Synthesis of glycosides of a muramoyldipeptide with chromone aglycones

Article abstract of DOI:10.1023/A:1017694224906

The synthesis of β-(2-methyl-3-phenylchromonyl-7)- and β-[2-methyl-3-(3,4-trimethylenedioxy) phenylchromonyl-7]glycosides of the methyl ester of N-acetylmuramoyl-L-alanyl-D-isoglutamine, new derivatives of a muramoyldipeptide with chromone aglycones, is described. The starting arylglycosides of N-acetylglucosamine are prepared by glycosylation of 7-hydroxychromone derivatives with the peracetate of α-glucosamine chloride catalyzed by a crown ether. The synthesized β-aryl-4,6-O-isopropylidene-N-acetylmuramic acids are condensed with the dipeptide and deprotected to give the desired glycopeptides.

Full text of DOI:10.1023/A:1017694224906

Chemistry of Natural Compounds, Vol. 37, No. 1, 2001
SYNTHESIS OF GLYCOSIDES OF A MURAMOYLDIPEPTIDE
WITH CHROMONE AGLYCONES
V. O. Kur yanov,1 T. A. Chupakhina,1 A. E. Zemlyakov,1
V. Ya. Chirva,1 V.V. Ishchenko,2 and V. P. Khilya2
UDC 547.963.057
The synthesis of
-(2-methyl-3-phenylchromonyl-7)- and
-[2-methyl-3-(3,4-trimethylenedioxy)
phenylchromonyl-7]glycosides of the methyl ester of N-acetylmuramoyl-L-alanyl-D-isoglutamine, new
derivatives of a muramoyldipeptide with chromone aglycones, is described. The starting arylglycosides of
N-acetylglucosamine are prepared by glycosylation of 7-hydroxychromone derivatives with the peracetate
of -glucosamine chloride catalyzed by a crown ether. The synthesized -aryl-4,6-O-isopropylidene-N-
acetylmuramic acids are condensed with the dipeptide and deprotected to give the desired glycopeptides.
Key words: methyl ester of O-[(2-methyl-3-phenylchromonyl-7)-2-acetamido-2-deoxy- -D-glucopyranosidyl-3]-D-
lactoyl-L-alanyl-D-isoglutamine, methyl ester of O-{[-2-methyl-3-(3,4-trimethylenedioxy)phenylchromonyl-7]-2-acetamido-2-
deoxy- -D-glucopyranosidyl-3}-D-lactoyl-L-alanyl-D-isoglutamine, synthesis.
The high antitumor activity of the conjugate of the methyl ester of N-acetylmuramoyl-L-valyl-D-isoglutamine with
ubiquinone Q derivatives created great interest in synthetic combinations of muramoyldipeptide (MDP) with various biologically
active compounds [1]. Derivatives of benzo- and naphthoquinones [2], phenolic compounds [3], acridine [4], and salicylic and
nicotinic acids [5] are widely used as modifiers. In most instances the primary hydroxyl of MDP is modified. The -glycosides
of MDP with aliphatic, aromatic, and phenolic aglycones that we synthesized possessed high immunostimulatory activity [6].
Therefore, it seemed promising to modify the compounds with MDP at the -O-glycoside bond.
In continuation of the studies of the relationship between the MDP glycoside structures and their biological activity
and in order to find new promising immunomodulators, we synthesized -(2-methyl-3-phenylchromonyl-7)- and -[2-methyl-3-
(3,4-trimethylenedioxy)phenylchromonyl-7] glycosides of the methyl ester of N-acetylmuramoyl-L-alanyl-D-isoglutamine, new
glycoside MDP derivatives with chromone aglycones. We chose 7-hydroxychromone derivatives as the aglycone because of their
wide spectrum of biological activities [7].
