Note: Descriptions are shown in the official language in which they were submitted.
CA 02302144 2000-03-27
TITLE: HYDROXYPHENYL DERIVATIVES WITH
HIV INTEGRASE INHIBITORY PROPERTIES
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a hydroxyphenyl derivatives which have HIV
integrase
inhibitory properties that have been characterized by specific structural and
physicochemical
features. This inhibitory property may be advantageously used, for example, to
provide
medicinals (e.g. compositions) with antiviral properties against HIV viruses,
including the
HIV-1 and HIV-2 viruses, i.e. the hydroxyphenyl derivatives including
pharmaceutical
compositions thereof may be used to inhibit the activity of HIV integrase.
BACKGROUND OF THE INVENTION
The HIV (human immunodeficiency virus) retrovirus is the causative agent for
AIDS (acquired
immunodeficiency syndrome). Thus the HIV-1 retrovirus primarily uses the CD4
receptor (a 58
kDa transmembrane protein) to gain entry into cells, through high-affinity
interactions between
the viral envelope glycoprotein (gp 120) and a specific region of the CD4
molecule found in T-
lymphocytes and CD4 (+) T-helper cells (Lasky L. A. et al., Cell vol. 50, p.
975 - 985 (1987)).
HIV infection is characterized by a period immediately following infection
called
"asymptomatic" which is devoid of clinical manifestations in the patient.
Progressive HIV-
induced destruction of the immune system then leads to increased
susceptibility to opportunistic
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infections, which eventually produces a syndrom called AIDS-related complex
(ARC)
characterized by symptoms such as persistent generalized lymphadenopathy,
fever, weight loss,
followed itself by full blown AIDS. After entry of the retrovirus into a cell,
viral RNA is
converted into DNA, which is then integrated into the host cell DNA. The
reverse transcriptase
encoded by the virus genome catalyzes the first of these reactions (Haseltine
W. A. FASEB J.
vol 5, p. 2349 - 2360 ( 1991 )). At least three functions have been attributed
to the reverse
transcriptase: RNA-dependent DNA polymerase activity which catalyzes the
synthesis of the
minus strand DNA from viral RNA, ribonuclease H (RNase H) activity which
cleaves the RNA
template from RNA-DNA hybrids and DNA-dependent DNA polymerase activity which
catalyzes the synthesis of a second DNA strand from the minus strand DNA
template (Goff S.
P. J. Acq. Imm. Defic. Syndr. Vol 3, p. 817 - 831 (1990)). The double stranded
DNA produced
by reverse transcriptase, now called provirus, is then able to be inserted
into host genomic DNA.
At the end of reverse transcription, the viral genome now in the form of DNA
is integrated into
host genomic DNA and serves as a template for viral gene expression by the
host transcription
system, which leads eventually to virus replication (Roth et al.,1989). The
preintegration complex
consists of integrase, reverse transcriptase, p17 and proviral DNA (Bukrinsky
M. L, Proc. Natn.
Acad. Sci. USA vol. 89 p.6580 - 6584 (1992)). The phosphorylated p17 protein
plays a key role
in targeting the preintegration complex into the nucleus of host cell (Gallay
et al., 1995).
The primary RNA transcripts made from the provirus are synthesized by the host
cell RNA
polymerase II which is modulated by two virus-encoded proteins called tat and
rev. The viral
proteins are formed as polyproteins.
Post-translational modifications of viral polyproteins include processing and
glycosylation of Env
(envelope) proteins, and myristylation of the N-terminal residue of the p 17
protein in the Gag and
Gag-Pol polyproteins. The latter two precursors correspond to structural
proteins and viral
enzymes. The viral protease is involved in processing polyproteins Gag and Gag-
Pol into mature
proteins, a step essential for virus infectivity.
A number of synthetic antiviral agents have been designed to block various
stages in the
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replication cycle of HIV. These agents include compounds which interfere with
viral binding
to CD4 T-lymphocytes (for example, soluble CD4), compounds which block viral
reverse
transcriptase (for example, didanosine and zidovudine (AZT)), budding of
virion from the cell
(interferon), or the viral protease (for example Ritonavir and Indinavir).
Some of these agents
proved ineffective in clinical tests. Others, targeting primarily early stages
of viral replication,
have no effect on the production of infectious virions in chronically infected
cells.
Furthermore, administration of many of these agents in effective therapeutic
doses has led to
cell-toxicity and unwanted side effects, such as anemia, neurotoxicity and
bone marrow
suppression. Anti-protease compounds in their present form are typically large
and complex
molecules of peptidic nature that tend to exhibit poor bioavailability and are
not generally
consistent with oral administration. These compounds often exhibit side
effects such as
nausea, diarrhea, liver abnormalities and kidney stones.
Accordingly, the need exists for compounds that can effectively inhibit the
action of the third
1 S viral enzyme called integrase, for use as agents for treating HIV
infections.
The terms HIV integrase and integrase as used herein are used interchangeably
and refer to
the integrase enzyme encoded by the human immunodeflciency virus type 1 or 2.
In
particular this term includes the human immunodeficiency virus type 1
integrase.
SUMMARY OF THE INVENTION
The present invention provides an hydroxyphenyl derivative selected from the
group
consisting of a compound of formula
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/(Hal)e OH
(HOB,
W N H-X ~ OH
O
OH
(HO
N C\H / OH
II
S
O
HO N~ /IC
C N
~H)e
as
HO
(Hal)r
and when a compound of formula I comprises a carboxylic acid group
pharmaceutically
acceptable salts thereof and when a compound of formula I comprises an amino
group
pharmaceutically acceptable ammonium salts thereof, wherein n is 1, 2 or 3, a
is l, 2 or 3,
Hal represents a halogen atom (e.g. Cl, Br, F or I), p is 0, 1 or 2, r is 0, 1
or 2, X and X' each
independently represents a single bond, a saturated straight or branched
hydrocarbon group of
1 to 4 carbon atoms or a straight or branched hydrocarbon group of 2 to 4
carbon atoms
comprising a carbon to carbon double bond, Ra represents H or -CH3, and Raa
represents H or
-CH3; W, may for example, represent an amino acid residue or fragment (in
particular alpha-
amino acid residues) such as for example a residue based on tyrosine, DOPA,
hydroxyproline, serine, threonine, histidine, valine, phenylalanine, lysine,
alanine, glycine,
glutamic acid, aspartic acid, arginine, asparagine, glutamine, leucine,
lysine, isoleucine,
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proline, tryptophan, methionine, cysteine, cystine, thyroxine, meta-tyrosine,
sarcosine, other
alpha-methyl amino acids such as alpha-methyl DOPA, as well as other 3-
substituted
tyrosines, and the like.
W, for the above formula I, may, for example, be derived from natural or
unnatural alpha-
amino acids. The term unnatural alpha-amino acid refers to alpha - amino acids
which do not
occur in nature but which can be derived from naturally occurring alpha -
amino acids or other
chemical reagents by methods known to those skilled in the art.
W may, for example, represent a group of formula
O O
-A-~-LA~-~J k-~
wherein k is 0 or 1, A and A' each independently represents a group of formula
~a ~b ~a ~b
N- I - ~ - N- I -CHz- ,
R R
R b
~a ~b
or - N
N - C-CHZCHz-
-~R
R c
Ra represents H or -CH3, Rb represents H or -CH3, R~ represents H or OH, R is
selected
from the group consisting of H, CH3-, (CH3)ZCH-, (CH3)zCHCHz-, CH3CHZCH(CH3)-,
C6HSCH2-, CH3SCHzCHz-, HOzCCHz-, HZNC(O)CH2-, HOZCCHZCHz-, HZNC(O)CHzCH2-,
HZNCHZCH2CHzCH2-, HOCHZ-, CH3CH(OH)-, HSCHZ- , HOZC-, benzyloxycarbonyl,
benzyloxycarbonylmethyl,
CH2-
HN~ N
~ CNHCH 2CH2CH2-
N/ NH2 H CH2_
H
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HO I I
HO ~ / CH2- HO ~ / CH2- H ~ / O ~ / CH2-
( lal)f I I
(Hal)9~ / OH
/ ~~- and
HO
-CHz(CH2)qCCNH(CHZ)q H ~ OH
wherein Hal is as defined above and f is 0, 1 or 2, g is 0, 1 or 2, each q is
independently 0 or
l and provided that n and p may not at the same time represent 3 and a and r
may not at the
same time represent 3.
The group of structure
HO \ ~CH2
( ~ al)t
may in particular for example be a fluoride substituted structure of formula
F
HO \ / CH2-
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Similarly, the group of structure
(Hal)g~
CH2_
HO
may in particular for example be a fluoride substituted structure of formula
F \ / CH2-
HO
As mentioned, when a compound of formula I comprises a carboxylic acid group
the
polyhydroxy compounds may be any pharmaceutically acceptable salt thereof and
when a
compound of formula I comprises an amino group the polyhydroxy compounds may
be any
pharmaceutically acceptable ammonium salt thereof.
The present invention provides, where appropriate, salts (e.g. derived from
appropriate bases
or acids) which include but are not limited to alkali metal (e.g., sodium,
potassium, cesium,
etc.. ) salts, alkaline earth metal (e.g., magnesium) salts, and ammonium
salts such as acid
addition salts of amines (e.g. ammonium chloride salts) as well as quaternary
ammonium salts
of for example N - (R")4+ type wherein R" is an organic residue.
The pharmaceutically acceptable salts of the compounds of this invention
include those
derived from pharmaceutically acceptable inorganic and organic acids and
bases. Examples of
such acid salts include: acetate adipate, alginate aspartate benzoate,
benzenesulfonate,
bisulfate, butyrate, citrate, camphorate, camphorsulfonate,
cyclopentanepropionate,
digluconate, dodecylhydrogen-sulfate, dodecylsulfate, ethanesulfonate,
formate, fumarate,
glucoheptanoate, glycerophosphate, glycollate, hemisulfate, heptanoate,
hexanoate,
hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate,
maleate,
CA 02302144 2000-03-27
malonate, methanesulfonate, 2-naphthylsulfonate, nicotinate, nitrate, oxalate,
pamoate,
pectinate, perchlorate, persulfate, 3-phenylpropionate, phosphate, picrate,
pivalate,
propionate, salicylate, succinate, sulfate, tartrate, thiocyanate, tosylate,
and undecanoate.
This invention also envisions the quaternization of any basic nitrogen
containing groups of the
compounds disclosed herein. The basic nitrogen can be quaternized with any
agents known to
those of ordinary skill in the art including, for example, lower alkyl
halides, such as methyl,
ethyl, propyl and butyl chloride, bromides and iodides; dialkyl sulfates
including dimethyl,
diethyl, dibutyl and diamyl sulfates; long chain halides such as decyl,
lauryl, myristyl and
stearyl chlorides, bromides and iodide; and arylalkyl halides including benzyl
and phenethyl
bromides. Water or oil-soluble or dispersible products may be obtained by such
quaternization.
This invention also envisions the presence of an ester groups) such as for
example on the
acidic end of an appropriate amino acid fragment(s), such as glutamic acid and
aspartic acid
as having some anti-integrase activity as such as acting as pro-drugs, i.e.
capable of hydrolysis
of the ester moiety to liberate in the systemic circulation the acid, also
possessing anti-
integrase activity. For example, the ether oxygen of an ester compound may be
attached or
linked to benzyl, a lower (branched or straight) alkyl (e.g. C,-C6 alkyl) such
as methyl, a lower
cycloalkyl (e.g. C3-C, cycloalkyl) such as cyclohexyl, and the like.
Alternatively, an esters)
may be derived from a carboxylic acids) and one or more hydroxyl groups, such
as for
example an hydroxyl group on a phenyl ring. A carboxylic acid may, for
example, comprise
an acyl group having from 2 to 8 carbon atoms; the acyl group may for example
comprise
lower alkyl of 1 to 6 carbon atoms, lower cycloalkyl of from 3 to 7 carbon
atoms, etc..
In addition, this invention further envisions the presence of structures
having an amide
functionality such as, for example, on the carboxylic end located on the side
chain of such
acids. These amides, such as simple primary, secondary or tertiary amides,
possess activity of
their own. In addition, it is possible to couple such acids with dopamine to
yield compounds
of interest. The amino moiety of an amide compound may for example be -NH2, -
NH(C~-C6
alkyl), or -N(C,-C6 alkyl)Z, a pyrrolidine residue, a piperidine residue, a
morpholine residue
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and the like
In any event, it is also to be understood that the present invention relates
to any other
compound having a structure such that, upon administration to a recipient, it
is capable of
providing (directly or indirectly) a compound of this invention or an
antivirally active
metabolite or residue thereof. Thus the compounds of this invention may be
modified by
appending appropriate functionalities to enhance selective biological
properties. Such
modifications are known in the art and include those which increase biological
penetration
into a given biological system (e.g., blood, lymphatic system, central nervous
system),
increase oral availability, increase solubility to allow administration by
injection, alter
metabolism and alter rate of excretion.
The present invention in particular provides a dopamine derivative selected
from the group
consisting of a compound of formula Ia
OH
Ra O
(HO)~ I Ia
N C
C/ ~N ~ OH
II H
O R
and when a compound of formula Ia comprises a carboxylic acid group
pharmaceutically
acceptable salts thereof and when a compound of formula Ia comprises an amino
group
pharmaceutically acceptable ammonium salts thereof, wherein n, Ra and R are as
defined
above.
The present invention also provides a dopamine derivative selected from the
group consisting
of a compound of formula Ib
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H
O O~C/N
(HO)~
C ~ ~ Ib
N ~OH
OH
Rd
wherein n is as defined above (e.g. n may in particular be 1 or 2), and Rd is
selected from the
group consisting of H and OH.
The present invention further relates to dipeptide derivatives i.e. to
compounds of formula I
defined above wherein k is 1. The present invention in particular provides an
hydroxylphenyl
derivative wherein for the compound of general formula I above, W represents a
group of
formula
O O
a b a b
- C N -C
N ~ ~
R R
wherein n is as defined above (e.g. n may in particular be 1 or 2), p is as
defined above (p may
in particular be 0), each Ra is independently as defined above, each Rb is
independently as
defined above, and each R is independently as defined above; more
particularly, for example,
for each R, f may be 0 or 1 and g may be 0 or 1.
The compounds of this invention contain one or more asymmetric carbon atoms
and thus may
occur as racemates and racemic mixtures, single enantiomer, diastereomeric
mixtures and
individual diastereoisomers. All such isomeric forms of these compounds are
expressly
included in the present invention. Each stereogenic carbon may be of the R or
S configuration.
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The amino acid residues may, for example, in any event, be of L, D or DL form,
preferably of
L form; thus for example the amino acid residue (i.e. V~ may be a L-a-amino
residue, a D
a-amino residue, or a DL-a-amino residue.
Accordingly, the present invention father provides a dopamine derivative
selected from the
group consisting of a compound of formula Ic
OH
Ra O
(HO)~ I IC
H OH
O R
and when a compound of formula Ic comprises a carboxylic acid group
pharmaceutically
acceptable salts thereof and when a compound of formula Ic comprises an amino
group
pharmaceutically acceptable ammonium salts thereof,
wherein n is l, or 2, Ra and R are as defined above (e.g. f and g may be 0 or
1 and the
respective group Hal thereof may be fluorine (F)).
The present invention furthermore provides a dopamine derivative selected from
the group
consisting of a compound of formula Id
Ra O R
(HO)~
H
CAN CAN Id
O R Ra O
OH
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and when a compound of formula Id comprises a carboxylic acid group
pharmaceutically
acceptable salts thereof and when a compound of formula Id comprises an amino
group
pharmaceutically acceptable ammonium salts thereof, wherein n is 1, or 2, each
Ra is
independently as defined above, and each R is independently as defined above;
more
particularly, for example, for each R, f may be 0 or 1 and g may be 0 or 1.
The compounds of the present invention including where applicable their
pharmaceutically
acceptable derivatives have an affinity for integrase, in particular, HIV
integrase. Therefore,
these compounds are useful as inhibitors of such integrase, i.e. they are in
particular useful as
HIV integrase inhibitors. These compounds can be used alone or in combination
with other
therapeutic or prophylactic agents, such as antivirals, antibiotics,
immunomodulators or
vaccines, for the treatment or prophylaxis of viral infection.
According to the present invention, the compounds of this invention are
capable of inhibiting
HIV viral replication in human CD4+ T-cells, by inhibiting the ability of HIV
integrase to
integrate the double stranded DNA into host genomic DNA for further virus
replication by the
host cell machinery (Sakai H., J. Virol. Vol. 67 p. 1169 - 1174 (1993)). These
novel
compounds can thus serve to reduce the production of infectious virions from
acutely infected
cells, and can inhibit the initial or further infection of host cells.
Accordingly, these
compounds are useful as therapeutic and prophylactic agents to treat or
prevent infection by
HIV-1 and related viruses, which may result in asymptomatic HIV-1 infection,
AIDS-related
complex (ARC), acquired immunodeficiency syndrome (AIDS), AIDS-related
dementia, or
similar diseases of the immune system.
Thus the present invention also provides a pharmaceutical composition
comprising a
pharmaceutically acceptable carrier and a pharmaceutically effective amount of
at least one
hydroxyphenyl derivative as defined above. The pharmaceutical compositions may
be used
to inhibit integrase, including HIV integrase, thus providing protection
against HIV infection.
The expression " pharmaceutically effective amount" is to be understood herein
as referring to
an amount effective in treating HIV infection in a patient. The term
prophylactically effective
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amount refers to an amount effective in preventing HIV infection in a patient.
As used herein,
the term patient refers to a mammal, including a human. The expressions
"pharmaceutically
acceptable carrier" (or adjuvant) and "physiologically acceptable vehicle" are
to be understood
as referring to a non-toxic carrier or adjuvant that may be administered to a
patient, together
with a compound of this invention, and which does not destroy the
pharmacological activity
thereof. These factors will be discussed in more detail below.
The compounds of this invention may be readily prepared using conventional
techniques from
commercially available and cheap starting materials. The relative ease of
synthesis of the
products described in this invention represents a marked advantage for the
large scale
preparation of these compounds. In general, the derivatives of the present
invention may be
readily obtained from amino acids through sequences recognized by those
knowledgeable in
the art as straightforward, requiring readily available reagents and easy
techniques. Using
standard techniques, amino acids may be transformed to the desired HIV
integrase inhibitors
according to approaches as shown in Scheme 1, Scheme 2 and Scheme 3 which are
discussed
below. The preparation of dipeptide derivatives may be accomplished by solid
phase peptide
synthesis; this type of process is generically illustrated in scheme 4 below
(see example 42
below).
Scheme 1 illustrates example steps for the preparation of a derivative in
accordance with the
present invention:
Note:
a) For scheme l, PG and PG' may be any suitable (known) removable
protecting group for respectively protecting the amine functional group and
the
carboxylic acid functional group(s). PG may, for example, be Boc i.e. tert-
butoxycarbonyl and PG' may, for example, be tent-Butyl, 2,6-C12Bz1 or Bzl,
i.e. a functional group of the following formula
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b) For scheme 1 R~ may, for example, be CH3-, Bz100CCH2CH2-,
HZNC(O)CHZCHZ-; R,a may, for example, be CH3-, HOOCCHZCHZ-,
HzNC(O)CHZCHz-
Step 1)
0 0
H H
N N \
PG ( e.g. Boc) \OH PG~ ~~G' (e.g. O-Bz)
R~ R~
compound 1 compound 2
Step 2)
0 0
N H2N
PG~ \O-PG' ~ \O-PG'
R~ Ri
compound 2 compound 3
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Step 3)
H \ O
HZN
OH + ~O-PG'
Rz v
R~
O
compound 4 compound 3
(Rz = H or OH)
H
0
H
N
Rz ~ ~O~G'
O R~
compound 5
Step 4)
0
H
N
F -O
R1a
cor~ound 5 cor~ound 6
15
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Step 5)
H ~ OH
o I
N +
R2 / OH H2N / OH
O Rya
compound 6 compound 7
H ~ O ~ OH
H
R / N N I /
z " H OH
O Rya
compound 8
In accordance with Scheme l, illustrated above, different pharmacophores may
be attached to
the amino acid via the N terminal. Thus, for step 1 compound 1 is treated so
as to protect the
carboxylic acid functional group by means of a suitable protecting group PG';
for example
compound 1 may be a Boc-amino acid which is benzylated with benzyl bromide to
yield
compound 2 in the form of a benzyl ester using cesium carbonate in DMF
according to the
method of S.-S. Wang et al (J. Org. Chem. vol 49 p. 1286 (1977)). For step 2
the amino
protecting group PG is removed to provide compound 3 having a free amino
functional group;
for example the removal of the Boc group from compound 3 may be carried out by
stirring in
a mixture of TFA and methylene chloride (1:1 (v/v)). For step 3 the amino
compound 3 is
then coupled with an hydroxylated benzoic acid (compound 4) with EDC and HOBT
in DMF
providing the desired coupled product compound 5. For step 4 compound 5 is
treated to
remove the protecting group PG' to yield compound 6 having a free carboxylic
acid group; for
example the benzyl protecting group PG'may be removed by hydrogenolysis using
10% Pd/C
as catalyst to yield compound 6 having a free carboxylic acid group. Finally
for step 5
compound 6 is coupled with dopamine (compound 7) to provide the desired
derivative,
namely compound 8.
