Synthesis and cytotoxic activity of conjugates of muramyl and normuramyl dipeptides with batracylin derivatives

Article abstract of DOI:10.1021/jm021067v

The synthesis of MDP (muramyl dipeptide) or nor-MDP (normuramyl dipeptide) conjugates modified at the peptide part with batracylin (BAT) or batracylin derivatives is described. Batracylin was synthesized by our modified method (Scheme 3). The synthesis of BAT via this modified route now appears to be feasible on a multigram scale. Preliminary screening data obtained at the National Cancer Institute (NCI, Bethesda, MD) have revealed that the conjugates did not expose any cytotoxic activity even at 10^-4-10^-8 M or μg/mL. During tests performed at Medical University of Gdansk, Poland, two analogues 11c and 11e reduced the proliferation of Ab melanoma cells in vitro compared with batracylin alone (Table 2, Figure 1).

Full text of DOI:10.1021/jm021067v

978
J . Med. Chem. 2003, 46, 978-986
Syn th esis a n d Cytotoxic Activity of Con ju ga tes of Mu r a m yl a n d Nor m u r a m yl
Dip ep tid es w ith Ba tr a cylin Der iva tives
Krystyna Dzierzbicka,*,† Piotr Trzonkowski,‡ Przemysław Sewerynek,§ and Andrzej Mys´liwski‡
Department of Organic Chemistry, Gdansk University of Technology, 11/ 12 G. Narutowicza Street, 80-952 Gdansk, Poland,
and Department of Histology and Immunology and Intercollegiate Faculty of Biotechnology, Medical University of Gdansk,
Debinki 1 Street, 80-210 Gdansk, Poland
Received October 10, 2002
The synthesis of MDP (muramyl dipeptide) or nor-MDP (normuramyl dipeptide) conjugates
modified at the peptide part with batracylin (BAT) or batracylin derivatives is described.
Batracylin was synthesized by our modified method (Scheme 3). The synthesis of BAT via this
modified route now appears to be feasible on a multigram scale. Preliminary screening data
obtained at the National Cancer Institute (NCI, Bethesda, MD) have revealed that the
conjugates did not expose any cytotoxic activity even at 10-4-10-8 M or µg/mL. During tests
performed at Medical University of Gdansk, Poland, two analogues 11c and 11e reduced the
proliferation of Ab melanoma cells in vitro compared with batracylin alone (Table 2, Figure 1).
In tr od u ction
in nonproliferating cells. The role of acetylation in the
genotoxicity of the BAT was evaluated in Salmonella
typhimurium strains expressing various levels of N- and
O-acetyltransferase activity. The results demonstrate
that the mutagenicity of BAT is directly related to
N-acetyltransferase activity.12 These data suggest that
the genotoxic effects of BAT require the free amine while
the antitumor effects are independent of the amino
group. The limitations associated with the chemothera-
peutic potential of BAT, along with the large dose levels
required for anticancer activity and high toxicity, espe-
cially in rats, therefore turn our attention to the
synthesis of its analogues.13-15 Up to now, several BAT
analogues have been synthesized with modifications in
positions C8 (Cl, Br, NO2, CH3, NH2) and C7 (Cl).16
Recently, three patents on different types of BAT
analogues were published.17-19 BAT and its analogues
are poorly soluble in water. To increase their solubility
in water, they were acylated with amino acids, dipep-
tide, and tripeptide17 or coupled to sugar moieties.18
Continuing our program of syntheses of MDP (mu-
ramyl dipeptide) and nor-MDP (normuramyl dipeptide)
conjugates with anticancer active compounds,20-23 we
present syntheses of MDP and nor-MDP analogues that
are modified at the C-terminus of the peptide residue
by the formation of an amide bond between the iso-
glutamine carboxylic group and the amine group of the
respective BAT or N-(Nn-amino acid)-BAT derivatives.
In this paper we describe a modified method of batra-
cylin (BAT) synthesis (Scheme 3). The synthesized
conjugates have been submitted to the National Cancer
Institute (NCI, Bethesda, MD) and the Medical Uni-
versity of Gdansk, Poland, for testing of cytotoxic
activity.
MDP (N-acetylmuramyl-L-alanyl-D-isoglutamine), the
minimal structure of the Mycobacterium cell wall,
retains most of its biological activity, in particular the
adjuvant activity stimulates nonspecific resistance to
microbial infections, exhibits some residual anticancer
and antiviral potency, and also acts synergistically with
many drugs including antibiotics and anticancer
agents.1,2 Immunoactive properties of muramyl peptides
depend on the stimulation or inhibition of the biosyn-
thesis of many cytokines.3 As we have already shown,
MDP and some of its analogues are able to increase the
anticancer activity of compounds possessing acridine
rings.4 On the other hand, batracylin, 8-aminoisoindolo-
[1,2-b]quinazolin-1,2-(10H)-one (BAT), displays antitu-
mor activity in vivo against murine leukemia P-388 and
colon adenocarcinoma 38 in mice sublines with acquired
resistance to adriamycin, cisplatin, and methotrexate.5,6
Oral administration of batracylin is effective against
other murine solid tumors including pancreatic ductal
adenocarcinoma, colon adenocarcinoma no. 51, and
hepatoma 129.7 BAT is inactive against B16 melanoma,
CD8F1 mammary carcinoma, L1210 leukemia, Lewis
lung carcinoma, and human MX-1 mammary xenograft.5
Recently, the chemotherapeutic mechanism of BAT was
shown to differ from that leading to genotoxicity.8,9 BAT
acts as a topoisomerase II inhibitor and induces un-
scheduled DNA synthesis (UDS) of nonproliferating
cells. Susceptibility to BAT toxicity was species-depend-
ent.10,11 The greater sensitivity of the rat has been
associated with a high plasma concentration of the
N-acetyl metabolite of BAT. The authors suggested that
the presence of the metabolite may be associated with
its acute toxicity and initiation of the genotoxic response
Ch em istr y
* To whom correspondence should be addressed. Telephone:
(48 58) 347-27-36. Fax: (48 58) 347-26-94., E-mail: kd@chem.pg.gda.pl.
† Gdansk University of Technology.
Conjugates of MDP and nor-MDP with BAT or BAT
derivatives [N-(Nn-amino acid)-BAT] were synthesized
according to Scheme 1. The protected MDP or nor-MDP
1 were synthesized as described previously.24,25 These
substrates were subjected to a cautious hydrolysis at
‡ Department of Histology and Immunology, Medical University of
Gdansk.
§ Intercollegiate Faculty of Biotechnology, Medical University of
Gdansk.
10.1021/jm021067v CCC: $25.00 © 2003 American Chemical Society
Published on Web 02/19/2003

Conjugates of MDP and nor-MDP
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6 979
Sch em e 1
Ta ble 1. MDP or nor-MDP Conjugates of Batracylin 11a -h
The reaction involves heating of substrates in dioxane
for 4 h under mild conditions, and the yield of BAT was
56%. However, the preparation of 2,5-diaminobenzy-
lamine is difficult. This substance is very unstable and
is not commercially available. Roservear and Wilshere16
described a BAT synthesis using ethyl N-(4-acetami-
dophenyl)carbamate, in which the reaction with N-
(hydroxymethyl)phthalimide, according to the Czerniak-
Einhorn reaction, provides ethyl [4-acetamido)-2-
(phthalimidomethyl)phenyl]carbamate 8 (Scheme 2).
The Czerniak-Einhorn reaction is an electrophilic
substitution, where the proton donor N-(hydroxymeth-
yl)phthalimide is the electrophilic reagent. The reaction
takes place in the presence of sulfuric acid. After acid
hydrolysis of acetyl and urethane protecting groups and
because of alkalinization with ammonia to promote
cyclization, BAT was obtained. The N-(hydroxymethyl)-
phthalimide is commercially available, but the synthesis
of unsymmetrically protected phenylenediamine [ethyl
4-(acetamido)phenylcarbamate] consists of many steps
and is difficult.16 This method is the most common
method for BAT synthesis in which p-nitroaniline or
acetanilide is used as the substrate. In this method we
modified the reduction of the nitro group of compound
5 using Raney nickel and 100% hydrazine hydrate
(Scheme 2). The third method of BAT synthesis involves
catalytic reduction of 8-nitroisoindolo[1,2-b]quinazolin-
12(10H)-one in the presence of glacial acetic acid.15
Thorough analysis of the above-referred methods of BAT
synthesis served as basis for some of our modifications.
However, the question arises of why unsymmetrical
protection was, up to now, used for BAT synthesis.
In our modified method, 1,4-phenylenediamine 12 was
used with symmetrically protecting acetyl or urethane
groups (Scheme 3). A symmetrically protected 1,4-
phenylenediamine derivative could undergo the Czer-
niak-Einhorn reaction, and after hydrolysis of the
protecting groups, BAT would result. The N,N′-diacetyl-
1,4-phenylenediamine 13 or N,N′-diethoxycarbonyl-1,4-
compda
R
X
n
molecular formula mp (°C) yield (%)
11a
CH3 Ala
CH3 Ala
C
C
C
C
C
C
C
C
41H48N7O11
(814.0)
44H52N8O12
(884.0)
43H50N8O12
(870.0)
46H56N8O12
(912.0)
43H52N7O11
(842.0)
47H59N8O12
(927.0)
47H58N8O12
(926.0)
48H61N8O12
(941.0)
223-227
192-199
127-133
134-138
196-202
211-216
224-229
144-151
50
52
55
56
54
57
58
56
11b
11c
11d
11e
11f
11g
11h
2
H
H
Ala
Ala
5
CH3 Val
CH3 Val
3
5
5
H
Val
CH3 Pro
a
1H NMR spectra of all compounds were in compliance with
the expected ones. Elemental analyses (C, H, N) of all compounds
agreed within (0.4% of theoretical values.
the carboxyl group with 90% TFA at room temperature
to give compounds 2, followed by a formation of an
amide bond at the C-terminus with the BAT 9 or with
its derivatives 10a -c. The acylation was performed
using DPPA (diphenyl azidophosphate) as a coupling
agent in the presence of TEA (when using hydrochlo-
ride). Treating compounds 1 with 90% TFA for 20 min
caused removal of the protecting tert-butyl and 4,6-
benzylidene groups. There is no earlier information on
this acidolysis of compounds 1. The final products
11a -h were purified with radial chromatography and
preparative TLC. The composition of the conjugates was
1
confirmed by H NMR (500 MHz) spectroscopy, elemen-
tal analysis, and TLC qualitative amino acid analysis.