The starting peracetyl glycosides of N-acetylglucosamine (2a and -b) were prepared from -D-chloroglucosamine (1)
by solid-liquid phase-transfer catalysis. The 7-hydroxy-2-methyl-3-phenyl- and 7-hydroxy-2-methyl-3-(3,4-trimethylenedioxy)
phenylchromones [8] were glycosylated by an equimolar amount of glycosylating agent at room temperature in acetonitrile in
the presence of anhydrous K CO and 15-crown-5 (20 mol%). The products (2a and -b) were isolated by crystallization in
2
3
yields of 66 and 76%, respectively. The PMR spectra of 2a and -b contain signals of the framework protons, three O-acetyls,
and the acetamide of the hydrocarbon (for assignments, see Experimental). Signals of the aglycone protons were identified, in
particular, singlets of methyls at 2.31 and 2.30 ppm and multiplets of 8 and 6 aromatic protons at 6.88-7.91 and 6.87-7.85 ppm,
respectively. This confirms the structures. The -configuration of the glycoside bond is consistent with the presence of doublets
of the anomeric protons at 5.18 and 5.21 ppm with spin—spin coupling constant (SSCC) 8.5 Hz.
Compounds 2a and -b were deacetylated according to Zemplen. The -glycol in the resulting triols 3a and -b was
closed by 2,2-dimethoxypropane to give the isopropylidene protection. The structure of acetals 4a and -b was confirmed by PMR
spectroscopy. The hydrocarbon portions of the spectra contain characteristic signals: two singlets of methyl protons of the
1) V. I. Vernadskii Tavricheskii National University, ul. Yaltinskaya 4, Simferopol , Ukraine, 95007, fax (0652) 23
23 10; 2) Taras Shevchenko Kiev National University, ul. Vladimirskaya 60, Kiev-33, Ukraine, 01033, fax (044) 235 12 73.
Translated from Khimiya Prirodnykh Soedinenii, No. 1, pp. 35-38, January-February, 2001. Original article submitted February
22, 2001.
0009-3130/01/3701-0039$25.00 ©2001 Plenum Publishing Corporation
39

isopropylidene group at 1.46-1.51 ppm, a 3H singlet of the N-acetyls at 2.14 and 2.12 ppm, a broad doublet of the C(3)-OH
groups at 4.50 and 4.60 ppm, and a doublet of the glycoside protons at 4.55 and 4.66 ppm, respectively.
OH
OAc
OAc
O
O
O
ArOH
K CO
Me C(OMe)
2
OAr
HO
HO
MeOH
AcO
AcO
2
AcO
AcO
OAr
NHAc
2a, b
TsOH
MeONa
2
3
NH
Ac
NHAc
Cl
1
3a, b
Me
Me
O
O
Me
O
OAr
O
Me
HOSu, DCC
NaH
O
O
OAr
O
CH CHBrCOOH
3
NHAc
L
D
-Ala- -Glu-OMe
Me
HO
NHAc
COOH
4a, b
5a, b
Me
OH
O
Me
O
O
+
HO
O
OAr
OAr
H O, H
2
O
O
NHAc
NHAc
Me
Me
L
D
L
D
CO- -Ala- -Glu-OMe
CO- -Ala- -Glu-OMe
6a, b
7a, b
O
Me
O
O
Me
Ar =
a:
O
O
O
4a
b
Alkylation of the free hydroxyl in derivatives and - with NaH and (S)-2-bromopropanoic acid was performed in
5a
b
dioxane and gave (R)-N-acetylmuramic acids and - . Successive condensation of the acids with the methyl ester of L-alanyl-
D-isoglutamine using N-hydroxysuccinimide and acid hydrolysis of the isopropylidene protection gave the desired glycopeptides
7a
b
7a
b
and - . The presence in the PMR spectra of and - of a singlet of the methyl-ester protons ( 3.58 ppm), two multiplets
of isoglutamine -methylenes ( 1.76 and 1.75 and 2.02 and 2.00 ppm), a triplet of isoglutamine -methylene group
( 2.30 ppm), and two doublets of alanine and lactyl methyls ( 1.27 and 1.29 ppm) confirm that the lactylpeptide was
incorporated. Removal of the acteyl protection was confirmed by the presence of signals for protons of the two hydroxyls: a
doublet at 5.41 and 5.44 ppm [C(4)-OH] and a broad triplet at 4.67 ppm [C(6)-OH].
