16); amino acid analysis: arginine was
detected; RP-HPLC purity, 97%.
Arg(Tos)-PQ.TFA (8)sThe removal of the Boc group was ac-
complished with 50% TFA in methylene chloride. The reaction was
monitored by TLC (chloroform:methanol:acetic acid, 95:5:3, v/v/v).
After total deprotection, the solvents were eliminated and the PQ
derivative was dissolved in water and lyophilized.
Boc-Lys(Cl-Z)-Arg(Tos)-PQ (9)sCoupling of Boc-Lys(Cl-Z)-OH with
8 produced 9. Isolation of 9 from the reaction medium was ac-
complished as described for 6.
Lys-Arg-PQ (10)sDeblocking was proceeded by treatment of 9 with
HF/anisol (5%) for 1.5 h. HF and anisol were eliminated with high
vacuum. The product was dissolved in water, lyophilized, and
purified: yield, 86%; mp, 100-102 °C; Rf (A) ) 0.13; Rf (B) ) 0.11;
mass spectrometry: m/ e (intensity, %) ) 59.1; 84.9 (100); 123 (74.18);
155 (60.15); 232 (54.96); 259.2(18.61); 303.1 (19.36); 356.1 (46.05);
414.2 (5.45); 1H NMR (DMSO-d6): 8.52-8.50 (dd, 1H, Het-H); 8.08-
8.05 (dd, 1H, Het-H), 7.70-7.67 (ml, 1H, NH), 7.60-7.67(ml, 1H, NH),
7.44-7.38 (q, 1H, Het-H), 6.47-6.46 (d, 1H, Het-H), 6.24 (m, 1H, Het-
H), 3.80 (s, 3H, OCH3), 3.34 (m, H2O), 2.79-2.72(t, CH-NH2), 1.53-
1.42 (m, 1H, NH), and 1.19-1.16 (d, 3H, CH3); 13C NMR (DMSO-d6,
ppm); 170.08 (C17), 158.97 (C6), 156.70 (C22), 144.59 (C8), 144.24 (C2),
134.80 (C9), 134.53 (C10), 129.80 (C4), 122.11 (C3), 96.24 (C5), 91.78
(C7), 54.97 (C11), 46.95 (C15, C12), 33.38 (C13), 26.85 (C20), and 20.18
(C16); amino acid analysis: Lys (0.96), Arg (1.00); RP-HPLC purity,
90%.
Boc-Phe-Arg(Tos)-PQ (11)sThis derivative was obtained by cou-
pling Boc-Phe-OH (1.2 mmol) with 8 (1.0 mmol) in DMF and TEA in
an acetone-dried ice bath. The pH of the solution was adjusted to
7-8, and HOBt (1.2 mmol) and DIC (1.2 mmol) were added. The pH
was corrected to 7-8 with NMM. The reaction was carried out
overnight at room temperature and monitored by TLC (chloroform:
methanol:acetic acid, 95:5:3, v/v/v). Then, 0.2 M Na2CO3 solution was
added to the reaction mixture and the product was extracted with
ethyl ether and washed with 0.2 M citric acid solution. After being
dried over anhydrous Na2SO4, the organic layer was filtered and
evaporated.
Phe-Arg-PQ (12)sAfter 2 deblocking reaction with HF/5% anisol
for 1.5 h, the deprotected product was lyophilized and submitted to
RP-HPLC purification and analysis: yield, 60,1%; mp, 158-160 °C
(dec.); Rf (A) ) 0.58; Rf (B) ) 0.57; mass spectrometry: m/ e (intensity,
%) ) 84.9 (12.95), 91 (31.70), 120 (22.53), 128 (4.46), 201 (100), 259.2
(18.00), 303.1 (3.52), 470.3 (0.51); 1H NMR (DMSO-d6): 8.52-8.50
(dd, 1H, Het-H), 8.08-8.04 (dd, 1H, Het-H), 7.98 (ml, 1H, NH), 7.65
(ml, 1H, NH), 7.44-7.38 (q, 1H, Het-H), 7.21-7.19 (m, 5H, Ar-H),
6.46-6.45 (d, 1H, Het-H), 6.25-6.24 (d, 1H, Het-H), 6.12-6.08 (d,
1H, NH), 3.80 (s, 3H, OCH3), 1.53-1.45 (m, CH2), and 1.19-1.16 (d,
3H, CH3); 13C NMR (DMSO-d6, ppm): 170.70 (C17), 158.97 (C6), 156.70
(C22), 144.59 (C8), 144.24 (C2), 134.81 (C9), 134.52 (C10), 129.57 (C4),
129.32 (C28, C30), 128.16 (C27, C31), 126.35 (C26), 122.11 (C3), 96.11
(C5), 91.61 (C7), 54.97 (C11), 46.94 (C12, C15), 33.38 (C13), 29.05 (C19),
25.88 (C20), 24.95 (C14) 20.19 (C16), and 18.62 (C19); amino acid
analysis, Phe (1.11), Arg (1.00); RP-HPLC purity, 99%.
