Antitumor Activity of Muramylpeptide Conjugates
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22 3613
and 4.68 (2d, J ) 12.5 Hz, J ) 12.5 Hz, 2H, CH2C6H5), 4.76
(d, J ) 3.5 Hz, 1H, H-1), 6.68 (d, J ) 9.9 Hz, 1H, C2-H), 7.24-
7.3 (m, 5H, C6H5), 7.40 (t, J ) 7.4 Hz, 1H, C7-H), 7.08 and
7.57 (2s, 2H, CONH2), 7.78 (t, J ) 7.3 Hz, 1H, C6-H), 7.92 (d,
J ) 7.2 Hz, 1H, C5-H), 8.21(d, J ) 8 Hz, 1H, C8-H), 8.38 (d,
J ) 9.9 Hz, 1H, C3-H), 11.91 (t, J ) 5 Hz, 1H, NHCH2), 12.38
(s, 1H, N10-H). Anal. (C45H55N7O15) C, H, N.
times. The crude product was purified using preparative TLC
in solvent system A. The yields and melting points of 10a and
10b are given in Table 3.
9-[N-[(L)-m eso-2,6-Dia m in op im elyl-(D)-m eth yl ester -(L)-
L-a la n yl-D-isoglu t a m in yl(â-a m in oet h yl-â-a m in oet h yl)]-
am in o]-1-n itr oacr idin e dih ydr och lor ide (10a): yield 0.028
1
g (41%); mp 133-136 °C; H NMR (DMSO) δ 1.2 (d, J ) 6.8
Th e gen er a l p r oced u r e for th e syn th esis of com -
p ou n d s 4a -z (Table 2) was published previously.25,26 The
structures of the compounds were confirmed by their spectral
data (1H NMR)44 and by elemental analyses.26
Hz, 3H, CH3-Ala), 1.4-1.8 (m, 6H, (CH2)3), 1.5 (m, 1H, CH2
NHCH2), 1.4-1.9 and 1.95-2.05 (2m, 2H, âCH2-isoGln), 2.3
(m, 2H, γCH2-isoGln), 2.95 and 3.15 (2m, 4H, CH2NHCH2),
3.4 and 3.5 (2m, 4H, COOCH2CH2NHCH2CH2), 3.62 (s, 3H,
COOCH3), 3.85, 3.95, and 4.2 (4m, RCH-Ala, RCH-isoGln, 2CH-
A2pm), 6.2 (br, 2H, 2NH3+-A2pm), 6.75 (t, J ) 7.4 Hz, 1H, C3-
H), 7.1 and 7.3 (2s, 2H, CONH2-isoGln), 7.12-7.45 (m, 4H,
C4,5,6,7-H), 7.5 (d, J ) 7.2 Hz, 1H, C8-H), 7.6 (d, J ) 8.4
Hz, 1H, γNH-isoGln), 7.7 (d, J ) 6.8 Hz, 1H, NH-Ala), 7.85
(d, J ) 7.8 Hz, 1H, C2-H), 8.2 (d, J ) 8.3 Hz, 1H, NH-isoGln),
10.8 (s, 1H, N10-H). Anal. (C33H46N10O8. 2HCl) C, H, N.
1-[N-[(L)-m eso-2,6-Dia m in op im elyl-(D)-m et h yl est er -
(L)-L-a la n yl-D-isoglu ta m in yl(γ-a m in op r op a n oyl)]a m in o]-
4-n itr o-9(10H)-a cr id in on e d ih yd r och lor id e (10b): yield
0.032 g (46%); mp 117-119 °C; 1H NMR (DMSO) δ 1.2 (d, J )
6.8 Hz, 3H, CH3-Ala), 1.4-1.8 (m, 6H, (CH2)3), 1.4-1.9 and
1.95-2.05 (2m, 2H, âCH2-isoGln), 1.6 (m, 2H, CH2(2)-Pr), 2.3
(m, 2H, γCH2-isoGln), 3.1 (m, 2H, CH2(1)-Pr), 3.45 (m, 2H, CH2-
(3)-Pr), 3.6 (s, 3H, COOCH3), 3.85, 3.95, and 4.2 (4m, RCH-
Ala, RCH-isoGln, 2CH-A2pm), 6.3 (br, 2H, 2NH3+-A2pm), 6.7
(d, J ) 9.8 Hz, 1H, C2-H), 7.1 and 7.3 (2s, 2H, CONH2-isoGln),
7.35 (m, 1H, C7-H), 7.7 (d, J ) 6.8 Hz, 1H, NH-Ala), 7.75 (m,
1H, C6-H), 7.9 (d, J ) 8.3 Hz, 1H, γNH-isoGln), 7.95 (d, J )
5.4 Hz, 1H, C5-H), 8.0 (d, J ) 8.3 Hz, 1H, NH-isoGln), 8.2 (d,
J ) 6.8 Hz, 1H, C8-H), 8.35 (d, J ) 9.8 Hz, 1H, C3-H), 11.8
(t, 1H, Pr-NH-Acr), 12.4 (s, 1H, N10-H). Anal. (C32H43N9O9·
2HCl) C, H, N.
