Note: Descriptions are shown in the official language in which they were submitted.
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Chemical Compounds
The present invention relates to benzaldehyde derivatives which are useful as
anticancer
agents, antiviral agents, immunopotentiators and/or as agents which may be
used for
combating illnesses which arise due to an elevated cell proliferation and/or
for combating
auto immune diseases. Some of the compounds of this invention are novel per
se.
Most of the presently used anticancer agents are cytotoxic in their action.
Although these
agents have shown good results in treatment of some cancers like lymphoma,
leukaemia
and testicular cancer, they often produce severe and unacceptable side-effects
limiting the
possibility for an effective treatment. Furthermore, in several types of
cancer like in solid
tumours (carcinoma), chemotherapy has so far proven to be of limited value
since
established cytostatic drug seldom improves the prognosis for the patient. The
ability of
cancer cells to develop resistance against cytotoxic products is also a main
reason for the
failure in their use in the treatment of solid tumours. There is thus a great
need for new
anticancer agents having fewer side effects and having a more selective action
on
malignant cells.
It is known among other from EP-0215395, JP-6326441 l, JP-8800940, JP-55069510
and
EP-0283139 that benzaldehydes and derivatives thereof exhibit a selective
anticancer
effect:
Aldehydes react with a range of O, S or N nucleofilic entities like hydroxy
groups,
sulfhydryl groups and amino groups to form carbonyl condensation products like
acetals,
mercaptals, aminals, etc. However, with primary amines, the reaction normally
take the
form of Schiffs base (imine) adduct formation. It is well known that in vivo
Schiff s base
formation is involved in key biochemical processes like transamination,
decarboxylation
and other amino acid modifying reactions mediated by pyridoxal phosphate, the
action of
aldolase on fructose di-phosphate in the glycolysis and the condensation of
retinal with
rhodopsin in the process of vision. It is also known that carbonyl
condensation reactions
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2
are involved in transmembrane signalling events, for example in generating an
immune
response.
The formation of imines proceeds through a two-stage mechanism: The addition
of the
amino nucleofile to the carbonyl group to form a carbinolamine (aminohydrin)
intermediate followed by a dehydration step to generate the C=N double bond.
Both steps
are reversible, but are facilitated at different pH values. As a consequence,
the reaction
occurs according to a characteristic bell-shaped pH/rate profile with the
highest over-all
reaction rates being found at moderate acidities.
OH
I
R- \ -f- H2N-R~ R- I - I -R~ R- I -N-R~ -f- H20
H H H H
aldehyde amine carbinotamine imine
However, the formation of Schiff s bases are known to take place readily also
in
physiological conditions, and many carbonyl condensation reactions are well
known in
vivo (E. Schauenstein et. al., Aldehydes in biological systems. London, Pion
Ltd. 1977).
The Schiff s base tend to be a reactive species itself and is prone to further
reaction
resulting in the addition of nucleofilic agents to the double bond. For
certain
sulfur-containing amines, in particular the amino acids cystein and
methionine, and for
glutathione, the initially formed Schiff s base can undergo reversible
internal cyclization in
which the sulfhydryl group adds to the imine to form thiazolidine carboxylate
(M.
Friedman, The chemistry and biochemistry of the sulfhydryl group in amino
acids,
peptides and proteins, Oxford, Pergamon Press, 1973).
/O // /S-CHz
R- ~ ~- HzN- H_ ~ R- ~ ~ -~ H20
H ~H OH H CH /O
2 /
SH
OH
aldehyde cystein thiazolidine carboxylic acid
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Evidence for reactions between carbonyl compounds and free amino groups of
proteins to
form reversible Schiff s base linkages was reported by G. E. Means and R. E.
Feeney
(Chemical Modification of Poteins, pp. 125 - 138, San Francisco, Holden-Day,
1971).
Aromatic aldehydes are in general more reactive than saturated aliphatic
aldehydes, and
Schiffs bases can be formed even without removal of the water formed during
the
reaction. (R. W. Layer Chem. Rev. 63 (1963), 489 - 510). This fact is
important when
considering formation of Schiff s bases under physiological conditions. Using
haemoglobin as a source of amino groups Zaugg et. al. (J. Biol. Chem. 252
(1977), 8542 -
8548) have shown that aromatic aldehydes have a two- to threefold increased
reactivity
over aliphatic aldehydes in Schiffs base formation. An explanation for the
limited
reactivity of alkanals could be the fact that in aqueous solution at neutral
pH a very large
excess of free aldehyde is required to shift the equilibrium in favour of
Schiffs base
formation (E. Schauenstein et. al., Aldehydes in biological systems. London,
Pion Ltd.
1977).
Benzaldehyde and salicylaldehyde readily form Schiffs base imines with
membrane
amino groups, and high equilibrium constants have been measured for
benzaldehyde
reacting with amines (J. J. Pesek and J. H. Frost, Org. Magnet. Res. 8 (1976),
173 - 176;
J. N. Williams Jr. and R. M. Jacobs Biochim Biophys Acta. 154, (1968) 323 -
331). With
salicylaldehyde, the imine could achieve extra stabilisation due to hydrogen
bonding
between the lone-pair electrons of the imine nitrogen and the orto hydroxyl
group (G. E.
Means and R. E. Feeney, Chemical Modification of Poteins, pp. 125 - 138, San
Francisco, Holden-Day, 1971; J. M. Dornish and E. O. Pettersen Biochem.
Pharmac. 39
(1990), 309 - 318).
We have previously shown by radio labelling images that benzaldehyde do not
enter the
cell, but adhere to the cell membrane (Dornish, J.M. and Pettersen, E.O.:
Cancer Letters
29 (1985) 235-243). This is in agreement with an earlier study, showing that
benzaldehyde interacted with the membrane proteins of E. coli (K.Sakaguchi et.
al. Agric.
Biol. Chem., (1979), 43, 1775-1777). It was also found that pyridoxal and
pyridoxal-5-phosphate both protect the cells against the cytotoxic anti-cancer
agent
cis-DDP. Cis-DDP exerts its action in the nucleus within the cell. While
pyridoxal in
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principle could penetrate the lipophilic cell membrane, this possibility is
blocked for
pyridoxal-5-phosphate because of the ionic phosphate group on the latter.
Pyridoxal-5-phosphate thus have to exert its protective effect by acting from
outside the
cell membrane. A spectral shift in the absorbance of pyridoxal-5-phosphate to
lower
wavelengths observed simultaneously is consistent with Schiff s base adduct
formation
between the aldehyde and cell membrane amino groups (J. M. Dornish and E. O.
Pettersen, Cancer Lett. 29, (1985), 235 -243).
These findings suggest that aldehydes bind to amines and other nucleofilic
entities on the
cell membrane to form Schiff s bases and other condensation products. It is
known that
stimulation of cell growth is mediated by a cascade of events acting from
outside the cell
membrane. In the same way, the derivatives in the present patent application
may act by
forming adducts with ligands on the cell membrane, triggering impulses inside
the cell
with significance on cell growth parameters like protein synthesis and
mitosis, and on the
expression of tumour suppressor genes and immune responses. Since the
condensation
reactions are reversible, cellular effects can be modulated as a result of a
shift in
equilibrium involving ligating species. The presence of dynamic equlibria at a
chemical
level is consistent with the reversible and non- toxic way of action observed
with the
benzaldehyde derivatives.
Inhibition of the protein synthesis exerted by benzaldehyde derivatives is
very well
studied in vitro in our research group. In solid tumours the reduced protein
synthesis may
result in a lack of vital proteins which lead to cell death. In normal cells
there is a potential
capacity for protein synthesis which is higher than in most cancer cells of
solid tumours.
This is demonstrated by comparison of the cell cycle duration in normal stem
cells, which
is often below lOh, and thus shorter than that of most cancer cells of solid
tumours, which
is typically 30-150h (see Gustavo and Pileri in: The Cell Cycle and Cancer.
Ed. : Baserga,
Marcel Dekker Inc., N.Y. 1971, p 99). Since cells, as an average, double their
protein
during a cell cycle, this means that protein accumulation is higher in growth-
stimulated
normal cells than in most types of cancer cells.
Keeping in mind this difference between normal and cancer cells, there is
another
difference of similar importance: while normal cells respond to growth-
regulatory stimuli,
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cancer cells have reduced or no such response. Thus, while normal cells, under
ordinary
growth conditions, may have a reserve growth potential, cancer cells have
little or no such
reserve. If a protein synthesis inhibition is imposed continuously over a long
period of
time on normal cells as well as on cancer cells, the two different types of
cells may
5 respond differently: Normal tissue may make use of some of its reserve
growth potential
and thereby maintain normal cell production. Cancer tissue however, have
little or no such
reserve. At the same time the rate of protein accumulation in most cancer
cells is rather
low (i.e. protein synthesis is only slightly greater than protein
degradation). Therefore the
protein synthesis inhibition may be enough to render the tumour tissue
unbalanced with
respect to protein accumulation, giving as a result a negative balance for
certain proteins.
During continuous treatment for several days this will result in cell
inactivation and
necrosis in the tumour tissue while normal tissue is unharmed.
To date, the most tested compound inducing reversible protein synthesis
inhibition and
displaying anti-cancer activity is 5,6-benzylidene-d,-ascorbic acid
[zilascorb(ZH)~. The
protein synthesis inhibiting activity of this prior art compound is described
in detail by
Pettersen et.al. (Anticancer Res., vol. 11, pp. 1077-1082, 1991) and in EP-
0283139.
Zilascorb(ZH) induces tumour necrosis in vivo in human tumour xenografts in
nude mice
(Pettersen et al., Br. J. Cancer, vol. 67, pp. 650-656, 1993). In addition to
zilascorb(zH),
the closest prior art compound related to cancer treatment is
4,6-O-Benzylidene-D-glucopyranose (Compound 1 ) . These two compounds are
known to
possess a general anti-cancer activity and have been tested in clinical trials
against a
number of cancer diseases. However, no particular cancer afflicted organs or
tissues
projected as more suitable for treatment with these compounds, and commercial
development was not justified.
We have now surprisingly found that benzaldehyde derivatives of sugars of the
hexose
type, (including 4,6-O-(Benzylidene-dl)-D-glucopyranose, Compound 2) give an
unexpected strong effect on cancer in certain organs or tissues. We cannot yet
explain the
mechanism for this selectivity, but we believe that this is connected to the
affinity of the
sugar moiety of the derivatives to certain cells or tissues.
We have found that certain of our new products like for instance Compound 8,
(2-acetamido-4,6-O-(benzylidene-dl)-2-deoxy-D-glucopyranose) gives an
unexpected
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good effect in a nude mice model (see Example 3, Table 1). 3 of 8 mice were
free of
tumours, which is an unusual result in similar experiments on immunosupressed
species.
The reason for this effect could be that the acetamido moiety has a high
affinity to
hyaluronic acid receptors. It is known that malignant tumours are rich on
hyaluronic acid
and hence rich on corresponding receptors.
We have also found in our experiments that the deuterated analogue of these
compounds
are substantially more effective than the corresponding proton analogues. This
difference
in effect is very striking in our experiment on cell adhesion (see Example 5
and also
Example 8). When a hydrogen atom is substituted by the twice as heavy
deuterium
isotope, the kinetic properties of the molecule are altered as the rate in
breaking the C-D
bond is lowered compared to breaking the C-H bond. It is known among others
from M.I.
Blake et.al., J. Pharm. Sci. 64 (1975), 367-391 that deuteration of drugs may
alter their
pharmacological function.
It is also known in the art (EP 0 283 139 and Anticancer Res. 15: 1921-1928
(1995)) that
when the acetal proton in 4,6-O-benzylidene-D-glucopyranose is substituted
with
deuterium (Compound 1 versus Compound 2), this can affect both the protein
synthesis
and the cell surviving fraction measured in vitro. We believe that one
possible explanation
for this D-isotope effect at a chemical level is related to slower oxidation
of deuterated
benzaldehyde to inactive benzoic acid, resulting in a longer half-life of the
deuterated
active ingredient at a cellular level. However, to demonstrate a significant
difference in
the surviving fraction of NHIK 3025 cells exposed to Compounds 1 resp. 2, drug
concentrations of more than 6 mM must be applied. The difference in protein
synthesis
inhibition was very small when these cells were exposed at 1-10 mM
concentration.
The inventors now performed a completely different kind of experiment: The
adhesion
force between NHIK 3025 cells and the substratum was measured after pre-
incubation of
the cells in solutions of Compounds 1 and 2 (see Example 5). Even at 1 mM
concentration, an astonishing D-isotope effect was shown. Surprisingly,
Compound 2
significantly reduced the adhesion force to 1/3 relative to control, whereas
Compound 1
did not lead to significant reduction. The inventors believe that Compound 2
may have
interfered with the biosynthesis of integrins, reducing the cell's ability to
attach to the
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substratum. Integrins are structural trans-membrane proteins crucial for
binding cells to
the extracellular matrix and for cell-cell interactions. Inhibiting the
function of the
integrins could thus directly affect the metastasising ability of cancer
cells. The
experiment indicate that integrines could be especially sensitive to protein
synthesis
inhibition. Thus, Compound 2 could well be used for prevention of metastatic
processes in
cancer development.
The chemical induced carcinogenesis has a similar mechanism as the
cancerogenesis
induced by certain virus types like hepatitis B and C, certain papilloma
virus, certain
herpes virus etc. Especially this will be the case in the development of liver
cancer in
hepatitis B and C infected patients. It is therefore presumable that a
prophylactic treatment
of these patients with products of this invention could prevent or delay the
development of
liver cancer. Also the fact that these products show a low toxic profile would
make them
suitable for such a treatment.
It is known from UK Patent application 9026080.3 that benzaldehyde compounds,
previously known as anti cancer agents may be used for combating diseases
resulting from
an abnormally elevated cell proliferation. Such compounds also exert an effect
on cells
having an abnormally elevated cellular proliferation rate, and accordingly the
compounds
may be used for treatment of diseases such as psoriasis, inflammatory
diseases, rheumatic
diseases and other auto immune disorders like Ulcerous colitt and Morbus
Crohn, and
allergic dermatologic reactions.
Dermatologic abnormalities such as psoriasis are often characterised by rapid
turnover of
epidermis. While normal skin produces about 1250 new cells/day/cm2 of skin
consisting of
about 27,000 cells, psoriatic skin produces 35,000 new cells/day/cmz from
52,000 cells.
The cells involved in these diseases are however "normal" cells reproducing
rapidly and
repeatedly by cell division. While the renewal of normal skin cells takes
approximately
311 hours, this process is elevated to take about 10 to 36 hours for psoriatic
skin.
Today psoriasis, inflammatory diseases, rheumatic diseases and other auto
immune
disorders are treated with corticosteorids, NSAIDs and in serious cases, with
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immunosupressive agents like cytostatica and cyclosporins. All these drugs can
give
serious side-effects. It is thus a great need for products giving less side-
effects.
It is known that aromatic aldehydes and certain acetal derivatives thereof
have a
growth-inhibitory effect on human cells which is by its nature reversible.
Growth
inhibition induced by these compounds is primarily due to a reduction in the
protein
synthesis by cells. (Pettersen et al., Eur.J. Clin. Oncol., vol. 19, pp. 935-
940, 1983 and
Cancer Res., vol. 45, pp. 2085-2091, 1985). The inhibition of protein
synthesis is only
effective as long as these agents are present in the cellular
microenvironment. The
synthesis of cellular protein is, for instance, rapidly restored to its normal
level following
removal of the agent from the cells (i.e. within 1 h in most cases).
This leads to the effect that the normal cells are left without damage after
treatment with
the above compounds.
The ability of cells to transmit signals via cell-contact ( adhesion )
dependent mechanisms
has been studies over many years. These mechanisms are especially important
for the
reactivity of circulating cells like lymphocytes, macrophages etc., and also
for instance for
methastatic cells to be anchored in tissues to establish new tumours. The
possibility to
alter the adhesion characteristic of cells which are monitoring the immune or
inflammatory response might be of great therapeutic value for the treatment of
many
diseases like rheumatoid arthritis, psoriasis, psoriatic arthritis, lupus
erythematosis, acne,
Bechterew's arthritis, progressive systemic sclerosis (PSS), seborrhea and
other auto
immunic disorders like Ulcerous colitt and Morbus Crohn.
The immune system is carefully designed to identify and eliminate any material
recognised as non-self, whether it originates from a bacteria-, virus- or
protozoal infection,
or abnormal cells like cancer. In order to provide a specific response to the
huge range of
biotic variation represented by the invaders, the immune system has to be
highly
diversified. However, over-stimulation of this finely tuned system can lead to
various
allergic- and inflammatoric reactions and cause auto-immune diseases. The
rejection of
beneficial transplants is also difficult to overcome. It is therefore a big
therapeutic
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challenge to modulate the immune system, either by up-regulating or down-
regulating a
specific response.
