Note: Descriptions are shown in the official language in which they were submitted.
CA 02353462 2001-05-31
WO 00/34231 PCT/US99/29339
MOLECULAR SCAFFOLDS ACTING AS TEMPLATES COMPIltISING CARBAMATE LINKAGES
TECHNICAL FIELD
This invention pertains generally to valency molecules. The invention also
relates
I O to the field of valency platform molecules which act as scaffolds to which
one or more
molecules may be covalently tethered to form a conjugate. More particularly,
the present
invention pertains to valency molecules which comprise a carbamate linkage
(z.e., -O-
C(=O)-N<). In one aspect, the present invention pertains to valency platforms
comprising
carbamate linkages, which molecules have the structure of any one of Formulae
I, II, or III,
shown in Figure 1. In one aspect, the present invention pertains to valency
platforms
comprising carbamate linkages, which molecules have the structure of any one
of Formulae
IV, V, or VI, shown in Figure 8. The present invention also pertains to
methods of
preparing such valency molecules, conjugates comprising such valency
molecules, and
methods of preparing such conjugates.
1
CA 02353462 2001-05-31
WO 00/34231 PCT/US99129339
BACKGROUND
A "valency platform" is a molecule with one or more (and typically multiple)
attachment sites which can be used to covalently attach biollogically active
molecules of
interest to a common scaffold. The attachment of biologically active molecules
to a
common scaffold provides multivalent conjugates in which multiple copies of
the
biologically active molecule are covalently linked to the same platform. A
"defined" or
"chemically defined" valency platform is a platform with df;fined structure,
thus a defined
number of attachment points and a defined valency. A defined valency platform
conjugate
is a conjugate with defned structure and has a defined number of attached
biologically
active compounds. Examples of biologically active molecules include
oligonucleotides,
peptides, polypeptides, proteins, antibodies, saccharides, polysaccharides,
epitopes,
mimotopes, drugs, and the like. In general, biologically active compounds
interact
specifically with proteinaceous receptors.
Certain classes of chemically defined valency platforms, methods for their
preparation, conjugates comprising them, and methods for tlhe preparation of
such
conjugates, have been described in the U.S. Patents Nos. 5,1.62,515;
5,391,785; 5,276,013;
5,786,512; 5,726,329; 5,268,454; 5,552,391; 5,606,047; andl 5,663,395.
The valency platforms of the present invention reflect a new class of valency
platforms which comprise a carbamate linkage, as shown, for example, in
Formulae I, II,
and III in Figure 1 and in Formulae IV, V, and Vi in Figure 8.
SUMMARY OF THE INVENTION
One aspect of the present invention pertains to a vale:ncy platform compound
having
the structure of one of the following formulae:
2
CA 02353462 2001-05-31
WO 00/34231 PCTNS99129339
Formula I
0
R~ J-w~ IC-N C~-O-Z
y'
RN
2_y,
Formula II
0 0
R~ J-'C-N G~-O-CI-N C~2--O-Z
N 2 ~ 1
[ RN ] 2_Y Y
2_Y~
Formula III
0 0 0
R~ J-IC-N G'-O-IC-N G2-O-IC-IV G3-O-Z
I N ~ Y3
RN ~ 2_Ya Y2
RN ~ 2_Y2 Y~
2_Y~
wherein:
n is a positive integer from 1 to 10;
y' , yz, and y3 are independently 1 or 2;
J independently denotes either an oxygen atom or a covalent bond;
R~ is selected from the group consisting of:
hydrocarbyl groups having from 1 to 20 carbon atoms;
organic groups consisting only of carbon, oxygen, and hydrogen atoms, and
having from 1 to 20 carbon atoms;
organic groups consisting oiZly of carbon, oxygen, nitrogen, and hydrogen
atoms, and having from 1 to 20 carbon .atoms;
organic groups consisting only of carbon, oxygen, sulfur, and hydrogen atoms,
and having from 1 to 20 carbon atoms;
each G', G2, and G3 is independently selected from the group consisting of:
hydrocarbyl groups having from 1 to 20 carbon atoms;
3
CA 02353462 2001-05-31
WO 00/34231 PCTIUS99/29339
organic groups consisting only of carbon, oxygen, and hydrogen atoms, and
having from 1 to 20 carbon atoms;
organic groups consisting only of carbon, oxygen, nitrogen, and hydrogen
atoms, and having from 1 to 20 carbon atoms;
each RN is independently selected from the group consisting of:
hydrogen;
linear or branched alkyl groups having from I to 15 carbon atoms;
alkyl groups comprising an alicyclic structure and having from 1 to 15 carbon
atoms;
aromatic groups having from 6 to 20 carbon atoms;
heteroarornatic groups having from 3 to 20 carbon atoms;
each Z is independently selected from the group consisting of:
-H
-C(=O)OR~''~
-C(=O)RES~R
-C(=O)NR"Ra
wherein:
each R~~~ is organic groups comprising from 1 to about 20 carbon atoms;
each RES'~~ is organic groups comprising from 1 to about 20 carbon atoms;
each group -NR"Rg is independently selected from the group consisting of:
-NHz
-NHRA
-NR"RB
-NR'°'B
wherein each monovalent R" and R~ and each divalent R'~ is independently an
organic group comprising from:I to 20 carbon atoms, and further comprising a
reactive conjugating functional group.