N-ter t-Bu toxycar bon yl-L-valyl-D-isoglu tam in yl(Nr-ben -
zyloxyca r bon yl)-L-lysin e Ben zyl Ester (13). To a solution
containing 0.31 g (0.85 mmol) of Boc-L-Val-D-isoGln-OH (11)
in 2 mL of DMF cooled to -15 °C were added 0.1 mL (0.85
mmol) of NMM and 0.11 mL (0.85 mmol) of isobutyl chloro-
formate were. Three minutes later a cooled solution of 0.45 g
(0.85 mmol) of TsOH‚L-Lys(N-Z)-OBn and a mixture of 0.096
mL (0.85 mmol) of NMM in 2 mL of DMF were added. The
reaction was stirred for 5 h at -15 °C, then 0.5 mL of 2.5 M
KHCO3 was added, and stirring was continued at 0 °C for 30
min. Then the reaction mixture was poured into 200 mL of
saturated aqueous sodium chloride solution, and the solid was
filtered off and recrystallized from ethanol-hexane to give
13: 0.51 g (87%) yield; mp 184-86 °C, Rf ) 0.68 (solvent
system B); 1H NMR (DMSO) δ 0.9 and 0.92 (2d, J ) 7.3 Hz, J
) 7.3 Hz, 6H, CH3-Val), 1.35 (s, 9H, (CH3)3), 1.7 (m, 1H, CH-
Val), 1.7-1.9 (m, 6H, (CH2)3-Lys), 1.8-1.9 and 1.95-2.05 (2m,
2H, âCH2-isoGln), 2.3 (m, 2H, γCH2-isoGln), 3.2 (m, 2H, ꢀCH2-
Lys), 3.9, 3.95, and 4.0 (3m, 3H, RCH-Val, RCH-isoGln, RCH-
Lys), 5.05 and 5.1 (2s, 4H, 2CH2C6H5), 6.9 (d, J ) 10 Hz, 1H,
BocNH), 7.1 and 7.3 (2s, 2H, CONH2-isoGln), 7.35 (m, 10H,
C6H5), 7.65 (d, J ) 8.5 Hz, 1H, NH-Lys), 7.9 (d, J ) 8 Hz, 1H,
NH-isoGln), 8.0 (t, J ) 9 Hz, 1H, NH-Lys).
N-ter t-Bu toxycar bon yl-L-valyl-D-isoglu tam in yl(Nr-ter t-
bu toxyca r bon yl)-L-lysin e (14). The protected tripeptide 13
(0.36 g, 0.52 mmol) was hydrogenated in the presence of 0.02
g of 10% Pd/C in 35 mL of methanol for 3 h. The reaction
mixture was filtered and the solvent evaporated. The residue
was dissolved in 2 mL of DMF, and 0.06 mL (0.5 mmol) of
TEA and 0.12 g (0.6 mmol) of (Boc)2O were added. The mixture
was stirred for 24 h at room temperature. The reaction was
next cooled to 0 °C, acidified with 1 N HCl to pH 2-3, and
extracted with ethyl acetate (3 × 30 mL). The acetate solution
was washed with water and saturated sodium chloride solution
and dried with magnesium sulfate, and the solvent was
evaporated. The product was crystallized from the ethyl
acetate-hexane mixture to give 14: 0.198 g (67%) yield; mp
145-147 °C, Rf ) 0.87 (solvent system D); 1H NMR (DMSO) δ
0.9 and 0.92 (2d, J ) 7.3 Hz, J ) 7.3 Hz, 6H, CH3-Val), 1.35
(s, 18H, (CH3)3), 1.4-1.9 (m, 6H, (CH2)3-Lys), 1.7 (m, 1H, CH-
N-Ben zyloxyca r b on yl-(D)-N-ter t-b u t oxyca r b on yl-(L)-
m eso-2,6-d ia m in op im elyl-(D)-m eth yl ester -(L)-L-a la n yl-D-
isoglu ta m in e Ben zyl Ester (7). To a solution of 3.3 g (7.5
mmol) of selectively protected diaminopimelic acid 5 in 24 mL
of acetonitrile cooled to -15 °C were added 0.84 mL (7.5 mmol)
of NMM and 0.96 mL (7.5 mmol) of isobutyl chloroformate.