Scheme 2 (which is divided below into scheme 2a and scheme 2b) illustrates
example steps
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for an alternate method for the preparation of a derivative in accordance with
the present
invention:
Note:
a) For scheme 2a, PG, as mentioned above, may be any suitable (known)
removable protecting group for protecting the amine functional group. PG may,
for example, be Boc i.e. tert-butoxycarbonyl
b) For scheme 2a, R3 may, for example, be (CH3)ZCHCHz-, CH3SCHZCHz-, or
a functional group of the following formula
CH2-
N
H
25
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Scheme 2a:
Step 1
O
H ~ OH
N
PG~ OOH
(e.g. Boc) R H2N / OH
3
compound 1 compound 9
OH
O \
H
N
PG~ N / OH
H
R3
compound 10
Step 2:
OH
O ~ \
H
N
PG~ N / OH
H
R3
compound 10
OH
O
HZN
N OH
H
R3
compound 11
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Step 3:
O ~ OH H
HzN ~ / + / OH
OH Rz
R3 O
compound 11 ~mPound 4
(Rz = H or OH)
H ~ O ~ OH
H
/ N /
Rz H O H
O R3
compound 12
15
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Scheme 2b:
Step 1
OH
H2N OH
compound 1a compound 9a
O ~ ~ OH
N /
-N OH
H
Kd compound 10a
Step 2:
OH
NG O ~ /
-N OH
H
Kd compound 10a
1~
OH
O
H
N
~N OH
H
compound 11a
d
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Step 3:
O ~ OH HO
_ I / + I / OH
H OH Rz
O
compound 11a impound 4
Rd (RZ = H or OH)
Rd
HO
/ N ~ OH
R2
O N / OH
O H
compound 12a
The second approach illustrated in scheme 2 above proceeds by the C-terminal
first with the
subsequent coupling taking place at a later stage after the removal of the
amino blocking
group. Thus, for step 1 of scheme 2a compound 1 (e.g. a Boc amino acid) is
coupled with
dopamine (compound 9) using EDC and HOBT as coupling reagents in DMF to obtain
compound 10. For step 2 of scheme 2a compound 10 is treated to remove the
protecting or
blocking group PG to obtain compound 11; for example, the removal of a Boc
group may be
performed by stirring compound 10 in a mixture of TFA and methylene chloride
at room
temperature for a short period of time. For step 3 of scheme 2a compound 11
may then be
coupled with the appropriate hydroxybenzoic acid (compound 4) using the
EDC/HOBT
coupling conditions to obtain compound 12. The desired product compound 12 may
then be
deprotected if needed or appropriate by hydrogenolysis using 10% Pd/C as
catalyst for those
amino acid with functionality on the side chain. Scheme 2b may proceed in an
analogous
fashion.
Scheme 3 illustrates yet another example method for the preparation of a
derivative in
accordance with the present invention
Note:
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a) For scheme 3, PG and PG" may be any suitable (known) independently
removable protecting group for respectively protecting different functional
groups including a nitrogen atom. PG may, for example, be Boc i.e. tert-
butoxycarbonyl and PG" may, for example, be Fmoc, i.e. 9-
fluorenylmethoxycarbonyl
b) For scheme 3, R4 may, for example, be -HNCHZCHZCHZ- or a group of
formula
~N~CH -
2
and RS may, for example, be HZNCHZCHZCHz- or a group of formula
N
20
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Step 1
O OH
N i\
PG"~ OH
(e.g. Fmoc) R HzN OH
4
LPG
compound 13 compound 9
O I ~ OH
H
PG~. /N N / OH
H
R4
LPG
compound 14
Step 2
OH
p ~ \
H
N
PG"/ N / OH
H
RQ
LPG
compound 14 O ~ OH
HzN
~N OH
H
~\
PG
compound 15
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Step 3:
O \ OH H \ Rz=HorOH
z H / pH + R ~ / OH
Ra \ z
PG
compound 15 compound 4
H / OH
O ~ \
\ ~ N
Rz N / OH
H
O Ra
\PG compound 16
Step 4:
HO / OH
\ I N O I \
H
R2 N / OH
H
O Ra\
PG compound 16
HO / OH
a I\
\I
R2 N / OH
H
O Rs
compound 17
In scheme 3 illustrated above the starting amino acid (compound 13) is
provided with a pair of
independently removable protecting groups PG and PG"; the amino group may have
a
protecting group (PG") such as Fmoc for example. On the other hand if the
group R4 includes
a primary or secondary amino component a protecting group PG may likewise be
attached to
the nitrogen atom of such an amino component; PG may, for example, be Boc or
tert-
butoxycarbonyl. Thus, for step 1 of scheme 3 compound 13 is coupled with
dopamine
(compound 9) using EDC and HOBT as coupling reagents in DMF to obtain compound
14.
For step 2 compound 14 is treated to remove the protecting or blocking group
PG" to obtain
CA 02302144 2000-03-27
compound 15. For step 3 compound 15 may then be coupled with the appropriate
hydroxybenzoic acid (compound 4) using the EDC/HOBT coupling conditions to
obtain
compound 16 . For step 4 compound 15 is treated to remove the protecting group
PG to yield
compound 17.
Scheme 4 illustrates in a generic fashion an example method for the
preparation of a dipeptide
derivative in accordance with the present invention (see example 42 below for
a more specific
description of a process for making a dipeptide derivative):
Step 1
HO ~ OH
O O
H
OH + ~N N / OH
H
O RX RX'
c o m p o a n d 18 compound 19
1$
OH
O RX O
H
N H / OH
HO ~ O RX
compound 20
(RX and RX' independently have the values set forth for R herein).
The compounds listed in Table 1 were prepared by following Scheme 1 or Scheme
2 (see
examples below); the numbers) in brackets after each root amino acid name is
the numer(s) of
an examples) below. Their activities are also listed in the same table
demonstrating their
potential usefulness.
26
i
CA 02302144 2000-03-27
Table 1. Anti-integrase activity (ICSO) of amino acid derivatives in
accordance
with formula Ic above
Root Amino acid Anti-integrase activity
(ICSO)
(i.e. W for formula I is the 4-hydroxyderivative 3, 4-dihydroxy
fragment derivative
thereof) ,uM ,uM
Glycine (ex. 15) 100
L-Glutamic (ex. 21 & 22 - step 64 11
B )
L-Glutamic-4-O-benzyl (ex. 23 ) 26
L-Tyrosine (ex. 11 & 12) 88 8
L-Tryptophan (ex. 29 & 30) 245 17
L-Proline (ex. 27 & 28) >200 80
L-Leucine (ex. 25 & 26) >200 45
L-Phenylalanine (ex. 13 & 14) >200 45
L-Serine (ex. 18) 100
L-Methionine (ex. 31 ) 100
L-Dopa (ex. 16) 8
D-Tyrosine (ex. 10) 67
D-Tyrosine-O-benzyl (ex. 10 - step 42
D)
L-Alanine (ex. 19 & 20) 160 71
L-Histidine (ex. 33) 0.1
DL-3-Fluoro-Tyrosine (ex. 48) 1.4
L-Glutamine benzyl ester (ex. 49) 2.2
DL-m-Tyrosine (ex. 50) 1.3
Dipeptide derivatives were also prepared and are listed in Table 2 ; the
numbers) in Table 2
with respect to each product structure name therein indicated a number of an
example.
27
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Table 2. Anti-integrase activity (ICSO) of dipeptide derivatives of I
Product Xa Y Anti-integraseEx. No.
activity (ICSO)
~M
S Xa- Tyr-Tyr-Y3,4-dihydroxybenzoylOH 177 36
Xa- Tyr-Tyr-Y3,4-dihydroxybenzoyl3,4-dihydroxy-17 51
phenethylamino
Xa-Gly-Tyr-Y3,4-dihydroxybenzoylOH > 200 39
Xa-Tyr- 3,4-dihydroxybenzoyl3,4-dihydroxy-20 41
Asp(OBn)-Y phenethylamino
Xa-Tyr-Gly-Y3,4-dihydroxybenzoylOH > 200 37
As can be appreciated by the skilled artisan, the above synthetic schemes are
not intended to
comprise a comprehensive list of all means by which the compounds described
and claimed in
this application may be synthesized. Further methods will be evident to those
of ordinary skill
in the art.
For the purposes of Table 1 (and Table 2) the HIV-1 integrase inhibition assay
was carried out
following a known procedure (Burke, Jr. T. R. et al., J. Med. Chem. 38, 4171-
4178 (1995)). A
suitable radiolabeled duplex substrate corresponding to the US end of the HIV
LTR was used.
The novel compounds of the present invention are excellent ligands for
integrase, particularly
HIV-1, and most likely HIV-2 and HTLV-1 integrase. Accordingly, these
compounds are
capable of targeting and inhibiting an early stage event in the replication,
i.e. the integration of
viral DNA into the human genome, thus preventing the replication of the virus.
In addition to their use in the prophylaxis or treatment of HIV infection, the
compounds
according to this invention may also be used as inhibitory or interruptive
agents for other
28
CA 02302144 2000-03-27
viruses which depend on integrases, similar to HIV integrases, for obligatory
events in their life
cycle. Such compounds inhibit the viral replication cycle by inhibiting
integrase. Because
integrase is essential for the production of mature virions, inhibition of
that process effectively
blocks the spread of virus by inhibiting the production and reproduction of
infectious virions,
particularly from acutely infected cells. The compounds of this invention
advantageously
inhibit enzymatic activity of integrase and inhibit the ability of integrase
to catalyze the
integration of the virus into the genome of human cells.
The compounds of this invention may be employed in a conventional manner for
the treatment
or prevention of infection by HIV and other viruses which depend on integrases
for obligatory
events in their life cycle. Such methods of treatment, their dosage levels and
requirements may
be selected by those of ordinary skill in the art from available methods and
techniques. For
example a compound of this invention may be combined with a pharmaceutically
acceptable
adjuvant for administration to a virally infected patient in a
pharmaceutically acceptable
manner and in an amount effective to lessen the severity of the viral
infection. Also, a
compound of this invention may be combined with pharmaceutically acceptable
adjuvants
conventionally employed in vaccines and administered in prophylactically
effective amounts to
protect individuals over an extended period of time against viral infections,
such as HIV
infection. As such, the novel integrase inhibitors of this invention can be
administered as agents
for treating or preventing viral infections, including HIV infection, in a
mammal. The
compounds of this invention may be administered to a healthy or HIV-infected
patient either as
a single agent or in combination with other antiviral agents which interfere
with the replication
cycle of HIV. By administering the compounds of this invention with other
antiviral agents
which target different events in the viral replication cycle, the therapeutic
effect of these
compounds is potentiated. For instance, the co-administered antiviral agent
can be one which
targets early events in the life cycle of the virus, such as cell entry,
reverse transcription and
viral DNA integration into cellular DNA. Antiviral agents targeting such early
life cycle events
include, didanosine (ddI), zalcitabine (ddC), stavudine (d4T), zidovudine
(AZT), polysulfated
polysaccharides, sT4 (soluble CD4) -- which blocks attachment or adsorption of
the virus to
host cells -- and other compounds which block binding of virus to CD4
receptors on CD4-
bearing T-lymphocytes. Other retroviral reverse transcriptase inhibitors, such
as derivatives of
29
CA 02302144 2000-03-27
AZT, may also be co-administered with the compounds of this invention to
provide therapeutic
treatment for substantially reducing or eliminating viral infectivity and the
symptoms
associated therewith. Examples of other antiviral agents include ganciclovir,
dideoxycytidine,
trisodium phosphonoformiate, eflornithine, ribavirin, acyclovir, alpha
interferon and
trimenotrexate. Additionally, non-ribonucleoside inhibitors of reverse
transcriptase, such as
TIBO or nevirapine, may be used to potentiate the effect of the compounds of
this invention, as
may viral uncoating inhibitors, inhibitors of trans-activating proteins such
as tat or rev, or
inhibitors of the viral protease. These compounds may also be co-administered
with other
inhibitors of HIV integrase.
Combination therapies according to this invention exert a synergistic effect
in inhibiting HIV
replication because each component agent of the combination acts on a
different site of HIV
replication. The use of such combinations also advantageously reduces the
dosage of a given
conventional anti-retroviral agent that would be required for a desired
therapeutic or
prophylactic effect as compared to when that agent is administered as a
monotherapy. These
combinations may reduce or eliminate the side effects of conventional single
anti-retroviral
agent therapies while not interfering with the anti-retroviral activity of
those agents. These
combinations reduce potential of resistance to single agent therapies, while
minimizing any
associated toxicity. These combinations may also increase the efficacy of the
conventional
agent without increasing the associated toxicity. Preferred combination
therapies include the
administration of a compound of this invention with AZT, 3TC, ddI, ddC or d4T.
Alternatively, the compounds of this invention may also be co-administered
with other HIV
protease inhibitors such as Ro 31-8959 (Roche), L-735,524 (Merck), XM 323
(Dupont Merck)
and A-80,987 (Abbott) to increase the effect of therapy or prophylaxis against
various viral
mutants or members of other HIV quasi species.
We prefer administering the compounds of this invention as single agents or in
combination
with retroviral reverse transcriptase inhibitors, such as derivatives of AZT
or HIV aspartyl
protease inhibitors. We believe that the co-administration of the compounds of
this invention
with retroviral reverse transcriptase inhibitors or HIV aspartyl protease
inhibitors may exert a
CA 02302144 2000-03-27
substantial synergistic effect, thereby preventing, substantially reducing, or
completely
eliminating viral infectivity and its associated symptoms.
The compounds of this invention can also be administered in combination with
immunomodulators (e.g., bropirimine, anti-human alpha interferon antibody, IL-
2, GM-CSF,
methionine enkephalin, interferon alpha, diethyldithiocarbante, tumor necrosis
factor,
naltrexone and rEPO); antibiotics (e.g., pentamidine isethionate) or vaccines
to prevent or
combat infection and disease associated with HIV infection, such as AIDS and
ARC.
When the compounds of this invention are administered in combination therapies
with other
agents, they may be administered sequentially or concurrently to the patient.
Alternatively,
pharmaceutical or prophylactic compositions according to this invention may be
comprised of a
combination of an integrase inhibitor of this invention and another
therapeutic or prophylactic
agent.
Although this invention focuses on the use of the compounds disclosed herein
for preventing
and treating HIV infection, the compounds of this invention can also be used
as inhibitory
agents for other viruses that depend on similar integrases for obligatory
events in their life
cycle. These viruses include, but are not limited to, other diseases caused by
retroviruses, such
as simian immunodeficiency viruses, HTLV-I and HTLV-II.
Pharmaceutical compositions of this invention comprise any of the compounds of
the present
invention, and pharmaceutically acceptable salts thereof, with any
pharmacentically acceptable
carrier, adjuvant or vehicle. Pharmaceutically acceptable carriers, adjuvants
and vehicles that
may be used in the pharmaceutical compositions of this invention include, but
are not limited to
ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as
human serum
albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium
sorbate, partial
glyceride mixtures of saturated vegetable fatty acids, water, salts or
electrolytes, such as
protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,
sodium
chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl
pyrrolidone, cellulose-
based substances, polyethyleneglycol, sodium carboxymethylcellulose,
polyacrylates, waxes,
31
CA 02302144 2000-03-27
polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool
fat.
The pharmaceutical compositions of this invention may be administered orally,
parenterally by
inhalation spray, topically, rectally, nasally, buccally, vaginally or via an
implanted reservoir.
We prefer oral administration or administration by injection. The
pharmaceutical compositions
of this invention may contain any conventional non-toxic pharmaceutically
acceptable carriers,
adjuvants or vehicles. The term "parenteral" as used herein includes
subcutaneous,
intracutaneous, intravenous, intramuscular, infra-articular, intrasynovial,
intrasternal,
intrathecal, intralesional and intracranial injection or infusion techniques.
The pharmaceutical compositions may be in the form of a sterile injectable
preparation, for
example, as a sterile injectable aqueous or oleaginous suspension. This
suspension may be
formulated according to techniques known in the art using suitable dispersing
or wetting agents
(such as, for example, Tween 80)-and suspending agents. The sterile injectable
preparation may
also be a sterile injectable solution or suspension in a non-toxic
parenterally acceptable diluent
or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable
vehicles and
solvents that may be employed are mannitol, water, Ringer's solution and
isotonic sodium
chloride solutions. In addition, sterile, fixed oils are conventionally
employed as a solvent or
suspending medium. For this purpose, any bland fixed oil may be employed
including synthetic
mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride
derivatives are useful in
the preparation of injectables, as are natural pharmaceutically-acceptable
oils, such as olive oil
or castor oil, especially in their polyoxyethylated versions. These oil
solutions or suspensions
may also contain a long-chain alcohol diluent or dispersant, such as Ph. Helv.
or a similar
alcohol.
The pharmaceutical compositions of this invention may be orally administered
in any orally
acceptable dosage form including, but not limited to, capsules, tablets, and
aqueous suspension
and solutions. In the case of tablets for oral and carriers which are commonly
used include
lactose and corn starch. Lubricating agents, such as magnesium stearate, are
also typically
added. For oral administration in a capsule form, useful diluents include
lactose and dried corn
starch. When aqueous suspensions are administered orally, the active
ingredient is combined
32
CA 02302144 2000-03-27
with emulsifying and suspending agents. If desired, certain sweetening and/or
flavoring and/or
coloring agents may be added.
The pharmaceutical compositions of this invention may also be administered in
the form of
suppositories for rectal administration. These compositions can be prepared by
mixing a
compound of this invention with a suitable non-irritating excipient which is
solid at room
temperature but liquid at the rectal temperature and therefore will melt in
the rectum to release
the active components. Such materials include, but are not limited to, cocoa
butter, beeswax,
and polyethylene glycols.
Topical administration of the pharmaceutical compositions of this invention is
especially useful
when the desired treatment involves areas or organs readily accessible by
topical application.
For application topically to the skin, the pharmaceutical composition should
be formulated with
a suitable ointment containing the active components suspended or dissolved in
a carrier.
Carriers for topical administration of the compounds of this invention
include, but are not
limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol,
polyoxyethylene
polyoxypropylene compound, emulsifying wax and water. Alternatively, the
pharmaceutical
compositions can be formulated with a suitable lotion or cream containing the
active compound
suspended or dissolved in a carrier. Suitable carriers include, but are not
limited to mineral oil,
sorbitan monostearate, polysorbate 60, cetyl esters wax cetearyl alcohol, 2-
octyldodecanol,
benzyl alcohol and water. The pharmaceutical compositions of this invention
may also be
topically applied to the lower intestinal tract by rectal suppository
formulation or in a suitable
neat formulation. Topically-transdermal patches are also included in this
invention.
The pharmaceutical compositions of this invention may be administered by nasal
aerosol or
inhalation. Such compositions are prepared according to techniques well-known
in the art of
pharmaceutical formulation and may be prepared as solutions in saline
employing benzyl
alcohol or other suitable preservatives, absorption promoters to enhance
bioavailability,
fluorocarbons, and/or other solubilizing or dispersing agents known in the
art.
Dosage levels of between about 0.01 and about 25 mg/kg body weight per day,
preferably
33
CA 02302144 2000-03-27
between about 0.5 and about 25 mg/kg body weight per day of the active
ingredient compound
are useful in the prevention and treatment of viral infection, including HIV
infection. Typically,
the pharmaceutical compositions of this invention will be administered from
about 1 to about 5
times per day or alternatively, as a continuous infusion. Such administration
can be used as a
chronic or acute therapy. The amount of active ingredient that may be combined
with the
carrier materials to produce a single dosage form will vary depending upon the
patient treated
and the particular mode of administration. A typical preparation will contain
from about 5% to
about 75% active compound (w/w). Preferably, such preparations contain from
about 20% to
about 50% active compound.
Upon improvement of a patient's condition, a maintenance dose of a compound,
composition
or combination of this invention may be administered if necessary.
Subsequently, the dosage or
frequency of administration, or both, may be reduced, as a function of the
symptoms, to a level
at which the improved condition is retained. When the symptoms have been
alleviated to the
desired level, treatment should cease, at least in principle. Patients may,
however, require
intermittent treatment on a long-term basis, upon any recurrence of disease
symptoms,
especially for AIDS.
As the skilled artisan will appreciate, lower or higher doses than those
recited above may be
required. Specific dosage and treatment regimen for any particular patient
will depend upon a
variety of factors, including the activity of the specific compound employed,
the age, body
weight, general health status, sex, diet, time of administration, rate of
excretion, drug
combination, the severity and course of the infection, the patient's
disposition to the infection
and the judgment of the treating physician.
The compounds of this invention are also useful as commercial reagents which
effectively bind
to integrases, particularly HIV integrase. As commercial reagent, the
compounds of this
invention, and their derivatives, may be used to block integration of a target
DNA molecule by
integrase, or may be derivatized to bind to a stable resin as a tethered
substrate for affinity
chromatography applications. These and other uses which characterize
commercial integrase
inhibitors will be evident to those of ordinary skill in the art.
34
CA 02302144 2000-03-27
DETAILED DESCRIPTION OF THE INVENTION
In order that the invention herein described may be more fully understood, the
following
detailed description is set forth. In the description, the following
abbreviations are used:
Desi n~ Reagent or Fra, ment
Et ethyl
Trityl triphenylmethyl
I O Ala DL, D - or L-alanine
Asn DL, D- or L-asparagine
Cys DL, D- or L-cysteine
Gly glycine
Gln DL, D- or L-glutamine
His DL, D- or L-histidine
Ile DL, D- or L-isoleucine
Leu DL, D- or L-leucine
Met DL, D- or L-methionine
Phe DL, D- or L-phenylalanine
Pro DL, D- or L-proline
Ser DL, D- or L - serine
Thr DL, D- or L-threonine
Trp DL, D- or L-tryptophan
Val DL, D- or L-valine
Boc tent-butoxycarbonyl
EDC I -(3-dimethylaminopropyl)-3-ethylcarbodiimide
BOP benzotriazol-1-yloxytris(dimethylamino)phosphonium
hexafluorophosphate
TFA trifluoroacetic acid
EtOAC ethyl acetate
DMF dimethylformamide
CA 02302144 2000-03-27
AZT zidovudine
IL-2 interleukin-2
rEPO recombinant erythropoietin
EtOH ethyl alcohol
MeOH methyl alcohol
THF tetrahydrofuran
CHZCIz dichloromethane
Clz-Bzl 2,6-dichlorobenzyl
tent-Bu tent-butyl
Bzl benzyl
NMP N methylpyrrolidone
CHC13 chloroform
Combinations of substituents and variables envisioned by this invention are
only those that
result in the formation of stable compounds.
The term stable, as used herein, refers to compounds which possess stability
sufficient to allow
manufacture and administration to a mammal by methods known in the art.
Typically, such
compounds are stable at a temperature of 40 °C or less, in the absence
of moisture or other
chemically reactive conditions, for at least a week.
EXAMPLES
In order that this invention be more fully understood, the following examples
are set forth. These
examples are for the purpose of illustration only and are not to be construed
as limiting the scope
of the invention in any way.
Materials and Methods
Analytical thin layer chromatography (TLC) was carried out with 0.25 mm silica
gel E. Merck 60
Fzsa Plates and eluted with the indicated solvent systems. Preparative
chromatography was
36
CA 02302144 2000-03-27
performed by flash chromatography, using silica gel 60 (EM Science) with the
indicated solvent
systems and a positive nitrogen pressure to allow proper rate of elution.