EXPERIMENTAL
Melting points were determined on a PTP apparatus; optical rotation at 20-22oC, on a Polamat-A polarimeter. H NMR
1
spectra were obtained on a Varian VXR-300 (300 MHz) spectrometer with TMS internal standard. Chemical shifts are given
in ppm ( -scale). TLC was performed on Sorbfil-AFV-UV plates (Sorbpolymer, Russia). Spots were developed with H SO
2
4
(5%) in ethanol with heating to 200-300oC. Solvent systems were: 1) chloroform—ethylacetate-propanol-2 (25:5:1),
2) chloroform—propanol-2 (3:1), 3) chloroform—propanol-2 (17:3), 4) chloroform—propanol-2 (15:1). Column
chromatography used Merck 240-400 mesh silica gel.
(2-Methyl-3-phenylchromonyl-7)-2-acetamido-3,4,6-tri-O-acetyl-2-deoxy- -D-glucopyranoside (2a). A solution
of 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy- -D-glucopyranosyl chloride (1, 1,7 g, 4.65 mmol) [9] in acetonitrile (20 mL) was
treated with 7-hydroxy-2-methyl-3-phenylchromone (1.17 g, 4.65 mmol), finely ground anhydrous K CO (640 mg, 4.65 mmol),
2
3
and 15-crown-5 (185 L, 0.93 mmol) and stirred at room temperature until the glycosylating agent completely disappeared (TLC
monitoring in system 1). The precipitate was filtered off. The filtrate was evaporated. The solid was dissolved in CHCl
3
40

(50 mL) and washed with KOH (1 N, 20 mL) and water (2×20 mL). The organic layer was separated, dried with anhydrous
Na SO , and evaporated. The solid was crystallized from propanol-2 to give 2a (2.0 g, 66%), mp 194-196oC, [ ]546 +2o
2
4
(c 1.0, CH Cl ). PMR (CD Cl , , ppm, J/Hz): 1.95, 2.04, 2.06, 2.08 (12H, NAc and 3 OAc, s), 2.31 (3H, s, Me), 3.75 (1H,
2
2
2
2
ddd, J5,6a = 2.5, J5,6b = 5.5, H-5), 4.12 (1H, ddd, J2,3 = 10, H-2), 4.17 and 4.24 (2H, dd, J6a,6b = 12, H-6a, H-6b), 5.06 (1H, dd,
J4,5 = 9.5, H-4), 5.18 (1H, d, J1,2 = 8.5, H-1), 5.22 (1H, dd, J3,4 = 9.5, H-3), 6.30 (1H, d, J2,NH = 8.5, NH), 6.88-6.96 (2H, m,
Ar-H), 7.32-7.52 (5H, m, Ph), 7.89 (1H, d, Ar-H).
[2-Methyl-3-(3,4-trimethylenedioxy)phenylchromonyl-7]-2-acetamido-3,4,6-tri-O-acetyl-2-deoxy- -D-glucopyranoside
(2b) was synthesized analogously from α-chloride 1 (2.0 g, 5.48 mmol) and 7-hydroxy-2-methyl-3-(3,4-trimethylenedioxy)
phenylchromone (1.7 g, 5.48 mmol): yield 2.4 g (76%), mp 233-235oC (dec.), [ ]546 +10o (c 1.0, CH Cl ).
2
2
PMR (CD Cl , , ppm, J/Hz): 1.96, 2.03, 2.05, 2.08 (12H, s, NAc and 3OAc), 2.20 (2H, m, OCH CH CH O), 2.30
2
2
2
2
2
(3H, s, Me), 3.80 (1H, ddd, J5,6a = 2.5, J5,6b = 5.5, H-5), 4.15 (1H, ddd, J2,3 = 10.5, H-2), 4.21 and 4.30 (2H, dd, J6a,6b = 12, H-6a,
H-6b), 4.25 (4H, t, 2 OCH ), 5.07 (1H, dd, J4,5 = 9.5, H-4), 5.21 (1H, d, J1,2 = 8.5, H-1), 5.22 (1H, dd, J3,4 = 9.5 Hz, H-3), 6.43
2
(1H, d, J2,NH = 9, NH), 6.87-6.92 (4H, m, Ar-H), 7.06 and 7.84 (2H, d, Ar-H).