Yields and melting points of the products are collected
in Table 1.
Batracylin 9 was synthesized by our modified method
(Scheme 3). There are several methods for the synthesis
of batracylin (BAT). Kabbe13 proposed the condensation
of 2,5-diaminobenzylamine with phthalic anhydride.

980 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6
Dzierzbicka et al.
Sch em e 2
phenylenediamine 14 were reacted with N-(hydroxym-
ethyl)phthalimide under standard conditions of the
Czerniak-Einhorn reaction. Protecting groups present
in the product mixture were hydrolyzed by the method
described.16 Concentrated sulfuric acid was used as the
hydrolyzing agent for 5 h at 100 °C. After alkalinization
with NH3, an aqueous solution of BAT in the form of
crystals was obtained. The yields after crystallization
with DMF were 65-70% (for acetyl groups) and 75-
80% (for urethane groups). The advantage of the method
consists of elimination of two steps carried out in
previously exploited methods and elimination of the
difficult and troublesome reduction of the nitro group
to the amino group. The synthesis of BAT via this
modified route (Scheme 3) now appears to be feasible
on a multigram scale. Batracylin derivatives [N-(Nn-Boc-
amino acid)-BAT] and hydrochloride salts 10a -c were
obtained making use of known methods of amide bond
formation: DCC and DMAP in anhydrous methylene
chloride (10b,c); EEDQ and anhydrous pyridine in
anhydrous methylene chloride (10a ).
that combining strong antitumor BAT with immuno-
modulators, e.g., MDP or nor-MDP, would improve its
pharmacological properties. Our synthesized conjugates
are more soluble in water than BAT alone. All the final
compounds 11a -h were tested for their cytotoxicity in
the National Cancer Institute (NCI, Bethesda, MD)
screening system based on 60 human tumor lines.26-28
This primary antitumor screen is designed to discover
selective, disease-specific drugs. The synthesized con-
jugates did not exhibit cytotoxic activity at 10-4-10-8
M. The results were contrary to what we had expected.
The influence of conjugates of MDP or nor-MDP with
BAT was also tested in vitro on the growth of the Ab
melanoma cells. These investigations were carried out
at the Department of Histology and Immunology, Medi-
cal University of Gdansk, Poland. Bomirski melanoma
variant Ab cells were maintained by serial passage in
hamsters using the suspension method of tumor trans-
plantation. The natural history and characteristics of
that melanoma are given by Bomirski30 and Slominski.31
Batracylin (BAT) alone did not influence the prolifera-
tion of Ab melanoma cells. Two of the examined conju-
gates 11c and 11e inhibited the proliferation of Ab
melanoma cells in vitro compared with batracylin or the
solvent. Additional post-hoc tests revealed that the
inhibiting effect exerted by 11c and 11e is mainly
dependent on the two highest concentrations of the
analogues: 0.05 and 0.1 mg/mL (Table 2 and Figure 1).
The reductive effect of low doses of the used conjugates
on the growth of the tumor cells could not be clearly
Resu lts a n d Discu ssion
The low water solubility of BAT limits its oral
administration. Toxicity of BAT, especially when rats
are tested, reduces its usefulness as a chemotherapeutic
agent when high doses must be used. It is very probable
that BAT can be very toxic for the human organism.
Therefore, syntheses of BAT analogues with improved
therapeutic characteristics are undertaken. We hoped

Conjugates of MDP and nor-MDP
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6 981
Sch em e 3
Ta ble 2. ANOVA Test: Between-Group Comparison of the
Serial Dilutions of the Examined Conjugates
(4:1:1) (A), CHCl3-MeOH (4:1) (B), CH2Cl2-(CH3)2CO (10:1)
(C), CH2Cl2-(CH3)2CO (50:50) (D). Elemental analyses were
performed by Laboratory of Elemental Analysis, Faculty of
Chemistry, University of Gdansk. Qualitative amino acid
analyses of the hydrolyzates of these compounds were ac-
complished on TLC plates. ESI mass spectrometry was per-
formed on a Mariner spectrometer. Benzyl 1-O-benzyl-4,6-O-
benzylidene-N-acetylmuramyl(or normuramyl)-L-alanyl(valyl
or prolyl)-D-isoglutaminate 1 was prepared as described.24,25
N-(4-Nitrophenyl)acetamide 5 is commercially available and
cheap.
compared pairs
F
P
BAT/DMSO
11d /DMSO
11h /DMSO
11e/DMSO
11c/DMSO
BAT/11d
BAT/11h
BAT/11e
BAT/11c
1.152
1.907
10.492
305.18
363.37
1.159
1.349
249.14
292.76
0.3435
0.3012
0.0835
0.0032
0.0027
0.3942
0.3819
0.0039
0.0033
N-(4-Am in op h en yl)a ceta m id e (6). An amount of 3.6 g (20
mmol) of N-(4-nitrophenyl)acetamide 5 was hydrogenated with
Raney Ni in 300 mL of dioxane in the presence of 2 mL of
100% hydrazine hydrate for 3 h. The reaction mixture was
filtered off, and the catalyst was rinsed with 100 mL of hot
dioxane. The filtrates were combined and the solvent was
evaporated to give 2.70 g of 4-aminoacetanilide (90%): mp
elucidated because all the experiments were performed
in vitro against Ab melanoma cells alone. MDP most
visibly affects the immune cells, so the MDP fragment
of the conjugates might indirectly block the tumor
growth by an activation of the immune system. How-
ever, only further in vivo tests on animals could verify
such hypothesis. The investigations will be carried out
at the Medical University of Gdansk, Poland.
1
161-162 °C (lit.32 mp 162 °C); H NMR (CDCl3) δ 2.1 (s, 3H,
COCH3), 3.25 (br s, 2H, NH2-), 6.52 (d, J ) 8.8 Hz, 2H, protons
of aromatic), 7.24 (d, J ) 8.8 Hz, 2H, protons of aromatic),
9.45 (br s, 1H, -NH-CO-).
Eth yl N-(4-Aceta m id op h en yl)ca r ba m a te (7). This was
prepared from N-(4-aminophenyl)acetamide 6 as
a solid
1
(78%): mp 210-212 °C (lit.33 mp 202.5 °C); H NMR (CDCl3)
δ 1.26 (t, J ) 7 Hz, 3H, -CH2CH3), 1.98 (s, 3H, COCH3), 4.3
(q, J ) 7 Hz, 2H, -CH2CH3), 6.52 (d, J ) 8.8 Hz, 2H, protons
of aromatic), 7.24 (d, J ) 8.8 Hz, 2H, protons of aromatic),
8.99 (br s, 1H, NH-COOEt), 9.8 (br s, 1H, NH-COMe).
Eth yl N-[4-Aceta m id o-2-(p h th a lim id om eth yl)p h en yl]-
ca r ba m a te (8). This was prepared from ethyl N-(4-acetami-
dophenyl)carbamate 7 as a solid (75%): mp 251-254 °C (lit.16
mp 258-260 °C); 1H NMR (DMSO-d6) δ 1.25 (t, J ) 7 Hz, 3H,
COOCH2CH3), 1.94 (s, 3H, COCH3), 4.10 (q, J ) 6.8 Hz, 2H,
COOCH2CH3), 4.72 (s, 2H, CH2N), 7.15 (brs, 1H, C3-aromatic),
7.2 (d, J ) 8 Hz, 1H, C6-aromatic), 7.64 (dd, J ) 2 Hz and J )
8 Hz, 1H, C5-aromatic), 8.9 (br s, 1H, NHCOOC2H5), 9.83 (br
s, 1H, NHCOCH3). Anal. (C20H19N3O5) C, H, N.
Exp er im en ta l Section
Melting points were determined with a Kofler block ap-
paratus and are uncorrected.
1H NMR spectra were measured in DMSO solutions with
Varian 500 and 200 MHz NMR spectrometers. Preparative
column chromatography and radial chromatography were
performed on silica gel (Kieselgel 60, 100-200 mesh) in solvent
systems specified in the text. All chemicals and solvents were
of reagent grade and were used without further purification.
The reactions were monitored by TLC on Merck F254 silica gel
precoated plates. The following solvent systems (by volume)
were used for TLC development: n-BuOH-H2O-AcOH

982 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6
Dzierzbicka et al.
F igu r e 1. In vitro activity of the conjugates 11c-e,h on the growth of the Ab melanoma cells.
8-Am in oisoin d olo[1,2-b]qu in a zolin -12(10H)-on e (BAT)
(9). This was prepared from ethyl N-[4-acetamido-2-(phthal-
imidomethyl)phenyl]carbamate 8 as a yellow solid (75%): mp
275-278 °C (lit. mp 270 °C16 and 287-288 °C13); 1H NMR
(DMSO-d6) δ 4.8 (s, 2H, B10), 5.55 (s, 2H, NH2), 6.4 (s, 1H,
B9), 6.48 (d, J ) 8.2 Hz, B7), 7.15 (d, J ) 8.2 Hz, B6), 7.67 (t,
J ) 7.4 Hz, 1H, B3), 7.74 (t, J ) 7.4 Hz 1H, B2), 7.83 (d, J )
7.4 Hz, 1H, B4), 7.92 (d, J ) 7.4 Hz, 1H, B1). Anal. (C15H11N3O)
C, H, N.