Detection of the
compounds was carried out by exposing eluted plates (analytical or
preparative) to UV light and/or
treating analytical plates with a 2% solution ofp-anisaldehyde in ethanol
containing 3% sulfuric
acid and 1 % acetic acid followed by heating.
Unless otherwise indicated, all starting materials were purchased from a
commercial source such
as Aldrich Co. or Sigma Co.
Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AMX 500
equipped with
a reversed or QNP probe. Samples were dissolved in deuterochloroform (CDC13),
deuteroacetone
(acetone-db) or deuterodimethylsulfoxide (DMSO-db) for data acquisition using
tetramethylsilane
as internal standard. Chemical shifts are expressed in parts per million
(ppm), the coupling
constants (J) are expressed in hertz (Hz) whereas multiplicities are denoted
as s for singlet, d for
doublet, dd for doublet of doublets, t for triplet, q for quartet, m for
multiplet, and br s for broad
singlet.
GENERAL PROCEDURES
Example 1. Preparation of N (tert-butoxycarbonyl)amino acids
To a solution of amino acid (1 eq.) in water and dioxane were added at room
temperature
triethylamine (1.3-1.5 eq.) and Boc-ON (l.l eq.) or di-tert-butyl-dicarbonate
(2 eq.). The
mixture was stirred at room temperature under argon for 3 to 5 h. The solution
was diluted with
water and extracted by ether at least six times. The aqueous layer was
acidified to pH ~ 2.5 with
cold 1N HCl to yield an oily layer. The mixture was extracted three times with
methylene
chloride. The combined organic extracts were washed with brine and dried over
magnesium
sulfate. After filtration, the filtrate was evaporated using a bath set at
30°C. The residue was
found to be of sufficient purity for the next reaction step.
37
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Example 2. Benzylation of N Boc amino acid
Three different solvent systems were used to achieve benzylation of acids or
hydroxyl groups.
a) methanol/water followed by DMF method
To a N Boc amino acid ( 1 eq.) in methanol was added cesium carbonate ( 1.4-
2.0 eq.) as a 20%
solution in water, and then the solution was evaporated to dryness. The
residue was dissolved
in dimethylformamide (DMF) and benzyl bromide (1.1-1.5 eq.) was added. The
mixture was
stirred at room temperature under argon overnight. The mixture was diluted
with water and
the organic layer was extracted with ethyl acetate. The combined organic
phases were washed
with brine and dried over magnesium sulfate. The solids were filtered off and
solvent was
evaporated under vacuum yielding a residue that was purified by silica gel
chromatography
using 20% ethyl acetate in hexane.
b) dimethylformamide method
To a N Boc amino acid (1 eq.) in dimethylformamide (DMF) were added cesium
carbonate
(1.4-2.0 eq.) and benzyl bromide (l.l-1.5 eq.). The reaction mixture was
stirred at room
temperature overnight under argon. A work-up and purification as previously
described in
example 2a yielded the desired product.
c) acetone method
To a N Boc amino acid (1 eq.) in acetone were added potassium carbonate (1.4-
2.0 eq.) and
benzylbromide ( 1.1-1.5 eq.). The reaction mixture was stirred at room
temperature for a period
of 3 - 5 h under argon. Work-up and purification as carried out in the
previous example 2a
afforded the desired product.
Example 3. Removal of the N tert-butoxycarbonyl (Boc) group
38
CA 02302144 2000-03-27
A solution of N tent-butoxycarbonyl amino acid ( 1 eq.) in a 1:1 mixture of
trifluoroacetic acid
(TFA) (10 eq.) and methylene chloride (CHZC12) was stirred at room temperature
for 15-30
min. The solvent and excess acid were removed under vacuum to yield the
desired product
that was used without further purification.
Example 4. Coupling reaction of hydroxylated benzoic acid with the NH part of
an
amino acid
To a mixture of 3-hydroxy- or 3,4-dihydroxybenzoic acid (1.5 eq.),
hydroxybenzotriazole
hydrate (HOBT) (1.6 eq.), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
(EDC) (1.6
eq.) in DMF was added a solution of product from example 3 (1 eq.) and
triethylamine or
diisopropylethylamine (1 eq.) in DMF. The mixture was stirred at room
temperature under
argon for either 6 h or overnight, monitoring the reaction by TLC. The
reaction mixture
was quenched by water and extracted three times with ethyl acetate. The
organic phases
were combined and washed with brine. After drying over magnesium sulfate, the
solution
was filtered and the solvent was evaporated under vacuum. The residue was
purified by
silica gel chromatography, eluting as indicated in each procedure.
Example 5. Cleavage of benzyl esters or benzyl ethers
The benzyl ester or benzyl ether of an amino acid derivative dissolved in
methanol was
hydrogenated over 10% Pd-C (less than 10% by weight of the weight the amino
acid
benzyl ester or ether) under 1 atmosphere of Hz for 1-2 h. The catalyst was
filtered off and
the filtrate was evaporated under vacuum to yield the desired product.
Example 6. Coupling reaction of dopamine with the COON of a substituted amino
acid
To a solution of substituted carboxylic acid (1 eq.) prepared as in example 5,
HOBT (1.5 eq.)
39
CA 02302144 2000-03-27
and EDC ( 1.5 eq.) in DMF at 0°C was added a solution of dopamine
hydrochloride (2 eq.) and
triethylamine or diisopropylethylamine (2 eq.) in DMF. The mixture was stirred
under argon
for 0.5 h and the mixture was allowed to reach room temperature and stirred
overnight. The
resulting mixture was diluted with water and extracted three times with ethyl
acetate. The
organic phases were combined and washed with brine. After drying over
magnesium sulfate,
the solution was filtered and the solvent was evaporated under vacuum. The
residue was
purified by silica gel chromatography, eluting agent as indicated in each
procedure.
Example 7. Removal of the N 9-fluorenylmethoxycarbonyl (Fmoc) group
A solution of N (9-fluorenylmethoxycarbonyl) amino acid (1 eq.) in 30%
diethylamine in
acetonitrile was stirred 15 min at room temperature. The solvent was removed
under vacuum
to yield the desired product that was used without further purification.
Example 8. Removal of the methyl ester group
Amino acid methyl ester (0.2 eq.) was dissolved in methanol at room
temperature 1N sodium
hydroxide (0.65 mL) was added, the mixture was stirred for 0.5 h and 1N HCl
(0.3 mL) was
added, maintaining the temperature at around 0°C. After removing the
methanol under
vacuum, a second portion of 1N HCl (0.3 mL) was added to adjust the pH at
~2.5. The
organic acid was extracted with CHZCIz, dried over magnesium sulfate and
concentrated in
vacuo, yielding the desired product that was used for the next step without
further purification.
Example 9. Coupling reaction of hydroxylated benzoic acid with the NH part of
an
amino acid using BOP reagent
The acid (O.1M in a 1-1 mixture of dioxan and dichloromethane) and BOP reagent
(1.0 eq.)
was stirred at room temperature under an inert atmosphere. The amine (1.2 eq.)
was directly
added followed by the base (triethylamine, 1.2 eq.). The reaction was stirred
for 3 to 16 h. The
suspension was then poured in an extraction vessel containing ethyl acetate
and 1N
CA 02302144 2000-03-27
hydrochloric acid and the organic layer washed with 3 portions of water before
drying over
magnesium sulfate. The solution was filtered and concentrated in vacuo before
purification
by flash chromatography.
Specific examples for the preparation of derivatives in accordance with the
present invention
Example 10. Preparation of the N [1V'-(3',4'-dihydroxybenzoyl)-D-tyrosyl]-
dopamine
Step A. Preparation of N-(tent-butoxycarbonyl)-D-tyrosine
The title compound was prepared from D-tyrosine (543 mg, 3.0 mmol), by
following the
procedure described in example 1. The product was isolated as a colorless
syrup (740 mg,
88% yield).
'H NMR (DMSO-db): 1.32 (s, 9H); 2.71 (dd, J = 3.3, 12.9, 1H); 2.89 (dd, J =
3.8, J = 12.3,
1 H); 4.00 (m, 1 H); 6.64 (d, J = 8.6, 2H); 6.95 (d, J = 8.0, 1 H); 7.03 (d, J
= 8.6, 2H); 9.18
(br s, 1 H); 12.46 (s, 1 H).
Step B. Preparation of N (tent-butoxycarbonyl)-O-benzyl-D-tyrosine benzyl
ester
The title compound was prepared from the product obtained in step A of this
example (650
mg, 2.3 mmol) according to the indications of example 2a. The crude product
was purified
by silica gel column chromatography using 5% MeOH/CHC13 to yield the desired
product
(650 mg, 61 %).
'H NMR (DMSO-db): 1.32 (s, 9H); 2.83 (dd, J = 9.8, 13.5, 1H); 2.93 (dd, J =
5.5, 13.8,
1 H); 4.17 (q, J = 7.1, 8.3, 1 H); 5.05 (s, 2H); 5.08 (s, 2H); 6.91 (d, J =
8.2, 2H); 7.13 (d, J =
8.2, 2H); 7.28-7.44 (m, lOH).
41
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Step C. N (3',4'-dihydroxybenzoyl)-O-benzyl-D-tyrosine benzyl ester
The title compound was prepared from the product obtained in step B of this
example (110
mg, 0.24 mmol) by the removal of the Boc group following the indications of
example 3.
The resulting unblocked derivative was then coupled with 3,4-dihydroxybenzoic
acid
according to the indications of example 4. The crude product was purified by
silica gel
column chromatography using 5% methanol/chloroform to yield the desired
product as a
white solid, mp. 140°C (dec.), (88 mg, 74%).
'H NMR (CDC13): 3.15 (m, 2H); 4.98 (s, 2H); 5.01 (m, 1H); 5.18 (m, 2H); 6.63
(d, J = 7.4,
1 H); 6.80 (d, J = 7.4, 2H); 6.85 (d, J = 8.9, 1 H); 6.92 (d, J = 7.4, 2H);
7.08 (d, J = 7.4, 2H);
7.26 (s, 1H); 7.31-7.41 (m, lOH).
Step D. N [N-(3',4'-dihydroxybenzoyl)-O-benzyl-D-tyrosyl]-dopamine
The title compound was prepared from the product of step C of this example
(200 mg, 0.39
mmol) by removing the benzyl ester group following the indications of example
5. The
resulting unblocked derivative was coupled with dopamine hydrochloride
according to the
indications of example 6. Purification by silica gel chromatography
(3%MeOH/EtOAc)
provided the desired product, (88 mg, 40%) as a white solid, mp. 131°C
(dec.).
'H NMR (DMSO-db): 2.60 (m, 2H), 3.03 (dd, J = 7.9, 13.7, 1 H); 3.16 (dd, J =
5.3, 14.2,
1 H); 3.36 (m, 2H); 4.79 (m, 1 H); 5.02 (s, 2H) 6.69 (d, J = 7.9, 1 H); 6.71
(s, 1 H); 6.85 (d, J
= 7.9, 1 H); 6.89 (d, J = 8.8, 2H); 7.19 (d, J = 6.1, 2H); 7.27 (t, J = 6.7, 1
H); 7.30 (s, 1 H);
7.34 (d, J = 7.4, 1H); 7.30-7.47 (m, SH); 7.58 (d, J = 7.7, 1H); 8.04 (br s,
4H).
Step E N [N-(3',4'-dihydroxybenzoyl)-D-tyrosyl]-dopamine
The title compound was prepared from the product of step D of this example (60
mg, 0.11
mmol) by following the indications in example 5. Purification by silica gel
42
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chromatography (100% EtOAc) provided the desired product, (21 mg, 43%) as a
white
solid, mp. 178°C (dec.).
'H NMR (DMSO-d6): 2.60 (m, 2H); 2,89 (dd, J = 8.0, 13.9, 1H); 3.11 (dd, J =
5.5, 13.9,
1 H); 3.3 6 (m, 2H); 4.74 (m, 1 H); 6.50 (d, J = 7.0, 1 H); 6.68 (d, J = 8.1,
1 H); 6.72 (d, J =
6.3, 1H); 6.73 (s, 1H); 6.83 (d, J = 7.5, 1H); 7.11 (d, J = 8.0, 2H); 7.38 (d,
J = 7.4, 2H);
7.26 (t, J = 6.7, 1 H); 7.48 (d, J = 7.6, 1 H); 8.04 (br s, SH).
Example 11. Preparation of N [N'-(p-hydroxybenzoyl)-L-tyrosyl]-dopamine
Step A. Np-hydroxybenzoyl-O-tent-butyl-L-tyrosine tent-butyl ester
The title compound was prepared from O-tent-butyl-L-tyrosine tent-butyl ester
(445 mg;
1.60 mmol) by following the indications of example 4. Purification by flash
chromatography eluting with 20% ethyl acetate in hexane provided 291 mg (51 %)
of the
title compound, mp. 106°C.
'H NMR: (CDC13) : 1.31 (s, 9H), 1.41 (s, 9H), 3.18 (d, J = 5.6, 2H), 4.91 (m,
1 H), 6.68 (d,
J = 7.3, 1 H), 6.82 (d, J = 8.0, 2H), 6.92 (d, J = 8.3, 2H), 7.07 (d, J = 8.0,
2H), 7.56 (d, J =
8.3, 2H), 8.41 br s, 1 H).
Step B. N [N-(p-benzoyl)-L-tyrosyl]-dopamine
The tert-butyl protecting group was removed by treatment of the product of
step A of this
example (21 mg, 0.05 mmol) with trifluoroacetic acid according to the
conditions in
example 3. The residue was treated without further purification with dopamine
hydrochloride according to the procedure of example 6, providing the title
product (16
mg, 53%), mp. 161°C.
'H NMR (acetone-db): 2.52 - 2.61 (m, 2H), 2.95 (dd, J = 14.4, 8.2, 1H), 3.10
(dd, J = 14.4,
43
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8.2, 1 H), 3.27 - 3.37 (m, 2H), 4.71 - 4.76 (m, 1 H), 6.47 (d, J = 8.4, 1 H),
6.63 - 6.70 (m,
4H), 6.82 (d, J = 6.9, 2H), 7.07 (m, 2H), 7.38 (d, J = 5.2, 1 H), 7.54 (d, J =
8.0, 1 H), 7.69
(d, J = 7.7, 2H), 7.4 - 7.9 (br s, 2H), 8.12 (br s, 1 H).
Example 12. Preparation of N [N'-(3',4'-dihydroxybenzoyl)-L-tyrosylJ-dopamine
Step A. N 3',4'-dihydroxybenzoyl-O-tent-butyl-L-tyrosine tent-butyl ester
The title compound was prepared from O-tent-butyl-L-tyrosine tent-butyl ester
(500 mg;
1.8 mmol) by following the indications of example 4. Purification by flash
chromatography eluting with 10% methanol in methylene chloride provided 511 mg
(80%)
of the title compound, mp. 122°C.
'H NMR: (CDC13) : 0.87 (s, 9H), 0.88 (s, 9H), 3.16 (m, 2H), 4.78 (d, J = 7.5,
1H), 6.85 (d,
J = 9.1, 1 H), 6.89 (d, J = 8.5, 2H), 7.21 (d, J = 8.5, 2H), 7.29 (d, J = 9.1,
2H), 7.42 (s, 1 H),
7.49 (d, J = 7.5, 2H), 8.29 br s, 1 H), 8.50 (br s, 1 H).
Step B. N [N-(3',4'-dihydroxybenzoyl)-L-tyrosyl]-dopamine
The tent-butyl protecting group was removed by treatment of the product of
step A of this
example ( 184 mg, 0.42 mmol) with trifluoroacetic acid according to the
indications of
example 3. The residue was treated without further purification with dopamine
hydrochloride
according to the procedure of example 6, providing the title product ( 128 mg,
66%), mp.
205°C.
'H NMR (acetone-db): 2.56 (m, 2H), 2.99 (dd, J = 13.6, 7.9, 1H), 3.11 (dd, J =
13.6, 5.3, 1H),
3.36 (m, 2H), 4.78 (m, 1 H), 6.46 (d, J = 7.8, 1 H), 6.64 (d, J = 7.8, 1 H),
6.68 s, 1 H), 6.70 (d, J
= 8.3, 2H), 6.80 (d, J = 8.6, 1 H), 7.09 (d, J = 8.3, 2H), 7.24 (d, J = 8.6, 1
H), 7.39 (s, 1 H), 7.55
(m, 1H), 7.65 (d, J = 7.6, 1H), 7.5 - 8.4 (br s, 5H).
44
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Example 13. Preparation of N [N-(p-hydroxybenzoyl)-L-phenylalanyl]-dopamine
Step A. Np-hydroxybenzoyl-L-phenylalanine benzyl ester
The title compound was prepared from L-phenylalanine benzyl ester (400 mg;
1.58 mmol) by
following the indications of example 4. Purification by flash chromatography
eluting with
10% ethyl acetate in hexane containing 1% acetic acid provided 323 mg (92%) of
the title
compound, mp. 163°C.
'H NMR: (CDC13) : 3.16 (dd, J = 12.8, 8.9, 1H), 3.24 (dd, J = 12.8, 8.9, 1H),
4.92 (m, 1H),
5.12 (s, 2H), 6.83 (d, J = 6.6, 2H), 7.15 - 7.31 (m, lOH), 7.70 (m, 3H), 8.90
(br s, 1H).
Step B. N [N-(p-benzoyl)-L-phenylalanyl]-dopamine
The product obtained in step A of this example (287 mg, 0.76 mmol) following
the
indications of examples 5 and 6, provided, after flash chromatography eluting
with 50% ethyl
acetate in dichloromethane containing 1% acetic acid, the desired product (301
mg, 94%), mp.
196°C.
'H NMR (acetone-d6): (two conformers) 2.48 (t, J = 7.6, 2H), 2.60 (t, J = 7.8,
2H), 2.93 (m,
1 H), 3.00 (dd, J = 13.3, 3.8, 1 H), 3.13 - 3.25 (m, 1 H), 3.32 (m, 1 H), 4.59
(m, 1 H), 6.41 (d, J =
7.1, 1 H), 6.57 (m, 2H), 6.76 (m, 2H), 7.12 (m, 1 H), 7.21 (d, J = 7.4, 2H),
7.26 (d, J = 7.5, 1 H),
7.65 (d, J = 7.4, 2H), 8.00 (t, J = 5.7, 1 H), 8.21 (d, J = 8.3 (1 H), 8.59
(s, 1 H), 8.69 (s, 1 H),
9.92 (s, 1H).
Example 14. Preparation of N [N-(3',4'-dihydroxybenzoyl)-L-phenylalanyl]-
dopamine
Step A. N 3,4-dihydroxybenzoyl-L-phenylalanine benzyl ester
The title compound was prepared from L-phenylalanine benzyl ester (400 mg;
1.57 mmol) by
CA 02302144 2000-03-27
following the indications of example 4. Purification by flash chromatography
eluting with
40% ethyl acetate in hexane containing 1 % acetic acid provided 323 mg (60%)
of the title
compound, mp. 155°C.
'H NMR: (CDC13) : 3.24 (dd, J = 13.6, 8.5, 1 H), 3.32 (dd, J = 13.6, 5.2, 1
H), 4.92 (m, 1 H),
5.03 (m, 1 H), 5.17 (s, 2H), 6.92 (d, J = 8.0, 1 H), 7.20 - 7.36 (m, 11 H),
7.53 (s, 1 H), 7.88 (d, J
= 7.3, 1 H), 8.57 (br s, 1 H).
Step B. N [N-(3',4'-benzoyl)-L-phenylalanyl]-dopamine
The product obtained in step A of this example (18 mg, 0.046 mmol) following
the
indications of examples 5 and 6, provided after flash chromatography eluting
with 50% ethyl
acetate containing 1 % acetic acid, the desired product ( 19 mg, 73%), mp. 201
°C.
'H NMR (acetone-db): 2.52 - 2.59 (m, 2H), 3.04 (dd, J = 13.7, 7.7, 1H), 3.18
(dd, J = 13.7,
7.7, 1H), 3.32 (m, 2H), 4.77 (dt, J = 6.3, 7.6, 1H), 6.45 (d, J = 7.1, 2H,
7.11 - 7.26 (m, 6H),
7.34 (s, 1 H), 7.38 (m, 2H), 7.52 (d, J = 7.6, 1 H), 7.98 (s, 4H).
Example 15. Preparation of N [1V'-(3',4'-dihydroxybenzoyl)-glycyl]-dopamine
Step A. N (3,4-dihydroxybenzoyl)-glycine tent-butyl ester
The title compound was prepared from glycine tent-butyl ester (400 mg; 2.57
mmol) by
following the indications of example 4. Purification by flash chromatography
eluting with
40% ethyl acetate in hexane containing 1% acetic acid provided 337 mg (52%) of
the title
compound, mp. 139°C.
'H NMR: (acetone -db) : 1.52 (s, 9H), 4.09 (d, J = 5.5, 2H), 6.95 (d, J = 7.3,
1 H), 7.42 (d, J =
7.3, 1 H), 7.55 (s, 1 H), 7.90 (br s, 1 H), 8.34 (br s, 1 H), 8.60 (br s, 1
H).
46
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Step B. N [N-(3',4'-benzoyl)-glycyl]-dopamine
The product obtained in step A of this example (277 mg, 1.03 mmol) following
the
indications of examples 3 and 6, provided, after flash chromatography eluting
with 30% ethyl
acetate in methylene chloride containing 1% acetic acid, the desired product
(163 mg, 45%),
mp. 155°C.
'H NMR (acetone-db): 2.58 (t, J = 7.1, 2H), 3.34 (dt, J = 7.1, 5.1, 2H), 3.99
(d, J = 5.3, 2H),
6.47 (d, J = 8.0, 1 H), 6.64 (d, J = 8.0, 1 H), 6.67 (s, 1 H), 6.8 5 (d, J =
57.7, 1 H), 7.31 (d, J =
7.7, 1 H), 7.44 (s, 1 H), 7.47 (t, J = 5 .1, 1 H), 7.94 (t, J = 5.0, 1 H), 7.4
- 8.0 (br s, 2H), 8.4 (br s,
2H).