In an immunological recognition process, a fragment of a foreign protein is
confined in the
groove of the class II MHC protein on the surface of an antigen presenting
cell (APC).
Attached to this MHC-antibody complex is also the receptor of a T helper cell.
To
activate a T helper cell, at least two signals must be provided: The primary
signal is
mediated by the antigen itself, via the class II MHC complex and augmented by
CD4
co-receptors. The second signal can be provided by a specific plasma-membrane
bound
signalling molecule on the surface of the APC. A matching co-receptor protein
is located
on the surface of the T-helper cell. Both signals are needed for the T-cells
to be activated.
When activated, they will stimulate their own proliferation by secreting
interleukin growth
factors and synthesising matching cell-surface receptors. The binding of
interleukins to
these receptors then directly stimulates the T-cells to proliferate.
In the 1980'ties, it was recognised that a cyclodextrin benzaldehyde inclusion
complex
could stimulate the immune system by augmentation of the lymphokine activated
killer
cells in a murine model (Y. Kuroki et. al., J. Cancer Res. Clin. Oncol. 117,
(1991), 109 -
114). Studies made in vitro have later revealed the nature of the chemical
reactions at the
APC-donor/T-cell receptor interaction site responsible for the second co-
stimulatory
signal, and that these take form of carbonyl-amino condensations (Schiff s
base
formation). Moreover, these interactions can be mimiced by synthetic chemical
entities.
These findings open up for new therapeutic opportunities for artificially
potentiating the
immune system. In WO 94/07479 use of certain aldehydes and ketons which forms
Schiff s bases and hydrazones with T-cell surface amino groups are claimed. In
EP
0609606 A1 the preferred immuno stimulating substance is
4-(2-formyl-3-hydroxyphenoxymethyl) benzoic acid (Tucaresol), a compound
originally
designed to cure sickle cell anaemia. This substance is administered orally
and is
systemically bioavailable. The potential of Tucaresol in curing a number of
diseases
including bacteria-, virus- and protozoal infections, auto-immune related
illness and
cancer is presently being investigated (H.Chen and J. Rhodes, J.Mol.Med (1996)
74:497-504) and combinational strategies where Tucaresol is administered
together with a
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vaccine to cure chronic hepatitis B, HIV and malignant melanoma are currently
under
development.
By measuring immuno parameters in vitro, and assessing effects in vivo, a bell
shaped
5 dose/response profile was revealed (H.Chen and J. Rhodes, J.Mol.Med (1996)
74:497-504). This otherwise somewhat unusual dose/response relationship can be
justified by assuming that at high concentration of the aldehyde drug will
saturate the
co-stimulatory ligands necessary for the effective binding of APC to the T-
cell and
therefor will be inhibitory. A dose sufficient for achieving a dynamic
equilibrium
10 providing co-stimulation without blocking intercellulary ligating, seems to
be optimal.
In general, aldehydes are intrinsically unstable due to oxidation.
4-(2-Formyl-3-hydroxyphenoxymethyl) benzoic acid (Tucaresol) which is
disclosed in
EP-0609606, is considerably more potent in vivo than in vitro. This may be
because of the
drug's susceptibility to oxidation in aqueous solutions in vitro (H.Chen and
J. Rhodes,
J.Mol.Med (1996) 74:497-504). Many aldehydes are too reactive to be
administered as
such, and benzaldehyde, even proven to be an active anti cancer drug in vitro,
is highly
irritating and unsuitable for direct in vivo application. In a biotic system,
the aldehyde
carbonyl group will react rapidly with nucleofilic entities predominantly
present in all
body fluids. These unwanted by-reactions could lead to fast drug
metabolisation and
difficulty in controlling serum level of the active drug. Controlling the drug
at a cellular
level within a narrow concentration window is crucial for achieving an
effective immune
potentiation. Tucaresol is orally administered as an unprotected aldehyde, and
one might
suspect drug deterioration and difficulties in controlling pharmacokinetics.
The benzaldehyde derivatives 4,6-benzylidene-D-glucose and the deuterated
analogue
(Compound 1 and 2) have proven to possess high bioavailability either
administered i.v. or
per oz. Bioavailability measured as serum level after oral administration of
Compound 2
to BALB mice was 93-99°70 (C.B. Dunsaed, J.M. Dornish and E.O.
Pettersen, Cancer
Chemother. Pharmacol. (1995) 35: 464-470). Moreover, the glucose moiety can
possess
affinity to receptors present at the cell surface, thereby improving drug
availability at a
cellular level. The free aldehyde can easily be released by hydrolysis of the
acetal,
making the carbonyl group available for Schiffs base formation at the target
ligands.
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In the present patent application, the aldehydes are derivatised with
biologically
acceptable carbohydrates like glucose, galactose and others to form acetals.
The sugar
moiety will thus contribute by improving stability and enhancing
bioavailability of the
aldehyde function to the target cells. This surprisingly leads to more
effective carbonyl
condensation reactions and easier controllable pharmacokinetics by using our
compounds
as compared with previously known compounds.
In order to compare Compound 2 with Tucaresol, cell inactivation and protein
synthesis
inhibition were measured in the presence of equal concentrations of the two
drugs. As can
be seen from Fig. 4 and Fig. 5, it was shown that Compound 2 was more
effective than
Tucaresol with respect to both measured parameters.
The immune stimulating effect of the invented compounds may also be used in
the
treatment of certain virus diseases in combination with other anti-viral
therapy like
anti-viral drugs or vaccines. Many virus types, after the first infection,
incorporate with
the cell nucleus and are inactive for a long period of time. Oncogenic viruses
like hepatitis
B and C, certain retro virus and certain papilloma virus may cause development
of cancer.
In these latent period it is very difficult to cure the virus infection. These
viruses can often
be triggered by immune responses to cause viremia, and in this stage make it
possible to
get rid of the virus infection. The ability of the benzaldehyde derivatives to
trigger the
immune response may be used in combination with antivirals or vaccines to
develop a
treatment for these diseases.
It is a main object of the invention to provide new compounds for prophylaxis
and/or
treatment of cancer and disorders related to the immune system.
Another object of the invention is to provide new compounds being able to
potentiate
immune responses giving a possibility to combat infectious diseases caused by
virus,
bacteria, fungus and other micro organisms.
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A third object of the invention is to provide compounds for prophylaxis or
treatment of
cancer and diseases related to immune disorders not giving toxic side-effects.
A fourth object of the invention is to provide compounds for prophylactic
treatment to
prevent the development of liver cancer in persons with Hepatitis B or C
infection.
A fifth object of the invention is to provide compounds for effective and
favourable
prophylaxis and/or treatment of cancer in tissues and cells having receptors
with affinity
to corresponding sugar moieties.
A sixth object of the invention is to provide compounds for treatment of
diseases related to
the immune system like psoriasis, bowel inflammations, arthritis, SLE, PSS
etc.
These and other objects by the invention are achieved by the attached claims.
The compounds of the present invention have the general formula (I):
0
Ar\ /
L >\
O R
(I)
wherein L is H or D;
Ar is phenyl or substituted phenyl with 1-3 substituents, the substituents
which are the
same or different, are selected from the group comprising alkyl with 1-20
carbon atoms,
cycloalkyl with 3-6 carbon atoms, fluoroalkyl with 1-6 carbon atoms, alkenyl
with 2-6
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carbon atoms, alkynyl with 2-6 carbon atoms, phenyl, halogen, nitro, cyano,
NHz, NHR',
N(R')z, NHC(O)R' or N[C(O)R']z wherein R' which is the same or different, is
alkyl with
1-20 carbon atoms, or fluoroalkyl with 1-6 carbon atoms, ORz or OC(O)Rz
wherein Rz is
H, D, alkyl with 1-20 carbon atoms, or fluoroalkyl with 1-6 carbon atoms, SRz,
CA(OR')z
or CA[OC(O)R']z wherein A is H or D, C(O)Rz, COORS wherein R3 is H or alkyl
with
1-20 carbon atoms, or fluoroalkyl with 1-6 carbon atoms or CON(R3) z wherein
R3 is the
same or different;
Y is selected from the atoms or groups comprising H, D, alkyl with 1-20 carbon
atoms,
cycloalkyl with 3-6 carbon atoms, fluoroalkyl with 1-6 carbon atoms, alkenyl
with 2-6
carbon atoms, alkynyl with 2-6 carbon atoms, fluoro, chloro, nitro, ORz,
OC(O)Rz, SRz,
NHz, NHR', N(R' )z wherein R' is the same or different, NHC(O)R' or N[C(O)R']z
wherein R' is the same or different;
R is H, D, alkyl with 1-20 carbon atoms, cycloalkyl with 3-6 carbon atoms,
fluoroalkyl
with 1-6 carbon atoms, alkenyl with 2-6 carbon atoms, alkynyl with 2-6 carbon
atoms, or
a pharmaceutical acceptable salt thereof.
It is understood that any stereoisomer according to formula (I) is comprised
in the present
invention.
Compounds 5, 6, 7, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23
and 24 (see table
on page 18-21 ) are new per se.
Detailed description of the invention
The invention is further explained below by examples and attached figures and
tables.
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Description of the fi ures
Fi.g. 1: The data represent an experiment where NHIK 3025-cells were treated
with
Compound 8 (O) or Compound 9 (~) for 20 hours at 37°C while attached to
plastic Petri
dishes. Surviving fraction means fraction of cells able to form a macroscopic
colony
following treatment. Each point represents the mean value of colony counts
from 5
parallel dishes. The standard errors are smaller than the size of the symbols.
Fig. 2: The data represent an experiment where NHIK 3025-cells were treated
with
Compound 5 (O) or Compound 7 (~) for 20 hours at 37°C while attached to
plastic Petri
dishes. Surviving fraction means fraction of cells able to form a macroscopic
colony
following treatment. Each point represents the mean value of colony counts
from 5
parallel dishes. Vertical bars indicate standard errors and are shown when
exceeding the
symbols.
F~. 3: The data represent an experiment where NHIK 3025-cells were treated
with
Compound 12 (~) for 20 hours at 37°C while attached to plastic Petri
dishes. Surviving
fraction means fraction of cells able to form a macroscopic colony following
treatment.
Each point represents the mean value of colony counts from 5 parallel dishes.
Vertical
bars indicate standard errors and are shown when exceeding the symbols.
Fib. 4~. The data represents an experiment where NHIK 3025-cells were treated
with
Compound 2 (0) or Tucaresol (~) for 20 hours at 37°C while attached to
plastic Petri
dishes. Surviving fraction means fraction of cells able to form a macroscopic
colony
following treatment. Each point represents the mean value of colony counts
from 5
parallell dishes. The standard errors are shown when exceeding the size of the
symbols.
Fig. 5: The rate of protein synthesis relative to untreated control of NHIK
3025-cells
treated with Compound 2 (~) or Tucaresol (1) for 1 hour at 37°C. The
rate of protein
synthesis was measuered by amount of [3H]-valine incorporated during the first
hour after
start of drug treatment. Protein synthesis rate was measured relative to the
total amount of
protein in the cells. Data are representative for one experiment performed in
quadruplicate. Standard errors are indicated when exceeding the size of the
symbols.
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Fig_ 6: Mean tumour growth curves of tumour line SK-OV-3 ovarian carcinoma
xenograft
implanted in nude mice are shown. Mice were treated daily i.v. with 1 mglkg
Compound 8
(1) and 7.5 mg/kg Compound 8 (1). ~, Control group received 0.9% NaCI. Each
data
5 point represents the mean tumour volume of 4 to 5 mice related to the tumour
volume at
day 1. Vertical bars represent standard error.
Fi_.~7-12 show morphologic appearance of SK-OV-3 tomours from each of the
following
3 groups: The placebo-treated group of animals (figures 7 and 8), the group
treated with 1
10 mg/kg/day of Compound 8 (figures 9 and 10) and the group treated with 7.5
mg/kg/day
(figures 11 and 12). Tumours were fixed in formalin, embedded in paraffin,
sliced in 6
mm slices and stained with haematoxylin and eosin. Magnification is 40 times.
15 Fig. 13: Mean spheroide volume growth curves of cell line T-47D breast
carcinoma are
shown. The spheroids were treated with 0.1 mM Compound 8 (1) and 1.0 mM
Compound 8 (1) dissolved in medium. ~ ,Control. Each data point represents the
mean
spheroid volume of 6 to 11 spheroids. Vertical bars represent standard error.
Fib. 14 shows microscopic photographs of sections of 3 differently treated
NHIK 3025
cell spheroids, one untreated control (A), one treated with 0.1 mM Compound 8
for 4 days
(B) and one treated with 1.0 mM Compound 8 for 4 days (C).
Fig. 15-18: The data show the fraction of nuclei within each of the interphase
stages, G1,
S and G2, having the RB-protein bound in the nucleus following treatment with
Compound 8.
Fig. 19-20: Rate of protein synthesis relative to that of control cells for
NHIK 3025 cells
(figure 19) and T-47D-cells (figure 20). Each point represent the mean of
measurements
from 4 parallell samples. Standard errors are indicated by vertical bars when
exceeding the
symbols.
Fig 21: Median adhesion forces for cells exposed to different benzaldehyde
derivatives.
The cells were exposed to a 1 mM concentration of Compounds 1 and 2.
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F~ Peripheral blood mononuclear cells and Superantigen in Ex Vivo 10 medium
were exposed to either benzaldehyde, deuterated benzaldehyde, Compound 2 or
zilascorb(ZH). The proliferation of peripheral blood mononuclear cells was
measured as
incorporation of tritiated thymidine at different drug concentrations.
Fi .~23~. NMRI mice were infected i.p. with spleen invading Friend
erythroleukemia virus.
Infected- and uninfected mice were treated i.p. daily with 5 mg/kg of either
Compound 2
or Compound 5. After treatment for 19 days, spleens were dissected out and
weighted.
F~ The effect of Compound 1, 2 and 5 on the liver invasion of human colorectal
tumour, C170HM2 is shown.
Fib Cell survival as measured by colony-forming ability for human cervix
carcinoma
cells, NHIK 3025, after treatment for 20h with either Compound 1 (O) or
Compound 13
(~) is shown.
Fig. 26: Cell survival as measured by colony-forming ability for human cervix
carcinoma
cells, NHIK 3025, after treatment for 20h with either Compound 1 (O) or
Compound 14
(~) is shown.
Fig-27: Rate of protein synthesis of human cervix carcinoma cells, NHIK 3025,
treated
with Compound 1 or Compound 21 as measured by amount of incorporated [3H]-
valine
during a pulse period of lh starting either immediately following addition of
test
compound (closed symbols) or starting 2h later (open symbols).
Fig. 28: Rate of protein synthesis of human cervix carcinoma cells, NHIK 3025,
treated
with Compound 2 or Compound 22 as measured by amount of incorporated [3H]-
valine
during a pulse period of lh starting either immediately following addition of
test
compound (closed symbols) or starting 2h later (open symbols).
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Fib Cell survival as measured by colony-forming ability for human cervix
carcinoma
cells, NHIK 3025, after treatment for 20h with either Compound 1 (~) or
Compound 21
(O) is shown.
Fig_ 30: Cell survival as measured by colony-forming ability for human cervix
carcinoma
cells, NHIK 3025, after treatment for 20h with either Compound 2 (O) or
Compound 22
(1) is shown.
Fig. 31: Cell survival as measured by colony-forming ability for human breast
carcinoma
cells, T47-D, after treatment for 20h with either L-glucose (~) or Compound 21
(O) is
shown.
Fig. 32: Airway responsiveness 24 hours after exposure to aerosolized
methacholine in
ovalbumin-sensitized mice challenged with saline (open bars and horizontally
striped
bars) or ovalbumin (solid bars and vertically striped bars) and treated with
solvent solution
or Compound 2. Results are expressed as arithmetic average ~SEM (n=9 per
group).
Fib. 33: Number of neutrophil cells recovered 24 hours after the last saline
(open bars) or
ovalbumin (solid bars) challenge in broncho-alveolar fluid of ovalbumin-
sensitized mice,
treated with solvent solution or Compound 2. Results are presented as
arithmetic average
~SEM (n=9 per group).
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Compound Chemical Structure Name
No.