N-(3-Am in op r op a n oyl)ba tr a cylin Hyd r och lor id e (10a ).
To a stirred solution of 0.283 g (1.5 mmol) of Boc-â-alanine in
170 mL of anhydrous methylene chloride, cooled to 4 °C, 0.72
g (3 mmol) of EEDQ and 0.37 g (1.5 mmol) of batracylin 9 in
dry pyridine (0.6 mL) were added. The stirring was continued
for 2 h at 4 °C and then at room temperature for 3 days (TLC
in system C or D). After evaporation of the solvents under
reduced pressure, the residue was purified using radial
chromatography in solvent D and N-(3-(1,1-dimethylethoxy-
carbonylamino)propanoyl)batracylin in 60% (0.28 g) yield was
obtained: mp 214-216 °C; 1H NMR (DMSO) δ 1.36 (s, 9H,
Boc), 2.74 (t, J ) 6.8 Hz, 2H, -NHCH2 CH2CONH-), 3.08 (q,
J ) 6.8 Hz, 2H, -NHCH2CH2CONH-), 4.86 (s, 2H, B10), 6.47
(brs, 1H, Boc-NH), 7.34 (d, J ) 8.8 Hz, 1H, B6), 7.52 (d, J )
8.6 Hz, 1H, B7), 7.57 (s, 1H, B9), 7.88 (t, J ) 7.4 Hz, 1H, B3),
7.80 (t, J ) 7.4 Hz 1H, B2), 7.89 (d, J ) 7.4 Hz, 1H, B4), 8.0
(d, J ) 7.4 Hz, 1H, B1), 10.2 (s, 1H, B8-NH).
solvent D, and N-(6-(1,1-dimethylethoxycarbonylamino)hex-
anoyl)batracylin in 75% (0.52 g) yield was obtained: mp 187-
190 °C; 1H NMR (DMSO) δ 1.22-1.42 (m, 4H, -NHCH2-
CH2CH2CH2CH2CONH-), 1.36 (s, 9H, Boc), 1.57 (q, J ) 7 Hz,
2H, -NHCH2CH2CH2CH2CH2CONH-), 2.3 (t, J ) 7 Hz, 2H,
-NHCH2CH2(CH2)2CH2CONH-), 2.88 (q, J ) 7 Hz, 2H,
-NHCH2CH2(CH2)2CH2CONH-), 4.9 (s, 2H, B10), 6.8 (brs,
1H, Boc-NH), 7.34 (d, J ) 8.8 Hz, 1H, B6), 7.52 (d, J ) 8.6
Hz, 1H, B7), 7.57 (s, 1H, B9), 7.88 (t, J ) 7.4 Hz, 1H, B3),
7.80 (t, J ) 7.4 Hz 1H, B2), 7.89 (d, J ) 7.4 Hz, 1H, B4), 8.0
(d, J ) 7.4 Hz, 1H, B1), 10.1 (s, 1H, B8-NH).
The obtained 0.2 g of N-(6-(1,1-dimethylethoxycarbonylami-
no)hexanoyl)batracylin was poured into a saturated solution
of hydrogen chloride in diethyl ether (10 mL) and left for 2 h
at 0 °C. Then the solution was evaporated and the residue was
washed several times with ethyl ether. The yellow hydrochlo-
ride salt of compound 10c was obtained: yield 0.19 g (90%);
1
ESI (MeOH) m/z 363.2 (MH)+; H NMR (DMSO) δ 1.30-1.40
(m, 2H, -NHCH2CH2CH2CH2CH2CONH-), 1.50-1.65 (m, 4H,
-NHCH2CH2CH2CH2CH2CONH-), 2.35 (t, J ) 7 Hz, 2H,
-NHCH CH2(CH2)2CH2 CONH-), 2.75 (t, J ) 7 Hz, 2H,
-NHCH22CH2(CH2)2CH2 CONH), 4.9 (s, 2H, B10), 7.38 (d, J )
8.8 Hz, 1H, B6), 7.57 (d, J ) 8.6 Hz, 1H, B7), 7.62 (s, 1H, B9),
7.78 (t, J ) 7.3 Hz, 1H, B3), 7.82 (t, J ) 7.3 Hz 1H, B2), 7.90
(d, J ) 7.4 Hz, 1H, B4), 7.90 (bs, 3H, NH3+), 8.05 (d, J ) 7.4
Hz, 1H, B1), 10.20 (s, 1H, B8-NH).
The obtained 0.2 g of N-(3-(1,1-dimethylethoxycarbonylami-
no)propanoyl)batracylin was poured into a saturated solution
of hydrogen chloride in diethyl ether (10 mL) and left for 2 h
at 0 °C. Then the solution was evaporated and the residue was
washed several times with ethyl ether. The yellow hydrochlo-
ride salt of compound 10a was obtained: yield 0.2 g (92%);
mp 258 °C (dec); ESI (MeOH) m/z 321.2 (MH)+; 1H NMR
(DMSO) δ 2.75 (t, J ) 6.8 Hz, 2H, -NHCH2CH2CONH-), 3.10
(t, J ) 6.8 Hz, 2H, -NHCH2CH2CONH-), 4.90 (s, 2H, B10),
7.34 (d, J ) 8.8 Hz, 1H, B6), 7.52 (d, J ) 8.6 Hz, 1H, B7), 7.57
(s, 1H, B9), 7.88 (t, J ) 7.4 Hz, 1H, B3), 7.80 (t, J ) 7.4 Hz
1H, B2), 7.90 (d, J ) 7.4 Hz, 1H, B4), 7.90 (bs, 3H, NH3+), 8.0
(d, J ) 7.4 Hz, 1H, B1), 10.3 (s, 1H, B8-NH).
N-(6-Am in oh exa n oyl)ba tr a cylin Hyd r och lor id e (10c).
To a stirred solution of 0.5 g (2 mmol) of compound 9 in 290
mL of anhydrous methylene chloride cooled to 0 °C, 0.92 g (4
mmol) of 6-(1,1-dimethylethoxycarbonylamino)hexanoic acid,
0.83 g (4 mmol) of DCC, and 0.04 g of DMAP ((dimethylamino)-
pyridine) were added. The stirring was continued for 4 h at 0
°C and then at room temperature for 4 days. The reaction was
monitored with TLC in system solvent D. The side product of
dicyclohexylurea was removed by filtration. The solvent was
evaporated under reduced pressure, the yellow residue was
purified with radial chromatography and eluted with system
The N-(4-aminobutanoyl)batracylin 10b was obtained in the
same manner. N-(4-(1,1-dimethylethoxycarbonylamino)butan-
oyl)batracylin: mp 222-224 °C; yield 82%; 1H NMR (DMSO)
δ 1.36 (s, 9H, Boc), 1.65 (q, J ) 7 Hz, 2H, -NHCH2CH2CH2-
CONH-), 2.3 (t, J ) 7 Hz, 2H, -NHCH2CH2CH2CONH-),
2.96 (q, J ) 7 Hz, 2H, -NHCH2CH2CH2CONH-), 4.9 (s, 2H,
B10), 6.9 (brs, 1H, Boc-NH), 7.34 (d, J ) 8.8 Hz, 1H, B6),
7.52 (d, J ) 8.6 Hz, 1H, B7), 7.57 (s, 1H, B9), 7.88 (t, J ) 7.4
Hz, 1H, B3), 7.80 (t, J ) 7.4 Hz 1H, B2), 7.89 (d, J ) 7.4 Hz,
1H, B4), 8.0 (d, J ) 7.4 Hz, 1H, B1), 10.1 (s, 1H, B8-NH).
The hydrochloride salt of N-(4-aminobutanoyl)batracylin
10b, mp 255 °C (dec), was obtained in 95% yield; ESI (MeOH)
1
m/z 335.3 (MH)+; H NMR (DMSO) δ 1.65 (q, J ) 7 Hz, 2H,
-NHCH2CH2CH2CONH-), 2.3 (t, J ) 7 Hz, 2H, -NHCH2-
CH2CH2CONH-), 2.98 (t, J ) 7.2 Hz, 2H, -NHCH2CH2CH2-
CONH-), 4.9 (s, 2H, B10), 7.34 (d, J ) 8.8 Hz, 1H, B6), 7.52
(d, J ) 8.6 Hz, 1H, B7), 7.57 (s, 1H, B9), 7.88 (t, J ) 7.4 Hz,
1H, B3), 7.80 (t, J ) 7.4 Hz 1H, B2), 7.91 (d, J ) 7.4 Hz, 1H,
B4), 7.91 (bs, 3H, NH3+), 8.0 (d, J ) 7.4 Hz, 1H, B1), 10.18 (s,
1H, B8-NH).
Gen er a l P r oced u r e for th e Syn th esis of 1-O-Ben zyl-
N-a cetylm u r a m yl(or n or m u r a m yl)-L-a la n yl(va lyl or p r o-
lyl)-D-isoglu ta m in e (2). A total of 2 mmol of compound 1 in
4 mL of 90% trifluoroacetic acid was left at room temperature

Conjugates of MDP and nor-MDP
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6 983
for 20 min. Then the solution was evaporated under reduced
pressure, and the residue was washed several times with ethyl
ether to give 2 as a white powder.