Example 16. Preparation ofN [N-(3',4'-dihydroxybenzoyl)-L-3,4-dihydroxy-
phenylalanyl]-
dopamine
Step A. N (tent-butoxycarbonyl)-O,O'-dibenzyl-3,4-dihydroxyphenyl-L-alanine
benzyl ester
The title compound was prepared from L-3,4-dihydroxyphenylalanine (DOPA) as
described in
examples 1 and 2b. In example 1, di-tert-butyl-dicarbonate (960 mg, 4.4 mmol)
was used
instead of Boc-ON to react with DOPA (790 mg, 4.0 mmol) with triethylamine
(600 mg, 6.0
mmol) as base. The product was used for the next step without purification
following the
indications of example 2b, using 280 mg, (0.94 mmol). Purification by flash
chromatography
using 15% EtOAc/hexane provided the title compound as white crystals (180 mg,
34% ), mp.
106-108°C.
1H NMR (CDC13): 1.41 (s, 9H); 3.00 (d, J = 4.9, 2H); 4.14 (s, 1 H); 4.94 (d, J
= 7.4, 1 H);
5.04-5.10 (m, 6H); 6.57 (d, J = 7.8, 1 H); 6.78 (d, J = 8.4, 1 H); 6.71 (s, 1
H); 7.26-7.43 (m,
15H).
Step B. N (3',4'-dihydroxybenzoyl)-O,O'-(dibenzyl)-L-3,4-
dihydroxyphenylalanine benzyl
47
CA 02302144 2000-03-27
ester
The title compound was prepared by cleaving the Boc protecting group of the
product
prepared in step A of this example (130 mg, 0.23 mmol) and coupling it with
3,4-
dihydroxybenzoic acid as described in example 3 and in example 4 respectively.
Purification
by flash chromatography using 5% MeOH/CHCl3 yielded the desired product as a
white solid
(86 mg, 60% ), mp. 215°C (dec.).
'H NMR (CDC13): 3.11 (m, J = 6.1, J = 14.1, 1 H); 3 .14 (dd, J = 6.7, 13.9, 1
H); 5.00 (s, 1 H);
5.06-5.13 (m, 6H); 6.53 (d, J = 8.0, 1 H); 6.65 (d, J = 7.6, 1 H); 6.69 (s, 1
H); 6.76 (d, J = 8.2,
1H); 6.82 (d, J = 8.1, 1H); 7,05 (d, J = 8.0, 1H); 7.25-7.47 (m, 15H); 7.47
(s, 1H).
Step C. N (3',4'-dihydroxybenzoyl)-L-3,4-dihydroxyphenylalanine
The title compound was prepared by the reduction of the compound obtained in
step B of this
example (64 mg, 0.11 mmol) according to the indications found in example 5.
The product
was purified by flash chromatography eluting with 10% MeOH/EtOAc, affording
the desired
product as a solid (27 mg, 73% yield), mp. 121°C (dec.).
'H NMR (DMSO-db): 2.98 (m, 2H); 4.44 (m, 1H); 6.53 (d, J = 7.9, 1H); 6.59 (d,
J = 7.6, 1H);
6.66 (s, 1 H); 6.75 (d, J = 8.2, 1 H); 7,17 (d, J = 7.9, 1 H); 7.24 (s, 1 H);
8.14 (d, J = 8.2, 1 H);
8.69 (s, 2H), 9.11 (s, 2H); 12.50 (s, H).
Step D. N [N-(3',4'-dihydroxybenzoyl)-L-3,4-dihydroxylphenylalanyl]-dopamine
The product from step C of this example was coupled with dopamine
hydrochloride
according to the procedure of example 6. Finally the O-benzyl ether protecting
groups were
hydrogenolyzed by following the indications of example 5. Purification by
flash
chromatography using 5% MeOH/EtOAc yielded the desired product ( 13.8 mg, 28%
), mp.
142°C (dec.).
48
CA 02302144 2000-03-27
'H NMR (DMSO-db): b 2.48 (m, 2H); 2.67 (m, 2H); 3.32 (m, 2H); 4.47 (m, J =
4.3, J = 9.1,
1 H); 6.42 (d, J = 7.0, 1 H); 6.51 (d, J = 7.4, 1 H); 6.58 (d, J = 7.5, 1 H);
6.59 (d, J = 8.4, 1 H);
6.66 (s, 1 H); 6.74 (d, J = 7.7, 1 H); 7,15 (s, 1 H); 7,17 (d, J = 7.6, 1 H);
7.22 (s, 1 H); 7.93 (t, J =
5.7, 1 H); 7.95 (d, J = 8.5, 1 H); 8.67 (br s, 6H, OH).
Example 17. Preparation of N [lV'-(3',4'-dihydroxybenzoyl)-traps-4-
hydroxyprolyl]-
dopamine
Step A. N (tert-butoxycarbonyl)-O-benzyl-traps-4-hydroxy-L-proline benzyl
ester
The title compound was prepared from traps-4-hydroxyproline as described in
example l and
2b. The Boc derivative was prepared with the following quantities: di-tert-
butyl-dicarbonate
(960 mg, 4.4 mmol), traps-4-hydroxyproline (260 mg, 2.0 mmol), triethylamine
(300 mg, 3.0
mmol). The product was used for the next step without purification. The
benzylation was
performed according to the indications of example 2b. Purification by flash
chromatography
using 20% EtOAc/hexane provided the title compound as syrup (368 mg, 48%).
'H NMR (CDC13): 1.35 and 1.45 (s, 9H); 2.06 (m, 1H); 3.62 (m, 1H); 3.72 (m,
1H); 4.41 (t, J
= 7.4, 1 H); 4.51 (m, 1 H); 5.15 (s, 4H); 7.34-7.37 (m, 1 OH).
Step B. N (3,4-dihydroxybenzoyl)-O-benzyl-traps-4-hydroxy-L-proline benzyl
ester
The title compound was prepared by cleaving the Boc group of the compound
obtained in step
A of this example (350 mg, 0.85 mmol) by following the indications of example
3, and
coupling it with 3,4-dihydroxybenzoic acid (196 mg, 1.27 mmol) according to
example 4.
Purification by flash chromatography using 5% MeOH/CHC13, provided the desired
product
as an oil (275 mg, 72.4% yield).
' H NMR (DMSO-db): 2.02 (m, 1 H); 2.24 (m, 1 H); 3.79 (d, J = 8.4,1 H); 3 .96
(d, J = 10.1, 1 H);
49
CA 02302144 2000-03-27
4.18 (s, 1 H); 4.59 (t, J = 8.4, 1 H); 5.16 (s, 4H); 6.77 (d, J = 7.9, 1 H);
6.87 (d, J = 7.7, 1 H);
6.98 (s, 1 H); 7.31-7.3 7 (m, 1 OH); 9.22 (br s, 1 H); 9.42 (br s, 1 H).
Step C. N [N-(3',4'-dihydroxybenzoyl)-O-benzyl-traps-4-hydroxyprolyl]-dopamine
The title compound was prepared from the product obtained in step B of this
example (420
mg, 1.0 mmol) by removal of the benzyl ester group as described in example 5.
The resulting
acid was then coupled with dopamine hydrochloride as described in example 6.
Flash
chromatography eluting with 1 % MeOH/EtOAc provided the desired product as a
syrup ( 100
mg, 20%).
'H NMR (DMSO-db): 1.88 (m, 1H); 2.28 (m, 1H); 2.53 (m, 2H); 3.16 (m, 2H); 3.58
(d, J =
11, 1 H); 3 .73 (d, J = 8.9, 1 H); 4.11 (s, 1 H) 4.3 5 (m, 2H); 4.48 (t, J =
7.7, 1 H); 6.44 (d, J = 7.7,
1 H); 6.5 8 (s, 1 H); 6.62 (d, J = 8.2, 1 H); 6.77 (d, J = 8.2, 1 H); 6.90 (d,
J = 7.9, 1 H); 7.00 (s,
1 H); 7.21-7.30 (m, SH); 7.95 (t, J = 5.3, 1 H); 8.62 (br s, 1 H); 8.72 (br s,
4H); 9.16 (br s, 1 H);
9.37 (br s, 1H).
Step D. N-[N-(3',4'-dihydroxybenzoyl)-traps-4-hydroxyprolyl]-dopamine
The title compound was prepared from the compound of step C of this example
(30 mg, 0.06
mmol) by following the indications of example 5. Purification by flash column
chromatography with 3% MeOH/EtOAc afforded the desired product (18 mg, 75%) as
white
crystals, mp. 59-62°C.
'H NMR (DMSO-db): 1.81 (m, 1H); 2.04 (m, 1H); 2.51 (m, 2H); 3.17 (m, 2H); 3.34
(d, J =
6.6, 1 H); 3 .69 (d, J = 8.3, 1 H); 4.21 (s, 1 H); 4.47 (t, J = 8.3, 1 H);
6.44 (d, J = 6.7, 1 H); 6.5 8 (s,
1 H); 6.61 (d, J = 7.7, 1 H); 6.76 (d, J = 8.2, 1 H); 6.90 (d, J = 8.0, 1 H);
7.01 (s, 1 H); 8.90 (br s,
4H); 7.90 (t, J = 5.3, 1H).
50
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Example 18. Preparation of N [N-(3',4'-dihydroxybenzoyl)-L-seryl]-dopamine
Step A. N (tent-butoxycarbonyl)-O-benzyl-L-serine benzyl ester
The title compound was prepared from N (tent-butoxycarbonyl)-O-benzyl-L-
serine, (300
mg,1.0 mmol) as described in example 2b. Purification by flash chromatography
eluting with
25% EtOAc/hexane provided the title compound as a syrup (370 mg, 96 %).
'H NMR (CDC13): 1.38 (s, 9H); 3.68 (m, 2H); 4.33 (q, J = 6.2, J = 6.0, 1H);
4.48 (m, 2H);
5.16 (m, 2H); 7.33 (m, lOH).
Step B. N (3,4-dihydroxybenzoyl)-O-benzyl-L-serine benzyl ester
The title compound was prepared by cleaving the Boc group of the compound
obtained in step
A of this example (325 mg, 0.84 mmol) and coupling it with 3,4-
dihydroxybenzoic acid as
described in examples 3 and 4 respectively. Purification by flash
chromatography eluting with
5% MeOH/CHCl3 provided the desired product (220 mg, 62 %).
'H NMR (DMSO-db): 3.83 (m, 2H); 4.52 (m, 2H); 4.74 (q, J = 6.3, J = 6.4, 1H);
5.16 (m, 2H);
6.78 (d, J = 8.4, 1H); 7.26 (d, J = 8.6, 1H); 7.28 (s, 1H); 7.29-7.32 (m,
lOH); 8.42 (d, J = 7.4,
1 H); 9.15 (br s, 1 H); 9.50 (br s, 1 H).
Step C. N [N'-(3',4'-dihydroxybenzoyl)-O-benzyl-L-Beryl]-dopamine
The title compound was prepared from the compound obtained in step B of this
example (160
mg, 0.38 mmol) according to the procedures described in example 5 and 6
respectively.
Purification by flash chromatography eluting with 2.5% MeOH/EtOAc provided the
desired
product (68 mg, 37%).
'H NMR (DMSO-db): 2.52 (m, 2H); 3.19 (m, 2H); 3.69 (d, J = 6.1, 2H); 4.49 (s,
2H); 4.64 (q,
S1
CA 02302144 2000-03-27
J = 5.6, J = 6.4, 1 H); 6.42 (d, J = 7.6, 1 H); 6.57 (s, 1 H); 6.60 (d, J =
8.2, 1 H); 6.77 (d, J = 8.3,
1H); 7.23 (d, J = 7.2, 1 H); 7.25 (s, 1 H); 7.29-7.327 (m, SH); 8.01 (d, J =
7.9, 1H); 8.02 (t, J =
5.4, 1 H); 8.62 (br s, 1 H); 8.72 (br s, 1 H); 9.12 (br s, l H); 9.47 (br s, l
H).
Step D. N [N-(3',4'-dihydroxybenzoyl)-L-seryl]-dopamine
The title compound was prepared from the compound obtained in step C of this
example as
described in example 5. The product was purified by flash chromatography
eluting with 5%
MeOH/EtOAc to provide the product (66%) as a solid, mp. 118°C
(dec.).
'H NMR (DMSO-db): 2.51 (m, 2H); 3.17 (m, 2H); 3.66 (d, J = 6.0, 2H); 4.39 (q,
J = 5.7, J =
6.5, 1 H); 6.43 (d, J = 7.9, 1 H); 6.57 (s, 1 H); 6.59 (d, J = 7.9, 1 H); 6.79
(d, J = 8.5, 1 H); 7.25
(d, J = 7.8, 1H); 7.32 (s, 1H); 7.86 (d, J = 7.8, 1H); 7.95 (t, J = 5.4, 1H);
9.00 (br s, SH).
Example 19. Preparation of N [N'-(p-hydroxybenzoyl)-L-alanyl)-dopamine
Step A. N-(tent-butoxycarbonyl)-L-alanine benzyl ester
The title compound was prepared from N-(tent-butoxycarbonyl)-L-alanine (567
mg, 3.0 mmol)
as described in example 2a. Purification by flash chromatography eluting with
20%
EtOAc/hexane provided the desired product as a syrup (800 mg, 96 %).
'H NMR (CDCl3): 1.38 (d, J =7.3); 1.43 (s, 9H); 4.36 (s, 1H); 5.07 (s, 1H);
5.15 (m, 2H);
7.35 (s, SH).
Step B. N (p-hydroxybenzoyl)-L-alanine benzyl ester
The title compound was prepared from the product prepared in step A of this
example (400
mg, 1.4 mmol) by removing the Boc group following the indications of example
3. The
52
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resulting product was then coupled with p-hydroxybenzoic acid according to
example 4.
Purification by flash chromatography eluting with 5% MeOH/CHCl3 provided the
desired
product as white crystals (270 mg, 64%), mp. 82-84°C.
'H NMR (DMSO-d6): 1.40 (d, J = 7.1, 3H); 4.49 (m, 1H); 5.13 (s, 2H); 6.80 (d,
J = 8.2, 2H);
7.35 (s, SH); 7.75 (d, J = 8.4, 2H); 8.53 (d, J = 6.8, 1H); 9.99 (s, 1H).
Step C. N [N'-(p-hydroxybenzoyl)-L-alanyl]-dopamine
The title compound was prepared from the compound obtained in step B of this
example
(150 mg, 0.50 mmol) following the indications of examples 5 and 6 for the
cleavage of the
benzyl ester and the coupling with dopamine hydrochloride. Purification by
flash
chromatography eluting with 5% MeOH/EtOAc provided the desired product (147
mg,
85%), mp. 127°C (dec.).
'H NMR (acetone-db): 1.28 (d, J = 7.1, 3H); 2.51 (m, 2H); 3.17 (m, 2H), 4.40
(m, 1H);
6.43 (d, J = 7.8, 1 H); 6.57 (s, 1 H); 6.59 (d, J = 8.0, 1 H); 6.80 (d, J =
8.0, 2H); 7.77 (d, J =
7.9, 2H); 7.86 (t, J = 5.1, 1 H); 8.12 (d, J = 7.4, 1 H); 8.16 (s, 1 H); 8.71
(s, 1 H); 9.95 (s,
1 H).
Example 20. Preparation of N [N-(3',4'-dihydroxybenzoyl)-L-alanyl]-dopamine
Step A. N-(3,4-dihydroxybenzoyl)-L-alanine benzyl ester
The title compound was prepared from N (tent-butoxycarbonyl)alanine benzyl
ester (400 mg,
1.4 mmol) by the removal of the Boc group following the indications of example
3. The
resulting compound was then coupled with 3,4-dihydroxybenzoic acid according
to
indications of example 4. Purification by flash chromatography eluting with 5%
MeOH/CHC13
provided the desired product as a syrup (180 mg, 41%).
'H NMR (DMSO-db): 1.40 (d, J =7.1, 3H); 4.47 (m, 1H); 5.13 (s, 2H); 6.78 (d, J
= 8.2, 1H);
53
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7.24 (d, J = 7.9, 1H); 7.32 (s, 1H); 7.35 ( s, 5H); 8.47 (d, J = 6.7, 1H);
9.12 (s, 1H), 9.48 (s,
1 H).
Step B. N [N-(3',4'-dihydroxybenzoyl)-L-alanyl]-dopamine
The title compound was prepared from the compound of step A of this example
(115 mg, 0.36
mmol) according to indications of example 5 and example 6 for the cleavage of
the benzyl
group and the coupling reaction with dopamine hydrochloride. Purification by
flash
chromatography eluting with 5% MeOH/EtOAc provided the desired product (56 mg,
15%),
mp. 205°C (dec.).
'H NMR (DMSO-d6): 1.40 (d, J = 7.1, 3H); 2.63 (t, J = 7.0, 2H); 3.38 (m, 2H);
4.58 (m, 1H);
6.52 (d, J = 6.6, 1 H); 6.67 (d, J = 7.9, 1 H); 6.71 (s, 1 H); 6.87 (d, J =
8.2, 1 H); 7.32 (d, J = 7.9,
1 H); 7.3 9 (s, 1 H); 7.45 (s, 1 H), 7.60 (d, J = 6.9, 1 H); 8.04 (s, 4H).
Example 21. Preparation of N [N-(p-hydroxybenzoyl)-b-(3-hydroxytyramine)-L-
glutamyl]-dopamine
Step A. N (tert-butoxycarbonyl)-b-benzyloxy-L-glutamic acid benzyl ester
The title compound was prepared from N-(tert-butoxycarbonyl)-b-benzyloxy-L-
glutamic acid
(169 mg, 0.50 mmol) as described in example 2a. Purification by flash
chromatography
eluting eith 20% EtOAc/hexane provided the title compound as white crystals
(186 mg, 87%),
mp. 71.5-74°C.
'H NMR (CDC13): 1.42 (s, 9H); 1.99 (m, 1H); 2.22 (m, 1H); 2.44 (m, 2H); 4.39
(s, 1H); 5.09
(s, 4H); 7.33 (s, lOH).
Step B. N-(p-hydroxybenzoyl)-b-benzyloxy-L-glutamic acid benzyl ester
54
CA 02302144 2000-03-27
The title compound was prepared from the product obtained in step A of this
example (350
mg, 0.80 mmol) by the removal of the Boc group following the indications of
example 3. The
resulting product was coupled withp-hydroxybenzoic acid according to the
indications found
in example 4. Purification by flash chromatography eluting with 15%
EtOAc/CHZCIz
provided the desired product as a syrup (161 mg, 43%).
'H NMR (DMSO-d6): 2.06 (d, J = 7.4, 1H); 2.15 (d, J = 5.8, 1H); 4.50 (m, 1H);
5.07 (s, 2H);
5.14 (s, 2H); 6.81 (d, J = 8.4, 2H); 7.34 (s, l OH); 7.77 ( d, J = 8.5, 2H);
8.51 (d, J = 7.5, 1 H);
10.02 (s, 1 H).
Step C. N [N-(p-hydroxybenzoyl)-8-(3-hydroxytyramine)-L-glutamyl]-dopamine
The title compound was prepared from the compound prepared in step B of this
example (116
mg, 0.26 mmol) according to indications found in examples S and 6 for the
cleavage of the
benzyl groups and the coupling reaction with dopamine hydrochloride.
Purification by flash
chromatography eluting with EtOAc provided the desired product (61.2 mg, 44%),
mp. 128°C
(dec.).
'H NMR (DMSO-db): 1.89 (m, 1H); 1.98 (m, 1H); 2.14 (t, J = 7.6, 2H); 2.50 (t,
J = 7.7, 4H~);
3.17 (m, 4H); 4.33 (m, 1H); 6.39-6.43 (m, 2H); 6.56-6.62 (m, 4H); 6.81 (d, J =
8.7, 2H);
7.77 (d, J = 8.1, 2H); 7.85 (t, J = 5.4, 1 H); 7.90 (t, J = 5.4, 1 H); 8.16
(d, J = 7.8, 1 H), 8.61 (s,
2H); 8.72 (s, 2H), 9.98 (s, 1 H).
Example 22. Preparation of N [N-(3',4'-dihydroxybenzoyl)-8-(3-hydroxytyramine)-
L-
glutamyl]-dopamine
Step A. N (3,4-dihydroxybenzoyl)-b-benzyloxy-L-glutamic acid benzyl ester
The title compound was prepared from N (tent-butoxycarbonyl)-b-benzyloxy-L-
glutamic acid
benzyl ester (350 mg 0.82 mmol) by the removal of the Boc group following the
indications
found in example 3. The resulting product was coupled with 3,4-
dihydroxybenzoic acid
CA 02302144 2000-03-27
according to the indications found in example 4. Purification by flash
chromatography eluting
with 5% MeOH/EtOAc provided the desired product as a syrup (169 mg, 36%).
'H NMR (CDC13): 2.15 (m, 1 H); 2.30 (m, 1 H); 2.49 (m, 2H); 4.81 (m, 1 H);
5.06 (s, 2H); 5.16
(s, 2H); 6.86 (d, J = 8.4, 2H); 7.12 ( d, J = 7.4, 1 H); 7.19 ( d, J = 7.4, 1
H); 7.26-7.31 (m, 1 OH);
7.46 (s, 1 H).
Step B. N [N-(3',4'-dihydroxybenzoyl)-b-(3-hydroxytyramine)-L-glutamyl~-
dopamine
The title compound was prepared from the compound obtained in step B of this
example (130
mg, 0.28 mmol) according to the indications of example 5 and 6 for the removal
of the benzyl
groups and the coupling with dopamine hydrochloride. Purification by flash
chromatography
eluting with EtOAc provided the desired product as a foam (23 mg, 15%).
1H NMR (DMSO-d6): 1.87 (m, 1H,); 1.96 (m, 1H); 2.12 (s, 2H); 2.48 (t, J = 7.6,
4H); 3.17
(m, 4H); 4.31 (m, 1 H); 6.40 (d, J = 7.6, 1 H); 6.42 (d, J = 7.3, 1 H); 6.56
(s, 1 H); 6.59 (d, J =
8.6, 1 H); 6.60 (s, 1 H); 6.62 (d, J = 7.7, 4H); 6.76 (d, J = 8.0, 1 H); 7.23
( d, J = 9.6, 1 H); 7.32
(s, 1 H); 7.85 ( t, J = 5.3, 1 H); 7.88 ( t, J = 5.9, 1 H); 8.05 (d, J = 7.8,
1 H), 8.60 (s, 2H); 8.71
(s, 2H), 9.10 (s, 1 H); 9.45 (s, 1 H).