H
O 4 6-O-Benzylidene-
O O D-glucopyranose
'-~~
/ HO
OH OH
D
O 4,6-O-(Benzylidene-d1 )-
O O D-glucopyranose
/ HO-~~
OH OH
Ph
H o
4,6-O-Benzylidene-
3 O D-galactopyranose
HO
O OH
H
O HO p Methyl 4,6-O-benzylidene-alpha-
D-mannopyranoside
OMe
D
4,6-O-(Benzylidene-d1 )-
/ HO 2-deoxy-D-glucopyranose
OH
H
p o 4,6-O-(4-Carbomethoxy-
~ benzylidene)-D-glucopyranose
Ho'-~~
O
/
OH OH
OMe
H
0 4 6-O-Benzylidene-2-deoxy-
7 I ~ o'_~ D-glucopyranose
HO
OH
D
o~_'~ 2-Acetamido-
Ho 4,6-O-(Benzylidene-d1)-
off 2-deoxy-D-glucopyranose
0
CH3
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Compound No. Chemical Structure Name
H
\ ~ 2-Acetamido-2-deoxy-
HO 4 6-O-(3-nitrobenzylidene)-
HN OH D-glucopyranose
NO2
O
CH3
Ph
p"O o 4 6-O-(Benzylidene-d1 )-
D-galactopyranose
o
HO
O OH
D
1 1 4,6-O-(Benzylidene-d1 )
\ o HO O D-mannopyranose
HO~
OH
Ph
H/\ O
0 2-Acetamido-4,6-O-
12 O benzylidene-2-deoxy-
alpha-D-galactopyranose
HO
HN
IOH
~O
CH3
H
13 ~ ~ O ~O 4 6-O-(3-Nitrobenzylidene)-
HO-\l~ D-glucopyranose
Ff ,/
O OH
NOZ
OH H
14 O 4 6-O-(2-Hydroxybenzylidene)-
O D-glucopyranose
/ HC~
O OH
OH H
~ O O 2-Deoxy-4,6-O-
(2-hydroxybenzylidene)-
H D-glucopyranose
OH
OH H
O 2-Acetamido-2-deoxy-
4 6-O-(2-hydroxybenzylidene)-
16
/ HO D-glucopyranose
N OH
Me" O
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Compound No. Chemical Structure Name
\
HO
4,6-O-(2-Hydroxybenzylidene)-
H p ~ D-galactopyranose
O
HO
HO pH
HO
2-Deoxy-4,6-O-
1$ H p (2-hydroxybenzylidene)-
D-galactopyranose
O
HO
OH
\
HO
H o 2-Acetamido-2-deoxy-
19 0 4 6-O-(2-hydroxybenzylidene)-
D-galactopyranose
HO
OH
O
CH3
OH H
O HO O 4 6-O-(2-Hydroxybenzylidene)-
20 ~ O D-mannopyranose
HC~
OH
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Compound no. Chemical structure Name
OH
21 ~ / O O 4,6-O-Benzylidene-
OH L-glucopyranose
H HO
OH
22 ~ / O p 4,6-O-(Benzylidene-d1)-
OH L-glucopyranose
D HO
O
H
H3C O 4,6-O-(2-Acetoxy-
23 \ O benzylidene)-
O O D-glucopyranose
/-~~
HO
OH OH
OH H
HO O 4,6-O-(2,3-Dihydroxy-
24 I ~ O--~~ benzylidene)-
HO D-glucopyranose
OH OH
Preparation
As is well known, aldehydes undergo acid facilitated condensation reactions
with alcohols
to generate acetals. Water is concomitantly formed as a co-product. The
reaction is
reversible, and in solution, an equilibrium mixture of aldehyde/alcohol and
acetal/water is
formed. The position of the equilibrium will mainly be determined by the
reactivity and
concentration of each species. In order to force the reaction towards
completion, one of
the products (acetal or water), is normally removed from the reaction mixture.
In the present patent application, various sugars, deoxysugars and aminosugars
are
condensed with aldehydes or aldehyde equivalents to form suger-acetal
derivatives.
Particularly preferred is a re-acetalisation strategy, where the aldehyde
protected as its
dimethyl acetal is used instead of the aldehyde itself. Methanol is then
formed as
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22
co-product. The reaction mixture is moderately heated at reduced pressure to
remove the
methanol once it is formed. In most cases, these reaction conditions will
drive the
equilibrium smoothly in favour of the acetal.
Acetalisation of sugars will normally lead to mixtures of regio- and stereo
isomers. Ring
contraction transformations may also occur, leading to mixtures of pyranoses
and
furanoses, and, in some cases, di-acetalisation adducts are formed. As a
consequence,
unless protection strategies are applied, very complex reaction mixtures are
often
encountered. However, surprisingly pure product fractions were prepared
following
appropriate work-up, especially by using liquid chromatography. Identification
of the
products were achieved by using GC-MS-spectroscopy and various NMR techniques.
The specific reaction conditions, solvent and catalyst used will in each
individual case
depend on the solubility and reactivity of the reactants and of the properties
of the product.
The catalyst may be a mineral acid, e.g. sulphuric acid, an organic acid, e.g.
para-toluene
sulfonic acid, an acidic ion exchanger resin, e.g. Amberlyst 15, a Lewis acid
mineral clay,
e.g. Montmorillonite K-10 or a resin supported super acid, e.g. Nafion NR 50.
The
reaction may conveniently be carried out in a dipolar, aprotic solvent such as
dimethyl
formamide, dimethyl acetamide, dimethyl sulfoxide, N-methyl pyrrolidone,
dimethoxyetane or the like. Para-toluene sulfonic acid in dimethyl formamide
constituted
the preferred and most applied reaction condition.
The compounds of formula (I) wherein L is deuterium may be prepared as
described
above, but starting with the dimethyl acetal of an aldehyde which is
deuterated in the
formyl position. The preparation of deutero-benzaldehyde may be performed by a
modified Rosenmund reduction using DZ gas in a deuterated solved, as described
in EP 0
283 139 B 1. Deuterated benzaldehyde derivatives with substituents in the
phenyl ring may
be prepared according to examples given in EP 0 493 883 A1 and EP 0 552 880
Al.
The following examples are illustrative of how the compounds of the present
invention
may be prepared.
Compound 1: 4,6-O-Benzylidene-D- lucopyranose
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23
This prior art compound was prepared as described for compound 2, starting
with
undeuterated benzaldehyde dimethylacetal. The identity was confirmed by 'H NMR
spectroscopy in DMSO-db:
8 rel. to TMS: 7.58-7.29 (5H, m, Ar-H), 6.83 (0.4H, d, OH-1-(3), 6.60 (0.6H,
d, OH-1-oc),
5.61 (1H, s+s, acetal-H), 5.25 (0.4H, d, OH-3-[3), 5.21 (0.4H, d, OH-2-(3),
5.62 (0.6H, d,
OH-3-a), 5.00 (0.6H, H-1-a), 4.82 (0.6H, d, OH-2-a), 4.49 (0.4H, t, H-1-(3),
4.18-4.02
( 1 H, m, H-6'-a+(3), 3.89-3.77 (0.6H, m, H-5-cc), 3.75-3.57 ( 1.6H, m, H-6"-
oc+~3 and
H-3-cc), 3.45-3.27 (2.5H, m, H-3-~3, H-4-a+(3, H-5-(3 and H-2-a) and 3.11-3.00
(0.4H, m,
H-2-(3).
Compound 2: 4,6-O-(Benzylidene-d,)-D- lg-ucop rah
Benzaldehyde-d, was prepared and converted to benzaldehyde dimethylacetal-d,
as
described in EP 0 283 139 B 1. The preparation of
4,6-O-(benzylidene-d,)-D-glucopyranose is also described in EP 0 283 139 B1,
but the
compound was this time prepared according to an alternative procedure with
priority
given to achieving high purity:
D(+)-Glucose (706 g, 3.92 mol), benzaldehyde dimethylacetal-d, (571 g, 3.73
mol), dry
DMF ( 1.68 kg) and para-toluene sulfonic acid (4.5 g, 24 mmol) were mixed in a
dry
distillation apparatus connected to a vacuum pump through a cold reflux
condenser. The
mechanical stirred mixture was warmed to max. 69°C at 30 Torr to distil
off methanol and
after 2 hours, 235 g was collected. The reflux condenser was then shut off and
the
temperature increased to max 73°C in order to distil off DMF. After 2
more hours, an
additional amount of 1385 g was collected and the distillation interrupted.
The residue was cooled to approx. 40°C and icelwater (2.9 L) added
within 5 min. The
temperature dropped below 0°C and a precipitate was formed, partly as
big lumps. The
mixture was transferred to a beaker and additional 8-9 L ice/water added in
order to make
the lumps fell apart and form a suspension. The suspension was filtered on two
notches
and the two filter cakes left over night on the filters with water jet vacuum
connected, each
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filter cake being flushed with NZ via an inverted funnel. The filter cakes
were spread on
two boards and dried at 32°C for 20 hours in a vacuum oven. The vacuum
was first set at
13 mbar, then regulated down to 1 mbar.
The crude product was recrystallised (in order to remove di-benzylidene
acetals) and
water-washed (to remove DMF and glucose) until these contaminants were
eliminated.
Accordingly, the crude product (500 g) was dissolved in hot dioxane (800 ml)
and the
solution added via a folded filter to boiling chloroform (9 L). The solution
was allowed to
cool, first to ambient temperature, then in an ice bath overnight. The
precipitate was
filtered off, dried for 2 hours on the filter (flushing with NZ as described
previously) and
dried further overnight at 31°C in vacuo on a rotavapor. The product
(142 g) was
suspended in ice/water ( 1 L), filtered on a nutch (washing with 200 ml
ice/water) and
dried on the filter overnight as described previously. It was then grounded,
sieved (0.5
mm grid size) and dried in vacuo for 5 hours at 31°C on a rotavapor.
The product (96 g)
once again was suspended in ice/water (500 ml), filtered (washing with 150 ml
ice/water)
and dried (7 hours under Nz flush). It was finally grounded on a mortar,
sieved (0.5 mm)
and dried in a vacuum oven.
The product was a white, finely divided powder of high purity, as analysed on
HPLC. The
yield was 95 g, 10°Io of the theoretical. NMR in DMSO-d6 indicated an a
to (3 anomeric
ratio of approx. 7:3.
1H- and'3C NMR (DMSO-db), 8 rel. to TMS: 7.55-7.28 (S.OOH, m, Ar-H), 6.85
(0.27H, d,
OH-1-(3), 6.58 (0.71H, d, OH-1-a), 5.24 (0.27H, d, OH-3-a), 5.19 (0.28H, d, OH-
2-~3),
5.61 (0.71H, d, OH-3-a), 4.99 (0.72H, H-1-a), 4.82 (0.71H, d, OH-2-a), 4.48
(0.29H, t,
H-1-(3), 4.20-4.04 (1.04H, m, H-6'-a+(3), 3.88-3.73 (0.78H, m, H-5-a), 3.73-
3.56 (1.72H,
m, H-6"-a+(3 and H-3-a), 3.46-3.21 (2.61H, m, H-3-b, H-4-a+(3, H-5-(3 and H-2-
a) and
3.09-2.99 (0.28H, m, H-2-(3); 137.881, 128.854, 128.042, 126.435 (Ar-C),
100.462
(acetal-C), 97.642 (C-1-(3), 93.211 (C-1-a), 81.729- (C-4-a), 80.897 (C-4-(3),
75.796
(C-2-(3), 72.906 (C-2-a and C-3-(3), 69.701 (C-3-a), 68.431 (C-6-a), 68.055-
(C-6-(3),
65.810 (_C-5-(3) and 62.032 (C-5-a).
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Compound 3: 4,6-O-Benzylidene-D-galactopyranose
D(+) Galactose ( 15.0 g, 0.083 mol) and dry DMF (80 ml) were mixed with
stirnng at 50°C
5 in a distillation apparatus. To the suspension thus formed, benzaldehyde
dimethylacetal
( 12.2 g, 0.083 mol) and para-toluene sulfonic acid (0.14 g) were added and
methanol/DMF slowly distilled off with a water jet. After 3 hours, most of the
galactose
was consumed and the remaining DMF removed on a rotavapor connected to a
vacuum
pump. The residue, which formed a very viscous syrup, was purified on a Lobar
C RP-8
10 column eluting with methanol/water l:l. The product fractions were freeze
dried.
GC of the TMS derivatives showed the product to consist mainly of two isomers.
On the
basis of'H-,'3C-, COSY-, DEPT- and C-H correlation NMR spectra, the product
was
identified as the cc and (3 anomers of the title compound.
'H- and'3C NMR (D20), 8 rel. to TMS: 7.49-7.27 (5H, m, Ar-~, 5.57 (1H, s,
acetal-H),
5.22 (0.5H, d, H-1-a), 4.56 (0.5H, d, H-1-(3), 4.23+4.18 (0.5H+0.5H, d+d, H-4-
a+~),
4.14-3.98 (2H, m, H-6-a+~), 3.94-3.79 ( 1.5H, m, H-2-a, H-3-a and H-5-a), 3.69-
3.49
(1.5H, m, H-2-(3, H-3-(3 and H-5-(3); 137.422, 129.981, 128.902, 126.639 and
126.590
(Ar-C), 101.325 (acetal-C), 96.540 (C-1-(3), 93.161 (C-1-oc), 76.581 (C-4-a),
76.093
(C-4-(3), 71.889 + 71.802 (C-2-(3+C-3-(3), 69.404 (C-6-a), 69.182 (C-6-(3),
68.566 +
68.057 (C-2-a + C-3-(3), 66.759 (C-5-(3) and 62.886 ~-5-cc).
Compound 4: Methyl 4,6-O-Benzylidene-oc-D-mannopyranoside
Methyl-oc-D-mannopyranoside (18.1 g, 0.093 mol), benzaldehyde dimethylacetal
(21.0 g,
0.138 mol) and dry DMF (90 ml) were mixed with stirnng at 50-55°C in a
distillation
apparatus. Para-toluene sulfonic acid (ca. 0.1 g) was added and 10 min.
thereafter a water
jet was connected to distil off methanol. After 4 hours, the reaction mixture
was
evaporated to form a white solid. The residue was washed with dibutyl ether,
filtered and
the filter cake dissolved in acetonitrile. A precipitation started and the
mixture left in a
refrigerator for 5 days. Thereafter, the precipitation was filtered off and
the filtrate
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evaporated. The residue was purified on a Lobar C RP-8 column, eluting with 30
%
acetonitrile in water. Product fractions from 4 separate runs were freeze
dried and
combined.
GC analysis of the TMS derivatives indicated the product to consist of 95 area
%
monoacetals. The monoacetals in turn consisted of 4 peaks integrating for 0.4,
3.2, 94.1
and 2.4 area %, respectively. On the basis of'H-,'3C-, COSY-, DEPT- and C-H
correlation NMR spectra, together with GC/MS spectroscopy, the dominating
species was
identified as the title compound.
'H- and'3C NMR (aceton-db), 8 rel. to TMS: 7.54-7.30 (SH, m, Ar-H), 5.60 (1H,
s,
acetal-~, 4.71 (1H, s, H-1), 4.34 (1H, broad s, O~, 4.22-4.02 (2H, m+broad s,
H-6'+O~, 3.94-3.82 (3H, m, H-2, H-3 and H-4), 3.80-3-60 (2H, m, H-5+H-6") and
3.39
(3H, s, CH ); 139.264, 129.396, 128.662 and 127.211 (Ar-~, 102.825 (C-1),
102.468
(acetal-~, 79.888 ~-4), 72.090 (-C-3), 69.308 (-C-6), 69.127 (C-2), 64.363 (_C-
5) and
54.921 (CH3).
Compound 5: 4,6-O-(Benzylidene-d,)-2-deox -~lucopyranose
Benzaldehyde-d, was prepared and converted to benzaldehyde dimethylacetal-d,
as
described in EP 0 283 139 B 1.
2-Deoxy-D-glucose ( 10 g, 60.9 mmol), dry DMF (35 ml), benzaldehyde
dimethylacetal-d,
(11.7 g, 76.4 mmol) andpara-toluene sulfonic acid (70 mg, 0.37 mmol) were
mixed under
NZ to give a white slurry. By warming to 45-50°C, a colourless solution
was formed
within 1/2 hour. A vacuum pump was connected to remove methanol through a
cooled
column (to prevent loss of benzaldehyde dimethylacetal-d,). The pressure was
regulated
stepwise from 70 mbar down to 20-30 mbar during 4.5 hours and the temp. was
maintained at 40-45°C. Thereafter the distillation was interrupted, the
apparatus rebuilt
without the column and DMF removed by short path distillation at 50-
55°C, max. vacuum.
The residue was a slightly yellow syrup.
1/4 of the syrup was dissolved in slightly alkaline (NaHC03) methanol/water
60/40 and
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purified on a Merck LiChroprep RP-8 reversed phase column eluting with
methanol/water
60/40. Product fractions were concentrated to remove methanol and freeze dried
to give a
white, fluffy solid. Products from four separate runs were combined to give
3.5 g, 23 % of
the theoretical yield.