) 6.9 Hz, OH-C4-Mur), 7.03 and 7.17 (2s, 2H, NH2-isoGln),
7.24-7.38 (m, 5H, Ph), 7.92 (d, J ) 8.3 Hz, 1H, NH-Mur), 8.32
(d, J ) 7.9 Hz, 1H, NH-isoGln), 10.4 (s, 1H, COOH).
1-O -B e n zy l-N -a c e t y lm u r a m y l-L -a la n y l-D -is o g lu t -
a m in e (2a ): yield 1.08 g (93%); mp 88-92 °C; 1H NMR
(DMSO) δ 1.22 (d, J ) 7.3 Hz, 3H, CH3-Mur), 1.25 (d, J ) 6.8
Hz, 3H, CH3-Ala), 1.7 (s, 3H, AcMur), 1.74 (m, 1H, âCH2-
isoGln), 1.92 (m, 1H, âCH2-isoGln), 2.1 (t, J ) 7.8 Hz, 2H,
γCH2-isoGln), 3.32-3.55 (m, 3H, H-C3,4,5-Mur), 3.56 (d, J
) 5.6 Hz, 1H, H-C6-Mur), 3.65 (d, J ) 10.8 Hz, 1H, H-C6-
Mur), 3.8 (m, 1H, H-C2-Mur), 4.1(m, 1H, RCH-isoGln), 4.3
(m, 2H, RCH-Mur, RCH-Ala), 4.45 (d, J ) 12.2 Hz, 1H, CH2-
Ph), 4.75 (d, J ) 12.2 Hz, 1H, CH2-Ph), 4.76 (d, J ) 3.4 Hz,
1H, H-C1-Mur), 5.5 (br, 1H, OH-C4-Mur), 7.05 and 7.35 (2s,
2H, NH2-isoGln), 7.4 (m, 5H, Ph), 7.66 (d, J ) 6.8 Hz, 1H, NH-
Ala), 8.12 (d, J ) 8.4 Hz, 1H, NH-Mur), 8.26 (d, J ) 8.4 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 N-[1-O-Ben zyl-
N-a cet yl(m u r a m yl or n or m u r a m yl)-L-(a la n yl, va lyl, or
pr olyl)-D-γ-isoglu tam in yl)-Nn-am in o acid]batr acylin (11a-
h ). To a stirred solution of compounds 2 (1 mmol) and 9 or 10
(1.1 mmol) in anhydrous DMF (5 mL), cooled to 0 °C, DPPA
(1.1 mmol) in DMF (5 mL) was added followed by the addition
of TEA (2.2 mmol) in DMF (2 mL) at 0 °C for 3 h and then 24
h at room temperature. After evaporation of the solvent, the
reaction mixture was purified using radial chromatography
and preparative TLC in solvent system D to afford compounds
11a -h . The yield and melting points of 11a -h are given in
Table 1.
N -[1-O -B e n zy l-N -a c e t y lm u r a m y l-L -a la n y l-D -γ-is o -
glu ta m in yl]ba tr a cylin (11a ): 1H NMR (DMSO) δ 1.22 (d,
J ) 7.3 Hz, 3H, CH3-Mur), 1.25 (d, J ) 6.8 Hz, 3H, CH3-Ala),
1.74 (m, 1H, âCH2-isoGln), 1.78 (s, 3H, AcMur), 1.92 (m, 1H,
âCH2-isoGln), 2.1 (t, J ) 7.8 Hz, 2H, γCH2-isoGln), 3.32-3.55
(m, 3H, H-C3,4,5-Mur), 3.56 (d, J ) 5.7 Hz, 1H, H-C6-Mur),
3.65 (d, J ) 10.7 Hz, 1H, H-C6-Mur), 3.8 (m, 1H, H-C2-Mur),
4.1(m, 1H, RCH-isoGln), 4.3 (m, 2H, RCH-Mur, RCH-Ala), 4.45
(d, J ) 12.2 Hz, 1H, CH2-Ph), 4.74 (d, J ) 12.2 Hz, 1H, CH2-
Ph), 4.76 (d, J ) 3.4 Hz, 1H, H-C1-Mur), 4.9 (s, 2H, B-10),
5.5 (br, 1H, OH-C4-Mur), 7.05 and 7.35 (2s, 2H, NH2-isoGln),
7.36 (d, J ) 8.6 Hz, 1H, B6), 7.4 (m, 5H, Ph), 7.5 (d, J ) 8.6
Hz, 1H, B7), 7.56 (s, 1H, B9), 7.66 (d, J ) 6.8 Hz, 1H, NH-
Ala), 7.76 (t, J ) 7.4 Hz, 1H, B3), 7.8 (t, J ) 7.4 Hz, 1H, B2),
7.9 (d, J ) 7.4 Hz, 1H, B4), 8.12 (d, J ) 8.3 Hz, 1H, NH-Mur),
8.2 (d, J ) 7.4 Hz, 1H, B1), 8.26 (d, J ) 8.3 Hz, 1H, NH-isoGln),
10.1(s, 1H, B8-NH). Anal. (C41H48N7O11) C, H, N.
1-O -B e n zy l-N -a c e t y l-n o r m u r a m y l-L -a la n y l-D -is o -
1
glu ta m in e (2b): yield 1.08 g (95%); mp 63-66 °C; H NMR
(DMSO) δ 1.20 (d, J ) 6.8 Hz, 3H, CH3-Ala), 1.83 (s, 3H,
AcMur), 1.68 (m, 1H, âCH2-isoGln), 1.95 (m, 1H, âCH2-isoGln),
2.20 (t, J ) 7.4 Hz, 2H, γCH2-isoGln), 3.32-3.45 (m, 3H,
H-C3,4,5-Mur), 3.50 (d, J ) 5.8 Hz, 1H, H-C6-Mur), 3.65 (d,
J ) 10 Hz, 1H, H-C6-Mur), 3.85 (m, 1H, H-C2-Mur), 4.08
(d, J ) 15.8 Hz, 1H, OCH2CO), 4.15 (m, 1H, RCH-isoGln), 4.20
(d, J ) 15.8 Hz, 1H, OCH2CO), 4.30 (m, 2H, RCH-Mur, RCH-
Ala), 4.44 (d, J ) 12.4 Hz, 1H, CH2-Ph), 4.67 (d, J ) 12.4 Hz,
1H, CH2-Ph), 4.70 (d, J ) 3.3 Hz, 1H, H-C1-Mur), 5.65 (br,
1H, OH-C4-Mur), 7.05 and 7.30 (2s, 2H, NH2-isoGln), 7.35
(m, 5H, Ph), 8.05 (d, J ) 6.6 Hz, 1H, NH-Ala), 8.18 (d, J ) 8.8
Hz, 1H, NH-Mur), 8.25 (d, J ) 8.8 Hz, 1H, NH-isoGln), 10.0
(s, 1H, COOH).
1-O-Ben zyl-N-a cet ylm u r a m yl-L-va lyl-D-isoglu t a m in e
(2c): yield 1.15 g (94%); mp 106-110 °C; H NMR (DMSO) δ
N -[1-O -B e n zy l-N -a c e t y lm u r a m y l-L -a la n y l-D -γ-is o -
glu ta m in yl]-(N2-a m in oa la n yl)ba tr a cylin (11b): 1H NMR
(DMSO) δ 1.22 (d, J ) 7.3 Hz, 3H, CH3-Mur), 1.25 (d, J ) 6.8
Hz, 3H, CH3-Ala), 1.74 (m, 1H, âCH2-isoGln), 1.80 (s, 3H,
AcMur), 1.90 (m, 1H, âCH2-isoGln), 2.1 (t, J ) 7.8 Hz, 2H,
γCH2-isoGln), 2.67 (t, J ) 6.8 Hz, 2H, -NHCH2 CH2CONH-),
3.08 (q, J ) 6.8 Hz, 2H, -NHCH2CH2CONH-), 3.32-3.55 (m,
3H, H-C3,4,5-Mur), 3.56 (d, J ) 5.7 Hz, 1H, H-C6-Mur), 3.65
(d, J ) 10.7 Hz, 1H, H-C6-Mur), 3.8 (m, 1H, H-C2-Mur), 4.1
(m, 1H, RCH-isoGln), 4.3 (m, 2H, RCH-Mur, RCH-Ala), 4.42
(d, J ) 12.2 Hz, 1H, CH2-Ph), 4.74 (d, J ) 12.2 Hz, 1H,
CH2-Ph), 4.76 (d, J ) 3.4 Hz, 1H, H-C1-Mur), 4.9 (s, 2H, B-10),
5.5 (br, 1H, OH-C4-Mur), 7.05 and 7.35 (2s, 2H, NH2-isoGln),
7.36 (d, J ) 8.6 Hz, 1H, B6), 7.4 (m, 5H, Ph), 7.5 (d, J ) 8.6
Hz, 1H, B7), 7.56 (s, 1H, B9), 7.66 (d, J ) 6.8 Hz, 1H, NH-
Ala), 7.76 (t, J ) 7.4 Hz, 1H, B3), 7.8 (t, J ) 7.4 Hz, 1H, B2),
7.9 (d, J ) 7.4 Hz, 1H, B4), 8.12 (d, J ) 8.3 Hz, 1H, NH-Mur),
8.2 (d, J ) 7.4 Hz, 1H, B1), 8.26 (d, J ) 8.3 Hz, 1H, NH-isoGln),
10.1 (s, 1H, B8-NH). Anal. (C44H52N8O12) C, H, N.