In one embodiment, said compound has the structure of Formula I. In one
embodiment, said compound has the structure of Formula II. In one embodiment,
said
compound has the structure of Formula III. In one embodiment, said compound
has the
4
CA 02353462 2001-05-31
WO 0013423I PCT/US99/29339
structure of Formula IV. In one embodiment, n is a positive integer from 2 to
4. In one
embodiment, y' , yz, and y3 are each 2. In one embodiment, J is an oxygen
atom. In one
embodiment, J is a covalent bond. In one embodiment, R~ is selected from the
group
consisting of hydrocarbyl groups having from 1 to 20 carbon atoms. In one
embodiment,
R~ is selected from the group consisting of:
-CHZ-;
-CHZCHZ-;
-CHZCHZCHz-;
i H2
-CH2-C-CH2
:,and
\ /
In one embodiment, R~ is selected from the group consisting of organic groups
consisting
only of carbon, oxygen, and hydrogen atoms, and having fi.°om 1 to 20
carbon atoms. In
one embodiment, R~ is:
cH2-cH2-o cw2--cH2-
p-1
wherein p is a positive integer from 2 to 20. In one embodiment, each G', G'-,
and G3 is
independently selected from the group consisting of hydrocarbyl groups having
from 1 to
carbon atoms. In one embodiment, each G', GZ, and G3 is -(CHZ)q wherein q is a
positive integer from 1 to 20. In one embodiment, each G'; GZ, and G3 is
independently
selected from the group consisting of organic groups consisting only of
carbon, oxygen,
20 and hydrogen atoms, and having from 1 to 20 carbon atoms. In one
embodiment, each G',
Gz, and G3 is:
CH2-CH2-O CH2--CH2-
p-1
S
CA 02353462 2001-05-31
WO 00/34231 PCT/US99129339
wherein p is a positive integer from 2 to 20. In one embodiment, R~' is
independently
selected from the group consisting of -H, -CH3, and -CHZC'.H3. In one
embodiment, each
group -NRARB is independently selected from the group consisting of
'N-H
II
-N N-C-O-CH2-
--N 'NH.HBr
O
-N\ N-C-CH2-X
~'NH~CH2~NHz
~n
O CH3
-NH-CH2CH2-NH-~-O--C-CH3
cH3 and
-NH~cH2cH2o~cH2cH2--NHZ
Another aspect of the present invention pertains to a valency platform
compound
having the structure of one of the following formulae:
Formula iV
0
R~ d-IC-N-G' O-Z
Y~
RN
CA 02353462 2001-05-31
WO 00/34231 PCT/US99/29339
Formula V
0 0
Rc J-C-N-Gt O-IC-N-Gz O-Z
N N
yt
Formula VI
0 0 0
R~ ~_"'IC-N-Gi O-IC-N-Gz O-1C-N,_G3 ,O-Z
N RN RN Y3
wherein:
n is a positive integer from 1 to 10;
y' , yz, and y3 are independently a positive integer i:rom 1 to 10;
J independently denotes either an oxygen atom or a covalent bond;
R~ is selected from the group consisting of
hydrocarbyl groups having from I to 20 carbon atoms;
organic groups consisting only of carbon, oxygen, and hydrogen atoms, and
I O having from I to 20 carbon atoms;
organic groups consisting only of carbon, oxygen, nitrogen, and hydrogen
atoms, and having from 1 to 20 carbon ;atoms;
organic groups consisting only of carbon, oxygen, sulfur, and hydrogen atoms,
and having from 1 to 20 carbon atoms;
15 each G', Gz, and G3 is independently selected from the group consisting of
hydrocarbyl groups having from 1 to 20 carbon. atoms;
organic groups consisting only of carbon, oxygen, and hydrogen atoms, and
having from 1 to 20 carbon atoms;
organic groups consisting only of carbon, oxygen, nitrogen, and hydrogen
20 atoms, and having from I to 20 carbon atoms;
each R~' is independently selected from the group consisring of
7
CA 02353462 2001-05-31
WO 00/34231 PCT/US99/29339
hydrogen;
linear or branched alkyl groups having from 1 to 15 carbon atoms;
alkyl groups comprising an alicyclic structure and having from 1 to 15 carbon
atoms;
aromatic groups having from 6 to 20 carbon atoms;
heteroaromatic groups having from 3 to 20 carbon atoms;
each Z is independently selected from the group consisting of:
-H
-C(=O)OR~
-C(=O)RESrsR
-C(=O)NR~RB
wherein:
each Rc"~ is organic groups comprising from 1 to about 20 carbon atoms;
each RES~R is organic groups comprising from 1 to about 20 carbon atoms;
1 S each group -NRARB is independently selected from the group consisting of
-NHZ
-NHR"
-NRARB
-NR"~
wherein each monovalent R" and R~ and each divalent R"B is independently an
organic group comprising from 1 to 20 carbon atoms, and further comprising a
reactive conjugating functional group.
In one embodiment, said compound has the structure ofFormula V. In one
embodiment, said compound has the structure of Formula VI. In one embodiment,
said
compound has the structure of Formula VII. In one embodiiment, n is a positive
integer
from 2 to 4. In one embodiment, yl , yz, and y3 are each 2. In one embodiment,
J is an
oxygen atom. In one embodiment; d is a covalent bond. In one embodiment, RC is
selected
from the group consisting of hydrocarbyl groups having from 1 to 20 carbon
atoms. In one
embodiment, R~ is selected from the group consisting of
_CHz_~
8
CA 02353462 2001-05-31
WO 00!34231 PCT/US99/29339
-CHZCHz-;
-CH2CH2CH2-;
i H2
-CHZ- i -CH2
H2
...~.- ; and
v i ~
In one embodiment, R~ is selected from the group consisting of organic groups
consisting
only of carbon, oxygen, and hydrogen atoms, and having from I to 20 carbon
atoms. In
one embodiment, R~ is:
CH2.-CH2..-p CH2-, CH2-
p-1
wherein p is a positive integer from 2 to 20. In one embodiment, each G', Gz,
and G3 is
independently selected from the group consisting of hydrocarbyl groups having
from I to
carbon atoms. In one embodiment, each G', G2, and G3 its selected from the
group
consisting of:
IH2.-~ IH2-~ lti2-
~'-~H ~-i-CHs ~-i-CH2CN3
CHZ- ~ CH2- ~ and Cti2
In one embodiment, each G', Gz, and G3 is independently selected from the
group
1 S consisting of organic groups consisting only of carbon, oxygen, and
hydrogen atoms, and
having from 1 to 20 carbon atoms. In one embodiment, each RN is independently
selected
from the group consisting of -H, -CH3, and -CHZCH~. In one embodiment, each
group
-NR"RB is independently selected from the group consisting; of:
"'-N \N-H
O
20 ~ - ~N-C-o-cH2-
9
CA 02353462 2001-05-31
WO 00/34231 PCT/US99/29339
---N \NH.Hl3r
-N\ /N-C-CH2-X
-NH~ CHz~Nld2
Jn
O CH3
-NH-CH2CH2-NW-C!-O__C_CH3
i
cH~ ~d
-NH-f-CH2CH20-f-CH2CIH2-NH2
/n
Another aspect of the present invention pertains to methods of preparing a
valency
platform compound, as described herein.
Another aspect of the present invention pertains to a conjugate comprising a
valency
platform compound, as described herein, covalently linked to one or more
biologically
active molecules. In one embodiment, said biologically active molecules are
selected from
the group consisting of oligonucleotides, peptides, polypeptides, proteins,
antibodies,
saccharides, polysaccharides, epitopes, mimotopes, and dnigs.
Another aspect of the present invention pertains to methods of preparing
conjugates,
as described herein.
As will be appreciated by one of skill in the art, feal;ures of one aspect or
embodiment of the invention are also applicable to other aspects or
embodiments of the
invention.
CA 02353462 2001-05-31
WO 00134231 PCTNS99/29339
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows certain valency platforms of the present invention,
specifically,
those having the structure of Formulae I, II, and III.
Figure 2 shows certain valency platforms of the present invention,
specifically,
some of those having the structure of Formula I.
Figure 3 shows certain valency platforms of the present invention, specif
cally,
some of those having the structure of Formula I.