Example 23. Preparation of N [N'-(3',4'-dihydroxybenzoyl)-8-benzyloxy-L-
glutamyl]-
dopamine
Step A. N [N-(tert-butoxycarbonyl)-8-benzyloxy-L-glutamyl]-dopamine
The title product was prepared by the reaction of dopamine hydrochloride with
N (tert-
butoxycarbonyl)-8-benzyloxy-L-glutamic acid (500 mg, 1.15 mmol) according to
the
indications of example 6. Purification by flash chromatography eluting with
25%
EtOAc/CHZC12 containing 1 % acetic acid provided the desired product (400 mg,
65%) as a
56
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solid, mp. 58°C.
'H NMR (acetone-db): 1.40 (s, 9H,); 1.95 (m, 1 H); 2.14 (m, 1 H); 2.46 (t, J =
7.3, 4H); 2.65 (t,
J = 7.0, 2H), 3.38 (m, 2H); 4.19 (m, 1H); 5.11 (s, 2H), 6.19 (d, J = 7.4, 1H);
6.55 (d, J = 8.2,
1 H); 6.74 (m, 2H); 7.29 - 7.3 3 (m, 5H), 7.43 (m, 1 H), 7.75 (br s, 1 H),
7.83 (br s, 1 H).
Step B. N [N-(3',4'-dihydroxybenzoyl)-b-benzyloxy-L-glutamyl]-dopamine
The title compound was prepared from the product of step A of this example
(400 mg, 0.84
mmol) by the removal of the Boc group following the indications of example 3.
The resulting
compound was then coupled with 3,4-dihydroxybenzoic acid according to
indications found in
exemple 4. Purification by flash chromatography eluting with EtOAc containing
1 % acetic
acid provided the desired product (124 mg, 28%) as a solid, mp 108°C.
'H NMR (acetone-db): 2.02 (m, 1H), 2.23 (m, 1H), 2.47 (m, 1H,); 2.50 (t, J =
7.0, 2H); 3.35
(m, 2H); 4.61 (m, 1 H); 5.05 (s, 2H), 6.48 (d, J = 7.6, 1 H); 6.64 (d, J =
7.6, 1 H); 6.68 (s, 1 H);
6.82 (d, J = 8.8, 1 H); 6.90 - 7.03 (m, 1 H); 7.25 - 7.31 (m, 5H), 7.44 (s, 1
H), 7.49 (m, 1 H), 7.66
(d, J = 7.8, 1 H); 7.5 - 8.8 (br s, 4H)
Example 24. Preparation of N [N'-(3',4'-dihydroxybenzoyl)-L-glutaminylJ-
dopamine
Step A. N (tent-butoxycarbonyl)-L-glutamine benzyl ester
The title compound was prepared from N (tent-butoxycarbonyl)-L-glutamine (250
mg, 1.0
mmol) as described in example 2b. Purification by flash chromatography eluting
with 5%
MeOH /EtOAc provided the desired product as crystals (320 mg, 96%), mp. 105.5-
107.5°C.
'H NMR (CDC13): 1.43 (s, 9H); 1.94 (m, 1H); 2.19 ( m, 1H); 2.27 (m, 1H); 4.36
(s, 1H); 5.15
(m, 2H); 5.37 (d, J= 7.3, 1H); 5.58 (s, 1H); 7.35 (m, 5H).
57
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Step B. N (3,4-dihydroxybenzoyl)-L-glutamine benzyl ester
The title compound was prepared from the product of step A of this example
(300 mg, 0.89
mmol) by the removal of the Boc group following the indications of example 3.
The resulting
compound was then coupled with 3,4-dihydroxybenzoic acid according to
indications found in
example 4. Purification by flash chromatography eluting with 5% MeOH/EtOAc
provided the
desired product as a syrup (154 mg, 46%).
'H NMR (DMSO-db): 1.96 (m, 1 H); 2.06 (m, 1 H); 2.21 (m, 2H); 4.40 (m, 1 H);
5.13 (s, 2H);
6.78 (d, J = 8.4, 1H); 6.82 (s, 1H); 7.24 ( d, J = 7.6, 1H); 7.31 ( s, 2H);
7.33-7.36 (m, SH,);
8.52 (d, J = 6.9, 1 H); 9.12 (s, 1 H); 9.49 (s, 1 H)
Step C. N [N-(3',4'-dihydroxybenzoyl)-L-glutamine]-dopamine
The title compound was prepared from the compound obtained in step B of this
example (110
mg, 0.3 mmol) according to the indications found in example 5 and example 6
for the removal
of the benzyl group and the coupling with dopamine hydrochloride. Purification
by flash
chromatography eluting with 7.5% MeOH/EtOAc provided the desired product as a
solid
(24.5 mg, 20%), mp. 164°C (dec.).
'H NMR (DMSO-db): 1.85 m, 1H); 1.95 m, 1H); 2.13 (s, 2H); 2.51 (s, 2H); 3.18
(m, 2H);
4.30 (m, 1 H); 6.41 (d, J = 7.9, 1 H); 6.59 (d, J = 7.8, 1 H); 6.58 (s, 1 H);
6.77 (d, J = 8.5, 1 H);
7.24 (d, J = 8.7, 1 H); 7.29 (s, 1 H); 7.31 (s, 2H); 7.89 (t, J = 5.3, 1 H);
8.12 (d, J = 7.6, 1 H),
8.61 (s, 1 H); 8.72 (s, 1 H), 9.10 (s, 1 H); 9.46 (s, 1 H).
Example 25. Preparation of N [N'-(p-hydroxybenzoyl)-L-leucyl]-dopamine
Step A. N (p-hydroxybenzoyl)-L-leucine methyl ester
The title compound was prepared from L-leucine methyl ester hydrochloride (182
mg, 1.0
58
CA 02302144 2000-03-27
mmol) as described in example 4. Purification by flash chromatography eluting
with 5%
MeOH/CHC13 provided the desired product as white crystals (138 mg, 52%), mp.
138-140°C.
'H NMR (DMSO-db): 0.88 (d, J = 6.5, 3H); 0.93 (d, J = 6.6, 3H); 1.55 (m, 1H);
1.68 (m, 1H);
1.75 (m, 1H); 3.36 (s, 3H); 4.47 (m, 1H); 6.82 (d, J = 8.3, 2H); 7.77 ( d, J =
8.3, 2H); 8.42 (d,
J = 7.7, 1 H); 9.99 (s, 1 H).
Step B. N-[N-(p-hydroxybenzoyl)-L-leucyl]-dopamine
The title compound was prepared from the compound obtained in step A of this
example (96
mg, 0.40 mmol) by saponification of the methyl ester group according to
example 8. The
resulting acid was coupled with dopamine hydrochloride as in example 6. Flash
chromatography eluting with 100% EtOAc provided the title compound (96 mg,
63%), mp.
161°C (dec.).
'H NMR (DMSO-db): 0.84 (d, J = 5.5, 3H); 0.90 (d, J = 6.5, 3H); 1.47 (m, 1H);
1.60 (m, 2H);
2,52 (m, 2H); 3.17 (m, 2H); 4.43 (m, 1H); 6.42 (d, J = 8.0, 1H); 6.58 (d, J =
6.1, 1H); 6.60 (s,
1 H); 6.81 (d, J = 9.1, 2H); 7.78 (d, J = 8.4, 2H); 7.88 (t, J = 5.5, 1 H);
8.09 (d, J = 8.5, 1 H);
8.61 (s, 1 H); 8.69 (s, 1 H); 9.95 (s, 1 H).
Example 26. Preparation of N [N'-(3',4'-dihydroxybenzoyl)-L-leucyl]-dopamine
Step A. N-[N'(tert-butoxycarbonyl)-L-leucyl]-dopamine
The title compound was prepared from N (tent-butoxycarbonyl)-L-leucine (187
mg, 0.75
mmol) by coupling with dopamine hydrochloride as in example 6. Flash column
chromatography eluting with 25% EtOAc/CHZC12 provided the title compound as a
syrup (195
mg, 71 %).
'H NMR (CDC13): 0.88 (t, J = 6.5, 6H); 1.42 (s, 9H); 1.59 (s, 2H); 2.64 (t, J
= 6.7, 2H); 3.44
59
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(m, 2H); 4.13 (s, 1 H); 5.21 (s, 1 H); 6.54 (d, J =7.9, 1 H); 6.66 (s, 1 H);
6.61 (s, 1 H); 6.78 (d, J =
7.7, 1 H).
Step B. N [N-(3',4'-dihydroxybenzoyl)-L-leucyl)-dopamine
The title compound was prepared from the product obtained in step A of this
example (176
mg, 0.48 mmol) by removing the Boc group following the indications of example
3. The
resulting unblocked product was coupled with 3,4-dihydroxybenzoic acid
according to
example 4. Purification by flash chromatography eluting with 5% MeOH/EtOAc
provided the
desired product (42 mg, 22%), mp. 132°C (dec.),
'H NMR (DMSO-db): 0.85 (d, J = 5.6, 3H); 0.89 (d, J = 5.2, 3H); 1.45 (m, 1 H);
1.60 (m, 2H);
2.50 (t, J = 7.2, 2H); 3.19 (m, 2H); 4.40 (m, 1 H); 6.41 (d, J = 7.8, 1 H);
6.57 (s, 1 H); 6.59 (d, J
= 7.6, 1 H); 6.75 (d, J = 7.5, 1 H); 7.24 (d, J = 10, 1 H); 7.31 (s, 1 H);
7.86 (t, J = 5.5, 1 H); 7.86
(d, J = 7.8, 1 H); 8.66 (s, 1 H); 9.08 (s, 1 H); 9.42 (s, 1 H).
Example 27. Preparation of N [N'-(p-hydroxybenzoyl)-L-prolyl)-dopamine
Step A. N [N-(tent-butoxycarbonyl)-L-prolyl]-dopamine
The title compound was prepared from N-(tent-butoxycarbonyl)-L-proline (108
mg, 0.50
mmol) in a coupling reaction with dopamine hydrochloride ( 189 mg, 1.0 mmol)
according to
example 6. Purification by flash column chromatography (5% MeOH/CHC13)
provided the
desired compound as a syrup (128 mg, 73%).
'H NMR (DMSO-db): 1.32 (s, 6H); 1.39 (s, 3H,); 1.72 (m, 2H); 1.76 (m, 1H);
2.06 (m, 1H);
2.50 (s 2H); 3 .13 (m, 1 H); 3.25 (m, 2H); 3.27 (m, 1 H); 3.98 (m, 1 H); 6.43
(d, J = 7.4, 1 H);
6.56 ( s, 1 H); 6.61 (d, J = 7.9, 1 H); 7.86 (s, 1 H); 8.31 (s, 1 H); 8.61 (s,
1 H); 8.71 (s, 1 H).
Step B. N [N-(p-hydroxybenzoyl)-L-prolyl]-dopamine
CA 02302144 2000-03-27
r
The title compound was prepared from the product obtained in step A of this
example by the
removal of the Boc group following the indications found in example 3. The
resulting
unblocked derivative was then coupled with p-hydroxybenzoic acid according to
the
indications found in example 4. Purification by flash chromatography eluting
with EtOAc
afforded the desired product (23 mg, 31 %), mp. 165°C (dec.).
'H NMR (DMSO-db): 1.74 (m, 2H); 1.85 (s, 1H); 2.10 (s, 1H); 2.50 (s 2H); 3.17
(s, 2H); 3.46
(s, 1 H); 3.57 (s, 1 H); 4.09 (s, 1 H); 6.43 (d, J = 6.8, 1 H); 6.58 (s, 1 H);
6.61 (d, J = 7.9, 1 H);
6.78 (d, J = 7.2, 2H); 7.43 (d, J = 7.0, 2H); 7.83 (s, 1 H); 8.63 (s, 1 H);
8.73 (s, 1 H); 9.88 (s,
1H).
Example 28. Preparation of N [1V'-(3',4'-dihydroxybenzoyl)-L-prolyl]-dopamine
The title compound was prepared from N (tert-butoxycarbonyl)-L-prolyl-dopamine
(150 mg,
0.43 mmol) by the removal of the Boc group following the indications found in
example 3.
The resulting unblocked derivative was then coupled with 3,4-dihydroxybenzoic
acid as in
example 4. Purification by flash chromatography eluting with EtOAc afforded
the desired
product (54 mg, 32%) as a solid, mp. 131°C (dec.).
'H NMR (DMSO-db): 1.73 (s, 2H); 1.85 (s, 1H); 2.09 (s, 1H); 2.50 (s 2H); 3.17
(s, 2H); 3.48
(s, 1 H); 3.56 (s, 1 H); 4.09 (s, 1 H); 6.43 (d, J = 6.7, 1 H); 6.58 (s, 1 H);
6.61 (d, J = 78.4, 1 H);
6.76 (d, J = 7.3, 1 H); 6.90 (s, 1 H); 7.00 (s, 1 H); 7.84 ( s, 1 H); 8.62 (s,
1 H); 8.72 (s, 1 H); 9.13
(s, 1 H); 9.33 (s, 1 H).
Example 29. Preparation of N [N'-(p-hydroxybenzoyl)-L-tryptophanyl]-dopamine
Step A. N [N-(tent-butoxycarbonyl)-L-tryptophanyl]-dopamine
The title compound was prepared from N (tert-butoxycarbonyl)-L-tryptophan (204
mg, 0.65
mmol) by coupling with dopamine hydrochloride according to the procedure of
example 6.
Purification by flash chromatography eluting with 2.5% MeOH/EtOAc provided the
title
61
CA 02302144 2000-03-27
compound as a syrup (215 mg, 75%).
'H NMR (DMSO-db): 1.31 (s, 9H); 2.46 (t, J = 7.4, 2H); 3.02 (m, 2H); 3.14 (m,
1H); 3.22(s,
1 H); 4.15 (m, 1 H); 6.43 (d, J = 7.6, 1 H); 6. 5 8 (s, 1 H); 6.62 (d, J =
7.5, 1 H); 6.66 (d, J = 8.1,
1 H); 6.97 (t, J = 7.5, 1 H); 7.05 (t, J = 7.3, 1 H); 7.10 (s, 1 H); 7.31 (d,
J = 7.7, 1 H); 7.58 (d, J =
7.7, 1 H); 7.86 (t, J = 4.7, 1 H); 8.62 (s, 1 H); 8.71 (s, 1 H); 10.77 (s, 1
H).
Step B. N [N-(p-hydroxybenzoyl)-L-tryptophanyl]-dopamine
The title compound was prepared from the product obtained in step A of this
example ( 175
mg, 0.40 mmol) by removing the Boc group following the indications of example
3. The
resulting unblocked derivative was then coupled with p-hydroxybenzoic acid
according to the
indications of example 4. Purification by flash chromatography eluting with 5%
MeOH/EtOAc afforded the desired product (125 mg, 68%) as a foam.
'H NMR (DMSO-db): 2.50 (t, J = 7.6, 2H); 3.11(m, 1H, CH2~2~); 3.18 (m, 2H)
3.24 (m, 1H);
4.65 (m, 1H); 6.43 (d, J = 8.1, 1H); 6.59 (s, 1H,); 6.61 (d, J = 8.1, 1H);
6.76 (d, J = 8.6, 2H);
6.98 (t, J = 7.4, 1 H); 7.05 (t, J = 7.6, 1 H); 7.17 (s, 1 H); 7.30 (d, J =
8.1, 1 H); 7.65 (d, J = 8.0,
1 H); 7.67 (d, J = 8.3, 2H); 8.03 (t, J = 5.3, 1 H); 8.12 (d, J = 8.2, 1 H);
8.69 (s, 2H); 9.95 (s,
1 H); 10.74 (s, 1 H).
Example 30. Preparation of N [N-(3',4'-dihydroxybenzoyl)-L-tryptophyl]-
dopamine
The title compound was prepared from N [(N-(tert-butoxycarbonyl)-L-tryptophyl]-
dopamine
(44 mg, 0.10 mmol) by the removal of the Boc group following the indications
of example 3.
The resulting unblocked derivative was then coupled with 3,4-dihydroxybenzoic
acid
according to the conditions found in example 4. Purification by flash
chromatography eluting
with EtOAc afforded the desired product (25 mg, 53%) as a yellow solid, mp.
119°C (dec.)
'H NMR (DMSO-d6): 2.47 (m, 2H); 3.13 (m, 1 H); 3.17 (m, 2H); 3.22 (m, 1 H,);
4.62 (m, 1 H);
6.43 ( d, J = 7.4, 1 H); 6.59 ( s, 1 H); 6.61 (d, J = 7.9, 1 H); 6.73 (d, J =
8.0, 1 H); 6.97 (t, J = 7.7,
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1 H); 7.04 (t, J = 7.3, 1 H); 7.15 ( s, 1 H); 7.29 (d, J = 8.1, 1 H); 7.24 (s,
1 H); 7.29 (d, J = 6.1,
1 H); 7.64 (d, J = 8.0, 1 H); 7.66 (t, J = 6.6, 1 H); 8.00 (d, J = 7.00, 1 H);
8.61 (s, 1 H); 8.72 (s,
1 H); 9.07 (s, 1 H); 9.44 (s, 1 H); 10.74 (s, 1 H);.
Example 31. Preparation of N [N-(3',4'-dihydroxybenzoyl)-L-methionyl]-dopamine
Step A. N [N-(tent-butoxycarbonyl)-L-methionyl]-dopamine
The title compound was prepared from N (tent-butoxycarbonyl)-L-methionine (250
mg, 1.0
mmol) by coupling with dopamine (380 mg, 2.0 mmol) according to example 6.
Purification
by flash chromatography eluting with 30% EtOAc/CHZCIz yielded the title
compound (230
mg, 60%).
'H NMR (DMSO-db): 1.38 (s, 9H); 1.73 (m, 1H); 1.80 (m, 1H); 2.02 (s, 3H); 2.40
(m, 2H);
2.51 (t, J = 7.6, 2H); 3.24 (m, 2H); 3.96 (m, 1 H); 6.43 (d, J = 7.6, 1 H,);
6.5 7 (s, 1 H); 6.61 (d, J
= 8.4, 1 H); 6.87 (d, J = 7.9, 1 H); 7.78 (t, J = 5.0, 1 H); 8.62 (s, 1 H);
8.70 (s, 1 H).
Step B. N [N-(3',4'-dihydroxybenzoyl)-L-methionine]-dopamine
The title compound was prepared from the product prepared in step A of this
example (150
mg, 0.40 mmol) by the removal of the Boc group following the indications found
in example
3. The resulting unblocked derivative was coupled with 3,4-dihydroxybenzoic
acid according
to the indications of example 4. Purification by flash chromatography eluting
with 5%
MeOH/EtOAc afforded the desired product (40 mg, 24%) as a solid, mp.
126°C (dec.).
'H NMR (DMSO-db): 1.93 (m, 2H); 2.05 (s, 3H); 2.42 (m, 1H); 2.47 (m, 1H); 2.53
(t, J = 7.8,
2H); 3 .17 (m, 2H); 4.43 (m, 1 H); 6.43 (d, J = 8.0, 1 H); 6.57 (s, 1 H,);
6.59 (d, J = 8.0, 1 H);
6.77 (d, J = 8.1, 1 H); 7.25 (d, J = 8. l, 1 H); 7.32 (s, 1 H); 7.89 (t, J =
5.6, 1 H); 8.08 (d, J = 7.9,
1 H); 8.67 (s, 4H).
Example 32. Preparation of N [1V'-(3',4'-dihydroxybenzoyl)-L-lysyl]-dopamine
63
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Step A. N [Na-(9-fluorenylmethoxycarbonyl)-NE"-(tent-butoxycarbonyl)-L-lysyl]-
dopamine
The title compound was prepared from Na (9-fluorenylmethoxycarbonyl)-NE'-(tert-
butoxycarbonyl)-L-lysine (230 mg, 0.50 mmol) by coupling with dopamine
hydrochloride as
described in example 6. Purification by flash chromatography eluting with 40%
EtOAc/CH,CIZ provided the desired product as white crystals, mp. 58-
61°C (280 mg, 93%).
'H NMR(DMSO-db): 1.20 (m, 1H); 1.23 (m, 1H); 1.33 (m, 2H); 1.48 (m, 1H); 1.55
(m, 1H);
2.52 (m, 2H); 2.89 (s, 2H); 3.15 (m, 1 H); 3.22 (m, 1 H); 3.89 (q, JN,~ = 8.5,
J = 6.8, 1 H); 4.21
(t, J = 6.1, 1 H); 4.22 (d, J = 7.4, 2H); 6.43 (d, J = 7.9, 1 H); 6.75 (s, 1
H); 6.61 (d, J = 7.6, 1 H);
6.74 (s, 1 H); 7.37 (d, J = 8.0, 1 H); 7.32 (t, J = 7.4, 2H,); 7.42 (t, J =
7.4, 2H); 7.72 (d, J = 6.6,
2H); 7.86 (t, J = 5.7, 1H); 7.88 (d, J = 7.5, 2H); 8.62 (br s, 1H); 8.71 (br
s, 1H).
Step B. N [N-(3',4'-dihydroxybenzoyl)-L-lysyl]-dopamine
The title compound was prepared from the compound prepared in step A of this
example (140
mg, 0.23 mmol) by the removal of the Fmoc group following the indications
found in example
7. The resulting unblocked derivative was coupled with 3,4-dihydroxybenzoic
acid according to
the indications of example 4. Purification by flash chromatography using EtOAc
provided the
desired product (85 mg, 41 %). The cleaving of the Boc group of the side chain
of the coupled
product (20 mg, 0.04 mmol) was achieved via an acid hydrolysis as described in
example 3. The
solvent and acid were removed under vacuum to afford the desired product (
14.4 mg, 86%),
mp.93.5°C (dec.).
'H NMR(DMSO-db): 1.17 (m, 2H); 1.52 (m, 2H); 1.68 (m, 2H,; 2.53 (m, 2H); 2.76
(s, 2H);
3.18 (m, 2H), 4.34 (q, J = 6.7, J = 8.2, 1 H); 6.43 (d, J = 8.0, 1 H); 6.57
(s, 1 H); 6.61 (d, J = 8.0,
1 H); 6.78 ( d, J = 8.2, 1 H); 7.24 ( d, J = 8.2, 1 H); 7.33 (s, 1 H); 7.91
(t, J = 4.7, 1 H); 7.97 (d, J
= 7.9, 1H); 8.68 (s, 4H).