GC analysis of the TMS-derivatives and NMR spectroscopy proved the product to
consists of a 1:1 mixture of the a and (3 anomers.
'H- and'3C NMR (DMSO-db), 8 (ppm) rel. to TMS: 7.52-7.28 (m, 5H, Ar-H I+II),
6.9-6.65 (broad s, 1/2 H, OH-1 II), 6.55-6.32 (broad s, 1/2 H, OH-1 I), 5.25-
5.12 (m, 1 H,
OH-3 II and H-1 I), 5.12-5.0 (d, 1/2 H, OH-3 II), 4.84-4.73 (dd, 1/2 H, H-1
II), 4.20-4.02
(m, 1H, H-6 I+II), 3.98-3.73 (m, 1H, H-3 I and H-5 I), 3.73-3.58 (m, 1.5H, H-
6' I+II and
H-3 II), 3.42-3.18 (2.5 H, H-4 I+II and H-5 II and H O), 2.10-1.86 (m, 1H, H-2
I+II) and
1.62-1.34 (m, 1H, H-2' I+II); 137.979, 137.926, 128.841, 128.036 and 126.432
(Ar-C
I+II), 101.5-100.0 (acetal-C I+II), 94.057 and 91.424 (C-1 I+II), 83.916 and
83.093 (C-4
I+II), 68.374 and 68.119 (C-6I+II), 66.889, 66.092, 64.174 and 62.604 (C-3
I+II and C-5
I+II) and 41.932 and 40.051 ~-2 I+II).
Compound 6: 4,6-O- (4-Carbomethoxybenz lid)-D-~luco~yranose
Methyl 4-formylbenzoate ( 100 g, 0.609 mol), methanol (91.5 g, 2.86 mol),
trimethyl
ortoformate (71 g, 0.67 mol) and conc. hydrochloric acid ( 165 ~l) were mixed
in a 500 ml
three-necked flask. The slurry transformed into a slightly yellow solution
within few
minutes and the temp. spontaneously increased from 15°C to 30°C.
After stirring for 15
min., the reaction mixture was refluxed at 58°C for another 25 min. and
then cooled to
10°C (ice/water). An alkaline solution was made by dissolving KOH (8.3
g) in metanol
(53 ml) and 7 ml of the solution added to the reaction mixture. After stirring
at 10°C for
25 min., the reactor was re-built for short path distillation and all
volatiles removed in
vacuo (water jet). The distillation was thereafter continued with a vacuum
pump and a
colourless oil collected at 112-114°C/0.5 mbar. The oil transformed
into a colourless
solid, m.p. 32-33°C, and was identified by NMR to be methyl 4-
formylbenzoate
dimethylacetal. The yield was 108.75 g, 85 % of the theoretical.
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28
D(+)-glucose (8.0 g, 44.4 mmol), dry DMF (25 ml), methyl-4-formylbenzoate
dimethylacetal ( 10.4 g, 49.5 mmol) and para-toluene sulfonic acid were mixed
at 50°C
under NZ to give a white suspension. The apparatus was connected to a vacuum
pump
through a vertical condenser and evaporation of methanol started at 80-100
mbar, 55°C.
The vacuum was gradually lowered to 40 mbar, maintaining the temp. at 55-
60°C. The
reaction mixture gradually clarified and finally became transparent. After 8
hours the
distillation was interrupted and the apparatus rebuilt for short path
distillation of DMF.
The residue was a slightly yellowish syrup.
The syrup was dissolved in a warm solution of 100 mg NaHCOs in 20 ml methanol
and 8
ml water and precipitated by adding 100 ml ethylacetate. The precipitate was
isolated
from the mother liquor by filtration, washed with cold water (4 x 15-20 ml)
and
transferred to a rotavapor flask. Humidity was removed by adding ethylacetate
and
evaporating twice. The product was finally dried under high vacuum. More
precipitate
was filtered off from the mother liquor, washed and dried to give a second
crop. The two
crops were combined to give 1.94 g pure product, 13 % of the theoretical
yield.
GC analysis of the TMS-derivatives showed two isomers in the ratio 2:1.
'H- and '3C NMR (DMSO-db), 8 (ppm) rel. to TMS: 7.99 and 7.61 (dd, 2+2H,
furfuryl-~, 6.87 (d, 0.67H OH-1 II), 6.59 (d, 0.28H, OH-1 I), 5.68 (s+s, 1 H,
acetal-H
I+II), 5.29 (d, 0.68H, OH-3 II), 5.21 (d, 0.67H, OH-2 II), 5.16 (d, 0.31 H, OH-
3 I), 5.00 (t,
0.30H, H-1 I), 4.85 (d, 0.28H, OH-2 I), 4.48 (t, 0.73H, H-1 II), 4.25-4.08 (m,
1.14H, H-6),
3.95-3.77 (m, 3.43H, OCH3 and H-5 I), 3.78-3.59 (m, 1.40H, H-3 I and H-6'),
3.49-3.23
(m, 3.59H, H-4 I and II, H-5 II, H-2 I and H-3 II), 3.10-2.98 (m, 0.72H, H-2
II); 165.978,
142.553, 142.553, 129.959,129.067, 126.763, 99.982, 99.812, 97.661, 93.238,
81.794,
81.794, 80.963, 75.808, 72.902, 69.658, 68.487. 68.110, 65.714, 61.952 and
52.253.
Compound 7: 4,6-O-Benzylidene-2-deoxy-D- lug-co~yranose
12-Deoxy-D-glucose ( 10.0 g, 60.9 mmol), dry DMF (34 ml), benzaldehyde
dimethylacetal
( 11.6 g, 76.2 mmol) and para-toluene sulfonic acid (70 mg, 0.37 mmol) were
mixed under
NZ to give a white slurry. The reaction mixture was stirred at room temp. for
30 min. and
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by warming to 45-50°C, the solid gradually dissolved. A vacuum pump was
connected to
remove methanol through a cooled column (to prevent loss of benzaldehyde
dimethylacetal) and the reaction continued for 4.5 hours. Thereafter the
distillation was
interrupted, the column removed and DMF distilled off through a short path at
50-55°C,
max. vacuum. The residue was .a slightly yellow syrup.
The syrup was dissolved in slightly alkaline (NaHC03) methanol/water 60/40 and
purified
on a Merck LiChroprep RP-8 reversed phase column, eluting with methanol/water
60/40.
Product fractions were concentrated to remove methanol and freeze dried to
give a white,
fluffy solid. Products from four separate runs were combined to give 3.18 g,
21 % of the
theoretical yield.
GC analysis of the TMS-derivatives and NMR spectroscopy proved the product to
consists of a l:l mixture of the a and (3 anomers.
'H NMR (DMSO-db), 8 (ppm) rel. to TMS: 7.52-7.30 (m, 5H, Ar-H I+II), 6.85-6.68
(broad s, 1/2 H, OH-1 II), 6.50-6.35 (broad s, 1/2 H, OH-1 I), 5.61 (s+s, 1H,
acetal-H
I+II), 5.23-5.12 (m, 1 H, OH-3 II and H-1 I), 5.12-5.02 (d, 1/2 H, OH-3 II),
4.84-4.74 (dd,
1/2 H, H-1 II), 4.20-4.04 (m, 1H, H-6 I+II), 3.98-3.74 (m, 1H, H-3 I and H-5
I), 3.74-3.57
(m, 1.5H, H-6' I+II and H-3 II), 3.42-3.18 (2.5 H, H-4 I+II and H-5 II and H
O), 2.08-1.88
(m, 1H, H-2 I+II) and 1.62-1.32 (m, 1H, H-2' I+II).
Compound 8: 2-Acetamido-4,6-O-benzylidene-d,-2-deox~ lg uco~yranose
Benzaldehyde-d, was prepared and converted to benzaldehyde dimethylacetal-d,
as
described in EP 0 283 139 B 1.
Benzaldehyde dimethylacetal-d, (8.7 g, 56.8 mmol), N-acetyl-D-glucosamine
(10.0 g,
45.2 mmol), dry DMF (30 ml) and para-toluene sulfonic acid (88 mg, 0.46 mmol)
were
mixed under Nz to give a white suspension. The reaction mixture was stirred at
50°C for
45 min, then a vacuum pump was connected through a vertical condenser and the
reaction
continued for 2 hours at 55°C/60-70 mbar. The apparatus was rebuilt to
remove DMF
through a short path and the distillation continued at 55-60°C, max.
vacuum for 1 more
hour. The residue was a yellow-white, soft solid.
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A solution was made by mixing NaHC03 ( 150 mg) in 30 ml methanol/water (3:2)
and the
residue neutralised by adding the solution. The creamy slurry thus formed was
filtered,
washing 2-3 times with a 1 % NaHCOs solution and several times with ether. The
product
5 was analysed to be sufficiently pure (GC) and dried in vacuo. The yield was
8.8 g, 63 %
of the theoretical.
GC analysis of the TMS derivatives indicated a 1:1 isomeric mixture. NMR
spectroscopy
in DMSO-d~ solution identified the product as a 3:1 anomeric mixture.
'H- and'3C NMR (DMSO-db), 8 rel. to TMS: 7.83 (s+s, 1H, NH) 7.51-7.28 (m, 6H,
Ar-H), 7.0-6.2 (broad s, 1H, OH-1), 5.65-5.05 (broad s, 1H, OH-3), 4.99 (d,
1H, H-1 I),
4.61 (d, 0.3H, H-1 II), 4.21-4.03, 3.92-3.67 and 3.51-3.22 (m, 6H, H-2, H-3, H-
4, H-5 and
H-6) and 1.85 (s+s, 3H, CH3); 169.452 (C=O), 137.818, 128.869, 128.035 and
126.438
(Ar-~, 100.505 (acetal-C), 96.056, 91.500, 82.471, 81.505, 70.549, 68.300,
67.961,
67.218, 65.906, 62.123, 58.038, 54.790 (sugar-~ and 23.123 and 22.674 (CH3).
Compound 9: 2-Acetamido-2-deoxy-4,6-O-(3-nitrobenzylidene)-D- Ig ucopyranose
3-Nitrobenzaldehyde ( 100 g, 0.66 mol), methanol (99 g, 3.1 mol), trimethyl
ortoformate
(77.3 g, 0.73 mol) and conc. hydrochloric acid ( 165 pl) were mixed in a 500
ml
three-necked flask to form a yellow slurry which transformed into a solution
within 5 min.
The reaction mixture was refluxed at ~ 50°C for 15 min. and then cooled
with ice/water to
10 °C. KOH (2.5 g) was dissolved in methanol (16 ml), and the reaction
mixture
quenched by adding 6.6 ml of this solution. Stirring was continued for 15 min
and the
reactor re-built for short path vacuum distillation. Volatile material (CH30H
+
HCOOCH3) was distilled off in a water jet, the distillation interrupted and a
vacuum pump
connected. The distillation was then continued and a yellow oil distilled off
at 93 - 97.5
°C/20 mbar. The oil was identified by NMR to be 3-nitrobenzaldehyde
dimethylacetal of
high purity. The yield was 127 g, 97.6 % of the theoretical.
3-Nitrobenzaldehyde dimethylacetal (5.5 g, 0.028 mol), N-acetyl-D-glucosamine
(5.0 g,
0.023 mol), para-toluene sulfonic acid (50 mg, 0.263 mmol) and dry DMF ( 15
ml) were
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mixed with stirring at 50°C to form a slightly yellow suspension. After
1/2 hour, a
vacuum pump was connected and the reaction continued at 56°C/50 mbar
for 11 hours.
The reaction mixture was evaporated and the residue partitioned between a
small volume
of slightly alkaline (NaHC03) water and chloroform. The water phase (forming a
cheese-like suspension) was re-extracted twice with chloroform and filtered,
washing
several times with water and ether. The product was dried in vacuo to form a
slightly
brownish powder. The yield was 570 mg, 7 % of the theoretical.
GC analysis of the TMS derivatives indicated the product to consist of two
isomers in a
2:1 ratio. NMR spectroscopy identified the product as a mixture of oc- and (3
anomers.
'H- and'3C NMR (DMSO-d6), 8 rel. to TMS: 8.4-8.1 (m, 2H, Ar-H), 8.05-7.79 (m,
2H,
Ar-~, 7.79-7.60 (t, 1H, N~, 6.80 (d, 1H, OH-1), 5.81 (s+s, 1H, acetal 1~, 5.30
and 5.18
(s+s, 1/2 H + 1/2 H, H-1), 4.30-4.10, 3.93-3.3 (m, 6H, H-2 - H-6) and 1.85 (d,
3H, CH3);
169.331 (C=O), 147.490, 139.544, 133.018, 129.862, 123.732, 120.884 (Ar-C),
99.000,
98.806 (acetal-C), 95.924, 91.406, 82.433, 81.454, 70.320, 68.240, 67.907,
67.020,
65.574, 61.833, 57.875, 54.609 (sugar-~, 23.014 and 22.557 (CH3).
Compound 10: 4,6-O-(Benzylidene-d,)-D- a,~pyranose
Benzaldehyde-d, was prepared and converted to benzaldehyde dimethylacetal-d,
as
described in EP 0 283 139 B 1.
D(+)-Galactose15.0 g,0.0833 mol) and dry DMF (80 ml) were stirred in a
distillation
apparatus at 45°C. Benzaldehyde dimethylacetal-d, ( 12.8 g, 0.0836 mol)
and para-toluene
sulfonic acid (0.14 g) were added and methanol and DMF slowly distilled off in
vacuo
(water jet). After 3 hours, a vacuum pump was connected and the remaining DMF
distilled off. Portions of the residue was dissolved in methanol/water (1:1)
containing
NaHC03 (11 mg/ml) and purified on a Lobar C RP-8 column, eluting with
methanol/water
( 1:1 ). Product fractions from 7 individual runs were freeze dried and
combined to form a
white, fluffy product. The yield was 6.62 g, 30 % of the theoretical.
GC- and NMR analysis showed the product to consist of a l: l anomeric ratio.
'H- and'3C
NMR (DMSO-d6), 8 rel. to TMS: 7.52-7.30 (m, SH, Ar-H), 6.62 (O.SH, d, OH-1-
(3), 6.32
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(O.SH, d, OH-1-a), 5.05 (O.SH, t, H-1-a), 4.85+4.69+4.49 (1H+O.SH+O.SH, m+d+d,
OH-2+OH-3), 4.35 (O.SH, t, H-1-(3), 4.12-3.92+3.81-3.71+3.69-3.59
+3.49-3.39+3.39-3.28 (3H+1H+O.SH+1H+2H m+m+m+m+m, H-2-H-6+H20); 138.753,
138.690, 128.607, 128.319, 127.913 and 126.3 (Ar-C), 99.345 (acetal-C), 97.307
and
93.178 (C-1), 76.738 and 76.158 (C-4), 72.101, 71.605, 68.947, 68.862 , 68.486
and
67.730 (C-2, C-3 and C-6) and 65.829 and 62.068 (C-5).
Compound 11: 4 6-O-(Benzylidene-d,)-D-mannopyranose
Benzaldehyde-d, was prepared and converted to benzaldehyde dimethylacetal-d,
as
described in EP 0 283 139 B 1.
D(+)-Mannose (15.0 g, 0.0833 mol) and dry DMF (70 ml) were stirred in a
distillation
apparatus at 40°C. Benzylidene dimethylacetal-d, and para-toluene
sulfonic acid (0.14 g)
were added to form a clear solution. A vacuum pump was connected and methanol
and
DMF slowly distilled off at 70-20 mbar, 45-50°C. After 3 hours, the
remaining DMF was
distilled of at max. vacuum, leaving a slightly yellowish syrup.
The residue was washed repeatedly with ether in order to remove lipophilic
species.
Portions of crude product were dissolved in slightly alkaline (NaHC03)
methanol/water
(3:2) and purified on a Lobar C RP-8 column, eluting with methanol/water
(3:2). GC of
the TMS derivatives indicated the product to consist of 4 isomers in the ratio
10:3:1:4. It
was re-eluted with methanol/water (1:4) to obtain a white, fluffy product
consisting of just
2 isomers in the ratio 70/30, as analysed by GC. The yield was 1.42 g, 6.4 %
of the
theoretical.
On the basis of 'H-, '3C-, COSY-, DEPT- and C-H correlation NMR, the chemical
structure was confirmed and the a- and (3 anomers found to equilibrate at a
1:8 ratio.
'H- and'3C NMR (DMSO-d6) of predominating isomer, 8 rel. to TMS: 7.5-7.28 (m,
SH,
Ar-H), 6.56 (d, 1 H, OH-1 ), 5.0-4.85 (m, 3H, H-1, OH-2 and OH-3 ), 4.10-4.02
(m, 1 H,
H-6), 3.63-3.40 (m, SH, H-2, H-3, H-4, H-5 and H-6'); 138.045, 128.825,
128.029,
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126.438 (Ar-C), 100.802 (acetal-C), 95.233 (C-1), 78.987 (C-4), 72.032 (C-3),
68.317
(C-6), 67.252 (C-2), 63.495 (C-5).