Figure 4 shows certain vaiency platforms of the present invention,
specifically,
some of those having the structure of Farrnula II.
Figure 5 shows certain valency platforms of the present invention,
specifically,
some of those having the structure of Formula II.
Figure 6 shows certain valency platforms of the present invention,
specifically,
some of those having the structure of Formula III.
1 S Figure 7 shows certain valency platforms of the present invention,
specifically,
some of those having the structure of Formula I.
Figure 8 shows certain valency platforms of the present invention,
specifically,
those having the structure of Formulae VI, V, and VI.
Figure 9 shows a synthetic scheme for a simple example of "care propagation"
to
obtain valency platforms of the present invention.
Figure I O shows synthetic schemes for the preparation of certain
intermediates
useful in the preparation of valency platforms of the present; invention.
Figures 1 lA and 1 IB show synthetic schemes for the preparation of valency
platform molecules of the present invention.
Figures 12A and 12B show synthetic schemes for the preparation of valency
platform molecules of the present invention.
Figure 13 shows a synthetic schemes for the preparation of valency platform
molecules of the present invention.
Figures 14A and 14B show synthetic schemes for the preparation of valency
platform molecules of the present invention.
11
CA 02353462 2001-05-31
WO 00134231 PCTNS99/29339
Figure 15 shows a synthetic schemes far the preparation of vaiency platform
molecules of the present invention.
Figures 16A and 16B show synthetic schemes for the preparation of valency
platform molecules of the present invention.
Figures 17A, 17B, 17C, and 17D show synthetic schemes for the preparation of
valency platform molecules of the present invention.
Figures 18A and 18B show synthetic schemes for tile preparation of valency
platform molecules of the present invention.
Figure i 9 shows a synthetic schemes for the preparation of valency platform
molecules of the present invention.
Figures 20A and 20B show synthetic schemes for the preparation of valency
platform molecules of the present invention.
Figure 21 shows the structure of examples of two c~~rbamate compounds 39b and
39c.
DETAILED DESCRIPTION OF THE INVENTION
Throughout this application, various publications, patents, and published
patent
applications are referred to by an identifying citation. The disclosures of
the publications,
patents, and published patent specifications referenced in this application
are hereby
incorporated by reference into the present disclosure in their entirety.
In one embodiment, valency molecules are provided that comprise branches,
wherein at each branch, the molecule branches into two or more arms. The arms
also may
comprise branches. The valency molecule further comprises terminal groups on
arms
extending from the branches. Exemplary terminal groups are reactive
conjugating
functional groups. This is illustrated in the Figures, for exarnple, by
compound _14 in
Figure 11B, which includes 6 branches and 8 terminal CBZ-protected amino
groups.
Thus, in one embodiment, provided is a composition comprising valency
molecules,
wherein each valency molecule comprises at least two branches, ~at least four
terminal
12
CA 02353462 2001-05-31
WO 00134231 PCT/US99/29339
groups, and at least 2 carbamate linkages; and wherein said valency molecules
have a
polydispersity less than about I.2, or for example, less than about 1.07. The
valency
molecules further can comprise, for example, at least 4 carlbamate linkages,
at least 4
branches and at least 8 terminal groups. The valency molecules rnay be
dendrimers.
The number of branches in the valency molecule may vary and may be, for
example, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 16, 32, 64, 100 or more branches.
The number of
branches can be, for example, 2-64, 2-32, 2-16, 4-64, 4-32, 8-64, or $-32. In
a further
embodiment, the number of branches may be, for example, at least 2, at least
4, at least 6,
or at least 8.
The number of carbamate linkages may vary. The valency molecule can include,
for example, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 15, 16, 18, 20, 24~, 29, 32, 64,
100 or more
carbamate linkages. The number of carbaxnate linkages can be, for example, 2-
64, 2-32, 2-
16, 4-64, 4-32, 8-64, or 8-32. In a further embodiment, the number of
carbamate linkages
may be, for example, at least 2, at least 4, at least 6, or at least 8.
Each valency molecule can comprise for example, 1 to 100, e.g, 1-SO terminal
groups. For example, the valency molecule may comprise 4, 6, 8, 9, 10, I2, 14,
15, 16, 18,
20, 21, 24, 29, or 32 or more terminal groups. The valency molecule, for
example, may
comprise at least 4 terminal groups, or at least 6 terminal groups, or at
least 8 terminal
groups. The valency molecule in one embodiment has, for example, 4-16, 4-32, 4-
64, 8-32,
8-64, 12-32 or 12-64 terminal groups. The said terminal groups are in one
embodiment
identical.
Examples of valency molecules include valency platform molecules. Valency
molecules can be made as described herein for the synthesis of valency
platform molecules.
13
CA 02353462 2001-05-31
WO 00/34231 PCT/US99/29339
A. Valency Platforms
In one aspect, the present invention pertains to valE;ncy platforms comprising
carbamate linkages and methods for the preparation of such platforms.
Particular advantages of the present invention include, but are not limited
to, ( I ) the
ease of synthesis of valency platform molecules, (2) the metabolic stability
of the
carbamate linkages in the valency platform, (3) the ability to adjust the
length and water
solubility of the "arms" of the valency platform by using, i:or example,
different
dialcoholamines, (4) the ability to further attenuate the properties of the
valency platform
by choice of the core group (e.g., attachment of solubilizing groups,
chromophores,
reporting groups, targeting groups, and the like).
In one embodiment, a composition is provided comprising valency platform
I 5 molecules, wherein each valency platform molecule comprises at least 2
carbamate
linkages and at least 4 reactive conjugating functional groups; and wherein
said valency
platform molecules have a polydispersity less than about I "2, or optionally a
polydispersity
less than about 1.07. The valency platform molecules of the composition may
comprise,
for example, at least 4 carbamate linkages and at least 8 reactive functional
groups. In one
embodiment, the valency platform molecules comprise at least 4 identical
reactive
conjugating functional groups. In another embodiment, the; valency platform
molecules
comprise, for example, 232 carbamate linkages and 4-64 reactive functional
groups. The
valency platform molecules optionally may be linked to one or more
biologically active
molecules, e.g., via the reactive conjugating functional groups.
The valency molecules, such as valency platform molecules have the advantage
of
having a substantially homogeneous (i. e., uniform) molecular weight (as
opposed to
polydisperse molecular weight), amd are thus "chemically defined".
Accordingly, a
population of these molecules (or conjugates thereof) are substantially
monodisperse, i. e.,
have a narrow molecular weight distribution. A measure of the breadth of
distribution of
molecular weight of a sample of a platform molecule (such as a composition
and/or
14
CA 02353462 2001-05-31
WO 00134231 PCT/US99129339
population of platform molecules) is the polydispersity of the sample.