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Example 33. Preparation of N [N-3',4'-dihydroxybenzoyl)-L-histidyl]-dopamine
Step A. N [Na'-(fluorenylmethoxycarbonyl)-N' ;m-(trityl)-L-histidyl]-dopamine
The title compound was prepared from Na (fluorenylmethoxycarbonyl)-N' ;m-
trityl- histidine (619
mg, 1.0 mmol) according to the indications found in example 6 with dopamine
hydrochloride.
Purification by flash chromatography eluting with 40% EtOAc/CH2Clz provided
the desired
product (390 mg, 52% yield).
'H NMR(DMSO-db): 3.16 (m, 1H); 4.14 (m, 1H); 4.18 (d, J = 7.4, 2H); 4.20 (t, J
= 5.4, 1H);
6.39 (d, J = 7.6, 1 H); 6.56 (s, 1 H); 6.60 (d, J = 7.8, 1 H); 7.03 (s, 6H);
7.07 (t, J = 7.4, 1 H);
7.23 (s, 1 H); 7.27 (m, 2H); 7.31 (s, 9H); 7.40 (m, 2H); 7.68 (m, 2H); 7.85
(d, J = 7.8, 1 H);
7.90 (d, J = 7.7, 2H); 7.95 (s, 1H); 8.63 (br s, 1H); 8.72 (br s, 1H).
Step B. N [N-(3',4'-dihydroxybenzoyl)-L-histidyl]-dopamine
The title compound was prepared from the compound prepared in step A of this
example (320
mg, 0.42 mmol) by the removal of the Fmoc group as described in example 7. The
resulting
unblocked derivative was coupled with 3,4-dihydroxybenzoic acid as in example
4. Purification
by flash chromatography eluting with 5% MeOH/EtOAc provided the desired
compound (85 mg,
41 %). For the removal of the trityl group located on the side chain, the
product (60 mg, 0.089
mmol) was hydrogenolyzed following the conditions found in example 5 affording
the desired
product (30 mg, 79%).
'H NMR(DMSO-db): 2.47 (t, J = 7.6, 2H); 2.96 (m, 2H); 3.17 (m, 2H); 4.57 (q, J
= 7.8, J,V,; _
6.7, 1 H); 6.41 (d, J = 9.1, 1 H); 6.57 (s, 1 H); 6.60 (d, J = 8.2, 1 H); 6.78
( d, J = 8.4, 1 H); 6.87
(s, 1 H); 7.20 (d, J = 7.8, 1 H); 7.28 (s, 1 H); 7.78 (s, 1 H); 7.88 (t, J =
5.7, 1 H); 8.23 (d, J = 7.9,
1 H); 8.65 (s, 2H); 9.15 (s, 1 H). 9.55 (s, 1 H).
Example 34. Preparation of N [N-(3',4'-dihydroxybenzoyl)-L-aspartyl]-dopamine
CA 02302144 2000-03-27
Step A. N tent-butoxycarbonyl-'y-cyclohexyl-L-aspartic acid benzyl ester
The title compound was prepared from N tert-butoxycarbonyl-L-aspartic acid 'y-
cyclohexyl
ester ( 1.0 g, 3.2 mmol) by an alkylation with benzyl bromide following the
indication of
example 2c. The resulting ester was obtained in 98% yield after purification
by flash
chromatography eluting with 15% EtOAc/hexane.
Step B. N-(3,4-dihydroxybenzoyl)-y-cyclohexyloxy-L-aspartic acid benzyl ester
The title compound was prepared from the compound prepared in step A of this
example by
the removal of the Boc group according to the indications found in example 3
and coupling
with 3,4-dihydroxybenzoic acid as indicated in example 4. Purification by
flash
chromatography eluting with 20% ethyl acetate/CHZC12 provided the title
compound (260 mg,
52%).
Step C. N-[N-(3',4'-dihydroxybenzoyl)-y-cyclohexyloxy-L-aspartyl]-dopamine
The title compound was prepared from the compound prepared in step B of this
example (259
mg, 0.59 mmol) by hydrogenolysis of the benzyl ester following the conditions
outlined in
example 5. The resulting product was then subjected to coupling with dopamine
hydrochloride according to example 6. Purification by flash chromatography
eluting with
ethyl acetate afforded the title compound in 49% yield.
Example 35. Preparation of N [N'-(3',4'-dihydroxybenzoyl)-sarcosyl]-dopamine
Step A. N-tent-butoxycarbonyl-sarcosine benzyl ester
The title compound was prepared from N-tert-butoxycarbonyl-sarcosine (2.0 g,
10.6 mmol) by
an alkylation with benzyl bromide following the indication of example 2c. The
resulting ester
(2.89 g; 98%) was obtained after purification by flash chromatography eluting
with 15%
66
CA 02302144 2000-03-27
EtOAc/hexane.
'H NMR (CDCl3): 1.43 (d, 9H), 2.94 (d, 3H), 3.97 (d, 2H), 7.36 (s, 5H)
Step B. N-(3,4-dihydroxybenzoyl)-sarcosine benzyl ester
The title compound was prepared from the compound prepared in step A of this
example by
the removal of the Boc group according to the indications found in example 3
and coupling
with 3,4-dihydroxybenzoic acid as indicated in example 4. Purification by
flash
chromatography eluting with 80% ethyl acetate/CHZC12 provided the title
compound (433 mg,
43%).
'H NMR (CDC13): 3.1 (d, 3H), 3.5 (s, 2H), 5.2 (d, 2H), 6.9 (m, 3H), 7.40 (m,
5H), 9.40 (br s,
2H).
Step C. N [N-(3',4'-dihydroxybenzoyl)-sarcosinyl]-dopamine
The title compound was prepared from the compound prepared in step B of this
example (278
mg, 0.88 mmol) by hydrogenolysis of the benzyl ester following the conditions
outlined in
example 5. The resulting product was then subjected to coupling with dopamine
hydrochloride according to example 6. Purification by flash chromatography
eluting with 5%
MeOH/CHZCl2/1% AcOH afforded the title compound in 50% yield.
'H NMR (CDC13): 2.60 (t, 2H), 2.9 (d, 3H), 3.2 (q, 2H), 3.3 (t, 2H), 6.4 - 7.0
(m, 6H), 9.5 (br
s, 4H).
Example 36. Preparation of N [N-(3',4'-dihydroxybenzoyl)-L-tyrosyl]-L-tyrosine
The dipeptide was prepared using the peptide synthesizer (ABI 430A) utilising
Wang resin
(0.5 mmol).
67
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Step A. HZN-L-tyrosyl-L-tyrosyl-resin
The N-Fmoc-L-tyrosine(t-Bu) - OH ( 1 mmol was activated over a period of 45
min with
HOBT (1.0 eq.) and DCC (1.0 eq.) in 5 mL of N-methylpyrrolidone (NMP). At the
same
time, in a separate flask, the Fmoc protecting group on the tyrosine amino
group bound to the
polymer was removed by two successive treatments of 15 min with a solution of
30%
piperidine in N methylpyrrolidone, followed by a series of washes with NMP.
The activated
ester was then filtered and added to the resin. The suspension was stirred for
2 h. The Fmoc
blocking group was then removed as previously described and the resin was
successively
washed with N methylpyrrolidone and CHzCl2.
Step B. N [N-(3',4'-dihydroxybenzoyl)-L-tyrosyl]-L-tyrosyl-resin
To the dityrosyl moiety bound on the resin (1 g) and 3,4-dihydroxybenzoic acid
(85 mg; app. 3
eq.) in DMF (5 mL) and CHZC12 (2 mL) were added BOP (benzotriazol-1-yloxy-tris-
dimethylamino)phosphonium hexafluorophosphate) (240 mg, app. 3 eq.) and
diisopropylethylamine (125 ~L; 4 eq.). The flask was stirred under nitrogen
for a period of 16
h. The resin was filtered off, washed successively with DMF, MeOH and CHZCl2,
yielding
518 mg of crude resin.
Step C. N [N-(3',4'-dihydroxybenzoyl)-L-tyrosyl]-L-tyrosine
A mixture of 0.5 mL water in 4.5 mL of trifluoroacetic acid was cooled to
0°C and was added
to the crude resin. The resulting suspension was stirred, allowing the mixture
to reach room
temperature over a period of 2 h. The mixture was then filtered and the resin
washed with 5
mL of acetic acid. The filtrate was evaporated to dryness in vacuo and the
residue was
purified by preparative HPCL, using a Supelcosil C-18 column, flow rate: 18
mLlmin;
gradient: 0.1%TFA from 0-30% acetonitrile over 40 min. Retention time: 21 min.
5 mg (4%)
of the title compound was recovered (4% yield).
68
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Example 37. Preparation of N-[N'-(3',4'-dihydroxybenzoyl)-L-tyrosyl]-glycine
Step A. N (3,4-dihydroxybenzoyl)-O-tert-butyltyrosine tert-butyl ester
The hydrochloric salt of L-O-tent-butyltyrosine tent-butyl ester (800 mg, 2.4
mmol) was
coupled with 3,4-dihydroxybenzoic acid by following the directions found in
example 9.
Purification by flash chromatography eluting with 40% ethyl acetate in hexane
afforded the
desired product (385 mg, 37%).
'H NMR (CDC13): 1.31 (s, 9H), 1.42 (s, 9H), 3.13 (dd, J = 5.8, 14.3, 1H), 3.17
(dd, J = 6.3,
14.3, 1 H), 4.85 (ddd, J = 5.8, 6.3, 4.0, 1 H), 6.77 (d, J = 7.4, 1 H), 6.5-
6.9 (br s, 0.5H) and 7.26
(s, 0.5H), 6.80 (d, J = 8.2,), 6.91 (d, J = 8.0, 2H,), 7.03 (d, J = 8.2, 1H),
7.08 (d, J = 8.0, 2H),
7.54 (s, 1H), 7.8-8.4 (br s, 1H).
Step B. N-[N-(3',4'-dihydroxybenzoyl)-L-O-tent-butyltyrosyl]-glycine
The product of step A of this example (375 mg) was stirred with TFA (10 mL)
for a period of
90 min. The mixture was evaporated to dryness in vacuo and several h with a
mechanical
pump providing a quantitative yield (200 mg) of the free acid. The acid was
then condensed
using the BOP procedure as found in example 9 with glycine tent-butyl ester
hydrochloride
salt (80 mg,1.5 eq.) and triethylamine ( 132 ~L, 3.0 eq.) for a period of 15
h. Purification by
flash chromatography eluting with 80% EtOAc in hexane afforded the desired
product (120
mg, 89%) as the tent-butyl ester. The ester was hydrolysed using 0.5 mL of
water and 7 mL of
TFA with stirring for 1 h. The mixture was then evaporated in vacuo and
purified by flash
chromatography eluting with 5% methanol in dichloromethane containing 1%
acetic acid to
provide the desired product (35 mg, 34%).
'H NMR (DMSO-db): 2.86 (dd, J = 10.8, 13.7,1H), 2.98 (dd, J = 1.4, 13.7, 1H),
3.66-3.92 (m,
2H), 6.61 (d, J = 8.2, 2H), 6.73 (d, J = 8.4, 1 H), 7.09 (d, J = 8.2, 1 H),
7.16 (d, J = 8.4, 1 H),
7.23 (s, 1H), 8.09 (d, J = 8.5, 1H), 8.26 and 8.38 (2t, J = 5.6, 2x 0.5H).
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Example 38. N [N-[N'-(3",4"-dihydroxybenzoyl)-L-tyrosylJ-glycyl]-dopamine
Step A. N Boc-glycyl-dopamine
The title compound was prepared by coupling dopamine hydrochloride with tert-
butoxycarbonylglycine (200 mg, 1.1 mmol) according to the procedure of example
9.
Purification by flash chromatography afforded the title compound (214 mg,
64%).
'H NMR (DMSO-db): 1.38 (s, 9H), 2.5-2.58 (m, 2H), 3.16-3.20 (m, 2H), 3.49 (d,
J = 5.7, 2H),
6.42 (d, J = 7.9, 1 H), 6.57 (s, 1 H), 6.62 (d, J = 7.9), 6.86 (d, J = 5.2; 1
H), 7.71-7.77 (m, 1 H),
8.62 (s, 1 H), 8.72 (s, 1 H).
Step B. N- [N-[N'-(3",4"-dihydroxybenzoyl)-L-tyrosy]-glycyl]-dopamine
The product obtained in step A of this example was hydrolyzed under the
conditions of
example 3 providing an intermediate that was coupled with N (3,4-
dihydroxybenzoyl)-L-
tyrosine under the conditions outlined in example 9. Purification by flash
chromatography
eluting with 5% methanol in ethyl acetate containing 1% acetic acid provided
the title
compound (30 mg, 19%).
'H NMR (DMSO-db): 2.52 (t, J = 7.8, 2H), 2.88 (dd, J = 9.9, 13.8, 1H), 2.97
(dd, J = 4.3, 13.8,
1 H), 3.16-3.21 (m, 2H), 3.61 (dd, J = 5.2, 16.5, 1 H), 3.70 (dd, J = 5.6,
16.5, 1 H), 4.46-4.53
(m, 1 H), 6.44 (dd, J = 1.5, 8.2, 1 H), 6.56 (d, J = 1.5, 1 H), 6.62 (d, J =
8.2, 1 H), 6.63 (d, J =
8.5, 2H), 6.74 (d, J = 7.9, 1 H), 7.09 (d, J = 8.5, 2H), 7.18 (dd, J = 1.7,
7.9, 1 H), 7.25 (d, J =
1.7, 1H), 7.76 (t, J = 5.4, 1H), 8.19-8.23 (m, 2H), 8.1-9.7 (br s, 5H).
Example 39. Preparation of N [N-(3',4'-dihydroxybenzoyl)-glycyl]-L-tyrosine
Step A. N-3,4-dihydroxybenzoyl-glycine
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CA 02302144 2000-03-27
Coupling of the hydrochloride salt of glycine tert-butyl ester with 3,4-
dihydroxybenzoic acid
was obtained by following the indications of example 9. Purification by flash
chromatography
afforded 288 mg (81%) of the title compound after eluting with 4% methanol in
methylene
chloride.
'H NMR (DMSO-db): 1.41 (s, 9H), 3.82 (d, J = 5.6, 2H), 6.76 (d, J = 8.0, 1 H).
7.20 (d, J = 8.0,
1 H). 7.28 (s, 1 H), 8.45 (t, J = 5.6, 1 H),9.13 (s, 1 H), 9.47 (s, 1 H).
Step B. ~'-[N-(3',4'-dihydroxybenzoyl)-glycyl]-L-tyrosine
The product obtained in step A of this example was hydrolyzed by stirring in
TFA (10 mL) far
a period of 90 min. Evaporation of the acid in vacuo afforded a product that
was then reacted
with O-tent-butyl-L-tyrosyl-dopamine using the conditions outlined in example
9. Purification
by flash chromatography eluting with 70% ethyl acetate in hexane provided 230
mg (67%) of
the O-tent-butyl derivative of the title compound.
'H NMR (CDC1;) d 1.28 (s, 9H), 1.30 (s, 9H), 3.00 (d, J = 5.4, 2H), 3.89-4.08
(m, 2H), 4.60-
4.67 (m, 1 H), 6.64 (d, J = 7.3, 1 H), 6.83 (d, J = 8.0, 2H), 6.97-7.05 (m, 1
H), 7.04 (d, J = 8.1,
2H), 7.09-7.18 (m, 2H), 7.32-7.41 (m, 1H).
Treatment of the tent-butyl derivative with TFA for a period of 2 h provided a
quantitative
yield of the title product.
'H NMR (DMSO-db): 2.75-2.93 (m, 2H), 3.73-3.87 (m, 2H), 4.35-4.44 (m, 1H),
6.62 (d, J =
2 5 8.1, 2H), 6. 76 (d, J = 8.5, 1 H), 6.98 (d, J = 8. I , 2H), 7.20 (dd, J =
1.6, J = 8.5, 1 H), 7.30 (d, J =
1.6, 1 H), 7.96 (d, J = 8.1, 0.5H) 8.18 (d, J = 7.7, 0.5H), 8.29-8.36 (m, 1
H), 9.00-9.60 (br s,
4H).
Example 40. N [N-[N'-(3",4"-dihydroxybenzoyl)-glycyl]-L-tyrosylJ-dopamine
71
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Step A. N-L-tyrosine-dopamine
The title compound was prepared from N-tert-butoxycarbonyl-O-2,6-
dichlorobenzyl-L-
tyrosine (2.50 g, 5.7 mmol) according to the indications found in example 6
with dopamine
hydrochloride. Purification by flash chromatography eluting with 50%
EtOAc/hexane
provided the desired product (3.03 g, 93%).
'H NMR (DMSO-db): 1.31 (s, 9H), 2.45-2.55 (m, 2H), 2.67 (dd, J = 10.2, 13.4,
1H), 2.86 (dd,
J = 4.1. I 3.4, 1 H), 3.10-3.18 and 3.20-3.29 (2m, 2H), 4.07 (ddd, J = 4.1, I
0.2, 8.5, 1 H), 5.17
(s, 2H), 6.43 (d, J = 7.8, 1 H), 6.59 (s, 1 H), 6.64 (d, J = 7.8, I H), 6.75
(d, J = 8.5, 1 H), 6.94 (d,
J = 8.2, 2H), 7.15 (d, J = 8.2, 2H), 7.45 (t, J = 8.1, 1H), 7.53 (d, J = 8.1,
2H), 7.87 (t, J = 4.9,
1 H). 8.00-9.15 (br s. 2H).
Step B. ,'~~ -[:~'-[:~~'-(3".4"-dihydroxybenzoyl)-glycyl]-L-tyrosyl]-dopamine
The product obtained in step A of this example was hydrolyzed by stirring in
TFA (10 mL) for
a period of 30 min. Evaporation of the acid in vacuo afforded a product, part
of which (148
mg. 0.70 mmol) was then reacted with A~-3,4-dihydroxybenzoyl-glycine using the
conditions
outlined in example 9. Purification by flash chromatography eluting with 5%
methanol in
ethyl acetate provided 115 mg (25%) of the O-(2,6-dichlorobenzyl derivative of
the title
compound. The latter protected dipeptide derivative (115 mg, 0.17 mmol) in 4
mL of
methanol was then hydrogenolyzed according to the procedure found in example
5.
Purification by flash chromatography eluting with 2% methanol in ethyl acetate
provided 60
mg (68%) of the desired product.
'H NMR (DMSO-db): 2.47 (t, J = 7.7, 2H), 2.65 (dd, J = 8.9, J = 13.6, 1 H),
2.83 (dd, J = 4.7,
13.6, 1 H), 3.08-3.23 (m, 2H), 3.71 (dd, J = 5.6, J = 16.2, 1 H), 3.83 (dd, J
= 5.8, 16.2, 1 H),
4.35 (ddd, J = 4.7, 8.9, 8.4, 1 H), 6.42 (d, J = 7.7, 1 H), 6.57 (s, 1 H),
6.59 (d, J = 8.3, 2H), 6.62
(d, J = 7.7, 1 H), 6.76 (d, J = 8.0, 1 H), 6.95 (d, J = 8.3, 2H), 7.21 (dd, J
= 1.7,8.0, 1 H), 7.30 (d,
J = 1.7, 1 H), 7.94 (d, J = 8.4, 1 H), 7.96 (t, J = 5.5, 1 H), 8.37 (t, J =
5.7, 1 H), 8.50-8.85 (br s,
72
CA 02302144 2000-03-27
2H), 9.05-9.22 (br s, 2H), 9.20-9.60 (br s, 1 H).
Example 41. N-[N'-[1V"-(3",4"-dihydroxybenzoyl)-L-tyrosyl]-L-y-O-benzyl-
aspartyl]-
dopamine
Step A. N-Boc-L-aspartyl-dopamine y-benzyl ester trifluoroacetate
Coupling of N-Boc-L-aspartic acid y-benzyl ester (1.50 g, 4.63 mmol) dopamine
hydrochloride by following the indications of example 6. Purification by flash
chromatography eluting with 50% ethyl acetate in hexane afforded 2.06 g (93%)
of the title
compound after eluting with 40% ethyl acetate in hexane.
'H NMR (DMSO-db): 1.37 (s, 9H), 2.45-2.52 (m, 2H) 2.57 (dd, J = 8.6, 15.9,
IH), 2.70-2.78
(m. 1 H), 3.10 - 3.25 (m. 2H), 4.28 - 4.35 (m, 1 H), 5.08 (s, 2H), 6.41 (d, J
= 7.9, 1 H), 6.56 (s,
1 H), 6.62 (d, J - 7.9, 1 H), 7.04 (d, J = 7.8, 1 H), 7.29 - 7,40 (m, 5H),
7.80 (s, 1 H), 8.62 (s, 1 H),
8.71 (s, 1 H).
Step B. ,~'-[~"-[l~"'-(3".4"-dihydroxybenzoyl)-L-tyrosyl]-L-'y-O-benzyl-
aspartyl]-dopamine
The product obtained in step A of this example (500 mg, 1.00 mmol) was
hydrolyzed by
following the indications of example 3. The resulting product was then coupled
with l~'-3,4-
dihvdroxybenzoyl-L-tyrosine as indicated in example 6. Purification by flash
chromatography
eluting with 5% methanol in methylene chloride containing 1% acetic acid
afforded 69 mg
(35%) of the desired title compound.
'H NMR (DMSO-db): 2 signal sets 2.45-2.67 (m, 3H), 2.75-2.98 (m, 3H), 3.10-
3.25 (m, 2H),
4.39-4.52 (m, 1 H), 4.57-4.69 (m, 1 H), 5.04-5.06 (m, 2H), 6.42 (d, J=7.6, I
H), 6.57-6.76 (m,
4H), 7.05-7.35 (m, 7H), 7.71 (t, J= 5.5, 1 H), 7.93 (t, J=5.5, 1 H), 8.12 (d,
J - 7.6, 1 H), 8.26 (d,
J = 7.7, 1 H), 8u.63 (s, 1 H), 8.73 (s, 1 H), 9.10 (s, 1 H), 9.20 (s, 1 H),
9.51 (s, I H).