(2-Methyl-3-phenylchromonyl-7)-3-acetamido-2-deoxy- -D-glucopyranoside (3a). A solution of acetate 2a (1.7 g,
2.9 mmol) in dry methanol (50 mL) was treated with NaOMe (0.5 mL, 0.1 N) in methanol. The precipitate that formed on
+
standing was filtered off and washed with cold methanol. The mother liquor was neutralized with cation exchanger KU-2 (H ).
The resin was filtered off. The filtrate was evaporated. Addition of ether produced an additional amount of crystals. Yield of
compound 3a, 1.15 g (86%), mp 163-165oC, [ ]546 -10o (c 1.0, DMF).
Acetate 2b (2.4 g, 3.7 mmol) was similarly deacetylated to give [2-methyl-3-(3,4-trimethylenedioxy)phenylchromonyl-
7]-2-acetamido-2-deoxy- -D-glucopyranoside (3b, 1.75 g, 90%), mp 148-150oC, [ ]546 -15o (c 1.0, DMF).
(2-Methyl-3-phenylchromonyl-7)-2-acetamido-2-deoxy-4,6-O-isopropylidene- -D-glucopyranoside (4a).
A
suspension of substance 3a (0.70 g, 1.54 mmol) and TsOH (15 mg) in 2,2-dimethoxypropane (5 mL) was heated with stirring
until boiling and treated with THF (2 mL) until completely dissolved. The reaction mixture was cooled after 1 h (TLC
monitoring in system 3), neutralized with pyridine, and evaporated. The precipitate was purified by column chromatography
(eluent CH Cl
CH Cl —propanol-2, 50:1) to give 4a (0.47 g, 62%), mp 152-154oC, [ ]546 -73o (c 1.0, CH Cl ). PMR
2
2
2
2
2
2
(CDCl , , ppm, J/Hz): 1.50 and 1.51 (6H, s, Me C), 2.14 (3H, s, NAc), 2.44 (3H, s, Me), 2.99 (1H, ddd, J5,6a = 2.5, J5,6b = 5.5,
3
2
H-5), 3.28 (1H, dd, J4,5 = 9.5, H-4), 3.57 (1H, dd, J3,4 = 9.5, H-3), 3.74 (2H, m, H-6a, NH), 3.87 (1H, ddd, J2,3 = 10, H-2),
4.00 (1H, dd, J6a,6b = 12, H-6b), 4.49 (1H, br. d, C-3-OH), 4.55 (1H, d, J1,2 = 8.5, H-1), 6.49 (1H, d, Ar-H), 6.77 (1H, dd, Ar-H),
7.40-7.58 (6H, m, Ar-H).
The method described above was used to produce from triol 3b (0.89 g, 1.57 mmol) [2-methyl-3-(3,4-
trimethylenedioxy)phenylchromonyl-7]-2-acetamido-2-deoxy-4,6-O-isopropylidene- -D-glucopyranoside (4b, 0.7 g, 73%), mp
155-157oC, [ ]
-58o (c 0.5, CH Cl ). PMR (CDCl , , ppm, J/Hz): 1.46 and 1.51 (6H, s, Me C), 2.12 (3H, NAc, s), 2.23
546
2 2 3 2
(2H, m, OCH CH CH O), 2.40 (3H, s, Me), 3.24 (2H, m, H-5, H-6a), 3.60 (1H, dd, J4,5 = 9.5, H-4), 3.75 (1H, dd, J3,4 = 9.5,
2
2
2
H-3), 3.87 (1H, ddd, J2,3 = 10, H-2), 3.94 (1H, dd, J5,6b = 5, J6a,6b = 12, H-6b), 4.27 and 4.38 (4H, m, 2 OCH ), 4.60 (1H, br.
2
d, C-3-OH), 4.66 (1H, d, J1,2 = 8, H-1), 6.77 (1H, d, J2,NH = 9, NH), 6.52 and 7.04 (2H, s, Ar-H), 6.97, 7.14, and 7.53 (3H, d,
Ar-H), 7.73 (1H, m, Ar-H).
Methyl Ester of O-[(2-Methyl-3-phenylchromonyl-7)-2-acetamido-2-deoxy- -D-glucopyranosidyl-3]-D-lactyl-L-
alanyl-D-isoglutamine (7a). A suspension of compound 4a (0.33 g, 0.67 mmol) in dry dioxane (20 mL) was stirred and treated
in portions with NaH (4 eq.). The reaction mixture was heated to 65oC, held at that temperature for 1 h, treated with 2-S-
bromopropionic acid (120 L, 1.34 mmol), held at 65oC for 3 h, and cooled. The excess of NaH was decomposed with ethanol.