Polydispersity is
used as a measure of the molecular weight homogeneity or nonhomogeneity of a
polymer
sample. Polydispersity is calculated by dividing the weight average molecular
weight
(Mw) by the number average molecular weight (Mn). The. value of Mw/Mn is unity
for a
perfectly monodisperse polymer. Polydispersity (Mw/Mn;) is measured by methods
available in the art, such as gel permeation chromatography. The
polydispersity (Mw/Mn)
of a sample of valency molecules is preferably less than 2, more preferably,
less than 1.5, or
less than I.2, Less than l.l, less than I .07, less than 1.02, or, e.g., about
1.05 to 1.5 or about
1.05 to I .2. Typical polymers generally have a polydispersity of 2-5, or in
some cases, 20
or more. Advantages of the law polydispersity property oi~the valency platform
molecules
include improved biocompatibility and bioava.ilability since the molecules are
substantially
homogeneous in size, and variations in biological activity due to wide
variations in
molecular weight are minimized. The low polydispersity molecules thus are
pharmaceutically optimally formulated and easy to analyze;.
Further there is controlled vaIency in a population of the valency molecules.
Thus,
in a population of valency platform molecules, for example, the number of
attachment sites,
e.g., reactive conjugating functional groups, is controlled a.nd defined. Each
valency
platform molecule can comprise for example, I to 100, e.g, 1-50 attachment
sites. For
example, the valency platform molecule may comprise 4, fi, 8, 9, 10, 12, 14,
15, 16, 18, 20,
21, 24, 29, or 32 or more attachment sites. The valency platform molecule, for
example,
may comprise at Least 4 attachment sites, or at least 6 atta.clhment sites, or
at least 8
attachment sites. The valency platform molecule in one ennbodiment has, for
example, 4-
16, 4-32, 4-64, 8-32, 8-64, 12-32 or 12-64 attachment sites. The said
attachment sites are in
one preferred embodiment identical.
The number of earbamate linkages may vary. The valency platform molecule can
include, for example, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 15, 16, 1.8, 20, 24,
29, 32, 64, 100 or
more carbamate linkages. The number of carbamate Linkages can be, for example,
2-64, 2-
32, 2-16, 4-64, 4-32, 8-64, or 8-32. In a further embodiment, the number of
carbamate
Linkages may be, for example, at least 2, at least 4, at least ~6, or at least
8.
IS
CA 02353462 2001-05-31
WO 00/34231 PCT/US99/29339
The valency platform molecule can comprise various combinations of the
carbamate
linkages and attachment sites such as reactive functional groups depending on
the method
of preparation, for example, 2-32 , e.g., 2-if carbamate linkages; and 4-64;
e.g., 4-32
reactive functional groups.
Formula I
In one embodiment, the present invention pertains. to a valency platform
having the
structure of Formula I, as shown in Figure 1.
Formula I
0
R~ J--IC-N G~-fl__Z
Yi
2-y'
n
In Formula I, n is a positive integer from 1 to 10, snore preferably from 1 to
5. In
one embodiment, n is a positive integer from 2 to 10, more preferably from 2
to 5. In one
1 S embodiment, n is 1. In one embodiment, n is 2. In one embodiment, n is 3.
In one
embodiment, n is 4.
In Formula I, y' is I or 2, and the subscript "2-y'" its therefore I or 0,
respectively.
In Formula I, J independently denotes either an oxygen atom (i.e., -O-) or a
covalent
bond (i.e., no atom is present). When J is -O-, R~ is bound to the
corresponding sidechain
via a carbamate linkage {i.e., -O-C(=O)-N<). When J is a covalent bond, R~ is
bound to the
corresponding sidechain via an amide linkage (i.e., -C(=O;I-N<).
In Formula I, R~ denotes a "core group," that is, an organic group which forms
the
core of the valency platform, and to which one or more sid.echains is
attached. The valency
16
CA 02353462 2001-05-31
WO 00/34231 PCTlUS99/29339
of the core group is determined by n. If n is 1, then R~ is rnonovalent; if n
is 2, then Rc is
divalent; if n is 3, then RC is trivalent; if n is 4, then RC is tetravalent,
and so on.
In one embodiment, RC is a hydrocarbyl group (i.e., consisting only of carbon
and
hydrogen) having from 1 to 20 carbon atoms, more preferably from 1 to 10
carbon atoms,
still more preferably from 1 to 6 carbon atoms. In one embodiment, RC is
linear. In one
embodiment, R~ is branched. In one embodiment, R~ com;prises a cyclic
structure. In one
embodiment, R~ is cyclic. In one embodiment, R~ is fully saturated. In one
embodiment,
R~ is partially unsaturated. In one embodiment, R~ comprises an aromatic
structure. in one
embodiment, R~ is aromatic. In one embodiment, Rc is -CH2-. In one embodiment,
R~ is
-CH~CH,-. In one embodiment, R~ is -CHZCHZCHZ-. In one embodiment, R~ is:
f Hz
-CH2- i -CH2
H2
In one embodiment, R~ is:
v i ~
is
In one embodiment, R~ is an organic group consisting only of carbon, oxygen,
and
hydrogen atoms, and having from 1 to 20 carbon atoms, mare preferably from 1
to 10
carbon atoms, still more preferably from 1 to 6 carbon atoms. In one
embodiment, R~ is
derived from a polyalkylene oxide group. In one embodiment, R~ is derived from
a
polyethylene oxide group. In one embodiment, R~ is a divalent polyalkylene
oxide group.
In one embodiment, R~ is a divalent polyethylene oxide group. In one
embodiment, R~ is a
divalent polypropylene oxide group. In one embodiment, R~ is:
CH2-CH2-O CH2-CH2-
wherein p is a positive integer from 2 to about 200, rnore preferably from 2
to about
50, more preferably from 2 to about 20, more preferably from 2 to about 10,
more
17
CA 02353462 2001-05-31
WO 00/34231 PCT/US99/29339
preferably from 2 to about b. In one embodiment, p is 2. In one embodiment, p
is 3. In
one embodiment, p is 4. In one embodiment, p is 5. In one embodiment, p is 6.
In one embodiment, R~ is an organic group cansi;;ting only of carbon, oxygen,
nitrogen, and hydrogen atoms, and having from 1 to 20 carbon atoms, more
preferably from
1 to 10 carbon atoms, still more preferably from 1 to 6 carbon atoms. Examples
of such
core groups include, but are not limited to, those derive from the "core
compounds"
described below which consist only of carbon, oxygen, nitrogen, and hydrogen
atoms.