Compound 12: 2-Acetamido-4 6-O-benzylidene-2-deox~cc-D-~alactop ranose
Benzaldehyde dimethylacetal (2.0 ml, 14 mmol) followed by para-toluene
sulfonic acid
monohydrate ( 15 mg) were added to a stirred suspension of N-acetyl-D-
galactosamine
( 1.50 g, 6.77 mmol) in acetonitrile (37 ml). The reaction mixture was then
lowered into a
warm (60°C) oil bath and stirred under nitrogen for 3 h, during which
time a thick white
precipitate was seen to form. The reaction mixture was then filtered and the
solid washed
with cold dichloromethane (approx. 2 ml) followed by continued suction
filtration under a
stream of nitrogen. The white powder was then placed in a pre-weighed glass
vial and left
under vacuum (0.06 mbar) over 72 h to give the pure desired product as only
the a-isomer
(1.74 g, 83 %).
'H NMR 8H(300 MHz; d6-DMSO) 1.83 (3H, s, CH3), 3.80-4.17 (6H, m, H-2, H-3, H-
4,
H-5 and H-6), 4.65 ( 1 H, d, OH-3 ), 5.06 ( 1 H, t, H-1 ), 5.59 ( 1 H, s,
ArCH), 6.52 ( 1 H, d,
OH-1 ), 7.33-7.55 (5H, m, ArH) and 7.69 ( 1H, d, NH); '3C NMR 8~ {'H } (75
MHz;
D6-DMSO) 23 (CH3), 50, 62, 65, 69 and 76 (C-2, C-3, C-4 C-5 C-6), 91 (C-1),
100
(ArCH), 126, 128, 128 and 138 Carom. C) and 170 (C=O).
Compound 13: 4,6-O-(3-Nitrobenzylidene)-D- lg ucopyranose
3-Nitrobenzaldehyde dimethylacetal was prepared as described for Compound 9.
3-Nitrobenzaldehyde dimethylacetal (21.9 g, 0.11 mol), D(+)-glucose ( 16.0 g,
0.09 mol),
para-toluene sulfonic acid (100 mg, 0.5 mmol) and dry DMF (50 ml) were mixed
under
Nz and stirred at 58°C for 25 min. A vacuum pump was connected and
methanol and
DMF slowly distilled off via a cooled column at 55-60°C, 30-40 mbar for
4 h, 15 min.
The apparatus was rebuilt to remove most of the DMF through a short path and
the
distillation continued for 1.5 h. The residue was a slightly yellow syrup.
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The syrup was dissolved in slightly alkaline (NaHC03) methanol/water 60:40 and
purified
on a Lobar C RP-8 column, eluting with methanol/water 60:40. Product fractions
were
evaporated (to remove methanol), freeze-dried and combined to give 5 g of a
white, fluffy
solid. The product was re-purified eluting with methanol/water 40:60 to give
the title
compound of sufficient purity. The yield was 3.3 g, 12 % of the theoretical.
GC indicated
the product to consist of two isomers in a 70:30 ratio.
'H NMR (DMSO-d6), 8 rel. to TMS: 8.33-7.63 (SH, m, Ar-H), 6.89+6.60 (1H, d+d,
OH-1-I+II), 5.78 (1H, s+s, acetal-H- I+II), 5.34 (0.65H, d, OH-3-II),
5.75+5.71 (1.12H,
d+d, OH-2-II + OH-3-I), 4.99 (0.56H, m, H-1-I), 4.88 (0.32H, OH-2-I), 4.49
(0.74H, m,
H-1-II), 4.28-4.12 (1H, m, H-6'-I+II), 3.85-3.53 (2.27H, m, H-3-I, H-5-I and H-
6"-I+II),
3.49-3.32 (2.58H, m, H-2-I, H-3-II, H-4-I+II and H-5-II) and 3.12-2-98 (0.85H,
m H-2-II).
Compound 14: 4,6-O-(2-H dy rox~ylidene-D- lg ucopyranose
2-Hydroxybenzaldehyde (16.0 g, 0.13 mol), D-glucose (23.6 g, 0.13 mole) and
para-toluene sulfonic acid (catalytic amount) were mixed in DMF ( 100 ml). The
mixture
was heated to about 60 °C for 0.5 h to give a solution. The reaction
was monitored by
TLC analysis (silica gel, ethyl acetate). After 20 h at 20 °C the
mixture was heated twice
to 60 °C for 1 h and then evaporated at 60 °C at reduced
pressure to remove most of the
DMF. Ethyl acetate (approx. 100 ml) was added to give a precipitate. The
solution was
decanted off, analysed by TLC and evaporated at reduced pressure to give an
oil. The oil
was dissolved in ethyl acetate, silica gel (120 g, 200-500 Vim) added and the
solvent
evaporated. About half of the product was chromatographed on silica gel (550
ml, 30-60
Vim) with ethyl acetate as eluent. Fractions of 100 ml were collected and
fr.l5 to 25 were
evaporated at reduced pressure to give an oil (3.4 g), impured by substantial
amounts of
DMF. The product, only slightly soluble in chloroform, was precipitated by
addition of
about 20 ml of the same solvent. Washing with chloroform and drying gave a
solid (1.86
g). The rest of the product was chromatographed and precipitated accordingly
to give 2.43
g and further 1.6 g was recovered from the mother liquors by precipitation.
The total yield
was 5.85 g, 16 % of the theoretical.
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According to NMR and TLC analyses the product was impured by a few percent of
2-hydroxybenzaldehyde and glucose. Chromatography, as above, gave a product
essentially free from impurities. NMR spectroscopy showed the product to
consist of a
mixture of a and ~3 anomers. In DMSO-d6, the anomeric ratio was initially a/(3
= 2:1, but
5 changed over time. Also GC spectroscopy of the silylated derivatives
displayed two peaks
in a 2:1 ratio.
'H- and'3C NMR (DMSO-d6), 8 rel. to TMS: 9.50 (s, 1H, Ar-OH), 7.30 (m, 1H, Ar-
H-6),
7.09 (m, 1H, Ar-H-4) 6.80-6.68 (m, 2.34H, Ar-H-3 + Ar-H-5) + OH-1-(3), 6.48
(d, 0.67H,
10 OH-1-a), 5.71 (s, acetal-H-a+(3), 5.10 (t, 0.65H, OH-2-(3 + OH-3-(3), 4.97
(d, 0.63H,
OH-3-a), 4.90 (t, 0.66H, H-1-a), 4.71 (d, 0.65H, OH-2-a), 4.39 (t, 0.39H, H-1-
a),
4.10-3.93 (m, 1.13H, H-6'-a+(3), 3.80-3.67 (m, 0.71H, H-5-a), 3.60-3.45 (m,
1.73H,
H-3-a and H-6"-a+(3), 3.35-3.14 (m, 3.31H, H-4-a+/3, H-2-a, H-3-(3, H-5-(3 and
H O),
3.00-2.95 (m, 0.42, H-2-(3); 154.72, 154.69, 130.11, 127.85, 127.77, 124.44,
124.38,
15 118.97 and 115.69 (Ar-~, 97.95 (C-1-(3), 96.95 and 96.87 (acetal-C), 93.50
(C-1-a),
82.35 (C-4-a), 81.52 (C-4-~3), 76.04 (C-2-(3), 73.32 (C-3-~3), 73.19 (C-2-a),
70.04 (C-3-a),
68.98 (C-6-a), 68.61 (C-6-~3), 66.24 (C-5-(3) and 62.46 (C-5-a).
Compound 15: 2-Deoxy-4 6-O-(2-h d~~ylidene)-D- l~pyranose
A catalytic amount of para-toluene sulfonic acid was added to 2-
hydroxybenzaldehyde
(10.7 ml, 0.100 mol) and trimethyl ortoformate (11.0 ml, 0.100 mol). After 60
min.,
2-deoxy-D-glucose (16.4 g, 0.100 mol) and DMF (300 ml) were added and the
mixture
heated briefly to about 60°C. TLC analysis indicated the presence of
product after 10
minutes and a small amount of pyridine was added after 25 h. A sample was
withdrawn
and cromatographed (ethyl acetate/methanol 9:1) to give impure fractions.
Fractions with
fairly pure product were collected after chromatography of a small amount with
heptane/ethyl acetate (1:4). The rest of the reaction mixture was evaporated
under
vacuum, the residue dissolved in ethyl acetate, silica gel (200-500 pm) added
and the
solvent evaporated in vacuo. Chromatography (heptane/ethyl acetate 1:4)
yielded ca. 2 g
product with moderate purity.
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The modest yield may be suspected as a result of unnecessary long reaction
time and the
preparation was repeated, with addition of pyridine after 2.5 h. Work-up and
chromatography as above gave 2.4 g of impured product. The collected products
were
mixed and re-chromatographed (heptane/ethyl acetate 1:4) to give a white
solid. The yield
was 4.1 g, 7 % of the theoretical. NMR analysis showed a clean spectrum of the
expected
product, but also signals from a few percent of impurities (signals around 1
ppm; possibly
impurities introduced via solvents). Attempts to purify the product by
repeated
chromatography resulted in substantial loss of material and merely 1.2 g was
finally
isolated. NMR spectroscopy indicated the a:(3 ratio to be approximately 1:1.
'H- and'3C NMR (DMSO-db), 8 rel. to TMS: 9.56 (s, 1H, Ar-OH), 7.41-7.29 (m,
1H,
Ar-~, 7.18-7.06 (m, 1H, Ar-H), 6.86-6.70 (m, 2.54H, Ar-H + OH-1-~3), 6.40 (d,
0.50H,
OH-1-a), 5.83 and 5.80 (s + s, 1H, acetal-~, 5.17 (t, 0.51H, H-1-a), 5.11 (d,
0.46H,
OH-3-(3), 5.03 (d, 0.50H, OH-3-a), 4.79 (t, 0.46H, H-1-~3), 4.14-3.95 (m,
1.25H, H-6-a+(3)
+ EtOAc), 3.91-3.72 (m, 1.11H, H-3-a + H-5-a), 3.72-3.57 (m, 1.48H, H-3-(3 +
H-6'-a+(3), 3.36-3.16 (m, 1.39H, H-4-a+(3, H-5-~ + H20), 2.05-1.86 (m, 0.93H,
H-2-a+~
+ EtOAc) 1,63-1.47 (m, 0.51H, H-2'-a) and 1.47-1.32 (m, 0.48H, H-2'-(3);
154.71 and
154.66 (Ar-C-OH), 130.09, 127.88, 127.78, 124.54, 124.48, 118.96, 118.93,
115.67
(Ar-~, 97.06 and 97.00 (acetal-C), 94.36 (C-1-(3), 91.71 (C-1-a), 84.52 and
83.70 ~-4),
68.92 and 68.68 (C-6), 67.24 (C-3-~3), 66.52 (C-5-(3), 64.50 (C-3-a), 63.03 (C-
5-a) and
42.29 (C-2).
Compound 16: 2-Acetamido-2-deoxy-4 6-O-(2-h d~~ylidene)-D- lucop rah
To 2-hydroxybenzaldehyde ( 10.7 ml, 0.100 mol) and trimethyl ortoformate (
11.0 ml,
0.100 mol) was added a catalytic amount of para-toluene sulfonic acid. The
temperature
spontaneously raised to ca. 60 °C and the mixture was set aside for ca.
2 h. before addition
of N-acetyl glucosamine (20.3 g, 0.092 mol) and DMF ( 150 ml). The reaction
mixture
was stirred for 30 min. at 20 °C and then briefly heated to 50
°C. Most of the N-acetyl
glucosamine was dissolved and TLC-analysis indicated substantial conversion to
anticipated product. The reaction mixture was again briefly heated to ca. 50
°C and
volatile components evaporated at 20 °C/15 mmHg. The slightly turbid
raction mixture
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was kept at 20°C for 4 days. A small amount of pyridine was added and
most of the
solvents evaporated at 60 °C/15 mmHg to give an oil. The oil was added
to ethyl acetate
(350 ml), giving a small amount of precipitate, and the decanted solution was
evaporated
together with silica gel (200 g, 200-500 Vim).
A minor part of the product was chromatographed on silica (550 ml, 30-60 pm),
eluting
with ethyl acetate and collecting 100 ml fractions. After fraction 58, the
eluent was
changed to ethyl acetate/methanol 9:1 and the product collected within the
next 20
fractions. Evaporation of the product fractions gave 3.3 g of a solid. The
rest of the
product was chromatographed on silica gel with ethyl acetate/methanol 9:1 to
give further
21 g of solid product. DMF was removed by stirring the finely divided product
with ethyl
acetate (200 ml) for 2 h. Filtration, washing with ethyl acetate and drying
gave 16.7 g
pure product, 56 % of the theoretical yield. GC analysis of the silylated
derivatives
showed a 5:1 preponderance of one of the isomers. NMR analysis in DMSO-db
showed
the (3 isomer to exist in 4-5 fold excess over the oc isomer.
'H- and'3C NMR in (DMSO-db), 8 rel. to TMS: 9.59 (s, 1H, Ar-OH), 7.80 (d, 1H,
NH),
7.38 (t, 1H, Ar-~, 7.17 (t, 1H, Ar-H), 6.86-6.72 (m, 3H, Ar-H and OH-1-a+(3),
5.82 (s+s,
1H, acetal-~, 5.17 (d, 0.22H, OH-3-(3), 5.10-4.98 (m, 1.54H, OH-3-a and H-1-
a), 4.61 (t,
0.21H, H-1-(3), 4.17-4.10 (m, 0.24H, H-6'-(3), 4.10-4.03 (m, 0.78H, H-6'-a),
3.90-3.80 (m,
0.80H, H-5-a), 3.79-3.61 (m, 2.53H, H-6"-oc+(3, H-3-a, and H-2-a), 3.61-3.53
(m, 0.26H,
H-3-(3) and 3.46-3.28 (m, 3.58H, H-2-(3, H-4-oc+(3, H-5-(3 and H O); 169.81,
169.77
(_C=O), 154.70, 130.15, 127.86, 127.75, 124.41, 124.36, 118.97 and 115.70 (Ar-
~, 97.00
and 96.86 (acetal-C), 96.34 (C-1-~3), 91.81 (C-1-a), 83.08 and 82.12 (C-4),
70.92 and
67.58 (C-3), 68.86 and 68.52 (C-6), 66.34 and 62.58 (C-5), 58.33 and 55.08- (C-
2), 23.46
and 23.00 (CH3).
Compound 17: 4,6-O-(2-Hydroxybenzylidene)-D- ag lacto~ rah
A catalytic amount of para-toluene sulfonic acid was added to 2-
hydroxybenzaldehyde
( 10.7 ml, 0.100 mol) and trimethyl ortoformate ( 11.0 ml, 0.100 mol). After
stirring for 60
min., D-galactose ( 18.0 g, 0.100 mol) and DMF (300 ml) were added and the
reaction
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mixture heated briefly to about 60 °C. The reaction mixture was
homogeneous within a
few min. and the presence of product evident from TLC analysis. A small amount
of
pyridine was added after 20 h., most of the solvent removed, and the residue
applied on
silica gel as described previously. Column chromatography (ethyl
acetate/methanol 9:1 )
gave high yield of the desired product (ca. 17 g), impured only by DMF.
Repeated
chromatography gave 4.9 g pure product, together with ca. 10 g impured by a
few percent
DMF. NMR spectroscopy showed the oc to (3 ratio to be 77:23. Also GC analysis
of the
silylated derivatives showed two peaks in a similar ratio.
'H- and'3C NMR (DMSO-db), 8 rel. to TMS: 9.28 (s, 1H, Ar-OH), 7.43-7.34 (m,
1H,
Ar-H~, 7.19-7.13 (m, 1H, Ar-H), 6.85-6.76 (m, 2H, Ar-H), 6.63 (d, 0.23H, OH-1-
(3), 6.30
(d, 0.76H, OH-1-oc), 5.75 (s, 1H, acetal-~, 5.06 (t, 0.76H, H-1-cc), 4.84 (d,
0.23H,
OH-2-~3), 4.79 (d, 0.22H, OH-3-(3), 4.60 (d, 0.76, OH-3-a), 4.54 (d, 0.78, OH-
2-a), 4.34
(t, 0.23H, H-1-(3), 4.13-3.87 (m, 3H, H-4-a+~3 and H-6-a+(3), 3.79-3.70 (m,
1.55H, H-3-a
and H-5-cc), 3.66-3.59 (m, 0.78H, H-2-a), 3.45-3.36 (m, 0.49H, H-3-(3 and H-5-
(3) and
3.36-3.27 (m, 0.95H, H-2-(3 and H O); 154.76, 154.70, 129.96, 128.08, 128.03,
125.18,
125.06, 118.95, 118.88 and 115.69 (Ar-C), 97.57 (C-1-(3), 96.68 and 96.49
(acetal-C),
93.49 (C-1-cc), 77.19 and 76.62 (C-4), 72.36 and 71.88 (C-2-(3 and C-3-(3),
69.30 and
69.19 (C-6), 68.79 and 67.99 (C-2-oc and C-3-a), 66.10 and 62.33 (C-5).