Ala-PQ (3)sRemoval of the Boc group was accomplished with TFA
50% in methylene chloride. The reaction was monitored by TLC
(chloroform:methanol:acetic acid, 95:5:3, v/v/v). After total deprotec-
tion, the solvents were eliminated and the PQ derivative was dissolved
in water and lyophilized: yield, 59% (oil); Rf (A) ) 0.47; Rf (B) ) 0.46;
mass spectrometry: M+ ) 330.2, M+1 )331.2, m/ e (intensity,%) )
70 (6.34), 159 (9.8), 201 (100), 259.2 (3.5), 287.1 (3.83); 1H NMR
(DMSO-d6): 8.54-8.51 (dd, 1H, Het-H), 8.36 (m, 1H, NHCO), 8.09-
8.05 (d, 1H, Het-H), 7.45-7.39 (q, 1H, Het-H), 6.48-6.46 (d, 1H, Het-
H), 6.26 (s, 1H, Het- H), 3.81 (s, 3H, OCH3), 3.13 (ml, 3H, CH-CH3),
2.87 (s, 2H,CH2-NH2), 1.53 (ml, 4H, CH2-CH2), 1.32-1.27 (dd, 3H,
C
(17)H3), and 1.21-1.18 (d, 6H, C(16)H3); 13C NMR (DMSO-d6, ppm):
169.19 (C17), 159.03 (C6), 144.50 (C8), 144.20 (C2), 135.01 (C9), 134.37
(C10), 129.65 (C4), 122.13 (C3), 96.34 (C5), 91.78 (C7), 54.99 (C11), 48.21
(C18), 46.91 (C15, C12), 33.19 (C13), 25.70 (C14), 20.18 (C16) and 17.18
(C19); amino acid analysis: alanine was detected; RP-HPLC purity,
99%.
Boc-Phe-Ala-PQ (4)sDerivative 4 was obtained by reacting Boc-
Phe-OH (1.0 mmol) with 3 (1.2 mmol) according to the method used
in the synthesis of derivative 2. The deprotection was accomplished
to obtain Phe-Ala-PQ (5): yield, 52% (oil); Rf (A) ) 0.59; Rf (B) )
0.56; mass spectrometry: M+ ) 477.4, M+1 ) 478.4, m/ e (intensity,
1
%) ) 91 (22.82), 201(100), 259.2 (3.64), 330.3 (0.62), 462.3 (0.70); H
NMR (DMSO-d6): 8.52-8.50 (dd, 1H, Het-H), 8.11 (ml, 1H. NH),
8.09-8.08 (d, 1H, Het-H), 8.048-8.40 (d, 1H, Het-H), 7.44-7.42 (q,
1H, 1H, Het-H), 7.22 (m, 5H, Ar-H), 6.47-6.45 (d, 1H, Het-H), 6.25
(d, 1H, Het-H), 3,80 (s, 3H, OCH3), 3.54 (m, CH-CH3, H2O), 3.04 (m,
2H, CH2-NH2), 1.53-1.45 (m, 4H, CH2-CH2), and 1.20-1.17 (d, 6H,
CH-CH3); 13C NMR (DMSO-d6, ppm): 171.16 (C20), 167.41 (C17),
159.00 (C6), 144.60 (C8), 111.20 (C2), 134.81 (C9, C10), 129.59 (C4),
129.45 (C25, C27), 128.45 (C24, C28), 127.11 (C23), 122.11 (C3), 96.16
(C5), 91.65 (C7), 55.14 (C21), 54.98 (C11), 48.34 (C18), 46.97 (C15, C12),
33.34 (C13), 25.90 (C14), 20.21 (C16), and 18.62 (C19); amino acid
analysis, Phe (1.00), Ala (1.15); RP-HPLC purity, 99%.
Boc-Arg(Tos)-PQ (6)sPQ (1) (1.0 mmol) was dissolved in DMF and
TEA in an acetone-dried ice bath. Boc-Arg(Tos)-OH (1.2 mmol) was
then added and the pH of the solution was adjusted to 7-8. Next,
HOBt (1.2 mmol) and DIC (1.2 mmol) were added and the pH was
again adjusted to 7-8 with NMM. The reaction was carried out
overnight at room temperature and monitored by TLC (chloroform:
methanol:acetic acid, 95:5:3, v/v/v). Then, 0.2 M Na2CO3 solution was
added to the reaction mixture and the product was extracted with
ethyl ether and washed with 0.2 M citric acid solution. After drying
over Na2SO4, the solvents were eliminated and the crude compound
was dissolved in water and lyophilized.
Arg-PQ (7)sRemoval of the protective groups Boc and Tos was
carried out by HF treatment in 5% anisol for 1.5 h. The acid excess
and the anisol were eliminated with high vacuum. The resulting
residue was dissolved in water and lyophilized. The purification was
performed by RP-HPLC and the purified compound was character-
Biologica l In Vitr o TestssTrypanosomessTrypomastigote forms
of Trypanosoma cruzi, Y strain, were used in the in vitro assays. The
organism was maintained in continuous culture in Dulbecco modified
Eagle medium36 supplemented with NaHCO3 (1.2 g/L), penicillin
(500 000 U/L) and streptomycin (100 mg/L) (DME medium), contain-
ing 10% of heat-inactivated fetal bovine serum at 37 °C.
Cell Culture AssayssThe in vitro testing was performed by seeding
24/2 mL wells tissue culture slides with 2 × 104 Rhesus monkey
kidney epithelial cells (Macaca mullata), LLC-MK2 (provided by
Instituto Adolfo Lutz, Sa˜o Paulo, Brazil) maintained in DME supple-
mented with 5% heat-inactivated fetal bovine serum. Slides were
incubated at 37 °C in an atmosphere containing 5% CO2 in air.
Journal of Pharmaceutical Sciences / 1129
Vol. 86, No. 10, October 1997