In one embodiment, Rc is an organic group consisting only of carbon, oxygen,
sulfur, and hydrogen atoms, and having from 1 to 20 carbon atoms, more
preferably from 1
to 10 carbon atoms, still more preferably from 1 to 6 carbon atoms. Examples
of such core
groups include, but are not limited to, those derive from tJhe "core
compounds" described
below which consist only of carbon, oxygen, sulfur, and hydrogen atoms.
In Formula I, G' denotes an organic "linker group,." In one embodiment, G' is
a
hydrocarbyl group {i. e., consisting only of carbon and hydrogen) having from
I to 20
caxbon atoms, more preferably from 1 to 10 carbon atoms, still more preferably
from 1 to
carbon atoms. In one embodiment, G' is linear. In one ernbadiment, G' is
branched. In
one embodiment, G' comprises a cyclic structure. In one embodiment, G' is
cyclic. In one
embodiment, G' is fully saturated. In one embodiment, G' is partially
unsaturated. In one
embodiment, G' comprises an aromatic structure. In one embodiment, G' is
aromatic. In
one embodiment, G' is divalent. In one embodiment, RC i.s -{CHZ)q wherein q is
a positive
integer from 1 to about 20, more preferably from 1 to about 10, more
preferably from 1 to
about 6, more preferably from 1 to about 4. In one embodiment, G' is -CFIz-.
In one
embodiment, G' is -CHZCHZ-. In one embodiment, G' is -CHZCHzCH2-.
In one embodiment, G' is an organic group consisting only of carbon, oxygen,
and
hydrogen atoms, and having from 1 to 20 carbon atoms, rxrore preferably from 1
to I O
carbon atoms, still more preferably from 1 to 6 carbon atoms. In one
embodiment, G' is
derived from a polyalkylene oxide group. In one embodiment, G' is a divalent
18
CA 02353462 2001-05-31
WO 00/34231 PCT/US99/29339
polyalkylene oxide group. In one embodiment, G' is a divalent polyethylene
oxide group.
In one embodiment, G' is a divalent polypropylene oxide group. In one
embodiment, G' is:
CH2-CH2-O CH2--CH2-
p-1
wherein p is a positive integer from 2 to about 200, more preferably from 2 to
about
50, more preferably from 2 to about 20, more preferably from 2 to about 10,
more
preferably from 2 to about b. In one embodiment, p is 2. In one embodiment, p
is 3. In
one embodiment, p is 4. In one embodiment, p is 5. In one; embodiment, p is 6.
In one embodiment, G' is an organic group consisting only of carbon, oxygen,
I O nitrogen, and hydrogen atoms, and having from 1 to 20 carlbon atoms, more
preferably From
I to 10 carbon atoms, still more preferably from I to 6 carbon atoms.
In Formula I, RN denotes a nitrogen substituent, moue specifically, an amino
substituent. In one embodiment R~', if present, is hydrogen (i.e., -H). In one
embodiment,
R~', if present, is a linear or branched alkyl group having from I to I S
carbon atoms, more
preferably from 1 to 10 carbon atoms, more preferably from I to 6 carbon
atoms. In one
embodiment, RN, if present, is an alkyl group comprising an alicyciic
structure and having
from I to I 5 carbon atoms, more preferably from 1 to 10 carbon atoms, more
preferably
from I to 6 carbon atoms. In one embodiment, RN, if present, is or comprises
an aromatic
group. In one embodiment, RN, if present, is or comprises a~ heteroaromatic
group. In one
embodiment, RN, if present, is or comprises an aromatic group having from 6 to
20 carbon
atoms, more preferably from b to 1 S carbon atoms, more preferably from 6 to
I0 carbon
atoms. In one embodiment, RN, if present, is or comprises a heteroarornatic
group having
from 3 to 20 carbon atoms, more preferably from 3 to 1 S carbon atoms, more
preferably
from 3 to I0 carbon atoms. In one embodiment, R~ is selected from the group
consisting of
-H, -CH,, and -CH,CH3.
In Formula I, Z denotes a terminal group, which is independently selected from
the
group consisting of: -H (which yields a terminal alcohol group), -C(=O)ORc~'~
(which
i9
CA 02353462 2001-05-31
WO 00/34231 PCT/US99/29339
yields a terminal carbonate group), -C(=O)RES~ (which yield a terminal ester
group), and
-C{=O)NR"RB {which yields a terminal carbamate group).
In the above formulae, each -Rc~'~ is a carbonate substituent or an activated
carbonate substituent. Many carbonate substituents are well known in the art,
including, for
example, organic groups comprising from 1 to about 20 carbon atoms, including,
for
example, primary, secondary, and tertiary, substituted and unsubstituted,
alkyl and aryl
groups having from I to about 20 carbon atoms. Other examples of carbonate
groups
include those described herein for Rte. Still other examples of carbonate
groups include
those described below for activated carbonates.
In the above formulae, each RES~R is an ester substituent or an activated
ester
substituent. Many ester and activated ester substituents are well known in the
art,
including, for example, organic groups comprising from 1 to about 20 carbon
atoms,
including, for example, primary, secondary, and tertiary alkyl and aryl groups
having from
1 to about 20 carbon atoms. Other examples of carbonate groups include those
described
herein for RN. Examples of R~s~R include, but are not limited to, -CH3 (ta
give an acetate
group), -CH~SH (to give a mercaptoacetate group), and -C:HZC6H5, to give a
benzoate
group).
In one embodiment, Z is -NR"RH and denotes an amino group. The amino group
may be unsubstituted, in which case, R~ and RB are both hydrogen (i. e., -
NR"R$ is -NHZ).
The amino group may be monosubstituted, in which case R$ is hydrogen (i.e., -
NR"RH is -
NHRA). The anuno group may be disubstituted. In this case, RA and R$ may be
separate
moieties, as in -NR"RB, or R" and RB may be covalently linked together and
form a divalent
substituent, denoted R~ (i.e., -NR"RB is -NR"g). Thus, in one embodiment, each
group -
NRARH is independently selected from the group consisting; of -NHZ, -NHRA, -
NHR"RB,
and -NR's, wherein each monovalent R~ and RB and each divalent R'~ is
independently an
organic group comprising from 1 to 20 carbon atoms, and :further comprising a
reactive
conjugating functional group. In one embodiment, each group -NRARB is
independently
CA 02353462 2001-05-31
W~ 00/34231 PCT/US99I29339
selected from the group consisting of -NHRA, -NHRARB,, and -NR~$. When not
hydrogen,
R", RB, and R~B, preferably comprise a reactive conjugating functional group.