N-[1-O-Ben zyl-N-a cet yln or m u r a m yl-L-a la n yl-D-γ-iso-
glu ta m in yl]ba tr a cylin (11c): 1H NMR (DMSO) δ 1.25 (d,
J ) 6.8 Hz, 3H, CH3-Ala), 1.72 (m, 1H, âCH2-isoGln), 1.74 (s,
3H, AcMur), 1.94 (m, 1H, âCH2-isoGln), 2.1 (t, J ) 7.8 Hz,
2H, γCH2-isoGln), 3.32-3.55 (m, 3H, H-C3,4,5-Mur), 3.56 (d,
J ) 5.7 Hz, 1H, H-C6-Mur), 3.64 (d, J ) 10.7 Hz, 1H, H-C6-
Mur), 3.8 (m, 1H, H-C2-Mur), 4.1 (m, 1H, RCH-isoGln), 4.12
(d, J ) 15.2 Hz, 1H, OCH2CO), 4.2 (d, J ) 15.2 Hz, 1H, OCH2-
CO), 4.3 (m, 2H, RCH-Mur, RCH-Ala), 4.45 (d, J ) 12 Hz, 1H,
CH2-Ph), 4.75 (d, J ) 12 Hz, 1H, CH2-Ph), 4.76 (d, J ) 3.4 Hz,
1H, H-C1-Mur), 4.9 (s, 2H, B-10), 5.5 (br, 1H, OH-C4-Mur),
7.05 and 7.35 (2s, 2H, NH2-isoGln), 7.36 (d, J ) 8.6 Hz, 1H,
B6), 7.4 (m, 5H, Ph), 7.5 (d, J ) 8.6 Hz, 1H, B7), 7.56 (s, 1H,
B9), 7.66 (d, J ) 6.8 Hz, 1H, NH-Ala), 7.76 (t, J ) 7.4 Hz,
1H, B3), 7.8 (t, J ) 7.4 Hz, 1H, B2), 7.9 (d, J ) 7.4 Hz, 1H,
B4), 8.12 (d, J ) 8.3 Hz, 1H, NH-Mur), 8.2 (d, J ) 7.4 Hz, 1H,
B1), 8.26 (d, J ) 8.3 Hz, 1H, NH-isoGln), 10.1 (s, 1H, B8-NH).
Anal. (C43H50N8O12) C, H, N.
1
0.84 (d, J ) 6.8 Hz, 3H, CH3-Val), 0.87 (d, J ) 6.8 Hz, 3H,
CH3-Val), 1.18 (d, J ) 6.7 Hz, 3H, CH3-Mur), 1.80 (s, 3H,
AcMur), 1.69 (m, 1H, âCH2-isoGln), 1.93 (m, 1H, âCH2-isoGln),
1.98 (m, 1H, âCH-Val), 2.06 (t, J ) 7.8 Hz, 2H, γCH2-isoGln),
3.32-3.55 (m, 3H, H-C3,4,5-Mur), 3.56 (d, J ) 5.6 Hz, 1H,
H-C6-Mur), 3.65 (d, J ) 10.8 Hz, 1H, H-C6-Mur), 3.8 (m,
1H, H-C2-Mur), 4.02 (m, 1H, RCH-Val), 4.1(m, 1H, RCH-
isoGln), 4.3 (m, 1H, RCH-Mur), 4.44 (d, J ) 12.4 Hz, 1H, CH2-
Ph), 4.70 (d, J ) 12.4 Hz, 1H, CH2-Ph), 4.80 (d, J ) 3.4 Hz,
1H, H-C1-Mur), 5.5 (br, 1H, OH-C4-Mur), 7.08 and 7.32 (2s,
2H, NH2-isoGln), 7.34-7.4 (m, 5H, Ph), 8.11 (d, J ) 8.4 Hz,
1H, NH-Mur), 8.22 (d, J ) 8.3 Hz, 1H, NH-isoGln), 8.35 (d, J
) 8.0 Hz, 1H, NH-Val), 10.2 (s, 1H, COOH).
1 -O -B e n z y l -N -a c e t y l n o r m u r a m y l -L -v a l y l -D -i s o -
1
glu ta m in e (2d ): yield 1.13 g (95%); mp 66-70 °C; H NMR
(DMSO) δ 0.84 (d, J ) 6.8 Hz, 3H, CH3-Val), 0.87 (d, J ) 6.8
Hz, 3H, CH3-Val), 1.18 (d, J ) 6.7 Hz, 3H, CH3-Mur), 1.80 (s,
3H, AcMur), 1.69 (m, 1H, âCH2-isoGln), 1.93 (m, 1H, âCH2-
isoGln), 1.98 (m, 1H, âCH-Val), 2.06 (t, J ) 7.8 Hz, 2H, γCH2-
isoGln), 3.32-3.55 (m, 3H, H-C3,4,5-Mur), 3.56 (d, J ) 5.6
Hz, 1H, H-C6-Mur), 3.65 (d, J ) 10.8 Hz, 1H, H-C6-Mur),
3.8 (m, 1H, H-C2-Mur), 4.02 (m, 1H, RCH-Val), 4.08 (d, J )
15.8 Hz, 1H, OCH2CO), 4.1 (m, 1H, RCH-isoGln), 4.20 (d, J )
15.8 Hz, 1H, OCH2CO), 4.3 (m, 1H, RCH-Mur), 4.44 (d, J )
12.4 Hz, 1H, CH2-Ph), 4.70 (d, J ) 12.4 Hz, 1H, CH2-Ph), 4.80
(d, J ) 3.4 Hz, 1H, H-C1-Mur), 5.5 (br, 1H, OH-C4-Mur),
7.08 and 7.32 (2s, 2H, NH2-isoGln), 7.34-7.4 (m, 5H, Ph), 8.11
(d, J ) 8.4 Hz, 1H, NH-Mur), 8.22 (d, J ) 8.3 Hz, 1H, NH-
isoGln), 8.35 (d, J ) 8.0 Hz, 1H, NH-Val), 10.2 (s, 1H, COOH).
1-O -B e n z y l-N -a c e t y lm u r a m y l-L -p r o ly l-D -i s o g lu t -
a m in e (2e): yield 1.16 g (92%); mp 69-74 °C; 1H NMR
(DMSO) δ 1.23 (d, J ) 6.4 Hz, 3H, CH3-Mur), 1.67-2.07 (m,
4H, Pro-3, Pro-4), 1.73 (m, 1H, âCH2-isoGln), 1.78 (s, 3H,
AcMur), 2.01(m, 1H, âCH2-isoGln), 2.21 (m, 2H, γCH2-isoGln),
3.35-3.51 (m, 3H, H-C3,4,5-Mur), 3.52 and 3.64 (m, 2H, Pro-
5), 3.50 (d, J ) 8.3 Hz, 1H, H-C6-Mur), 3.62 (d, J ) 10.5 Hz,
1H, H-C6-Mur), 4.19 (dt, J ) 5.2 Hz, J ) 7.8 Hz, 1H, RCH-
isoGln), 4.32 (m, 1H, Pro-2), 4.34 (d, J ) 12.2 Hz, 1H, CH2-
Ph), 4.58 (t, J ) 5.9 Hz, OH-C6-Mur), 4.65 (d, J ) 12.2 Hz,
1H, CH2-Ph), 4.83 (d, J ) 3.4 Hz, 1H, H-C1-Mur), 5.38 (d, J
N-[1-O-Ben zyl-N-a cet yln or m u r a m yl-L-a la n yl-D-γ-iso-
glu ta m in yl]-(N5-a m in oca p r oyl)ba tr a cylin (11d ): 1H NMR
(DMSO) δ 1.2-1.42 (m, 2H, -NHCH2CH2CH2 CH2CH2CONH-),
1.25 (d, J ) 6.8 Hz, 3H, CH3-Ala), 1.57 (q, J ) 7 Hz, 4H,
-NHCH2CH2 CH2CH2CH2CONH-), 1.72 (m, 1H, âCH2-isoGln),

984 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6
Dzierzbicka et al.
F igu r e 2. The 500 MHz 1H NMR spectrum of N-(1-O-benzyl-N-acetylmuramyl-L-valyl-D-γ-isoglutaminyl)batracylin 11e in DMSO.
1.82 (s, 3H, AcMur), 1.90 (m, 1H, âCH2-isoGln), 2.1 (t, J ) 7.8
Hz, 2H, γCH2-isoGln), 2.32 (t, J ) 7 Hz, 2H, -NHCH2CH2-
(CH2)2 CH2CONH-), 2.89 (q, J ) 7 Hz, 2H, -NHCH2CH2(CH2)2-
CH2CONH-), 3.32-3.55 (m, 3H, H-C3,4,5-Mur), 3.58 (d, J )
5.7 Hz, 1H, H-C6-Mur), 3.67 (d, J ) 10.7 Hz, 1H, H-C6-Mur),
3.8 (m, 1H, H-C2-Mur), 4.1 (m, 1H, RCH-isoGln), 4.14 (d, J
) 15.4 Hz, 1H, OCH2CO), 4.2 (d, J ) 15.4 Hz, 1H, OCH2CO),
4.3 (m, 2H, RCH-Mur, RCH-Ala), 4.46 (d, J ) 12 Hz, 1H, CH2-
Ph), 4.72 (d, J ) 12 Hz, 1H, CH2-Ph), 4.76 (d, J ) 3.4 Hz, 1H,
H-C1-Mur), 4.9 (s, 2H, B-10), 5.5 (br, 1H, OH-C4-Mur), 7.05
and 7.35 (2s, 2H, NH2-isoGln), 7.36 (d, J ) 8.6 Hz, 1H, B6),
7.4 (m, 5H, Ph), 7.5 (d, J ) 8.6 Hz, 1H, B7), 7.56 (s, 1H, B9),
7.66 (d, J ) 6.8 Hz, 1H, NH-Ala), 7.76 (t, J ) 7.4 Hz, 1H,
B3), 7.8 (t, J ) 7.4 Hz, 1H, B2), 7.9 (d, J ) 7.4 Hz, 1H, B4),
8.12 (d, J ) 8.3 Hz, 1H, NH-Mur), 8.2 (d, J ) 7.4 Hz, 1H, B1),
8.26 (d, J ) 8.3 Hz, 1H, NH-isoGln), 10.1(s, 1H, B8-NH). Anal.