Compound 18: 2-Deoxy-4 6-O-(2-h~ybenzylidene)-D- al~actopyranose
A catalytic amount of para-toluene sulfonic acid was added to 2-
hydroxybenzaldehyde
(0.65 ml, 6.1 mmol) and trimethyl ortoformate (0.63 ml, 6.1 mmol). After 1 h.,
2-deoxy-D-galactose ( 1 g, 0.61 mmol) and DMF (25 ml) were added and the
mixture
heated briefly to about 60 °C to give a homogeneous solution. TLC
analysis indicated
formation of product within 10 min. Pyridine was added after 2.5 h. and work-
up
performed as above. Chromatography with ethyl acetate gave poor separation and
the
eluent was changed to ethyl acetate/methanol 95:5 to give 98 mg (6 %) of
fairly pure
product. NMR spectroscopy indicated the presence of two isomers in a 3:2
ratio.
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'H NMR (DMSO-d6), 8 rel. to TMS: 9.26 (s, 1H, Ar-OH), 7.48-7.37 (m, 1H, Ar-H),
7.23-7.10 (m, 1H, Ar-~, 6.86-6.73 (m, 2H, Ar-~, 6.62 (d, 0.31H, OH-1-(3), 6.21
(d,
0.48H, OH-1-oc), 5.80 (s, 1H, acetal-H), 5.31 (s, 0.49H, H-1-a), 4.78 (d,
0.31H, OH-3-(3),
4.67 (d, 0.94H, OH-3-a + H-1-a), 4.07-3.79 (m, 3.49H, H-4-a+a, H-6-a+(3 + H-3-
a),
3.70 (m, 0.95H, H-5-oc + H-3-(3), 3.33 (m, H-5-(3 + H20), 1.89-1.77 (m, 0.53H,
H-2-cc),
1.77-1.62 (m, 0.90H, H-2-(3 + H-2'-(3) and 1.72-1.51 ( m, 0.53H, H-2'-cc);
154.79 and
154.76 (Ar-C-OH), 129.96, 128.12, 125.24, 125.15, 118.94, 118.88 and 115.69
(Ar-C),
96.62 and 96.45 (acetal-~, 94.21 (C-1-(3), 91.66 (C-1-a), 75.80 and 74.71 (C-
4), 69.86
and 69.61 (C-6), 67.47 (C-3-(3), 66.35 (-C-5'=(3), 63.43 (C-3-oc), 62,38 (C-5-
oc) and 37.09
and 34.23 (C-2).
Compound 19: 2-Acetamido-2-deoxy-4 6-O-(2-h~ybenzylidene)-D- app raise
A small amount of para-toluene sulfonic acid was added to 2-
hydroxybenzaldehyde (0.48
ml, 4.5 mmol) and trimethyl ortoformate (0.47 ml, 4.5 mmol). After 60 min., N-
acetyl
galactosamine ( 1.0 g, 4.5 mmol) and DMF (25 ml) were added and the reaction
mixture
heated briefly to 50 °C to give a homogeneous solution. The presence of
product was
indicated by TLC analysis after 15 min. A small amount of pyridine was added
after 2.5
h. and most of the DMF evaporated at reduced pressure. The product was applied
on
silica gel as described previosly and chromatographed with ethyl
acetate/methanol 9:1 to
give 444 mg (30 %) of the title compound. NMR- spectroscopy showed the
compound to
excist predominently as one isomer, probably the a isomer.
'H- and'3C NMR (DMSO-db), 8 rel. to TMS (predominant isomer): 9.34 (s, 1H, Ar-
OH),
7.63 (d, 1H, N~, 7.47-7.39 (m, 1H, Ar-~, 7.21-7.14 (m, 1H, Ar-~, 6.87-6.79 (m,
2H,
Ar-~, 6.52 (d, 0.96H, OH-1), 5.80 (s, 1H, acetal-~, 5.08 (t, 0.97H, H-1), 4.58
(d, 1H,
OH-3), 4.12 (d, 1H, H-4), 4.08-3.98 (m, 2H, H-2 + H-6), 3.98-3.89 (m, 1H, H-
6'),
3.89-3.79 (m, 1H, H-3), 3.78 (s, 1H, H-5) and 1.83 (s, 3H, CH ); 169.92 (C=O),
154.69
(Ar-C-OH), 130.00, 128.05, 125.15, 118.98 and 115.68 (Ar-C), 96.40 (acetal-C),
91.72
(_C-1), 76.45 (C-4), 69.37 (C-6), 65.75 (C-3), 62.27 (C-5), 50.57 (-C-2) and
23.09- (CH3).
Compound 20: 4,6-O-(2-Hydroxybenzylidene)-D-mannopyranose
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A catalytic amount of para-toluene sulfonic acid was added to 2-
hydroxybenzaldehyde
(10.7 ml, 0.100 mol) and trimethyl ortoformate (11.0 ml, 0.100 mol). After 60
min.
D-mannose (18 g, 0.100 mol) and DMF (300 ml) were added and the reaction
mixture
5 heated briefly to about 60 °C. The reaction mixture was almost
homogeneous after 20
min. and TLC analysis indicated the presence of product. After 2.5 h., a small
amount of
pyridine was added and most of the DMF evaporated at reduced pressure. The
residue
was dissolved in ethyl acetate (a small amount of methanol was added to get a
homogeneous system), silica gel (200-500 pm) added and the solvents evaporated
in
10 vacuo. A small amount of the crude product was chromatographed (ethyl
acetate/methanol 9:1) and the identity of the product confirmed by NMR
analysis. The
rest of the crude mixture was chromatographed to give 7.7 g product, impured
by large
amounts of DMF. Attempts to remove DMF by stirring the product with chloroform
followed by filtration gave 7.1 g of a solid product containing ca. 20 mol %
DMF. The
15 product was stirred for 20 h. with ca. 300 ml ethyl acetate and filtered to
give 1.7 g of
slightly reddish crystals, essentially free from DMF. Chromatography gave a
DMF-free
but slightly discoloured product. The filtrate was evaporated and the residue
purified by
chromatography to give additional 3.5 g of the product. The total isolated
yield was 4.7 g,
16 % of the theoretical. NMR- spectroscopy showed the compound to excist
20 predominently as the oc isomer (oc:(3 ~ 85:15).
'H- and 13C NMR (DMSO-d6), 8 rel. to TMS (predominant isomer): 9.51 (s, 1H, Ar-
OH),
7.42-7.29 (m, 1H, Ar-H), 7.22-7.11 (m, 1H, Ar-H), 6.83-6.72 (m, 2H, Ar-~, 6.52
(d,
0.73H, OH-1), 5.79 (s, 0.72, acetal-~, 4.96-4.79 (m, 2.88H, H-1, OH-2 + OH-3),
25 4.04-3.98 (m, 0.58H, H-6)3.83-3.68 (m, 2.52H, H-3, H-4 + H-5) and 3.68-3.54
(m, 2.84H,
H-2 + H-6'); 154.65 (Ar-C-OH), 130.07, 127.84, 124.56, 118.92 and 115.69 (Ar-
~, 97.29
(acetal-C), 95.51 (C-1), 79.55 (C-4), 72.38 (C-2), 68.88- (C-6), 67.53 (C-3)
and 63.87
(C-5).
30 Compound 21: 4 6-O-Benzylidene-L-glucopyranose
L(-)-Glucose (5.0 g, 27.8 mmol), benzaldehyde dimethylacetal (4.66 g, 30.6
mmol) and
para-toluene sulfonic acid (32 mg, 0.17 mmol) were mixed in dry DMF (20 ml) in
a
distillation apparatus. A water pump was connected through a short path to
remove
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methanol and DMF in vacuo. The colourless suspension dissolved within 1/2 h at
55 °C
and the resulting solution stirred at 120 mbar for 1/2 h while gradually
increasing the
temperature to 65 °C. The vacuum was increased to maximum and the
reaction mixture
evaporated further for 45 min. The temp. increased to 75 °C at the end
of the destillation.
The residue was a slightly yellowish sirup, which was neutralised by adding
NaHCO~ (29
mg) and allowed to cool.
The crude product was dissolved in methanol ( 10 ml) and purified on a
reversed phase
RP-8 column, eluting with methanol/water 1:1. Product fractions were combined
and
evaporated to remove methanol. The residual solution was further diluted with
water and
freeze dried. White, fluffy solid from three separate runs were collected to
yield a total of
2.42 g, 32.5 % of the theoretical.
GC chromatography of silylated samples indicated the product to consist of two
isomers
(a and ~3 anomers) in a 35/65 ratio. The'H NMR shifts in DMSO-db were similar
to those
of 4,6-O-benzylidene-D-glucopyranose: 7.51-7.30 (5H, m, Ar-~, 6.86 (0.6H,
broad s,
OH-1-(3), 6.58 (0.3H, broad s, OH-1-a), 5.58 (0.9H, s+s, acetal-H-a+(3), 5.23
(0.7H, d,
OH-3-[3), 5.20 (0.6H, d, OH-2-~3), 5.11 (0.4H, d, OH-3-a), 5.00 (0.4H, H-1-a),
4.82 (0.3H,
d, OH-2-a), 4.47 (0.7H, d, H-1-(3), 4.21-4.08 (1H, m, H-6'-a+~3), 3.87-3.73
(0.4H, m,
H-5-a), 3.73-3.59 ( 1.3H, m, H-6"-a+(3 and H-3-a), 3.46-3.22 (3.7H, m, H-3-(3,
H-4-a+(3,
H-5-(3 and H-2-a) and 3.09-2.99 (0.6H, m, H-2-(3).
Compound 22: 4,6-O-(Benzylidene-d, ~-L- lg ucopyranose
L-Glucose (5.14 g, 28.6 mmol) was warmed in DMF (20 ml) to 95 °C until
a clear
solution was formed. The reaction flask was then transferred to a water bath
at 65 °C and
para-toluene sulfonic acid (33 mg, 0.17 mmol) was added. Benzylidene dimethyl
acetal-d, (4.7 ml, 31 mmol) was then added dropwise by syringe over 20 minutes
to the
stirred glucose solution under a regulated water pump vacuum of 80 mbar. The
DMF was
then evaporated under vacuum (2 mbar) at 65 °C to give a very pale
yellow oil to which
was added NaHC03 (345 mg) followed by stirring for 5 minutes. Warm water (67
°C, 15
ml) was added with stirring (magnetic bead) to the oil at 65 °C and
then the flask was
shaken in the warm water bath until the oil appeared to have dissolved. The
reaction flask
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was then placed under a stream of cold water for approx. 5 minutes. After only
one or two
minutes an amorphous mass formed. The aqueous mixture was placed in an ice-
water
bath and left to stand for 40 minutes. A white precipitate formed during this
time which
was isolated by vacuum filtration (decanted from the amorphous material),
washed with
cold spring water (25 ml) followed by cold iso-propanol (5 °C, 2 x 5
ml) and dried under a
stream of nitrogen to give 1.85 g of a dry white powder. This product was
silylated and
analysed by gas chromatography and appeared to be 99°Io pure desired
product.
IH NMR, 8 (DMSO-db) rel. to TMS: 7.55-7.25 (m, SH, Ar-H), 6.85 (s, 0.48 H, OH-
1-(3),
6.55 (s, 0.33H, OH-1-a), 5.25 (d, 0.48 H, OH-3-(3), 5.20 (d, 0.49 H, OH-2-(3),
5.10 (d,
0.35 H, OH-3-a), 4.98 (d, 0.35 H, H-1-a), 4.82 (d, 0.34 H, OH-2-a), 4.48 (d,
0.51 H,
H-1-~3), 4.20-4.05 (m+m, 0.53 H +0.42 H, H-6' a,+(3), 3.85-3.73 (m, 0.44 H, H-
5-oc),
3.72-3.57 (m, 1.27 H, H-6"-oc+(3 and H-3-oc), 3.45-3.20 (m, 7.8 H, H-3-(3, H-4-
cc+(3, H-5-~
and H-2-cc) and 3.10-2.98 (m, 0.56 H, H-2-(3).
Compound 23: 4,6-O-(2-Acetoxybenz~lidene)-D- lg ucop, rah
2-Acetoxybenzaldehyde is prepared, either by acetylation of 2-
hydroxybenzaldehyde or
by reduction of 2-acetoxybenzoyl chloride.
A catalytic amount of para-toluene sulfonic acid is added to an equmolar
mixture of
2-acetoxybenzaldehyde and trimethyl ortoformate. After stirring for 1 h., an
equal molar
amount of D-glucose and DMF are added and the mixture heated to about 60
°C. The
conversion is followed by TLC chromatography and when an equilibrium is
reached, the
reaction is quenched by the addition of a small amount of pyridine. Most of
the DMF is
evaporated at reduced pressure and the residue applied on silica as described
previously.
This is purified by chromatography, the product fractions isolated and
evaporated and the
compound analysed by NMR spectroscopy.
Compound 24: 4,6-O-(2 3-Dih d~ benzylidene)-D- lucopyranose
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The title compound is prepared and purified as described previously, starting
from
2,3-dihydroxybenzaldehyde. The identity is confirmed by NMR spectroscopy.
Biological Experiments
Example 1
Biological materials and methods used to demonstrate the effect.
Cell Culturing Techniques
Human cells, NHIK 3025, originating form a cervical carcinoma in situ
(Nordbye, K., and
Oftebro, R. Exp. Cell Res., 58: 458, 1969, Oftebro R., and Nordbye K., Exp.
Cell Res., 58:
459-460, 1969) were cultivated in Eagel's Minimal Essential Medium (MEM)
supplemented with 15% foetal calf serum (Gibco BRL Ltd). Human breast
carcinoma
cells, T-47D, (Keydar, I. et al., Eur. J. Cancer, vol 15, pp. 659-670, 1979)
were cultivated
in medium RPMI-1640 supplemented with 10% foetal calf serum, 0.2 u/ml insulin,
292
mg/ml L-glutamine, 50 u/ml penicillin, 50 mg/ml streptomycin. The cells are
routinely
grown as monolayers at 37°C in tissue culture flasks. In order to
maintain cells in
continuos exponential growth, the cells were trypsinised and recultured three
times a
week.
Cell Survival
Cell survival was measured as the colony forming ability. Before seeding, the
exponentially growing cells were trypsinised, suspended as single cells and
seeded
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directly into 5 cm plastic dishes. The number of seeded cells was adjusted
such that the
number of surviving cells would be approximately 150 per dish. After about 2 h
incubation at 37°C, the cells had attached to the bottom of the dishes.
Drug treatment was
then started by replacing the medium with medium having the desired drug
concentration.
Following drug treatment the cells were rinsed once with warm (37°C)
Hank's balanced
salt solution before fresh medium was added. After 10 to 12 days at
37°C in a
C02-incubator, the cells were fixed in ethanol and stained with methylene blue
before the
colonies were counted.
Fig. 1-3 show cell surviving fraction for NHIK 3025-cells treated for 20 hours
with either
Compound 8 and 9 (Fig. 1), Compound 5 and 7 (Fig. 2) or Compound 12 (Fig. 3).
The
data indicate that all compounds induce cell inactivation in a drug dose range
similar or
better to that of zilascorb(ZH) (Pettersen et al., Anticancer Res. vol. 11,
(1991), pp.
1077-1082).
It can be seen from Fig. 4 that Compound 2 incuces greater cell inactivation
than
Tucaresol.
Example 2
Protein Synthesis
The rate of protein synthesis was calculated as described previously (Ronning,
O. W. et
al., J. Cell Physiol., 107: 47-57, 1981). Briefly, cellular protein was
labelled to satuarion
during a minimum 2 day preincubation with ['4C]valine of constant specific
radioactivity
(0.5 Ci/mol). In order to keep the specific radioactivity at a constant level,
a high
concetration of valine ( 1.0 mM) was used in the medium. At this concetration
of valine,
the dilution of ['4C]valine by intracellular valine and proteolytically
generated valine will
be negligible (Ronning, O. W., et al., Exp. Cell Res. 123: 63-72, 1979). The
rate of protein
synthesis was calculated from the incorporation of [3H]valine related to the
total
['4C]radioactivity in protein at the beginning of the respective measurement
periods and
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expressed as percentage per hour (Ronning, O. W. et al., J. Cell Physiol.,
107: 47-57,
1981).