The term "reactive conjugating functional group" is used herein to refer to
reactive
functional groups which facilitate conjugation, for example, with a
biologically active
molecule. Examples of such reactive conjugating functional groups include, but
are not
limited to, the following:
-OH
-SH
-NCO
--NCS
-NHRsus
RA~KX
--RmKHgX
O
°C-H
O
-C-X
.~~-Rs
O
°C-CH2X
O
-C-CRB=C(R8)2
O
-O-C-X
O
-C-OH
° ~,_ QRESTER
~~-O-~-Rs
21
CA 02353462 2001-05-31
WO 00/34231 PCT/US99/29339
NRsue
o n
-c-o-c-NHRsu~
-~'N'N-Rsua
-N-N-RsuB
O
-N
.
O
NRsus
-C-QRsue
O
.~S'ORALKX
11
O
O
I I
-S-X
II
O
O
-'~_"CRBy~RB)2
-O-NH2
O
NH-C-NH-NH2
iH2 iH
-CH-CH2
In the above reactive conjugating functional groups, each X is independently
F, Cl,
Br, I, or other good leaving group; each R~'J" is independeni;ly an alkyl
group, such as a
linear or branched alkyl or cycloalkyl group having from 1 1:o about 20 carbon
atams; each
RS"s is independently H or an organic group, such as a linear or branched
alkyl group, or a
cycioalkyl group having from 1 to about 20 carbon atoms, an aryl group having
from 6 to
about 20 carbon atoms, or an alkaryl group having from 7 to about 30 carbon
atoms; each
RESTER is independently an organic group having from 1 to albout 20 carbon
atoms,
including, for example, primary, secondary, and tertiary alkyl and aryl groups
having from
1 to about 20 carbon atarns; and, each R$ is independently a organic group,
such as an
22
CA 02353462 2001-05-31
WO 00/34231 PCT/US99129339
organic group comprising 1 to 50 atoms selected from the group consisting of
C, H, N, O,
Si, P, and S.
In a preferred embodiment, the reactive conjugating functional group is an
amino
S group or a protected amino group. In one embodiment, th.e group -NR"R$
comprises an
amino group, and has the structure:
-N/ \N-Hi
In one embodiment, the group -NR"RB comprises a protected amino group, and has
the structure:
/~ I!
- ~N-G-O-CH2
which is often conveniently abbreviated using "CBZ" to denote
"carbobenzyloxy":
- ~N-Ge,z
U
In one embodiment, the group -NR"RB comprises a hydrobromide salt of an amino
group, and has the structure:
- ~NH.HBr
U
In one embodiment, the group -NRARH comprises a haloacetyl group (where X
denotes Cl, Br, or I), and has the structure:
~--~ 0
-N/ \N- IC-CH2-X
In one embodiment, the group -NR"RB comprises an amino group, and has the
structure:
-NH~CHZ~NFI2
~n
wherein n is a positive integer .from 1 to about 20, preferably from 1 to
about 10,
preferably from 1 to about 5. In one embodiment, the group -NR~R$ comprises a
protected
amino group, and has the structure:
23
CA 02353462 2001-05-31
WO 00/34231 PCTIUS99/29339
i Hs
-NH-CH2CH2-NH-C-(J-C-CH3
i
CH3
which is often conveniently abbreviated using "BOC" to denote "tert-
butoxycarbonyl":
-NH-CH2CH2-NH--BOC
In one embodiment, the group -NR"RB comprises an amino group, and has the
structure:
-NH-f-CH2CH20-t-CH2CH2-NH2
~n
wherein n is a positive integer from 1 to about 20, :preferably from 1 to
about 10,
preferably from 1 to about 5.
Examples of valency platforms having the structure of Formula I are shown in
Figures 2, 3, and 7. In Figure 2, the top structure has n=l and y'=I and the
bottom
structure has n=2 and y'=1. In Figure 3, the top structure l'~as n=1 and y'=2
and the bottom
structure has n=2 and y'=2. In Figure 7, the structure has :n---4 and y'=2.
The number of
I 5 terminal groups -NRaRB is given by "n*y'." When "n*y'" is 4, the structure
may
conveniently be referred to as a "tetrameric" structure. When "n*y'" is 8, the
structure may
conveniently be referred to as a "octameric" structure. When "n*y'" is I6, the
structure
may conveniently be referred to as a "hexadecameric" stn~cture.
Examples of compounds having the structure of Formula I where Z is -H include,
but are not limited to, compounds 21, 24, 27a, 29, 32, and 38, described in
the Examples
below.
Examples of compounds having the structure of Formula I where Z is -
C(=O)OR~~'~ include, but axe nat limited to, compounds 22, 25, 27, 30, 33, and
39
described in the Examples below.
24
CA 02353462 2001-05-31
WO OOI34231 PCT/US99/29339
Examples of compounds having the structure of Formula I where Z is -NR"RB
include, but are not limited to, compounds 23, 23a, 26, 26a, 31, 31a, 34, 34a,
40, 41, 42,
and 51, described in the Examples below.
Formula iI
In one embodiment, the present invention pertains to a valency platform having
the
structure of Formula II, as shown in Figure 1.
Formula ll
0 0
R° J-~C-N G'-O-IC-!J G2-o-"Z
"~ '~ ,
[ R" ~ 2-)~ Y
2-y~
1~
In Formula II, n, R~, J, R", Ra, y', R~', G' , and Z acre as defined above for
Formulae
I. In Formula II, yz and GZ are as defined above for y' and G', respectively.
Examples of valency platforms having the structure of Formula II are shown in
Figures 4 and 5. In Figure 4, the top structure has n=1, y'==2, and y2=1 and
the bottom
structure has n=i, y'=2, and yz=2. In Figure S, the structure has n=2, y'=2,
and y'-=2. The
number of terminal groups -NR~RB is given by "n*y'*y2." When "n*y'*y2" is 4,
the
structure may conveniently be referred to as a "tetrameric" structure. When
"n*y'*y'-" is 8,
the structure may conveniently be referred to as a "octamexic" structure. When
''n*y'*y2" is
16, the structure may conveniently be referred to as a "hex:adecameric"
structure.
Examples of compounds having the structure of Formula II where Z is -H
include,
but are not limited to, compounds 35, 43a, and 49a, described in the Examples
below.
CA 02353462 2001-05-31
WO 00134231 PCTIUS99/29339
Examples of compounds having the structure of Formula II where Z is -
C{=O)ORc"'~ include, but are not limited to, compounds :3Sa, 43, and S0,
described in the
Examples below.
S Examples of compounds having the structure of Formula II where Z is -NR~RB
include, but are not limited to, compounds 14, 1 S, 20, ZOa, 28, 28a, 36, 36a,
44, 44a, and
4S, described in the Examples below.
Formula III
In one embodiment, the present invention pertains to a valency platform having
the
structure of Formula III, as shown in Figure 1.