(C46H56N8O12) C, H, N.
N-[1-O-Ben zyl-N-acetylm u r am yl-L-valyl-D-γ-isoglu tam i-
n yl]ba tr a cylin (11e): 1H NMR (DMSO) δ 0.88 and 0.89 (2d,
J ) 6.3 Hz, 6H, -CHCH(CH3)2CO-), 1.19 (d, J ) 6.7 Hz, 3H,
CH3-Mur), 1.80 (m, 1H, âCH2-isoGln), 1.85 (s, 3H, AcMur), 2.01
(m, 1H, -CHCH(CH3)2 CO),2.08 (m, 1H, âCH2-isoGln), 2.36
(t, J ) 7.8 Hz, 2H, γCH2-isoGln), 3.41 (dt, J ) 6.3 Hz, J ) 8.5
Hz, 1H, H-C4-Mur), 3.55 (m, 3H, H-C3,5,6-Mur), 3.56 (d, J
) 5.7 Hz, 1H, H-C6-Mur), 3.67 (m,1H, H-C6-Mur), 3.95 (dt,
J ) 3.5 Hz, J ) 10.3 Hz, 1H, H-C2-Mur), 4.05 (t, J ) 7.5 Hz,
1H, -CHCH(CH3)2CO-), 4.21 (dt, J ) 5.3 Hz, J ) 8.7 Hz, 1H,
RCH-isoGln), 4.29 (q, J ) 6.8 Hz, 1H, RCH-Mur), 4.45 (d, J )
12.2 Hz, 1H, CH2-Ph), 4.65 (t, J ) 5.6 Hz, 1H, OH-C6-Mur),
4.68 (d, J ) 12.2 Hz, 1H, CH2-Ph), 4.60 (d, J ) 3.5 Hz, 1H,
H-C1-Mur), 4.90 (s, 2H, B-10), 5.82 (d, J ) 6.3 Hz, 1H, OH-
C4-Mur), 7.15 and 7.37 (2s, 2H, NH2-isoGln), 7.37 (d, J ) 8.6
Hz, 1H, B6), 7.25-7.4 (m, 5H, Ph), 7.52 (d, J ) 8.6 Hz, 1H,
B7), 7.57 (s, 1H, B9), 7.76 (t, J ) 7.4 Hz, 1H, B3), 7.81 (t, J )
7.4 Hz, 1H, B2), 7.89 (d, J ) 7.4 Hz, 1H, B4), 8.12 (d, J ) 9.6
Hz, 1H, NH-Mur), 8.0 (d, J ) 7.4 Hz, 1H, B1), 8.29 (d, J ) 7.9
Hz, 1H, NH-isoGln), 8.38 (d, J ) 7.9 Hz, 1H, NH-Val), 10.11
(s, 1H, B8-NH). Anal. (C43H52N7O11) C, H, N.
Ph), 4.70 (d, J ) 12.5 Hz, 1H, CH2-Ph), 4.76 (d, J ) 3.4 Hz,
1H, H-C1-Mur), 4.9 (s, 2H, B-10), 5.5 (br, 1H, OH-C4-Mur),
7.05 and 7.35 (2s, 2H, NH2-isoGln), 7.36 (d, J ) 8.6 Hz, 1H,
B6), 7.4 (m, 5H, Ph), 7.5 (d, J ) 8.6 Hz, 1H, B7), 7.56 (s, 1H,
B9), 7.76 (t, J ) 7.4 Hz, 1H, B3), 7.8 (t, J ) 7.4 Hz, 1H, B2),
7.9 (d, J ) 7.4 Hz, 1H, B4), 8.12 (d, J ) 8.3 Hz, 1H, NH-Mur),
8.2 (d, J ) 7.4 Hz, 1H, B1), 8.26 (d, J ) 8.3 Hz, 1H, NH-isoGln),
8.38 (d, J ) 8 Hz, 1H, NH-Val), 10.1 (s, 1H, B8-NH). Anal.
(C47H59N8O12) C, H, N.
N -[1-O-Be n zyl-N -a ce t yln or m u r a m yl-L-va lyl-D-γ-iso-
glu ta m in yl](N5-a m in oca p r oyl)ba tr a cylin (11g). 1H NMR
(DMSO) δ 0.88 and 0.9 (2d, J ) 6.3 Hz, 6H, -CHCH(CH3)2-
CO-), 1.2-1.4 (m, 2H, -NHCH2CH2CH2CH2CH2CONH-),
1.56 (q, J ) 7 Hz, 4H, -NHCH2CH2CH2CH2CH2CONH-), 1.71
(m, 1H, âCH2-isoGln), 1.85 (s, 3H, AcMur), 1.90 (m, 1H, âCH2-
isoGln), 2.0 (m, 1H, -CHCH(CH3)2CO-), 2.1 (t, J ) 7.8 Hz,
2H, γCH2-isoGln), 2.3 (t, J ) 7 Hz, 2H, -NHCH2CH2-
(CH2)2CH2CONH-), 2.86 (q, J ) 7 Hz, 2H, -NHCH2CH2(CH2)2-
CH2 CONH-), 3.32-3.55 (m, 3H, H-C3,4,5-Mur), 3.55 (d, J
) 5.7 Hz, 1H, H-C6-Mur), 3.67 (d, J ) 10.7 Hz, 1H, H-C6-
Mur), 3.8 (m, 1H, H-C2-Mur), 4.04 (t, J ) 7.5 Hz, 1H,
-CHCH(CH3)2CO-), 4.1 (m, 1H, RCH-isoGln), 4.12 (d, J )
15 Hz, 1H, OCH2CO), 4.22 (d, J ) 15 Hz, 1H, OCH2CO), 4.3
(m, 1H, RCH-Mur), 4.45 (d, J ) 12 Hz, 1H, CH2-Ph), 4.72 (d,
J ) 12 Hz, 1H, CH2-Ph), 4.76 (d, J ) 3.4 Hz, 1H, H-C1-Mur),
4.9 (s, 2H, B-10), 5.5 (br, 1H, OH-C4-Mur), 7.05 and 7.35 (2s,
2H, NH2-isoGln), 7.36 (d, J ) 8.6 Hz, 1H, B6), 7.4 (m, 5H, Ph),
7.5 (d, J ) 8.6 Hz, 1H, B7), 7.56 (s, 1H, B9), 7.76 (t, J ) 7.4
Hz, 1H, B3), 7.8 (t, J ) 7.4 Hz, 1H, B2), 7.9 (d, J ) 7.4 Hz,
1H, B4), 8.12 (d, J ) 8.3 Hz, 1H, NH-Mur), 8.2 (d, J ) 7.4 Hz,
1H, B1), 8.26 (d, J ) 8.3 Hz, 1H, NH-isoGln), 8.38 (d, J ) 8
Hz, 1H, NH-Val), 10.1 (s, 1H, B8-NH). Anal. (C47H58N8O12) C,
H, N.