It can be seen from Fig. 5 that Compound 2 induces greater protein synthesis
inhibition
5 than Tucaresol.
Example 3
10 Experiments on human xeno~rafts in nude mice
Drugs were tested in the treatment of three human cancer xenografts implanted
into
female, athymic mice. The cell lines used are SK-OV-3 ovarian carcinoma, A-549
lung
carcinoma and Caco-2 colorectal carcinoma. They were purchased from the
American
15 Type Culture Collection and cultivated shortly in vitro before being
implanted into nude
mice. The tumour lines were passaged as s.c. implants in nude mice. Mice which
should
be used in experiments were 8-9 weeks of age at the time of tumour
implantation. Small
tumour pieces were implanted s.c. on the left flank of the animals. Animals
with growing
tumours (tumour volumes 25-110 mm3) were randomly assigned to drug-treated or
20 control groups, with the average tumour size among the groups being
approximately
equal. The antitumour activity was measured by tumour volume growth curves and
histological evaluation of some tumours. During treatment tumours were
measured 2 times
a week by measuring two perpendicular diameters using calipers. Tumour volume
was
estimated by the formula: volume = (length x width2)/2. The tumour volume
growth
25 curves were generated by standardising the tumour sizes in the different
groups by
obtaining relative tumour volume (RV) calculated by the formula RV = Vx/V 1,
where Vx
is the tumour volume at day x and V 1 is the initial volume at the start of
the treatment (day
1 ), and plotting the mean volume with standard errors for each treatment
group as a
function of time. An exponential curve was fitted to the relative tumour
volume growth
30 data and the interval during which the tumour volume in each group
increased to twice its
volume, tumour volume doubling time (TD), was determined from the fitted curve
(loge2/k, where k is the estimated rate constant for the process.)
Histological evaluation
was based on macroscopic examination of the tumour and light microscopic
examination
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of small tumour sections (6-8 mm thick) embedded in paraffin and stained with
hematoxylin and eosin.
In the tablel tumour volume doubling time (TD) in human tumour xenografts
grown in
nude mice, treated daily i.v. with drugs and doses as indicated, are shown.
15 Table 1:
Type of Drug Dose TD SE Treatment
tumour (mg/kg days
da )
A549 control 13 1 37
Com . 90 16 1 52
2
Com . 90 17 1 52
5
Com . 1 21 1 42
8 *
Caco-2 control 27 1 81
Com . 20 33 1 81
5 *
SK-OV-3 control 20 1 67
Com . 1 28 1 67
8 *
Com . 7,5 31 1 56
8 *
Com .10 1 36 2* 67
Com .10 7,5 17 1 56
SK-OV-3 control 25 1 67
Com . 5 28 1 67
5
*significant difference between treated group and control group, p<0.05.
In Fig. 6 mean tumour growth curves of the tumour line SK-OV-3 ovarian
carcinoma
xenograft implanted in nude mice, where the mice were treated daily with 1
mg/kg and 7.5
mg/kg of Compound 8 are shown. The curves show a significant growth inhibitory
effect
for both doses.
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Fig. 7-12 show microscopic photographs of tumours from each group. These
photographs
indicate a general finding of this compound, namely that there is a difference
with respect
to tumour cell necrosis between control tumours and those treated with
Compound 8.
Since the treated tumours are necrotized by the treatment the drug effects are
in reality
even stronger than that shown by the growth curves.
Example 4
Multicellular spheroids and activation of retinoblastoma~rotein (pRB)
Spheroids were initiated by transferring suspended single cells to a 25 cm2
tissue culture
flask containing 12 ml of medium. The flask was then placed on a tilting board
(MIXER
440, Swelab Instrument) inside a walk-in incubator room at 37 °C.
Tilting rate was
adjusted to 10 tilts per 18 s. During tilting the suspended cells were
prevented from
attaching to the bottom of the flask. Instead many cells were able to attach
to each other
forming small aggregates of cells containing typically 50-100 cells each after
about 24 h
tilting. The small aggregates were then transferred to another 25 crnz tissue
culture flask.
In this case the bottom of the flask was on beforehand covered (i.e. coated)
with a thin
layer of 1.3% sterilised agar (Bacto-Agar, Difco Laboratories, USA). The cell
aggregates
sedimented on top of the agar layer and were unable to attach to this. The
cells within the
aggregates, however, attached to each other, started cell division. After 1
week the
aggregates had doubled their volume several times and had become rounded like
spheroids. During this period medium was changed 3 times per week and the
spheroids
were transferred to new agar-coated flasks once each week.
When spheroids had reached a size of about 400 pm in diameter (after 2 to 3
weeks
cultivation) they were transferred to small micro wells, with 1 spheroid per
well together
with 1 ml medium. It was made sure that all selected spheroids were of about
the same
size. The wells were also coated with agar in order to avoid attachment of
spheroids to the
bottom of the wells. The various wells were supplemented with new medium
containing
the test substance in the chosen concentration and thereafter the diameter of
each
individual spheroid was measured once each day. This was done by microscopy,
using
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phase contrast optics and a grating of known line separation in one of the
oculars (distance
between two neighbouring lines. In each group there were 8-12 parallel
spheroids.
Relative spheroid volume (volume at day n divided by volume at day 1 ) was
calculated for
each individual spheroid each day and growth cures were plotted with mean
relative
volume for all spheroids in a group as a function of time after start of
treatment.
In the table 2 T-47D spheroid volume doubling time (TD) treated for 259 hours
with
Compound 8 at doses indicated, are shown. Table 2 shows volume doubling times
of
spheroids of T-47D-cells either untreated (dose = OmM) or treated continuously
with
0.1 or 1.0 mM Compound 8 in the medium. Compound 8 is shown to increase the
spheroid doubling times (i.e. inhibiting spheroid growth) in a dose-dependent
manner,
since the effect is clearly stronger with 1.0 mM than with 0.1 mM of the drug.
Table 2:
Dose TD SE
0 752
0.1 mM 853
l.OmM 987*
* significant difference between treated group and control group p<0.05.
In Fig. 13 mean spheroid volume growth curves of cell line T-47D breast
carcinoma
where the spheroids were treated with 0.1 mM and 1.0 mM Compound 8 dissolved
in
medium, are shown.
With NHIK 3025-cell spheroids the increase with time of spheroid volume was
not found
to be reduced in the same manner as was found with T-47D cell spheroids of.
Instead the
spheroids treated with Compound 8 disintegrated after relatively short time
treatment (7 to
10 days). In order to elucidate the reason for this strong effect we performed
an
experiment treating spheroids of NHIK 3025-cells for only 4 days and then
preparing
histological sections of the spheroids. The results of this experiment is
shown in Fig. 14.
Fig. 14 shows microscopic photographs of sections of 3 differently treated
NHIK 3025
cell spheroids, one untreated control (A), one treated with 0.1 mM Compound 8
for 4 days
(B) and one treated with 1.0 mM Compound 8 for 4 days (C). Spheroids were
fixed in 4%
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formaldehyde and embedded in paraffin before 6mm thick sections were made and
stained
with haematoxylin and eosin.
Both spheroids treated with Compound 8 show considerable central areas where
the cells
have an appearance indicating that they are in apoptosis. Apoptotic figures
were not found
in any of the control spheroids, but was widespread in the Compound 8-treated
spheroids.
In both types of cells treated with Compound 8 there is a clear drug effect
although
different in the one cell type as compared to the other. In spheroids of T-47D
cells there is
a reduced volume growth in drug-treated spheroids which is drug-dose
dependent. In
spheroids of NHIK 3025 cells there is no reduction in spheroid volume growth
due to the
drug treatment, rather the volume increases faster in treated spheroids as
compared to
control spheroids over a period of 9 days. However, in these spheroid there is
a substantial
fraction of cells undergoing apoptosis in the drug-treated spheroids, so that
debris from
dead cells in this case constitutes much of the spheroid volume. The rapid
increase in
volume for these spheroids is probably due to changes in osmotic pressure
following lysis
of cell fragments. Due to the swelling these spheroids became unstable and
disintegrated
after about 9 days.
The reason for the difference in response between the two cell types can not
be stated with
certainty. There is, however, an important genetical difference between the
two cell types
with respect to regulation of cell growth and -proliferation which may be part
of the
reason for the difference. T-47D cells express functional pRB, the
retinoblastoma protein,
which is a normal tumour suppressor gene that is important in regulating cell-
cycle
progression in normal cells. This gene is often defect in cancer cells, and
NHIK 3025 cells
are among those with a defect pRB-function. We have observed that pRB may be
activated to arrest cells under conditions of stress even when cells have
entered the
S-phase of the cell cycle, indicating that this gene may protect cells against
the
inactivating effects of a stress in combination with DNA-synthesis (see
Amellem,
Sandvik, Stokke and Pettersen, British Journal of Cancer 77 (1998) 862-872).
In the
present study the benzaldehyde derivative acts as a growth-inhibitory stress
influence.
Thereby it is possible that The T-47D-cells are protected by their functional
pRB, and
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therefore do not induce apoptosis whereas NHIK 3025-cells having a defect pRB-
function
are unable to avoid apoptotic death.
In order to test activation of pRB we used two different cell types which both
have a
5 normal expression of pRB, T-47D- and MCF-7-cells. The nucleii-bound pRB-
protein was
measured by means of flow cytometry. Coincident measurement of DNA was also
performed and data were presented as two-parametric DNA versus pRB-histograms.
Fixation and staining methods were performed as described by Amellem, Sandvik,
Stokke
and Pettersen, British Journal of Cancer 77 ( 1998) 862-872. Briefly,
detergent-extracted
10 cells were prepared by resuspending cells in 1.5 ml of low-salt detergent
buffer. The
extracted nucleii were fixed in 4% paraformaldehyde for lh before pRB was
bound with
the PMG3-245 monoclonal antibody (Pharmingen) which recognise both the under-
and
hyperphosphorylated forms of the protein. The pRB antibody was
streptavidin-FITC-stained and DNA was stained with Hoechst 33258. The nucleii
were
15 measured in a FACStartp'°S flow cytometer (Becton-Dickinson)
equipped with two argon
lasers (Spectra Physics) tuned to 488 nm and UV respectively.
The data of Fig. 15-18 show the fraction of nuclei within each of the
interphase stages,
G1, S and G2, having the RB-protein bound in the nucleus following treatment
with
20 Compound 8. When bound this way pRB is considered to regulate cells out of
the cell
cycle, i.e. to take over cell-cycle control. (see Amellem, ~., Stokke, T.,
Sandvik, J.A. &
Pettersen, E.O.: The retinoblastoma gene product is reversibly
dephosphorylated and
bound in the nucleus in S and G2 phase during hypoxic stress. Exp. Cell Res.
227 (1996)
106-115.) Data are shown for two types of human breast cancer cells, MCF-7
(Fig. 15 and
25 16) and T-47D (Fig. 17 and 18) and for drug treatment times of 24h (Fig. 15
and 17) and
48h (Fig. 16 and 18). For both cell types Compound 8 at concentrations above
0.5 mM
induces increased fractions of nuclei with bound pRB in all interphase stages.
The effect
increases with increasing treatment time, and is thus far more pronounced
after 48 than
after 24h of treatment. This is taken to indicate that the substance activates
cell-cycle
30 regulatory action by pRB, resulting in reduced cell-cycle progression. The
drug dose
dependence is complicated, however, showing a maximum effect for Compound 8
doses
around 1 mM and a decrease for higher drug doses. Thus, we see a bell-shaped
dose
response curve in much the same way as was shown for Compound 10 in the animal
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experiment on SK-OV-3 xenograft in nude mice (see table 1). Although it is so
far not
known why the pRB-activation is reduced for Compound 8 doses above 1 to 1.5 mM
it is
interesting to notice that we have found a relatively strong protein synthesis
inhibition
with this drug at these doses. Following 24 h treatment of NHIK 3025-cells
with 1.5 mM
Compound 8 the rate of protein synthesis is 70 % of that of control cells (see
Fig. 19).
In Fig. 20 it is shown that protein synthesis following 24h treatment with
either 1.5 or 2.5
mM Compound 8 is 75 or 50% respectively, but increases back to normal in about
6h after
removal of the drug. Possibly cell-cycle inhibition that inevitably follow as
a result of the
reduced protein synthesis inhibition (see Running, Q~.W., Lindmo, T.,
Pettersen, E.O. &
Seglen, P.O.: Effect of serum step-down on protein metabolism and
proliferation kinetics
of NHIK 3025 cells. J. Cell Physiol. 107 (1981) 47-57.) is in itself over-
ruling the
regulatory effects of pRB at concentrations in the range 1.5 to 2.5 mM.
Example 5
Cell adhesion measurements
Cell adhesion forces were measured using the manipulation force microscope (G.
Sagvolden. Manipulation force microscope. Ph.D. thesis, University of Oslo,
1998, and
G. Sagvolden, I. Giaever and J. Feder. Characteristic protein adhesion forces
on glass and
polystyrene substrates by atomic force microscopy. Langmuir 14(21), 5984-5987,
1998.).
Briefly, NHIK 3025 carcinoma cells were cultured in COz-independent medium
containing 15% fetal calf serum. The cells were exposed to a 1 mM
concentration of
Compound 1 or Compound 2 for 20 hours before they were released from the cell
culture
flasks using trypsin. The cells were kept in suspension, and seeded in medium
with
Compound 1 or Compound 2 on polystyrene tissue culture substrates 90 minutes
after the
trypsin reaction had been stopped. The cell-substrate adhesion forces were
measured by
displacing cells using an inclined atomic force microscope cantilever acing as
a force
transducer. One cell was displaced at a time and each cell was displaced only
once.
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The maximal force exerted on each cell was recorded as a function of the time
since the
cells were seeded on the substrate. The median force of a group of 19
measurements is
shown as a function of the mean time for cells exposed to Compound 1 or
Compound 2 in
Fig. 21, together with the adhesion forces of cells not exposed to these
compounds.
Compound 2 shows a large effect in reducing the adhesion force at this
concentration,
while Compound 1 shows no significant response. The effect of the compound is
mainly
to reduce the adhesion force of the cells, but not the time course of
adhesion.
The reduced ability to attach to the substrate may be related to the blocking
of
integrin-mediated anchorage of the cells. It has been shown that such blocking
may induce
programmed cell death in both hepatoma and melanoma cancers. (Paulsen JE, Hall
KS,
Rugstad HE, Reichelt KL and Elgjo K, The synthetic hepatic peptides
pyroglutamylglutamylglycylserylasparagine and
pyroglytamylglutamylglycylserylaspartic
acid inhibit growth of MH 1 C 1 rat hepatoma cells transplanted into buffalo
rats and
athymic mice. Cancer Res. 52 (1992) 1218-1221. and Mason MD, Allman R, and
Quibell
M, "Adhesion molecules in melanoma - more than just superglue?" J. Royal Soc.
Med. 89
(1992) 393-395.)
The adhesion force between NHIK 3025 cells and the substratum was measured
after
pre-incubation of the cells in solutions of Compounds 1 and 2. Even at 1 mM
concentration, an astonishing D-isotope effect was shown. Surprisingly,
Compound 2
significantly reduced the adhesion force to 1/3 relative to control, whereas
Compound 1
did not lead to significant reduction. The inventors believe that Compound 2
may have
interfered with the biosynthesis of integrins, reducing the cell's ability to
attach to the
substratum. Integrins are structural trans-membrane proteins crucial for
binding cells to
the extracellular matrix and for cell-cell interactions. Inhibiting the
function of the
integrins could thus directly affect the metastasising ability of cancer
cells. The
experiment indicate that integrines could be especially sensitive to protein
synthesis
inhibition. Thus, Compound 2 could well be used for prevention of metastatic
processes in
cancer development.
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Example 6
Experiments with Compound 2 and Compound 5 in NMRI mice infected with FRIEND
erythroleucaemia virus (FLV).
Virus: Eveline cells were supplied by prof. Gerhard Hunsman, Munich. We have
shown
that this virus, which originally was used as a source of Friend helper virus,
contain a
defect virus of the same size as Spleen Focus Forming Virus (SFFV) which
induces
erythroleukaemia in NMRI mice after a delay of 4-8 weeks.
Mice: NMRI mice came from old Bomholt Farm, Denmark, and were purchased via
SIFF.
The mice were received on May 6th and entered into the experiment on May 11
th. They
were then infected with 50 microlitres supernatant from Eveline culture,
intraperitonally.