Formula ill
O O O
R~ J-CI-N G'-O-iC-N G2-O-IC-IN G3-O-Z
. ~ ~~ N ~ Y3
RN ~ ~ RN ~ 2_f~ 2_Ys ~ t
2_y~ Y
1 S In Formula III, n, R~, J, R", RB, y', y2, RN, G', Gz, and Z are as defined
above for
Formulae I and II. In Formula III, y3 and G~ are as defined above for y1 and
G',
respectively.
Examples of a valency platform having the structure of Formula III is shown in
Figure 6. This structure has n=2, y'=2, y2=2, and y3=2. The number of terminal
groups
-NR~RB is given by "n*y'*y2*y3." When "n*y'*y2*y3" is 4, the structure may
conveniently
be referred to as a "tetrameric" structure. When "n*y'*yz*y3" is 8, the
structure may
conveniently be referred to as a "octameric" structure. When "n*y'*yz*y'" is
16, the
structure may conveniently be referred to as a "hexadecameric" structure.
2S
26
CA 02353462 2001-05-31
WO 00/34231 PCT/US99I29339
Formulae IV, V, and VI
In one embodiment, the present invention pertains to a valency platform having
the
structure of Formula IV, V, or VI, as shown in Figure $.
Formula 1V
0
R° J-'C-N-G' O-Z
Y~
Formula V
0 0
R° J-C-N-G' O-C-N-Gz O-Z
~N ~N
Y~
Formula VI
o o
R~ J-C ~ G~ O-IC- I -G2 O-C~-N-G3 O-Z
RN RN RN Y3
Y~
In these formulae, n, R~, J, R~, RH, RN, and Z are as defined above for
Formulae I
through III. Unlike the compounds of Formulae I through III, which may have
branch
points at nitrogen atoms, compounds of Formulae IV throul;h VI may have branch
points at
a G group, for example, at G', G2, or G3, and there may be one, two, three, or
more
branches, for example, y', y2, or y3 branches.
For Formulae IV through VII, G', GZ, and G3 are similar to G', Gz, and G3 for
Formulae I through III. In a preferred embodiments, these l;roups are
trivalent, tetravalent
or higher. In one embodiment, G', G2, and G3 are selected from the group
consisting of
27
CA 02353462 2001-05-31
WO 00/34231 PCT/US99/29339
IH2-~ IH2.-~ IH2_...
""' i H ~ - i -CH3 ~ - i -CH2CH3
CH2- ~ CH2- ~ and cH2
Also, for Formulae IV through VI, y', y2, and y3 acre positive integers from 1
to
about 10, more preferably from 1 to 5, more preferably from 1 to 4, more
preferably from 1
to 3, more preferably from 1 to 2:
Examples of compounds having the structure of Formula IV where Z is -H
include,
but are not limited to, compound 46, described in the Examples below.
Examples of compounds having the structure of Formula IV where Z is -
C(=O)OR~'~ include, but are not limited to, compound 4~7, described in the
Examples
below.
Examples of compounds having the structure of Formula V where Z is -H include,
but are not limited to, compound 47a, described in the Examples below.
Examples of compounds having the structure of Formula V where Z is -
C(=O)OR~"'~ include, but are not limited to, compound 48, described in the
Examples
below.
Examples of compounds having the structure of Formula V where Z is -NR"RB
include, but are not limited to, compounds 48a, 48b, and 4~8c, described in
the Examples
below.
In general, the number of termini may be calculated.as the product of n, y',
y2, y3,
etc., as discussed above. In one embodiment, this product is 2 or more. In one
embodiment, this product is more than 2. In one embodiment, this product is
more than 3.
In one embodiment, this product is 4. In one embodiment, this product is 6. In
one
28
CA 02353462 2001-05-31
WO 00/34231 PCTNS99/29339
embodiment, this product is 8. In one embodiment, this product is 16. In one
embodiment,
this product is 32.
In some embodiments, the valency platform molecule may be described as
"dendritic," owing to the presence of successive branch points. Dendritic
valency platform
molecules possess multiple termini, typically 4 or more termini. In one
embodiment, the
valency platform molecule is dendritic and has 4 termini, such as, for
example, compounds
23a, 2&a, 31a, 34a, 42a, described in the examples below. In one embodiment,
the valency
platform molecule is dendritic and has 8 termini, such as, for example,
compounds 1 S, 20a,
28a, 36a, 4S, 48c and S1, described in the examples below. In one embodiment,
the
valency platform molecule is dendritic and has 16 termini"
Note that ForrnuIae I through Vi are intended to encompass both "symmetric"
and
"non-symmetric" valency platforms. In one embodiment, the valency platfor~rrr
is
I S symmetric. In one embodiment, the valency platform is non-symmetric. For
example,
each of the "n" groups which are pendant from the core group, RC, may be the
same or may
be independently different.
"Higher generation" valency platforms (e.g., 4th generation, Sth generation}
are also
contemplated, which have corresponding formulae. For e~;ample, 4th generation
valency
platforms would have G4 and y4, 5th generation valency platforms would further
have G~
and ys, and so on for successive generations. Also, "hybrid" valency
platfor~rrrs are
contemplated, which would include linkages of the sort found in Formulae I
through III as
well as linkages of the sort found in Formulae IV through 'VI.
2S
B. Preparation of Valency Platforms
In one embodiment, the valency platforms of the present invention may be
prepared
from "core" compounds which comprise one or more (say, j°) hydroxy
groups (i.e., -OH).
For example, the hydroxyl groups on the core are converted to active carbonate
derivatives,
such as activated carbonate esters (for example, apara-nitrophenylcarbonate
ester) and
29
CA 02353462 2001-05-31
WO 00/34231 PCTJUS99/29339
subsequently reacted with a polyhydroxyamine compounds having j I hydroxy
groups to
provide a "first generation" carbamate with j' hydroxyl groups for each
original hydroxyl
group, for a total of j°*j' hydroxyl groups. The resulting hydroxy
groups may then also be
converted to activated carbonate derivatives, such as activated carbonate
esters and
subsequently reacted with a polyhydroxyamine compound. having j2 hydroxy
groups to
provide a "second generation" carbamate with j2 hydroxyl groups for each j'
hydroxyl
group, for a total of j°*j'*j2 hydroxyl groups. In this way, a
dendritic structure may be
constructed. The process can be terminated at any "generation" by treating the
terminal
activated carbonate derivatives, such as activated carbonate esters, with an
appropriately
functionalized compound (for example, an mono-protected diamine) to provide
whatever
functionality is desired at the termini.
In one embodiment, the valency platforms of the present invention may be
prepared
from a "segmental approach" in which "segments" are independently synthesized
and
subsequently attached to a "core" group
In another embodiment, an alternative, more eff cie:nt "core propagation"
process
has been developed in which a core group is modified in an iterative process
to generate a
dendritic structure. The core propagation approach involves fewer steps and is
preferred
over the segmental approach.