N-[1-O-Ben zyl-N-acetylm u r am yl-L-pr olyl-D-γ-isoglu tam i-
n yl](N5-a m in oca p r oyl)ba tr a cylin (11h ): 1H NMR (DMSO)
δ 1.2-1.42 (m, 2H, -NHCH2CH2CH2 CH2CH2CONH-), 1.22
(d, J ) 7.3 Hz, 3H, CH3-Mur), 1.65 and 1.96 (m, 2H, C3-Pro),
1.56 (q, J ) 7 Hz, 4H, -NHCH2CH2CH2CH2CH2CONH-), 1.68
(m, 1H, âCH2-isoGln), 1.75 (s, 3H, AcMur), 1.80 (m, 2H, C4-
Pro), 1.92 (m, 1H, âCH2-isoGln), 2.1 (t, J ) 7.8 Hz, 2H, γCH2-
isoGln), 2.32 (t, J ) 7 Hz, 2H, -NHCH2CH2(CH2)2CH2CONH-),
2.88 (q, J ) 7 Hz, 2H, -NHCH2CH2(CH2)2CH2CONH-), 3.32-
3.55 (m, 3H, H-C3,4,5-Mur), 3.48 and 3.60 (m, 2H, C5-Pro),
3.54 (d, J ) 5.7 Hz, 1H, H-C6-Mur), 3.62 (d, J ) 10.7 Hz, 1H,
H-C6-Mur), 3.8 (m, 1H, H-C2-Mur), 4.1 (m, 1H, RCH-isoGln),
4.3 (m, 1H, RCH-Mur), 4.31 (m, 1H, C2-Pro), 4.42 (d, J ) 12.5
Hz, 1H, CH2-Ph), 4.75 (d, J ) 12.5 Hz, 1H, CH2-Ph), 4.76
(d, J ) 3.4 Hz, 1H, H-C1-Mur), 4.9 (s, 2H, B-10), 5.5 (br,
1H, OH-C4-Mur), 7.05 and 7.35 (2s, 2H, NH2-isoGln), 7.36
(d, J ) 8.6 Hz, 1H, B6), 7.38-7.44 (m, 5H, Ph), 7.5 (d, J )
8.6 Hz, 1H, B7), 7.56 (s, 1H, B9), 7.76 (t, J ) 7.4 Hz, 1H,
N-[1-O-Ben zyl-N-acetylm u r am yl-L-valyl-D-γ-isoglu tam i-
n yl](N3-a m in obu ta n oyl)ba tr a cylin (11f): 1H NMR (DMSO)
δ 0.88 and 0.89 (2d, J ) 6.3 Hz, 6H, -CHCH(CH3)2CO-), 1.20
(d, J ) 7.3 Hz, 3H, CH3-Mur), 1.64 (q, J ) 7 Hz, 2H, -NHCH2
CH2CH2CONH-), 1.68 (m, 1H, âCH2-isoGln), 1.80 (s, 3H,
AcMur), 1.93 (m, 1H, âCH2-isoGln), 2.0 (m, 1H, -CHCH(CH3)2-
CO-), 2.1 (t, J ) 7.8 Hz, 2H, γCH2-isoGln), 2.32 (t, J ) 7 Hz,
2H, -NHCH2CH2CH2CONH-), 2.94 (q, J ) 7 Hz, 2H,
-NHCH2CH2CH2CONH-), 3.32-3.55 (m, 3H, H-C3,4,5-
Mur), 3.56 (d, J ) 5.7 Hz, 1H, H-C6-Mur), 3.67 (d, J ) 10.7
Hz, 1H, H-C6-Mur), 3.8 (m, 1H, H-C2-Mur), 4.1 (m, 1H,
RCH-isoGln), 4.06 (t, J ) 7.5 Hz, 1H, -CHCH(CH3)2CO-), 4.3
(q, J ) 6.8 Hz, 1H, RCH-Mur), 4.45 (d, J ) 12.5 Hz, 1H, CH2-

Conjugates of MDP and nor-MDP
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6 985
Ta ble 3. 1H NMR Data of N-(1-O-Benzyl-N-acetylmuramyl-L-
valyl-D-γ-isoglutaminyl)batracylin 11e
for 3 h. The reaction mixture was poured onto ice and mixed
with 250 mL of chloroform. The undissolved solids were filtered
out. The chloroform layer was evaporated to dryness. Crystal-
lization from acetic acid-water gave 7.1 g (54%) of ethyl N-{-
[2-(ph t h a lim idom et h yl)-4-et h oxyca rbonyla m in o]ph en yl}-
fragment
multiplicity
structure assignment
δ (ppm)
and J (Hz)
ROE
CH2φ
1
GlcN
C1
4.60
3.95
3.55
3.41
3.55
d 3.5
dt 3.5, 10.3
m
dt 6.3, 8.5
m
carbamate: mp 197-202 °C; H NMR (DMSO) δ 1.15 (t, J )
C2
C3
C4
C5
7 Hz, 3H, CH3), 1.25 (t, J ) 7 Hz, 3H, CH3), 4.05 (q, J ) 6.8
Hz, 2H, -CH2CH3), 4.10 (q, J ) 6.8 Hz, 2H, -CH2CH3), 4.7
(s, 2H, CH2 protons of phthalimidomethylene group), 7.1 (s,
1H, C5), 7.18 (d, J ) 8.5 Hz, 1H, C2), 7.42 (d, J ) 8.5 Hz, 1H,
C3), 7.86-7.94 (m, 4H, protons of phthalic group), 8.98 (brs,
1H, NH), 9.46 (s, 1H, NH). Anal. (C21H21N3O6) C, H, N.
8-Am in oisoin d olo[1,2-b]qu in a zolin -12(10H)-on e (BAT)
(9) P r ep a r ed by Ou r Meth od . Meth od A. This was pre-
pared from 1.13 g of N-[2-(phthalimidomethyl)-1,4-acetyl]-
phenylenediamine 15 and recrystallized from DMF to give a
yellow solid (0.56 g, 70%): mp 268-270 °C.
H2
dt 6.3; 8.5
m
C6
3.55, 3.67 m, m
m, m
d 6.3
t 5.6
d 9.6
s
C1, φ
CH2
C3, V2-NH
C4-OH
C6-OH
C2-NH
N-Ac
CH2
φ
H2
H3
V2
V3
5.82
4.65
8.12
1.85
d 6.3
t 5.6
d 9.6
s
Bz
4.45, 4.68 d, d 12.5
7.25-7.4
m
Khp
Val
4.29
q 6.8
d 6.7
t 7.5
m
1.19
4.05
2.01
Meth od B. This was prepared from 1 g of ethyl N-{[2-
(ph t h a lim idom et h yl)-4-et h oxyca r bon yla m in o]ph en yl]}-
carbamate 16 and recrystallized from DMF to give a yellow
solid (0.48 g, 80%): mp 269-272 °C; 1H NMR (DMSO-d6) δ
4.8 (s, 2H, B10), 5.55 (s, 2H, NH2), 6.4 (s, 1H, B9), 6.48 (d, J
) 8.2 Hz, B7), 7.15 (d, J ) 8.2 Hz, B6), 7.67 (t, J ) 7.4 Hz,
1H, B3), 7.74 (t, J ) 7.4 Hz 1H, B2), 7.83 (d, J ) 7.4 Hz, 1H,
B4), 7.92 (d, J ) 7.4 Hz, 1H, B1). Anal. (C15H11N3O) C, H, N.
In Vitr o Activity on th e Gr ow th of th e Ab Mela n om a
Cells. P r olifer a tion Test. In all tests, cells were incubated
for 12 h in an atmosphere of 5% CO2 at 37 °C on 96 plastic
flat-bottomed well plates (Corning, Science Products) in trip-
licate. Ab melanoma cells were dispensed as 1 × 104 cells per
well in 100 µL of F10 medium with 5% heat-inactivated fetal
calf serum (Gibco, Life Technologies Inc.). The cultures were
incubated with the following final concentrations of batracylin
and its derivatives: 0, 0.0001, 0.001, 0.01, 0.05, and 0.1 mg/
mL. Since the examined substances were dissolved in DMSO,
additional control cultures were performed only with the serial
dilutions of the solvent.
Color im etr ic MTT Assa y. After incubation, 3-(4,5-dim-
ethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
(Sigma Chemical Co.) was added to all the wells for a final
concentration of 1 mg/mL and the plates were incubated at
37 °C in an atmosphere of 5% CO2 for an additional 4 h. Then
the reaction was stopped by addition of 100 µL of 2-propanol.
Optical density was read at 570 nm on the automated plate
reader (FL600, Bio-Tec). The results were obtained and
analyzed in arbitrary units in absorbance mode using the
computer program Kineticalc 4 (Bio-Tec).
G2-NH
V4
0.88, 0.89 d, d 6.3
V2-NH
G2
G3
8.38
4.21
d 7.9
dt 5.3, 8.7
H2
CONH2
Glu
B
1.80, 2.08 m, m
2.36
8.29
G4
t 7.8
d 7.9
B8-NH
V2
G2
G2-NH
CONH2
B1 (B4)
B2 (B3)
B3 (B2)
B4 (B1)
B6
B7
B8-NH
B9
7.15, 7.37 s, s
8.00
7.81
7.76
7.89
7.37
7.52
10.11
7.57
4.90
d 7.4
t 7.4
t 7.4
d 7.4
d 8.6
d 8.6
s
B7, B9, G4
B10
B9
s
s
B10
B3), 7.8 (t, J ) 7.4 Hz, 1H, B2), 7.9 (d, J ) 7.4 Hz, 1H, B4),
8.12 (d, J ) 8.3 Hz, 1H, NH-Mur), 8.2 (d, J ) 7.4 Hz, 1H, B1),
8.26 (d, J ) 8.3 Hz, 1H, NH-isoGln), 10.1(s, 1H, B8-NH). Anal.
(C48H61N8O12) C, H, N.
N,N′-Dia cetyl-1,4-p h en ylen ed ia m in e (13). Meth od A.
This was prepared from 1,4-phenylenediamine 12 and glacial
acetic acid, as a solid (84.7%): mp 308-312 °C (lit.29 mp 255-
257 °C).
Meth od B. This was prepared from 1,4-phenylenediamine
12, acetic anhydride, and pyridine, as a solid (92%): mp 310-
313 °C (lit.29 mp 312-315 °C); 1H NMR (DMSO) δ 2.01 (s, 6H,
2CH3), 7.48 (s, 4H, protons of aromatic), 9.84 (s, 2H, 2NH).
N,N′-Dieth oxycar bon yl-1,4-ph en ylen ediam in e (14). This
was prepared from 1,4-phenylenediamine 12, as colorless
Sta tistica l An a lysis. Since the results satisfied the normal
distribution, statistical analysis was based on ANOVA tests.
Ack n ow led gm en t. This work was supported by the
Polish State Committee for Scientific Research (Grant
No. 4 P05F 017 16). We express our thanks to Dr. V. L.
Narayanan from NCI (Bethesda, MD) for the in vitro
screening. We are grateful to Danuta Laskowska for her
skillful technical assistance.
1
crystals (89%): mp 193-196 °C (lit.33 mp 196-196.5 °C); H
NMR (DMSO) δ 1.25 (t, J ) 6.6 Hz, 3H, CH3), 4.05 (q, J ) 6.6
Hz, 2H, -CH2CH3), 7.4 (s, 4H, protons of aromatic), 9.8 (s,
2H, 2NH).