After 24 hours the treatment was started. Compound 2 and Compound 5 were
dissolved in
sterile isotonic glycerol solution in a concentration corresponding to 5 mg
per kg when
giving 50 microlitres intraperitonally.
The experiment was set up as follows:
10 mice uninfected control
10 mice infected control
5 mice uninfected, treated with Compound 2
10 mice infected, treated with Compound 2
5 mice uninfected, treated with Compound 5
10 mice infected, treated with Compound 5
The mice were given injections intraperitonally once daily for 19 days. From
June 1st till
June 16th, when they were sacrificed, no treatment was given. On June 16th,
the mice
were sacrificed. Blood was withdrawn (for future analysis). The spleen was
removed and
weighed (see table 3 below). One bit of the spleen was frozen in nitrogen for
the purpose
of cutting thin slices and one bit was formaline fixated.
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Table 3:
uninfectedinfected Comp.2, Comp.2, Comp. Comp. S,
controls controls uninfectedinfected S, infected
uninfected
125 154 266 151 185 308
160 240 143 153 161 162
94 214 106 168 188 150
146 212 153 145 155 153
118 165 117 149 120 195
120 171 157 127 161.8 129
115 190 63.824 131 27.472 157
103 204 170 176
130 203 127 153
147 148 148 197
125.8 190.1 157 146.9 162 178 (aver.w)
20.5 24.5 63 15.228 27 50.206 (st.dev.)
The results can also be seen in Fig. 23.
As one can see, there is a significant difference in spleen weights in
infected animals
compared to uninfected controls. Weights of uninfected animals treated with
Compound 2
or with Compound 5 are above the weights of uninfected controls, even if this
is not
significant. One notices that infected animals which were treated with
Compound 2
actually have a lower average spleen weight compared to uninfected animals
which were
treated similarly (here, it is assumed that the outcome stems from one animal
in the
control group having a comparatively big spleen).
A histological examination revealed that the uninfected controls have a normal
spleen
anatomy. All animals in the infected untreated group have invasion of
pathological
leukaemia cells in the red pulpa. The spleens, both from the uninfected
Compound 2 - and
Compound 5 - treated animals, have hypertrofic germinal centra, which are
interpreted as
an expression of immune stimulation. One does not find leukemic chan es in
spleens from
the Compound 2 - treated infected group. In the Compound 5 - treated group,
the animal
with the biggest spleen (308 g) had leukemic changes while all in the infected
control
group had leukemic changes.
The results are encouraging taking into consideration the aggressive nature of
FLV in
mice and also when one compares the effect with that seen with azidothymidine
and other
anti-virus treatment.
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Example 7
Proliferation of peripheral blood mononuclear cells
5 The inventors performed an experiment where peripheral blood mononuclear
cells were
exposed to Superantigen together with benzaldehyde, deuterated benzaldehyde,
Compound 2 or zilascorb(zH). Superantigen is used as a very active standard
for
proliferation of T-cells and is presented via antigen presenting cells to T-
cells.
10 The experiment demonstrated (see figur 22) that by adding benzaldehyde,
deuterated
benzaldehyde or Compound 2, the proliferation of peripheral blood mononuclear
cells was
increased significantly in a bell-shaped, dose-dependent manner, whereas very
little effect
was observed with zilascorb(ZH). The fact that we are able to increase the
proliferation
signal from the Superantigen indicates that the compounds act by additional
15 co-stimulatory effects on the T-cells.
Example 8
Effect on liver invasive colorectal cancer in nude mice
20 Material and Procedures
The cell line evaluated, C 170HM2, is an established human colorectal cell
line
(S.A.Watson et al., Eur.J.Cancer 29A (1993), 1740-1745) and was derived
originally from
25 a patient's primary tumour. C 170HM2 cells were maintained in vitro in RPMI
1640
culture medium (Gibco, Paisley, UK) containing 10% (v/v) heat inactivated
foetal calf
serum (Sigma, Poole, UK) at 37°C in 5% COz and humidified conditions.
Cells from
semi-confluent monolayers were harvested with 0.025% EDTA and washed twice in
the
culture medium described above.
C170HM2 cells harvested from semi-confluent cell monolayers were re-suspended
at
1x10~/ml of sterile phosphate buffered saline, pH 7.4 [PBS] and injected in a
1 ml volume
into the peritoneal cavity of 20 MFl male nude mice (bred within the Cancer
Studies Unit
at the University of Nottingham). Mice were identified by an electronic
tagging system
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(RS Biotech DL2000 Datalogger). On day 10 following cell injection, the mice
were
randomly assigned to alter a placebo control group or experimental groups;-
Group l: Compound 1 5 mglkg
30 mg/kg
90 mg/kg
Group 2: Compound 2 5 mg/kg
30 mg/kg
90 mg/kg
Group 3: Compound 5 20 mg/kg
40 mg/kg
90 mg/kg
The drugs were dosed intravenously (iv) from day 10 and continue until therapy
termination. The experiment was terminated at day 40 post cell implantation.
Mice were
weighed at regular intervals throughout the pilot study.
At termination the liver was exposed, and visible liver tumours were counted
and their
total cross-sectional area measured. The tumours were also photographed. No
liquefaction of the tumours had occurred, thus they were dissected free from
the normal
liver tissue, weighed and fixed in formal saline. Peritoneal nodules were
dissected free
and the cross-sectional area and weight measured. Detailed pathological
assessment of the
tumours was performed.
The effect of Compound 1, 2 and 5 on the liver invasion of the human
colorectal tumour,
C170HM2 is shown in Fig. 24.
Example 9
Biological effects of Compound 13 compared with Compound 1
Cell Survival
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Fig. 25 shows cell survival as measured by colony-forming ability for human
cervix
carcinoma cells, NHIK 3025, after treatment for 20h with either Compound 1 (O)
or
Compound 13 (~). Cells were treated in open plastic Petri dishes incubated in
COZ-incubators at 37°C. The plotted survival values represent mean
values from 5
simultaneously and similarly treated dishes. Standard errors are indicated by
vertical bars
in all cases where they exceed the size of the symbols. The data indicate that
Compound
13 induce roughly a 10 times stronger inactivating effect than Compound 1 on a
dose
basis.
15 Example 10
Biological effects of Compound 14 compared with Compound 1
Cell Survival
Fig. 26 show cell survival as measured by colony-forming ability for human
cervix
carcinoma cells, NHIK 3025, after treatment for 20h with either Compound 1 (O)
or
Compound 14 (~). Cells were treated in open plastic Petri dishes incubated in
COZ-incubators at 37°C. The plotted survival values represent mean
values from 5
simultaneously and similarly treated dishes. Standard errors are indicated by
vertical bars
in all cases where they exceed the size of the symbols. The data indicate that
the two
compounds induce similar inactivating effect over the whole concentration
range up to 12
mM.
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Example 11
Biological effects of Compounds 21 and 22 compared with Compounds l, 2 and
L- lug core
Protein Synthesis
Fig. 27 show rate of protein synthesis of human cervix carcinoma cells, NHIK
3025, as
measured by amount of incorporated [3H]-valine during a pulse period of lh
starting either
immediately following addition of test compound (closed symbols) or starting
2h later
(open symbols). Test compounds, Compound 1 and Compound~2l, were present from
time
zero to the end of the pulses. Cells were pre-labeled with ['4C]-valine for at
least 4 days in
order to have all cellular protein labeled to saturation. Incorporated amount
of [3H] was
related to incorporated amount of ['4C] so that protein synthesis was
calculated as per cent
of the total amount of protein in the cells. Rate of protein synthesis is
given as per cent of
that in an untreated control. The plotted values for protein synthesis
represent mean values
from 4 simultaneously and similarly treated wells. Standard errors are
indicated by
vertical bans in all cases where they exceed the symbols. The data indicate
that
Compound 1 induces a protein synthesis inhibition which increases linearly
with
increasing concentration of drug while little or no effect is seen by Compound
21.
Fig. 28 show rate of protein synthesis of human cervix carcinoma cells, NHIK
3025, as
measured by amount of incorporated [3H]-valine during a pulse period of lh
starting either
immediately following addition of test compound (closed symbols) or starting
2h later
(open symbols). Test compounds, Compound 2 and Compound 21, were present from
time
zero to the end of the pulses. Cells were pre-labeled with ['4C]-valine for at
least 4 days in
order to have all cellular protein labeled to saturation. Incorporated amount
of [3H] was
related to incorporated amount of ['4C] so that protein synthesis was
calculated as per cent
of the total amount of protein in the cells. Rate of protein synthesis is
given as per cent of
that in an untreated control. The plotted values for protein synthesis
represent mean values
from 4 simultaneously and similarly treated wells. Standard errors are
indicated by
vertical barrs in all cases where they exceed the symbols. The data indicate
that both
Compound 2 and Compound 22 induces an effective inhibition of protein
synthesis at
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about the same level for both compounds. Both these two deuterated compounds
are more
effective than the corresponding undeuterated compounds shown in Fig. 27.
Cell Survival
Fig. 29 show cell survival as measured by colony-forming ability for human
cervix
carcinoma cells, NHIK 3025, after treatment for 20h with either Compound 1 (~)
or
Compound 21 (O). Cells were treated in open plastic Petri dishes incubated in
COZ-incubators at 37°C. The plotted survival values represent mean
values from 5
simultaneously and similarly treated dishes. Standard errors are indicated by
vertical bans
in all cases where they exceed the size of the symbols. From the data the dose
response
curves follow different shapes for the two compounds, indicating that Compound
21 is
more effective than Compound 1 in inactivating cells at low compound-
concentrations.
The differences in curve shapes indicate different mechanisms of cell
inactivation for
these two drugs.
Fig. 30 show cell survival as measured by colony-forming ability for human
cervix
carcinoma cells, NHIK 3025, after treatment for 20h with either Compound 2 (O)
or
Compound 22 (1). Cells were treated in open plastic Petri dishes incubated in
COZ-incubators at 37°C. The plotted survival values represent mean
values from 5
simultaneously and similarly treated dishes. Standard errors are indicated by
vertical bans
in all cases where they exceed the symbols. Compound 22 is more effective than
Compound 2 in inactivating cells, particularly in the low-dose region. For
example is cell
survival down to 50% following treatment with 0.5 mM of Compound 22 and 4 mM
of
Compound 2 respectively, indicating an 8-fold higher inactivating efficiency
of
Compound 22 compared to Compound 2 at this particular effect level. At a
survival level
of 10 % the difference is much smaller.
Fig. 31 show cell survival as measured by colony-forming ability for human
breast
carcinoma cells, T47-D, after treatment for 20h with either L-glucose (~) or
Compound
21 (O). Cells were treated in open plastic Petri dishes incubated in COZ-
incubators at
37°C. The plotted survival values represent mean values from 5
simultaneously and
similarly treated dishes. Standard errors are indicated by vertical bans in
all cases where
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they exceed the symbols. The data indicate that L-glucose has little or no
effect on cell
survival for concentrations up to at least 10 mM, the highest dose tested.
Compound 21
also has little effect on these cells for concentrations up to 2 mM, but
induces considerable
inactivating effect for higher concentrations and only one of 1000 cells
survive 20h in
5 presence of 8 mM of this compound.
Conclusion
10 Both the two L-glucopyranose derivatives tested (Compounds 21 and 22)
inactivate cells
more effectively than the corresponding D-glucopyranose derivatives (Compounds
1 and
2). L-glucose alone, however, does not induce any significant cell
inactivating effect for
concentrations tested here. Thus, it is in the context of a benzylidene
derivative this
increased effect of L as compared to D glucose is found.
20
Example 12
Testing of the effect of Compound 2 in ova-sensitized and challenged animals
Male Balb/C mice of 6 weeks old were sensitized with 7 i.p. injections of 10
~g
ovalbumin in 0,5 ml saline on alternate days. Three weeks after the last
injection, the mice
were exposed to 8 ovalbumin ( 2 mg/ ml ) or 8 saline aerosols on consecutive
days, 1
aerosol per day for 5 minutes. Two days prior to the first ovalbumin alt.
saline injection
treatment with Compound 2 was started, 5 mg per kg given daily i.p, for a
period of 10
days, nine animals given ovalbumin and nine animals given saline, the control
groups
were given saline. 24 hours post the last exposure, airway hyperresponsiveness
in
response to metacholine was measured in vivo using BUXCO set-up. After
measuring in
the BUXCO, the mice were put down, lungs lavaged and the isolated cells
washed,
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counted and differentiated. Blood was taken to isolate serum for determination
of total and
ova-specific IgE-levels. Thoracic lymph nodes were isolated from paratracheal
and
parabronchial region for determination of IFN-gamma, IL-4, IL-5 and IL-12. The
experiment contained 9 animals per group.
The number of cells (Fig. 32) in saline treated or ovalbumine sensitized mice
were not
affected by Compound 2 treatment, and there was no difference in the count of
the
macrophages, the lymphocytes or the eosinophils between the two groups.
Astonishingly, we found that the influx of neutrophils was inhibited by the
Compound 2
treatment (Fig. 33). The number of neutrophils in the lung lavage is the same
for the
control and the ovalbumin sensitized animals treated with Compound 2, whilst
the number
of neutrophils increases in ovalbumine sensitized animals treated with saline.
To our knowledge no other drug exert this kind of effect. The inhibition of
the neutrophil
influx could be of great value to medicine, as the tissue damage caused by
neutrophil
released lysosomal enzymes are thought to be of great importance in lung
emphysema
(also induced by smoking), in occupational asthma, inflammatory bowl diseases
(like
Morbus Crohn and Ulcerous colitt), rheumatoid arthritis and similar immunic
disorders.
This is a very surprising observation.
Conclusions
The benzaldehyde derivatives of this invention react with certain groups on
the cell
surface, e.g. with free amino groups to form Schiff s bases. As many cell
processes, like
protein synthesis, cell cycle, immune response etc. are controlled by signals
from the
cell-surface, these bindings will alter the behaviour of the cell. We have
also shown that
the benzaldehyde complex of the cell surface change the adhesion
characteristics of the
cell. We have shown that the compounds of this invention can be useful in new
therapies
to combat cancer, auto immune diseases, viral infections and possibly also
infections of
other microorganisms.
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We have found that the hexose derivatives of benzaldehydes are surprisingly
more
effective than derivatives of other carbohydrates in treating cancer in
certain organs like
liver, kidney and lung. We believe that this phenomenon is connected with
receptor
affinity of these organs to the sugar moiety of the derivatives.
Administration
The pharmaceutical compositions according to the present invention may be
administered
in anti-cancer treatment, anti-viral treatment or in treatment of diseases
which arise due to
abnormally elevated cell proliferation and/or for combating auto immune
diseases. This
pharmaceutical compositions may also be administered as immunopotentiators.
For this purpose the compounds of formula (I) may be formulated in any
suitable manner
for administration to a patient, either alone or in admixture with suitable
pharmaceutical
carriers or adjuvants.
It is especially preferred to prepare the formulations for systemic therapy
either as oral
preparations or parenteral formulations.
Suitable enteral preparations will be tablets, capsules, e.g. soft or hard
gelatine capsules,
granules, grains or powders, syrups, suspensions, solutions or suppositories.
Such will be
prepared as known in the art by mixing one or more of the compounds of formula
(I) with
non-toxic, inert, solid or liquid carriers.
Suitable parental preparations of the compounds of formula (I) are injection
or infusion
solutions.
When administered topically the compounds of formula (I) may be formulated as
a lotion,
salve, cream, gel, tincture, spray or the like containing the compounds of
formula (I) in
admixture with non-toxic, inert, solid or liquid carriers which are usual in
topical
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preparations. It is especially suitable to use a formulation which protects
the active
ingredient against air, water and the like.
The preparations can contain inert or pharmacodynamically active additives.
Tablets or
granulates e.g. can contain a series of binding agents, filler materials,
carrier substances
and/or diluents. Liquid preparations may be present, for example, in the form
of a sterile
solution. Capsules can contain a filler material or thickening agent in
addition to the active
ingredient. Furthermore, flavour-improving additives as well as the substances
usually
used as preserving, stabilising, moisture-retaining and emulsifying agents,
salts for
varying the osmotic pressure, buffers and other additives may also be present.
The dosages in which the preparations are administered can vary according to
the
indication, the mode of use and the route of administration, as well as to the
requirements
of the patient. In general a daily dosage for a systemic therapy for an adult
average patient
will be about 0.01 -SOOmg/kg body weight once or twice a day, preferably 0.5-
100 mg/kg
body weight once or twice a day, and most preferred 1-20 mg/kg weight once or
twice a
day.
If desired the pharmaceutical preparation of the compound of formula (I) can
contain an
antioxidant, e.g. tocopherol, N-methyl-tocopheramine, butylated
hydroxyanisole, ascorbic
acid or butylated hydroxytoluene.