Three minutes later a cold solution of 3.6 g (11.2 mmol) of TFA‚
NH2-Ala-D-isoGln-OBn in 15 mL of DMF and 1.25 mL (11.2
mmol) of NMM in 5 mL of DMF were added. The mixture was
stirred for 5 h at -15 °C, and then after addition of 4.7 mL of
2.5 M KHCO3, stirring was continued at 0 °C for 30 min. The
reaction mixture was diluted with 30 mL of ethyl acetate, and
the acetate solution was washed with the following cooled
liquids, solution of potassium hydrogen sulfate (10%), water,
solution of potassium hydrogen carbonate (10%), water, and
saturated sodium chloride solution, and then dried over
magnesium sulfate. After that ethyl acetate was removed with
a vacuum rotary evaporator, and the raw product was crystal-
lized from ethyl acetate. As a result 4.4 g (85%) of compound
7 was obtained: mp 150-153 °C; Rf ) 0.62 (in solvent system
B); 1H NMR (DMSO) δ 1.2 (d, J ) 6.8 Hz, 3H, CH3-Ala), 1.35
(s, 9H, (CH3)3), 1.4-1.8 (m 6H, (CH2)3), 1.8-1.9 and 1.95-2.05
(2m, 2H, âCH2-isoGln), 2.3 (m, 2H, γCH2-isoGln), 3.6 (s, 3H,
COOCH3), 3.85, 3.95, and 4.2 (4m, 4H, RCH-Ala, RCH-isoGln,
2CH-A2pm), 5.01 (s, 4H, 2CH2C6H5), 6.9 (d, J ) 10 Hz, 1H,
BocNH), 7.1 and 7.3 (2s, 2H, CONH2-isoGln), 7.35 (m, 10H,
C6H5), 7.7 (d, J ) 6.8 Hz, 1H, NH-Ala), 8.0 (d, J ) 8 Hz, 1H,
NH-isoGln).
Di-ter t-bu t oxyca r b on yl-(L)-m eso-2,6-d ia m in op im elyl-
(D)-m eth yl ester -(L)-L-a la n yl-D-isoglu ta m in e (8). The solu-
tion of protected tripeptide 7 (3.18 g, 4.3 mmol) in 30 mL of
methanol was hydrogenated in the presence of 0.15 g of 10%
Pd/C for 4 h. After removal of the catalyst from the reaction
mixture by filtration, the solvent was evaporated. The residue
was dissolved in 11 mL of DMF, and 0.58 mL (4.3 mmol) of
TEA and 1.1 g (4.73 mmol) of (Boc)2O were added. The mixture
was stirred for 24 h at room temperature. Then the reaction
mixture was cooled, acidified with 1 N HCl to pH 2-3, and
extracted with ethyl acetate (3 × 30 mL). The acetate solution
was washed with water and saturated sodium chloride solution
and dried over magnesium sulfate, and the solvent was
evaporated. The product was crystallized from ethyl acetate-
hexane mixture to give 2.2 g (81%) of compound 8: mp 179-
1
181 °C; Rf ) 0.76 (solvent system B); H NMR (DMSO) δ 1.2
(d, J ) 6.8 Hz, 3H, CH3-Ala), 1.35 (s, 18H, (CH3)3), 1.4-1.8
(m 6H, (CH2)3), 1.4-1.9 and 1.95-2.05 (2m, 2H, âCH2-isoGln),
2.3 (m, 2H, γCH2-isoGln), 3.6 (s, 3H, COOCH3), 3.85, 3.95, and
4.2 (4m, 4H, RCH-Ala, RCH-isoGln, 2CH-A2pm), 6.9 (2d, J )
10 Hz, 2H, BocNH), 7.1 and 7.3 (2s, 2H, CONH2-isoGln), 7.7
(d, J ) 6.8 Hz, 1H, NH-Ala), 8.0 (d, J ) 8 Hz, 1H, NH-isoGln),
10.05 (s, 1H, COOH).
Gen er a l P r oced u r e for th e Syn th esis of Com p ou n d s
10a ,b. To a solution of 0.06 g (0.09 mmol) of protected
tripeptide 8 in 2 mL of DMF cooled to -15 °C were added 0.01
mL (0.09 mmol) of NMM and 0.12 mL (0.09 mmol) of isobutyl
chloroformate. Three minutes later a cold solution containing
(0.09 mmol) of aminoacridine/aminoacridone derivative 9 in 2
mL of DMF was added. The reaction mixture was stirred at
-10 °C for 5 h and then at room temperature for 24 h. The
solvent was evaporated with a vacuum rotary evaporator. The
product was poured into an ethereal solution of hydrogen
chloride and left for 1 h at 0 °C. Then the solution was
decanted, and the residue was washed with ethyl ether several