The mixture was concentrated, poured into cold water (50 mL), and acidified with HCl (2 N) until the pH was 3-4. The
muramic acid was extracted with CHCl (3×20 mL). The extract was dried over anhydrous Na SO and evaporated. The solid
3
2
4
was crystallized by adding ether. Yield of 5a, 0.35 g (93%).
A solution of acid 5a (340 mg, 0.60 mmol) in dry dioxane (10 mL) was stirred and treated with N-hydroxysuccinimide
(76 mg, 0.66 mmol) and DCC (135 mg, 0.66 mmol). After 3 h the precipitate of dicyclohexylurea was filtered off and washed
with solvent. The filtrate was treated with the trifluoroacetate of the methyl ester of L-alanyl-D-isoglutamine [prepared by
treating the corresponding Boc-derivative with trifluoroacetic acid (218 mg, 0.66 mmol) and evaporating to dryness] and
triethylamine until the pH was 8. After the reaction was complete (TLC monitoring in system 4), the reaction mixture was
concentrated. The precipitate was dissolved in CHCl (30 mL). The organic layer was washed with water (2×10 mL), dried
3
over anhydrous Na SO , and evaporated. Addition of ether precipitated 6a (310 mg, 66%).
2
4
41

The alkylidene derivative 6a (270 mg, 0.35 mmol) was dissolved with heating on a boiling-water bath in acetic acid
(3 mL, 80%) and held at that temperature for 5 min (TLC monitoring in system 2). The solution was evaporated to dryness.
The solid was co-evaporated with toluene. Column chromatography (eluent CHCl
CHCl —propanol-2, 3:1) gave
3
3
glycopeptide 7a (130 mg, 51%), amorphous powder, [ ]546 +23o (c 1.0, CHCl —ethanol, 2:1). PMR (DMSO-d , , ppm, J/Hz):
3
6
1.27 and 1.29 (6H, d, 2 MeCH), 1.76 and 2.02 (2H, m, -CH -iGln), 1.80 (3H, s, NAc), 2.27 (3H, s, Me), 2.30 (2H, t, -CH -
2
2
iGln), 3.58 (3H, s, COOMe), 4.67 (1H, br. t, C-6-OH), 5.21 (1H, d, J1,2 = 8, H-1), 5.41 (1H, d, C-4-OH), 7.04 (1H, dd, Ar-H),
7.18 and 7.97 (2H, d, Ar-H), 7.27-7.44 (5H, m, Ar-H), 7.08 and 7.33 (2H, s, CONH -iGln), 7.50, 7.94, and 8.08 (3H, d, 3NH).
2
The method described above was used to synthesize from acetal 4b (220 mg, 0.46 mmol) the methyl ester of O-{[2-
methyl-3-(3,4-trimethylenedioxy)phenylchromonyl-7]-2-acetamido-2-deoxy- -D-glucopyranosidyl-3}-D-lactoyl-L-alanyl-D-
isoglutamine (7b, 85 mg), amorphous powder, [ ]546 +22o (c 0.67, CHCl —ethanol, 2:1). PMR (DMSO-d , , ppm, J/Hz):
3
6
1.27 and 1.29 (6H, d, 2 MeCH), 1.75 and 2.00 (2H, m, -CH -iGln), 1.80 (3H, s, NAc), 2.13 (2H, m, OCH CH CH O), 2.28
2
2
2
2
(3H, s, Me), 2.30 (2H, t, -CH -iGln), 3.58 (3H, s, COOMe), 4.67 (1H, br. t, C-6-OH), 5.20 (1H, d, J1,2 = 8, H-1), 5.44 (1H,
2
d, C-4-OH), 6.89 and 7.00-7.17 (5H, m, Ar-H), 7.98 (1H, d, Ar-H), 7.10 and 7.35 (2H, s, CONH -iGln), 7.50, 7.98, and 8.09
2
(3H, d, 3 NH).
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2.
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