73
CA 02302144 2000-03-27
Example 42. Preparation of N-[N-(3',4'-dihydroxyhydrocinnamoyl)-L-tyrosyl]-L-
tyrosine
Step A. N-3,4-dihydroxyhydrocinnamoyl-D-benzyloxy-L-tyrosine
O-benzyloxy-L-tyrosine benzyl esterp-toluenesulfonate salt (1.01 g. 1.9 mmol)
was coupled
with 3,4-dihydroxyhydrocinnamoic acid following the indications of example 4.
Purification
by flash chromatography eluting with 50% ethyl acetate in hexane afforded 855
mg (84%) of
the pure amide.
'H NMR (DMSO-db): 2.29 (t, J = 7.8, 2H), 2.55 (t, J = 7.8, 2H), 2.85 (dd, J =
8.5, 7.6, I H),
2.94 (dd. J = 6.1, 13.8, 1 H), 4.45 (ddd, J = 6.1, 8.9, 7.6, 1 H), 5.03 - 5.09
(m, 4H), 6.39 (d, J =
8.0, 1 H), 6.56 (s, 1 H), 6.61 (s, 1H), 6.88 (d, J = 8.2, 2H), 7.07 (d, J =
8.2, 2H), 7.27 (d, J = 7.0,
2H), 7.30 - 7.40 (m, 8H), 7.43 (d, J = 7.5, 1H), 8.29 (d, J = 7.6, IH), 8.61
(s, 1H), 8.70 (s, 1H).
Step B. ~'~-3,4-dihydroxyhydrocinnamoyl-L-tyrosine
The deprotection of the amide was carried out using the conditions outlined in
example 5.
Purification by flash chromatography eluting with 10% methanol in
dichloromethane afforded
190 mg (73%) of the title compound.
'H NMR (DMSO-db): 2.27 (t, J = 7.9, 2H), 2.53 (t, J = 7.9, 2H), 2.72 (dd, J =
9.4, 13.7, 1H),
2.89 (dd, J = 5.0, 13.7, 1 H), 4.33 (ddd, J = 5.0, 9.4, 8.2, 1 H), 6.39 (d, J
= 7.3, 1 H), 6.55 (s,
1 H), 6.59 (d, J = 7.3, 1 H), 6.64 (d, J = 8.1, 2H), 6.97 (d, J = 8.1, 2H),
8.04 (d, J = 8.2, 1 H), 8.4
- 8.9 (br s, 2H), 12 - 13 (br s, 1H).
Step C. N-[N-(3',4'-dihydroxyhydrocinnamoyl)-L-tyrosylJ-L-tyrosine
The product obtained in step B of this example (48 mg, 0.14 mmol) was coupled
with O-
benzyloxy-L-tyrosine benzyl esterp-toluenesulfonate salt following the
indications of
74
CA 02302144 2000-03-27
example 4. Purification by flash chromatography eluting with 4% methanol in
ethyl acetate
containing 1% acetic acid afforded 68 mg (100%) of the pure title compound.
'H .NMR (DMSO-db): 2.21 (t, J = 8.4, 2H), 2.47 (t, J = 8.4, 2H), 2.57 (dd, J =
9.9, 14.2, 1 H),
2.80 (dd, J = 7.9, 14Ø 1 H), 2.86 (dd, J = 3.3, 14.2, 1 H), 2.93 (dd, J =
4.9, 14.0, 1 H), 4.33
(ddd,. J = 4.9, 7.9, 7.6, 1 H), 4.44 (ddd, J = 3.3, 9.9, 8.2, 1 H), 6.36 (d, J
= 7.9, 1 H), 6.54 (s, 1 H),
6.59 (d, J = 7.9, 1H), 6.61 (d, J = 8.1, 2H), 6.65 (d, J = 7.9, 2H), 6.98 (d,
J = 8.1, 2H), 7.00 (d,
J = 7.9, 2H), 7.90 (d, J = 8.2, 1 H), 8.05 (d, J = 7.6, 1 H), 8.57 (s, 1 H),
8.60 - 8.80 (br s, 1 H),
9.11 (s, 1 H). 9.18 (s, 1 H), 12.1 - 12.9 (br s, 1 H).
Example 43. Preparation of N-[N-[N"-(3",4"-dihydroxyhydrocinnamoyl)-L-tyrosyl]-
L-
h~rosyt]-dopamine
The product obtained in step A of example 42 (100 mg, 0.29 mmol) was coupled
with O-2,6-
dichlorobenzyloxy-L-tyrosyl-dopamine salt following the indications of example
4. The crude
material (315 mg) was used as isolated and subjected to hydrogenolysis using
the conditions
of example 5. Purification by flash chromatography eluting with 7% methanol in
ethyl acetate
containing I % acetic acid afforded 100 mg (54%) of the pure title compound.
'H NMR (DMSO-db): 2.42 - 2.50 (m, 2H), 2.58 - 2.65 (m, 2H), 2.69 - 2.76 (m,
2H), 2.79 (dd,
J = 8.3, 14.0, 1 H), 2.85 (dd, J = 8.0, 13.8, I H), 2.96 (dd, J = 6.3, 14.0, 1
H), 3.03 (dd, J = 6.2,
13.9, 1 H), 3.29 - 3.42 (m, 2H), 4.47 - 4.53 (m, 1 H), 4.53 - 4.59 (m, 1 H),
6.52 - 6.59 (m, 2H),
6.69 - 6.80 (m, 8H), 7.02 (d, J = 8.2, 2H), 7.06 (d, 8.5, 2H),
Example 44. Preparation of N'-(3',4'-dihydroxyhydrocinnamoyl)-L-3,4-
dihydroxyphenylalanine
Step A. N-(3',4'-dihydroxyhydrocinnamoyl)-L-3,4-O-dibenzyloxyphenylalanine
benzyl ester
The title compound was prepared by cleaving the Boc protecting group of the
product
CA 02302144 2000-03-27
prepared in step B of the example 16 (310 mg, 0.80 mmol) and coupling it with
3,4-
dihydroxyhydrocinnamic acid as described in example 3 and in example 4
respectively.
Purification by flash chromatography using 5% MeOH/CH2Clz containing 1 %
acetic acid
yielded 220 mg (61%) of the desired product.
'H NMR (DMSO-db): 2.29 (t, J = 8.0, 2H), 2.55 (t, J = 8.0, 2H), 2.73 (dd, J =
8.5, 13.8, 1H),
2.82 (dd, 6.4, 13.8, 1 H), 4.41 ddd, J = 6.4, 8.5, 7.5, 1 H), 5.03 (d, J =
12.6, 1 H), 6.56 (s, 1 H),
6.60 (s, 1 H), 6.60 (d, J = 8.5, 1 H), 6.61 (d, J = 7.7, 1 H), 7.22 - 7.38 (m,
SH), 8.26 (d, J = 7.5,
1 H). 8.4 - 9.2 (br s, 4H).
Step B. N-(3',4'-dihydroxyhydrocinnamoyl)-L-3,4-dihydroxyphenylalanine
The title compound was prepared by the reduction of the compound obtained in
step A of this
example (135 mg, 0.30 mmol) according to the indications found in example 5.
The product
l ~ (90 mg. 83%) was obtained by filtering off the catalyst and evaporating
the filtrate to dryness.
'H NMR (DMSO-db): 2.27 (t, J = 8.0, 2H), 2.55 (t, J = 8Ø 2H), 2.6~ (dd, J =
8.6, 13.5, 1H),
2.66 (dd, 3.5, 13.5, 1 H), 4.26 ddd, J = 3.5, 8.6, 7.5, 1 H), 6.39 (d, J =
8.3, 1 H), 6.41 (d, J = 8.5,
1 H). 6.56 - 6.60 (m, 4H), 7.82 (d, J = 5.9, 1 H), 8.0 - 9.7 (br s, 4H).
Example 45. Preparation of N'-[N'-(3",4"-dihydroxyhydrocinnamoyl)-L-3',4'-
dihydroxyphenylalanyl]-L-3,4-dihydroxy~phenylalanine
Boc-L-3,4-di-O-benzyloxyphenylalanine benzyl ester (325 mg, 0.68 mmol) was
deprotected
by treatment with TFA as indicated in example 3, providing the desired L-di0-
benzyloxyphenylalanine benzyl ester that was coupled with Boc-L-
dihydroxyphenylalanine
following the indications of example 6. Purification by flash chromatography
eluting with 2%
methanol in methylene chloride afforded 275 mg (62%) of the dipeptide
intermediate.
Subsequent removal of the Boc group, again following the indications of
example 3, provided
the intermediate that was coupled with 3,4-dihydroxyhydrocinnamic acid as
indicated in
example 4. The coupled product was hydrogenolyzed as isolated according to the
indications
76
CA 02302144 2000-03-27
of example 5. Purification by flash chromatography eluting with 7% methanol in
ethyl acetate
containing 1 % acetic acid provided 75 mg (35%) of the title compound.
'H NMR (DMSO-db): 2.22 (t, J = 8.0, 2H), 2.46 - 2.53 (m, 3H), 2.74 (dd, J = 8.
l, 13.5, 1 H),
2. 80 - 2.85 (m, 1 H), 2.86 (dd, 5.3, 13 .5, 1 H), 4.31 ddd, J = 8.1, 5.3,
7.2, 1 H), 4.3 8 - 4.46 (m,
1 H), 6.37 (d, J = 7.8, 1 H), 6.43 - 6.49 (m, 2H), 6.54 (s, 1 H), 6.56 - 6.63
(m, 4H), 6.64 (s, I H),
7.93 (d. J = 8.3, 1 H), 8.01 (d, J = 7.2, 1 H), 8.5 - 8.9 (br s, 6H), 12.2 -
12.8 (br s, 1 H).
Example 46. Preparation of N [N-[3',4'-dihydroxyhydrocinnamoyl)-L-3,4-
dihydroxyphenylalanyl]-dopamine
Step A. ;~'-Boc-3,4-dihydroxyphenylalanyl-dopamine
:~'-Boc-L-3,4-dihydroxyphenylalanine (1.00 g, 3.36 mmol) was coupled with
dopamine
hydrochloride according to the indications of example 6. Purification by flash
chromatography eluting with 5% methanol in methylene chloride containing 1%
acetic acid
provided 1.17 g (83%) of the pure coupled product.
'H NMR (DMSO-db): 1.32 (s, 9H), 2.44 - 2.52 (m, 2H), 2.53 (dd, J = 9.7, 13.5,
1H), 2.71 (dd,
J = 4.3, 13.5, 1 H), 3.07 - 3.28 (m, 2H), 3.98 (ddd, J = 4.3, 9.7, 8.3, 1 H),
6.42 (d, J = 7.8, 1 H),
6.45 (d, J = 8.0, 1 H), 6.57 - 6.63 (m, 4H), 6.64 (d, J = 8.3, I H), 7.80 (t,
J = 5.0, 1 H), 8.0 - 9.8
(br s, 4H).
Step B. N-[N-[N'-[3",4"-dihydroxyhydrocinnamoyl)-L-3',4'-
dihydroxyphenylalanyl]-L-3,4-
dihydroxyphenylalanyl]-dopamine
The product of step A of this example (1.17 g, 2.79 mmol) was treated with TFA
as in
example 3 to remove the Boc protecting group. A portion of the product thus
obtained (260
mg, 0.60 mmol) vvas then coupled with 3,4-dihydroxyhydrocinnamic acid using
the conditions
as in example 4. Purification by flash chromatography eluting with 10%
methanol in ethyl
77
CA 02302144 2000-03-27
acetate containing 1 % acetic acid provided 113 mg (38%) of the desired
product.
'H NMR (DMSO-db): 2.26 (t, J = 7.7, 2H), 2.42 - 2.48 (m, 2H), 2.52 (t, J =7.7,
2H), 2.54 (dd,
J = 8Ø 13.6, 1 H), 2.73 (dd, J = 5.0, 13.6, 1 H), 3.05 - 3.24 (m, 2H), 4.31
(ddd, J = 5.0, 9.0,
8.3, 1H), 6.37 (d, J = 8.0, 1H), 6.39 - 6.43 (m, 2H), 6.55 - 6.63 (m, 6H),
7.79 (t, 4.9, 1H), 7.91
(d. J = 8.3, 1 H), 7.4 - 10.0 (br s, 6H).
Example 47. Preparation of N-[lV'-[1V"-(3",4"-dihydroxyhydrocinnamoyl)-L-3',4'-
dihydroxyphenylalanyl]-L-3,4-dihydroxyphenylalanyl]-dopamine
The product obtained in step A of example 46 (355 mg, 0.58 mmol) was
deprotected by
treating with TFA according to the indications of example 3. The product thus
obtained was
coupled with 3,4-dihydroxyhydrocinnamic acid according to the conditions of
example 4.
Purification by flash chromatography eluting with 5% methanol in ethyl acetate
containing 1
acetic acid provided 70 mg (18%) of the title compound.
'H NMR (DMSO-db): 2.22 - 2.28 (m, 2H), 2.45 (t, J = 6.8, 2H), 2.46 - 2.57 (m,
3H), 2.62 (dd,
J = 8.3, 13.6, 1 H), 2.71 - 2.80 (m, 2H), 3.06 - 3.21 (m, 2H), 4.25 - 4.32 (m,
1H), 4.32 - 4.40 (,
1 H), 6.33 - 6.46 (m, 4H), 6.54 - 6.67 (m, 8H), 7.74 (t, J = 5.0, 1 H), 7.83
(d, J = 8.0, 1 H), 8.00
(d. J = 8.0, 1 H). 2.8 - 4.7 (br s, 8H).
Example 48. Preparation of 1V [N'-(3',4'-dihydroxybenzoyl)-DL-3-
fluorotyrosinyl]-
dopamine
Step A. N-3',4'-dihydroxybenzoyl-O-benzyl-DL-3-fluorotyrosine benzyl ester
N-Boc-O-benzyl-DL-3-fluorotyrosine benzyl ester (524 mg, 1.09 mmol) was
deprotected by
treating with TFA as indicated in example 3, providing the crude intermediate
that was
coupled with 3,4-dihydroxybenzoic acid as indicated in example 4. Purification
of the
product eluting with 15% ethyl acetate in methylene chloride provided 205 mg
(34%) of the
78
CA 02302144 2000-03-27
desired amide product.
'H NMR (acetone-db): 3.17 (dd, J = 5.6, 5.0, 2H), 5.13 (m, 1H), 5.13 (d, J =
12, 2H), 5.23 (d, J
= 7.2, 2H), 6.58 - 7.40 (m, 16H).
Step B. ?~"-[(3',4'-dihydroxybenzoyl)-DL-3-fluorotyrosyl]-dopamine
The product obtained in step A of this example (179 mg, 0.32 mmol) was
unblocked by
hydrogenolysis according to example 5, providing a product that was then
reacted with
dopamine hydrochloride according to the indications of example 6. Purification
by flash
chromatography eluting with 10% methanol in ethyl acetate containing 1 %
acetic acid
provided 118 mg (71 %) of the title compound.
'H NMR (acetone-db): 2.62 (d, J = 6.0, 2H), 3.25 (dd> J = 5.3, 6.8, 2H), 3.38
(q, J = 6.2, 2H)
4.75 (q, J = 6, 1 H), 6.50 (d, J = 7.7, 2H), 6.60 (m, 2H), 6.83 (d, J = 8.3, 1
H), 6.89 (d, J = 8.9,
1 H). 6.90 (s, 1 H), 7.05 (d, J = 11.7, 1 H), 7.25 (d, J = 8.2, 1 H), 7.37 (s,
1 H).
Example 49. Preparation of N (3,4-dihydroxybenzoyl)-b-1V'-(3',4'-
dihydroxyphenethyl)-
L-glutamine a-benzyl ester
Step A. N-Boc-8-N-(3',4'-dihydroxyphenethyl)-L-glutamine benzyl ester
N-Boc-L-glutamic acid a-benzyl ester (800 mg, 2.37 mmol) was coupled with
dopamine
hydrochloride according to example 6. Purification by flash chromatography
eluting with 5%
methanol in ethyl acetate containing 1 % acetic acid yielded 896 mg (80%) of
white crystals.
'H NMR (acetone-db): 1.07 - 2.15 (m, 2H), 2.27 (t, J = 6.7, 2H), 2.63 (t, 7.2
, 2H)> 3.35 (q, T =
6.4, 2H), 4.20 (m, 1 H), 5.16 (q, J = 14.0, 2H), 6.45 (d, J = 5.8, 1 H), 6.53
(d, J = 7.8, 1 H), 6.70
(s, 1 H), 6.72 (d, J = 7.6, I H), 7.07 (s, 1 H), 7.30 - 7.40 (m, 5H), 7.62 -
7.71 (s, 2H).
79
CA 02302144 2000-03-27
Step B. N-(3,4-dihydroxybenzoyl)-8-N-(3',4'-dihydroxyphenethyl)-L-glutamine a-
benzyl ester
The product prepared in step A of this example (800 mg, 2.37 mmol) was
deblocked with
TFA as described in example 3. The product thus obtained was then coupled with
3,4-
dihydroxybenzoic acid according to the indications of example 4. Purification
by flash
chromatography eluting with 5% methanol in ethyl acetate containing 1 % acetic
acid yielded
896 mg (80%) of the title compound.
'H NMR (acetone-db): 2.15 - 2.26 (m, 2H), 2.39 (d, J = 4.7, 2H), 2.62 (t, 7.0,
2H), 3.35 (m,
2H), 4.62 (s, I H), 5.16 (q, J = 5.2, 2H), 6.50 (d, J = 7.8, 1 H), 6.70 (s, I
H), 6.90 (d, J = 7.9,
1 H), 7.03 (dd, J = 8.3, 8.7, 1 H), 7.31 (dd, J = 6.4, 8.9, 1 H), 7.35 - 7.41
(m, 6H), 7.68 (q, J =
9.5. 1 H). 8.2 (d, J = 6.6, 1 H).
Example 50. Preparation of N-[lV'-[(3',4'-dihydroxybenzoyl)-DL-n:-t~~rosyl]-
dopamine
Step A. Preparation of .~'-(tert-butoxycarbonyl)-DL-m-tyrosine
The title compound was prepared from DL-m-tyrosine (1.0 g, 5.5 mmol), by
following the
procedure described in example 1. The product was isolated as a colorless
syrup (1.18 g, 76%).
'H NMR (DMSO-db): 1.30 (s, 9H); 3.15 (dd, J = 3.1, 13.0, 1H); 4.10 (q, J =
7.2, 1H); 6.20 (d,
J = 8.0, 1 H); 6.50 - 6.80 (m, 4H), 7.2 (s, 1 H), I 0.0 (br s, 1 H).
Step B. Preparation of N-(tert-butoxycarbonyl)-O-benzyl-DL-m-tyrosine benzyl
ester
The title compound was prepared from the product obtained in step A of this
example (I.0 g,
3.56 mmol) according to the indications of example 2c. The crude product was
purified by
silica gel column chromatography using 5% MeOH/CHZCI~ to yield the desired
product (1.06
g, 65%).
CA 02302144 2000-03-27
'H NMR (CDC13): 1.45 (s, 9H); 3.15 (d, J = 3.3, 2H); 4.15 (q, J = 7.2, 1 H);
4.70 (J = 5.7, 1 H),
5.20 (d, J = 11, 4H), 6.71 (d, J = 6.8, 1 H); 6.8 (s, 1 H), 6.90 (d, 8.5, 1
H), 7.2 (t, J = 7.6, 1 H),
7.3 - 7.5 (m, 1 H).
Step C. N-(3,4-dihydroxybenzoyl)-O-benzyl-DL-m-tyrosine benzyl ester
The title compound was prepared from the product obtained in step B of this
example (520
mg, 1.09 mmol) by the removal of the Boc group following the indications of
example 3. The
resulting unblocked derivative was then coupled with 3,4-dihydroxybenzoic acid
according to
the indications of example 4. The crude product was purified by silica gel
column
chromatography using 10% ethyl acetate in methylene chloride to yield 205 mg
(34%) of the
desired product.
'H NMR (DMSO-db): 3.15 (d, J = 3.1, 2H); 4.20 m, 1H), 5.20 (m, 4H); 6.70 -
7.80 (m, 17H),
8.6 (d. J = 7.2. 2H).
Step D. N-[~~-(3',4'-dihydroxybenzoyl)-DL-m-tyrosyl]-dopamine
The title compound was prepared from the product of step C of this example
(208 mg, 0.56
mmol) by removing the benzyl ester group following the indications of example
5. The
resulting unblocked derivative was coupled with dopamine hydrochloride
according to the
indications of example 6. Purification by silica gel chromatography with 10%
MeOH in ethyl
acetate containing 1 % acetic acid provided the desired product, (26 mg, 14%).
'H NMR (DMSO-db): 2.62 (t, J = 5.9, 2H), 3.20 (m, 2H); 4.7 (s, 1 H); 6.70 -
7.5 (m, 12H),
8.5 (s, 1 H), 9.3 (br s, 4H).
Example 51. Preparation of N ~N'-~N"-(3",4"-dihydroxybenzoyl -L-tyrosyl]-L-
tyrosyl]-
dopamine
81
CA 02302144 2000-03-27
,~'-[N-(3',4'-dihydroxybenzoyl)-L-tyrosyl]-tyrosine (prepared as described in
example 36, step
C) (30 mg, 0.06 mmol) was coupled with dopamine hydrochloride according to the
indications of example 6. Purification by flash chromatography eluting with 5%
methanol in
ethyl acetate containing I% acetic acid provided 5 mg (14%) of the title
compound.
1H NMR (acetone-db): 2.57 (t, J=7.0, 2H), 2.84 (dd, J=7.8, 13.6, 1H), 2.87 -
303 (m, 2H), 3.08
(dd. J= 5.3, I 3.9, 1 H), 3.23 - 3.36 (m, 2H), 4.45 - 4.52 (m, I H), 4.58 -
4.66 (m, 1 H), 6.50 (dd,
J - 2.0, 7.8, 1 H). 6.65 (d, J=8.3, 2H), 6.69 (d, J - 7.8 1 H), 6.70 (d, J=
2.0, 1 H), 7.07 -7.11 (m,
1 H), 7. I 0 (d. J = 7.9, 2H), 7.21 (dd, J =1.9, 8.2, 1 H), 7.39 (d, J=1.9, 1
H), 7.40 - 7.46 (m, 1 H),
7.55 (d, J= 7.2, 1 H).