N -[2-(P h t h a lim id om e t h yl)-1,4-a ce t yl]p h e n yle n e d i-
a m in e (15). An amount of 0.96 g (5 mmol) of N,N′-diacetyl-
1,4-phenylenediamine 13 dissolved in 5 mL of concentrated
sulfuric acid at 50 °C and an amount of 1.78 g (10 mmol) of
N-(hydroxymethyl)phthalimide in portions were added for 1
h. After the reagent had dissolved, the solution was stirred at
50 °C for 3 h. The reaction mixture was poured onto ice and
mixed with 50 mL of ethanol. The precipitate formed was
collected by filtration. The yield of crystals was 1.13 g
(64.2%): mp 258-262 °C; 1H NMR (DMSO) δ 1.95 (s, 3H, CH3),
2.05 (s, 3H, CH3), 4.65 (s, 2H, CH2 protons of phthalimidom-
ethylenediamine), 7.16 (s, 1H, C5), 7.18 (d, J ) 8.5 Hz, 1H,
C2), 7.62 (d, J ) 8.5 Hz, 1H, C3), 7.84-7.96 (m, 4H, protons
of phthalic group), 9.58 (brs, 1H, NH), 9.84 (s, 1H, NH). Anal.
(C19H17N3O4) C, H, N.
Ap p en d ix
1
Table 3 and Figure 2 show additional H NMR data
of 11e.
Refer en ces
(1) Adam, A.; Lederer, E. Muramylpeptides: immunomodulators,
sleep factors and vitamins. Med. Res. 1984, 4, 111-152.
(2) Baschang, G. Muramylpeptides and lipopeptides: studies to-
wards immunostimulants. Tetrahedron 1989, 45, 6331-6360.
(3) Dzierzbicka, K.; Gozdowska, M.; Kołodziejczyk, A. M. Influence
of immunomodulators of the type MDP on secretion and activity
of cytokines. Postepy Biochem. 1998, 44 (3), 216-219.
(4) Dzierzbicka, K.; Kołodziejczyk, A. M.; Wysocka-Skrzela, B.;
Mys´liwski, A.; Sosnowska, D. Synthesis and antitumor activity
of conjugates of muramyldipeptides, normuramyldipeptide, and
desmuramyldipeptides with acridine/acridone derivatives. J .
Med. Chem. 2001, 44, 3606-3614.
Eth yl N-{[2-(P h th a lim id om eth yl)-4-eth oxyca r bon yl-
a m in o]p h en yl}ca r ba m a te (16). An amount of 8 g (17.3
mmol) of compound 15 was dissolved in 20 mL of concentrated
sulfuric acid at 50 °C and was combined with 6.14 g of
N-(hydroxymethyl)phthalimide, and the mixture was stirred

986 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6
Dzierzbicka et al.
(5) Plowman, J .; Paull, K. D.; Atassi, G.; Harrison, S. D.; Dykes, D.
J .; Kabbe, H. J .; Narayanan, V. L.; Yoder, O. C. Preclinical
antitumor activity of batracylin (NSC 320846). Invest. New
Drugs 1988, 6, 147-153.
(6) Waud, W. R.; Harrison, S. D.; Gilbert, K. S.; Laster, W. R.;
Griswold, D. P. Antitumor drug cross-resistance in vivo in a
cisplatin-resistant murine P388 leukemia. Cancer Chemother.
Pharmacol. 1991, 27, 456-463.
(7) Mucci-LoRusso, P.; Polin, L.; Bissery, M. C.; Valeriote, F.;
Plowman, J .; Luk, G. D.; Corbett, T. H. Activity of batracylin
(NSC 320846) against solid tumors of mice. Invest. New Drugs
1989, 7, 295-306.
(8) Stevens, G. J .; Meegalla, S. K.; Makhey, D.; LaVoie, E. J .;
McQueen, Ch. A. Genotoxicity of batracylin and several struc-
tural analogs. Proc. Am. Assoc. Cancer Res. 1993, 34, 158.
(9) Stevens, G. J .; McQueen, Ch. A. Species variation in the
genotoxicity of batracylin. Toxicol. Appl. Pharmacol. 1994, 126,
39-44.
(10) El-Hawari, M.; Stoltz, M. L.; Pallas, F. Species differences in
the toxic responses to batracylin (NSC 320846). Proc. Am. Assoc.
Cancer Res. 1989, 30, 626.
(20) Dzierzbicka, K.; Kołodziejczyk, A. M.; Wysocka-Skrzela, B.;
Kołodziejczyk, A. S. Synthesis of muramyl dipeptide conjugated
with acridine derivatives, showing anti-HIV-1 and anticancer
activity. Pol. J . Chem. 1994, 68, 37-45.
(21) Gozdowska, M.; Dzierzbicka, K.; Wysocka-Skrzela, B.; Kołodz-
iejczyk, A. M. Synthesis and in vitro anticancer activity of
conjugates of MDP with amino-acridine/acridone derivatives.
Pol. J . Chem. 1997, 71, 767-773.
(22) Dzierzbicka, K.; Gozdowska, M.; Wysocka-Skrzela, B.; Kołodz-
iejczyk, A. M. Synthesis and anticancer activites of new MDP
conjugates with amino-acridine/acridone derivatives. Acta Pol.
Pharm. 1999, 56, 20-24.
(23) Sosnowska, D.; Mys´liwski, A.; Dzierzbicka, K.; Kołodziejczyk,
A. The in vitro effect of new muramyl peptide derivatives on
cytotoxic activity of NK (Natural Killer) cells from hamsters
bearing Ab Bomirski melanoma. Biotherapy 1997, 10, 161-168.
(24) Lefrancier, P.; Choay, J .; Derrieu, M.; Lederman, I. Synthesis
of N-acetylmuramyl-L-alanyl-D-isoglutamine, an adjuvant of the
immune response and some N-acetylmuramyl peptide analogs.
Int. J . Pept. Protein Res. 1977, 9, 249-257.
(11) Ames, M. M.; Mathiesen, D. A.; Reid, J . M. Differences in
N-acetylation of the experimental antitumor agent batracylin
in the mouse and the rat. Invest. New Drugs 1991, 9, 219-225.
(12) Stevens, G. J .; LaVoie, E. J .; McQueen, Ch. A. The role of
acetylation in the mutagenicity of the antitumor agent, batra-
cylin. Carcinogenesis 1996, 17, 115-119.
(25) Gozdowska, M. Ph.D. Thesis, Gdansk University of Technology,
Gdansk, Poland, 1998.
(26) Monks, A.; Scudiero, D.; Skehan, P.; Shoemaker, R.; Paull, K.;
Vistica, D.; Hose, C.; Langley, J .; Cronise, P.; Vaigro-Wolff, A.;
Gray-Goodrich, M.; Campbell, H.; Mayo, J .; Boyd, M. Feasibility
of a high-flux anticancer drug screen using a diverse panel of
cultured human tumor cell lines. J . Natl. Cancer Inst. 1991, 83,
757-766.
(13) Kabbe, H. J . Heterocycles from diamines and diacyl compounds.
J ustus Liebigs Ann. Chem. 1978, 398-404.
(14) Luo, Y.; Ren, Y. F.; Chou, T. Ch.; Chen, A. Y.; Yu, Ch.; Liu, L.
F.; Cheng, C. C. A structure-activity relationship study of
batracylin analogues. Pharm. Res. 1993, 10 (6), 918-923.
(15) Meegalla, S. K.; Stevens, G. J .; McQueen, Ch. A.; Chen, A. Y.;
Yu, Ch.; Liu, L. F.; Barrows, L. R.; LaVoie, E. J . Synthesis and
pharmacological evaluation of isoindolo[1,2-b]quinazolinone and
isoindolo[2,1-a]benzimidazole derivatives related to the antitu-
mor agent. J . Med. Chem. 1994, 37, 3434-3439.
(27) Grever, M. R.; Schepartz, S. A.; Chabner, B. A. The National
Cancer Institute: Cancer Drug Discovery and Development
Program. Semin. Oncol. 1992, 19, 622-638.
(28) Information from NCI, Bethesda MD.
(29) Pfeiffer, F. R.; Case, F. H. The preparation of some pyrido and
pyridyl derivatives of phenazine and quinoxaline. J . Org. Chem.
1966, 31, 3384-3390.
(30) Bomirski, A.; Slominski, A.; Bigda, J . The natural history of a
family of transplantable melanomas in hamsters. Cancer Me-
tastasis Rev. 1988, 7, 95-118.
(31) Slominski, A.; Paus, R. Bomirski melanoma: a versatile and
powerful model of pigment cell and melanoma research. Int. J .
Oncol. 1993, 2, 221-228.
(32) Giffney, C. J .; O’Connor, Ch. J . Spectrophotometric determina-
tion of basicity constants. Part II. Acetanilides. J . Chem. Soc.,
Perkin Trans. 2 1975, 706-712.
(16) Roservear, J .; Wilshire, F. K. A comparison of the reactions of
some ethyl N-arylcarbamates with those of the corresponding
acetanilides. II Amidomethylation with N-hydroxymethylphthal-
imide. Aust. J . Chem. 1990, 43, 339-353.
(17) Stella, V.; Al-Razzak, L. A.; Kabbe, H. J . U.S. Patent 4,980,343,
1990.
(18) Lerchen, H. G.; Piel, N.; Lorentzen, J . P.; Antonicek, H. P.;
Kabbe, H. J . Fucose-marked batracylin glycopeptide conjugates
as antitumor agents. European Patent Office No. 0 501 250 A1,
1992; Chem. Abstr. 1993, 118, 60133k.
(33) Schiff, H.; Ostrogovich, A. Uramide, urethane und oxamathane
der p- und m-phenylendiamine. J ustus Liebigs Ann. Chem. 1896,
293, 371-388.
(19) Lerchen, H. G.; Von Dem Bruch, K.; Petersen, U.; Baum-Gartem,
J .; Piel, N.; Antonicek, H. P.; Weichel, W.; Sperzel, M.; Bremm,
K.; Dieter, K. Preparation of carbohydrate-modified cytostatic
agents. Patent No. PCT/EP96/01279, 1996; Chem. Abstr. 1997,
126, 31574q.
J M021067V