The compounds listed in Table 3 were prepared following similar procedures as
for the
preparation of the derivatives described above (see new examples below); the
numbers) in
brakets after each root amino acid name is the numbers) of an examples) below.
Their
activities are also listed in the same table demonstrating their potential
usefulness.
Table 3. Anti-integrase activity (IC;~) of amino acid derivatives in
accordance with
general formula I above
Root Amino acid Anti-integrase activity (ICso)
(example no.) Na-Caffeoyl Other Na-3,4-dihydroxyphenyl
derivative derivative
~M ,uM
L-Aspartic acid (ex. 52) 2.2
L-Tryptophan (ex. 53) 1.5
L-3,4-Dihydroxyphenylalanine (ex. 54 ) 0.62
L-3,4-Dihydroxyphenylalanine (ex. 55) 0.38
L-Tyrosine (ex. 56) 0:78
L-3,4-Dihydroxyphenylalanine (ex. S7) 0.106
82
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L-Cysteine (ex. 58) 0.21
L-Serine (ex. 59) 0.14
L-Glutamic acid (ex. 60) 0.105
L-Aspartic acid (ex. 61 ) 3.5
L-Aspartic acid (ex. 62) 3.5
L-Glutamic acid (ex. 63) 2.8
L-Tyrosine (ex. 64) 3
For the purposes of Table 3 the HIV-1 integrase inhibition assay was based on
a known
procedure {Hazuda, D. J. et al., Nucleic Acids Res. 22, 1121-1122 (1994)).
Example 52. Preparation of .'1'a-caffeoyl-Ny-(3-hydroaytyramine)-L-aspartic
acid
benzvl ester
Step A. Preparation of Na-tert-butoxycarbonyl-Ny-(3-hydroxytyramine)-L-
aspartic acid
benzvl ester
The title compound was prepared from commercially available Na-tert-
butoxycarbonyl-L-
aspartic acid benzyl ester (2.0 g, 6.0 mmol), by following the procedure
described in example
6. The product was isolated as a white solid (2 g, 76% yield).
'H NMR (acetone-db): 1.4 (s, 9H), 2.6 (t, J = 3.6, 2H), 2.7 and 2.80 (ABX, J =
8.5, 15.0, 2H),
3.3 (d, J = 3.2, 2H), 4.6 (br s, 1 H), 5.2 (q, J = 6.0, 2H), 6.3 - 7.5 (m, 1
OH), 7.68 - 7.72 (2 s,
2H).
Step B. Preparation of Na-caffeoyl-Ny-(3-hydroxytyramine)-L-aspartic acid
benzyl ester
The title compound was prepared from the product obtained in step A of this
example (958
mg, 2.0 mmol) according to the indications of example 3, for 2 h. The crude
intermediate was
coupled with caffeic acid (565 mg, 3.1 mmol) according to the indications of
example 4. The
83
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s
crude product was purified by flash chromatography using 30% AcOEt/CHC13 and 5
- 10%
MeOH/CHC13 to yield the desired product (432 mg, 40%).
'H NMR (DMSO-db): 2.5 (s, 2H), 2.6 - 2.7 (m, 2H), 3.2 (d, J = 1.7, 2H), 4.8
(q, J = 3.3, 1H),
S 5.1, (s, 2H), 6.3 - 7.0 (m, 13H), 8.0 (t, J = 2.6, I H), 8.4 (d, J = 3.8, 1
H), 8.5 - 9.4 (br s, 4H).
Example 53. Preparation of N (N-caffeoyl-L-tryptophanyl) dopamine
The title compound was prepared from N-(N-tent-butoxycarbonyl-L-tryptophanyl)
dopamine
obtained in step A of example no. 29 ( I .3 g, 2.8 mmol) according to the
indications of
example 3, for 2 h. The crude intermediate was coupled with caffeic acid (770
mg, 4.3 mmol)
according to the indications of example 4. The crude product was purified by
flash
chromatography using 30% AcOEtICH,CI,/1% AcOH and 5 - 10% MeOH/CH,CI,/I% AcOH
to yield the desired product (899 mg, 63%).
l~
'H NMR (DMSO-db): 2.4 (q, J = 3.7. 2H), 2.9 - 3.3 (m, 4H), 4.6 (q, J = 2.8, 1
H), 6.3 - 7.7 (m,
I 3H), 8.1 (t, J = 2.7, 1 H), 8.2 (d, J = 4.0, 1 H), 10.0 (br s, 4H), I 0.8,
(s, 1 H).
Example 54. Preparation of N-(N-caffeoyl-L-3,4-dihydroxyphenylalanyl) dopamine
The title compound was prepared from N-(l~"-tert-butoxycarbonyl-L-3,4-
dihydroxyphenylalanyl) dopamine obtained in step A of example no. 46 (878 mg,
2.0 mmol)
according to the indications of example 3, for 2 h: The crude intermediate was
coupled with
caffeic acid (546 mg, 3.0 mmol) according to the indications of example 4. The
crude product
was purified by flash chromatography using 30% AcOEt/CHZC12/1% AcOH and 10%
MeOH/CH,Cl2/l % AcOH to yield the desired product (407 mg, 41 %).
'H NMR (DMSO-db): 2.4 (s, 2H), 2.6 - 2.9 (m, 2H), 3.2 (m, 2H), 4.4 (m, 1H),
6.4 - 7.8 (m,
11H), 8.0 (m, 2H), 9.7 (br s, 6H).
84
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i
Example 55. Preparation of N-(N-caffeoyl-L-3,4-dihydroxyphenylalanyl)-3,4-
dihydroxybenzylamine
Step A. Preparation of N-(N-tert-butoxycarbonyl-L-3,4-dihydroxyphenylalanyl)-
3,4-
dihydroxybenzylamine
The title compound was prepared from Na-tert-butoxycarbonyl-L-3,4-
dihydroxyphenylalanine
(575 mg, 1.9 mmol), by following the procedure described in example 6, using
3,4-
dihydroxybenzylamine hydrobromide instead of dopamine hydrochloride. The crude
material
was purified by flash chromatography using 30, 50% AcOEt/CHZCIz/I% AcOH to
yield the
desired product as white crystals (457 mg, 56% yield).
'H NMR (DMSO-db): 1.3 (s, 9H), 2.5 - 2.8 (m, 2H), 4.2 (m, 3H), 6.4 - 6.8 (m,
7H), 8.2 (s,
1 H), 8.7 (br s, 4H).
Step B. Preparation of N-(IV"-caffeoyl-L-3,4-dihydroxyphenylalanyl)-3,4-
dihydroxybenzylamine
The title compound was prepared from the product obtained in step A of this
example (377
mg. 0.9 mmol) according to the indications of example 3, for 2 h. The crude
intermediate was
coupled with caffeic acid (246 mg, 1.35 mmol) according to the indications of
example 4.
The crude material was purified by flash chromatography using 50, 80%
AcOEtICH,CI,/l
AcOH to yield the desired product (120 mg, 28%).
'H NMR (DMSO-db): 2.6 - 2.9 (m, 2H), 4.2 (s, 2H), 4.6 (s, 1 H), 6.3 - 7.7 (m,
11 H), 8.0 ( d, J =
4.0, 1 H), 8.3 (d, J = 2.4, 1 H), 9.5 (br s, 6H).
Example 56. Preparation of N-[N'-(3',4'-dihydroxybenzoyl)-L-tyrosyl]-3,4-
dihydroxybenzylamine
3O
CA 02302144 2000-03-27
Step A. Preparation of N-(N-tert-butoxycarbonyl-L-tyrosyl)-3,4-
dihydroxybenzylamine
The title compound was prepared from commercially available Na-tert-
butoxycarbonyl-L-
tyrosine ( 1.5 g, 5.3 mmol), by following the procedure described in example
6. The product
was purified by flash chromatography using 30, 60% AcOEUCH2CIz to yield the
title product
as white crystals (1.9 g, 88% yield).
'H NMR (DMSO-db): 1.3 (s, 9H), 2.5 - 2.8 (m, 2H), 4.1 (t, J = 4.5, 2H), 6.4 -
7.0 (m, 7H), 8.2
(s, 1 H), 8.7 (br s, 2H), 9.2 (s, 1 H).
Step B. Preparation of ~'-[N-(3',4'-dihydroxybenzoyl)-L-tyrosyl]-3,4-
dihydroxybenzylamine
The title compound was prepared from the product obtained in step A of this
example (1.4 g.
3.3 mmol) according to the indications of example 3, for 2 h. The crude
intermediate was
coupled with 3,4-dihydroxybenzoic acid (758 mg, 5.0 mmol) according to the
indications of
example 4. The crude product was purified by flash chromatography using 50 -
80%
AcOEt/CH,CI,/1% AcOH to yield the title product as a white solid (600 mg,
40%).
'H NMR (DMSO-db): 2.7 - 3.0 (m, 2H), 4.2 (s, 2H), 4.6 (s, 1H), 6.3 - 7.7 (m,
lOH), 8.0 (d, J =
3.9, 1 H), 8.2 (d, J = 2.4, 1 H), 9.5 (br s, 5H).
Example 57. Preparation of N-[N-(3',4'-dihydroxyphenylacetyl)-L-3,4-
dihydroxyphenylalanyl] dopamine
N-(N-tert-butoxycarbonyl-L-3,4-dihydroxyphenylalanyl) dopamine ( 1.5 g, 3.4
mmol, example
no. 46, step A) was deprotected with TFA as described in example 3. The
product thus
obtained was then coupled with 3,4-dihydroxyphenylacetic acid (865 mg, 5.0
mmol)
according to the indications of example 4. Purification by flash
chromatography using 40 -
60% AcOEt/CHzCl2 containing 1% AcOH and 5% MeOH/CHZC12 containing 1% AcOH
yielded 120 mg (7%) of the title compound as white crystals.
86
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'H NMR (DMSO-db): 2.4 - 2.8 (m, 4H), 3.0 - 3.4 (m, 4H), 4.3 (s, 1H), 6.3 - 7.3
(m, 9H), 7.7 -
8.0 (m, 2H), 9.2 (br s, 6H).
Example 58. Preparation of N [N'-(3',4'-dihydroxybenzoyl)-L-cysteinyl]
dopamine
Step A. Preparation of N-[N-(3,4-dihydroxybenzoyl)-S-trityl-L-cysteinyl]
dopamine
Commercially available Na (9-fluorenylmethoxycarbonyl)-S-trityl-L-cysteine
(1.7 g, 2.9
mmol) was coupled with dopamine hydrochloride according to the indications of
example 6.
The crude N-[.~n-(9-fluorenylmethoxycarbonyl)-S-trityl-L-cysteinyl] dopamine
was
deprotected according to the indications of example 7. The resulting
intermediate was then
coupled with 3,4-dihydroxybenzoic acid (278 mg, 1.8 mmol) according to the
indications of
example 4. The final product was purified by flash chromatography using 20 -
50%
AcOEt/CH,C1, containing 1% AcOH to yield the desired derivative (644 mg, 35%)
as a
yellow crystals.
'H NMR (DMSO-db): 2.5 (m, 4H), 3.2 (m, 2H), 4.4 (s, 1H), 6.3 - 7.4 (m, 21H),
7.8 (s, 1H),
8.2 (d, J = 3.8. 1 H), 8.5 - 9.5 (4 s, 4H).
Step B. Preparation of N-[N-(3',4'-dihydroxybenzoyl)-L-cysteinyl] dopamine
The title compound was prepared from N-[N'-(3,4-dihydroxybenzoyl-S-trityl-L-
cysteinyl]
dopamine describe in step A (390 mg, 0.6 mmol) by following the indications of
example 3.
Purification by flash chromatography using 30 - 60% AcOEt/CHzCI, containing 1
% AcOH
gave 108 mg (45%) of the title compound as white crystals.
'H NMR (DMSO-db): 2.5 (m,4H), 3.2 (m, 2H), 4.4 (br s, 2H), 6.3 - 7.5 (m, 6H),
7.7 (s, 1H),
8.2 (d, J = 4.0, 1 H), 8.6 - 9.5 (4 s, 4H).
Example 59. Preparation of N-(N'-caffeoyl-L-Beryl) dopamine
87
CA 02302144 2000-03-27
Step A. Preparation of N-(N-tert-butoxycarbonyl-L-Beryl) dopamine
The title compound was prepared from Na-tert-butoxycarbonyl-L-serine (2.5 g,
12.0 mmol),
by following the procedure described in example 6. The crude material was
purified by flash
chromatography using 30% AcOEtICHZCI, and 5% MeOH/CHZCIz to yield the desired
product as white crystals (1.6 g, 40% yield).
'H NMR (DMSO-db): 1.4 (s, 9H), 2.5 (s, 2H), 3.1 - 3.3 (m, 2H), 3.5 (s, 2H),
3.9 (s, 1H), 4.8 (s,
1 H). 6.4 - 6.7 (m, 4H), 7.8 (s, 1 H), 8.6 and 8.7 (2 s, 2H).
Step B. Preparation of ,~'-(N-caffeoyl-L-seryl) dopamine
The title compound was prepared from the product obtained in step A of this
example (796
mg, 2.3 mmol) according to the indications of example 3, for 2 h. The crude
intermediate was
1 ~ coupled with caffeic acid (633 mg, 3.5 mmol) according to the indications
of example 4. The
crude material was purified by flash chromatography using 30% AcOEt/CH,C1, and
5 - 10%
MeOHICH,CI, to yield the desired product (282 mg, 30%) as yellow crystals.
'H NMR (DMSO-db): 2.5 (d, J = 4.0, 2H), 3.2 (s, 2H), 3.6 (s, 2H), 4.4 (s, 1H),
6.4 - 7.3 (m,
8H), 7.8 (s, 1 H), 8.0 (m, 1 H), 9.3 (br s, SH).
Example 60. Preparation of N-[N-caffeoyl-N8-(3-hydroxytyramine)-L-glutamylJ
dopamine
Step A. Preparation of Na tert-butoxycarbonyl-L-glutamic acid
The title compound was prepared from commercially available Na-tert-
butoxycarbonyl-L-
glutamic acid benzyl ester (1.0 g, 3.0 mmol), by following the procedure
described in example
5. The crude material was purified by flash chromatography using 30%
AcOEt/CH,C1,11 °io
AcOH to yield the desired product as a white powder (680 mg, 93% yield).
88
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Step B. Preparation of N-[l~"-tert-butoxycarbonyl-N8-(3-hydroxytyramine)-L-
glutamyl]
dopamine
Na-tert-butoxycarbonyl-L-glutamic acid (718 mg, 2.9 mmol) was coupled with
dopamine
according to the indications of example 6. The product was purified by flash
chromatography
using 15, 30% AcOEt/CH,C12 containing 1 % AcOH and 10% MeOHICHzCI, containing
1
AcOH to yield the desired product as a white powder (1.1 g, 76% yield).
'H NMR (DMSO-db): I .3 (s, 9H), 1.7 - 2.0 (m, 4H), 2.5 (s, 4H), 3.2 (m, 4H),
3.9 (d, J = 1.9,
1 H), 6.3 - 6.8 (m, 7H), 7.8 (d, J = 2.4, 2H), 9.5 (br s, 4H).
Step C. Preparation of N-[N-caffeoyl-NS-(3-hydroxytyramine)-L-glutamyl]
dopamine
The title compound was prepared from the product obtained in step B of this
example (721
mg, 1.4 mmol) according to the indications of example 3, for 2 h. The crude
intermediate was
coupled with caffeic acid (335 mg. 2.0 mmol) according to the indications of
example 4. The
crude product was purified by flash chromatography using 50, 60% AcOEtICH,CI,
and 10%
MeOH/CH,CI, to yield the desired product (484 mg, 60%) as yellow crystals.
'H NMR (DMSO-db): 1.7 - 2.0 (m, 2H), 2.1 (s, 2H), 2.5 (s, 4H), 3.2 (m, 4H),
4.3 (m, IH), 6.4
- 7.6 (m, 11 H), 8.0 (m, 3H), 9.4 (br s, 6H).
Example 61. Preparation of N (N-caffeoyl-Oy-benzyl-L-aspartyl) dopamine
Step A. Preparation of N-(N-tert-butoxycarbonyl-Oy-benzyl-L-aspartyl) dopamine
The title compound was prepared from commercially available Na-tert-
butoxycarbonyl-O~y-
benzyl-L-aspartic acid (3.0 g, 9.3 mmol), by following the procedure described
in example 6.
The product was isolated as a white solid (2.7 g, 64% yield).
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'H NMR (acetone-db): 1.5 (s, 9H), 2.76 (t, J = 3.5, 2H), 2.95 and 3.05 (ABX, J
= 5.5, 13.0,
4H), 4.6 (d, J = 3.0, 1 H), 5.2 (s, 2H), 6.4 - 7.6 (m, 1 OH), 8.5 (br s, 2H).
Step B. Preparation of N-(N-caffeoyl-Oy-benzyl-L-aspartyl) dopamine
The title compound was prepared from the product obtained in step A of this
example ( 1.0 g,
2.4 mmol) according to the indications of example 3, for 2 h. The crude
intermediate was
coupled with caffeic acid (640 mg, 3.5 mmol) according to the indications of
example 4. The
crude product was purified by flash chromatography using 30% AcOEt/CHC13 and
5%
MeOH/CHCI; to yield the desired product as a yellow powder (549 mg, 45%).
'H NMR (acetone-db): 2.6 (d, J = 2.7, 2H), 2.8 - 3.0 (m, 4H), 3.4 (d, J = 2.9,
2H), 4.9 (d, J =
3.2, 1 H). 5.1 (s, 2H), 6.4 - 7.6 (m, 11 H), 8.2 (br s, 6H).
Example 62. Preparation of N-(lV'-caffeoyl-L-aspartyl) dopamine
Step A. Preparation of ~'-(N-bent' loxycarbonyl-O~y-tert-butyl-L-aspartyl)
dopamine
The title compound was prepared from commercially available Na-
benzyloxycarbonyl-Oy-
tent-butyl-L-aspartic acid (2.5 g, 7.7 mmol), by following the procedure
described in example
6. The crude material was purified using 20, 50% AcOEt/CH,C1,. The product was
isolated
as a white solid (3.2 g, 91 % yield).
'H NMR (DMSO-db): 1.3 (s, 9H), 2.3 - 2.7 (m, 4H), 3.2 (s, 2H), 4.3 (d, J =
2.6, 1H), 4.8 and
5.3 (ABX, J = 5.6, 16.0, 2H), 6.3 - 7.4 (m, 8H), 7.5 (d, J = 4.0, 1 H), 7.9
(s, 1 H), 8.6 and 8.7 (2
xs,2xOH).
Step B. Preparation of N-(N-caffeoyl-Oy-tert-butyl-L-aspartyl) dopamine
N-(N-benzyloxycarbonyl-O~y-tort-butyl-L-aspartyl) dopamine (1.0 g, 2.3 mmol)
was
CA 02302144 2000-03-27
deprotected by hydrogenolysis as described in example 5. The product thus
obtained was then
coupled with caffeic acid (621 mg, 3.5 mmol) according to the indications of
example 4.
Purification by flash chromatography using 30 - 50% AcOEt/CH,CIz containing 1
% AcOH
yielded 519 mg (47%) of the title compound as yellow crystals.
'H NMR (DMSO-db): 1.3 (s, 9H), 2.4 - 2.7 (m, 4H), 3.2 (m, 2H), 4.7 (d, J =
6.8, 1H}, 6.4 - 7.4
(m, 8H), 8.0 (s, I H), 8.3 (d, J = 8.0, 1 H), 8.5 - 9.5 (br s, 4H).
Step C. Preparation of N-(N-caffeoyl-L-aspartyl) dopamine
The title compound was prepared from the product obtained in step B of this
example (333
mg. 0.7 mmol) according to the indications of example 3, for 2 h. The crude
product was
purified by flash chromatography using 50 - 99% AcOEt/CH,CI, containing 1 %
AcOH to
yield the desired product (200 mg, 60%).
'H NMR (DMSO-db): 2.4 - 2.8 (m, 4H), 3.2 (s, 2H), 4.7 (d, J = 2:6, 1H), 6.3 -
7.4 (m, 8H), 7.9
(s, 1 H), 8.3 (s, I H), 9.7 (br s, 4 x OH), 13.0 (br s, I H).
Example 63. Preparation of Na (3,4-dihydroxybenzoyl)-NS-(3-hydroxytyramine) L-
glutamic acid
The title compound was prepared from N-(3,4-dihydroxybenzoyl)-b-N'-(3,4-
dihydroxyphenethyl)-L-glutamine a-benzyl ester obtained in step B of example
no. 49 (266
mg, 0.5 mmol) according to the indications of example 5. The crude product was
purified by
flash chromatography using 30% AcOEt/CH,CIz /1 % AcOH and 10% MeOH/CH2C1, /I
AcOH to yield the desired product (208 mg, 95%) as white crystals.
'H NMR (DMSO-db): 1.9 - 2.4 (m, 4H}, 2.5 ( t, J = 7.0, 2H), 3.5 (d, J = 5.0,
2H), 4.2 (s, 1H),
6.3 - 7.4 (m, 6H), 8.2 (s, 1 H), 8.4 (d, J = 6.0, 1 H), 9.7 (br s, 4H), 12.0
(br s, 1 H).
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Example 64. Preparation of N-(N-caffeoyl-L-tyrosyl)-3,4-dihydroxybenzylamine
The title compound was prepared from N-(N-tert-butoxycarbonyl-L-tyrosyl)-3,4-
dihydroxybenzylamine (example 56, step A) (1.4 g, 3.3 mmol) according to the
indications of
example 3, for 2 h. The crude intermediate was coupled with caffeic acid (978
mg, 5.4 mmol)
according to the indications of example 4. The crude product was purified by
flash
chromatography using 40 - 80% AcOEt/CH,CIz containing 1% AcOH to yield the
title product
as yellow crystals (784 mg, 47%).
'H I~'MR (DMSO-db): 2.7 - 3.0 (m, 2H), 4.1 (s, 2H), 4.6 (s, 1H), 6.3 - 7.4 (m,
12H), 8.2 (s,
1 H), 8.4 (s, 1 H), 9.5 (br s, SH).
92