In another embodiment, the valency platforms of the present invention may be
prepared using solid phase synthesis from a hydroxyl containing resin. Such an
embodiment is illustrated in Figure 20. A hydroxyl group attached to a solid
phase by a
cleavable linker provides a way of building a dendrimeric scaffold using solid
phase
synthesis. The ability to prepare scaffolds on the solid phase can be
particularly useful for
the rapid synthesis of dendrimeric platforms with minimal purification. Also,
solid phase
dendrimeric platforms can be used to generate combinatorial Libraries of
multivalent
compounds.
30
CA 02353462 2001-05-31
WO 00/34231 PCT/US99/29339
In one preferred "core propagation" approach, the; synthesis typically begins
with an
alcohol containing "core compound." In principle, any hydroxyl-containing
compound can
be used. Examples of alcohol containing "care compounds" having one hydroxyl
group
{i.e., -OH) include, but are not limited to:
methanol,
CH3-OH
ethanol,
CH3-CHz-OH
prapanol,
CHswCHz-~CH2-OH
isopropanol,
OH
CH3-CH-CH3
methaxypalyethylene glycol,
CH30 CHz-CHz-O H
n
IS
Other examples of alcohol containing "core compounds" having one hydroxyl
group (i.e., -OH) include, but are not limited to, mono-hydroxylamines, such
as those
described below, for which the amino group may be in a protected form, for
example, using
a BOC or CBZ protecting group.
Examples of alcohol containing "core compounds" having two hydroxyl groups
(i.e., -OH) include, but are not limited to:
ethylene glycol,
HO-CHz-CHz-OH
diethylene glycol (also referred to as DEG),
HO-CHz-CHz-O-CHz-CHz-OH
triethylene glycol (also referred to as TEG),
HO-CHz-CH2-O-CH2-CH2-O-CI-~2-CHz-OH
31
CA 02353462 2001-05-31
WO 00/34231 PCT/US99/29339
tetraethylene glycol,
HO CHZ-CH2-O H
4
pentaethylene glycol,
HO CHz-CH2-O H
hexaethylene glycol,
HO CH2-CH2-O H
6
polyethylene glycol {also referred to as PEG), where n is typically from 1 to
about
20p,
HO CHZ-CH2-O H
n
I ,4-dihydroxymethylbenzene,
HOCH2 ~ ~ CH20H
Other examples of alcohol containing "core compounds" having two hydroxyl
groups (i.e., -OH) include, but are not limited to, primary or secondary
amines having two
I S hydroxyl groups, such as those described below. Again, the amino group may
be in a
protected form, for example, using a BOC or CBZ protecting group.
Examples of alcohol containing "core compounds" having three hydroxyl groups
{i.e., -OH) include, but are not limited to:
phluoroglucinol (also known as I,3,5-trihydroxybenzene),
OH
HO / OH
32
CA 02353462 2001-05-31
WO 00/34231 PCT/US99/29339
1,3,5-trihydroxymethylbenzene,
CH20H
HOCH2 ~ CH20H
1,3,5-trihydroxycyclohexane,
OH
HO OH
Other examples of alcohol containing "core compounds" having three or more
hydroxyl groups (i.e., -OH) include, but are not limited to, :primary or
secondary amines
having three hydroxyl groups, such as those described below. Again, the amino
group may
be in a protected form, for example, using a BOC or CBZ protecting group.
Examples of alcohol containing "core compounds" :having four hydroxyl groups
(i.e., -OH) include, but are not limited to:
pentaerythritol,
~ H20H
HOCH2- i -CH20H
CH20H
Further examples of alcohol containing "core compounds" include, but are not
limited to, those which comprise a sulfhydryl group (i.e., -SH), which may be
protected, for
example, with a trityl protecting group (i. e., as -S-Tr, that is, -S-
C(C6H5)3) or as a disulfide
(i. e., as -S-SR). Examples of core groups which have a protected sulfhydryl
group include,
but are not limited to, the following:
Tr-S-CH2CH2-OH
HO-CH2CH2-S-S-CH2CH2-OH
33
CA 02353462 2001-05-31
WO 00/34231 PCTIUS99/29339
i Hs
CH3-C-S-S CH2-CH2-O H
CH3
where n is from 1 to about 200, preferably from 1 to about 20.
In addition, hydroxyl groups on solid phase synthesis resins can be used as
core
groups to provide dendrimeric carbamate residues on solid phase which can be
used to
boost the valence of the resin or cleaved off the resin. For example, a Wang
resin of the
following form may be used:
CH20~~ CH20H
In one embodiment, a hydroxy containing core groaap may be prepared from a
corresponding carboxylic acid compound or halocarbonyl compound:
HN(CHZCH20H)2
pyridine or Et3N /CH2CH20H
R-COOH --a. R-COX ~~. R-CO-N
\GH2CH20H
Core compounds which possess amino or sulfl~ydryl groups, which may be
protected or unprotected, may be used to covalently attach the resulting
valency platform
molecule to other molecules of interest, via the core group rather than via
the termini, using
conjugation methods such as those described herein.
In one step, the hydroxyl groups of the alcohol containing core group are
converted
to active carbonate derivatives. The active carbonate derivative in one
embodiment has the
formula:
O
O C
X
where X is a leaving group such as CI, irnidazole or thiolate.
34
CA 02353462 2001-05-31
WD 00/34231 PCT/US99129339
The hydroxyl groups of the alcohol can be converted to active active
carbonates by
reaction of the hydroxyl groups of the alcohol containing core group with a
phosgene
equivalent. Phosgene equivalents with appropriate reactivity can be selected.
The
phosgene equivalent has, for example, the structure X~(CO)XZ where X~ and Xz
are both
Leaving groups. X, and XZ each independently can be chosen from typical
leaving groups
in acylation chemistry such as alkoxide, thiolate, halide, quid imidazole. In
one preferred
embodiment, the phosgene equivalent is 4-nitrophenylchloroformate. In another
embodiment, the phosgene equivalent is carbonyldiimida;aole. Other exemplary
phosgene
equivalents include phosgene, N,N'-succinimidylcarbonate, succinimidyl
2,2,2-trichloroethylcarbonate, bis-4-nitrophenylcaxbonate, triphosgene,
2,2,2-trichloroethyichloroformate, 4-nitrophenylchloroformate,
phenylchioroformate,
N-hydroxysuccinimidylchloroformate, trichloromethylchloroformate,
ethylchlorothiolformate, di-(1-benzotriazolyl)carbonate, and
4-nitrophenylsuccinimidylcarbonate.
Thus, in one embodiment, to form an active carbonate derivative in the
synthesis of
the valency platform molecule, an alcohol is reacted with 'the phosgene
equivalent to form
the activated carbonate by displacing X, . X, is chloride in. one preferred
embodiment. The
active carbonate derivative is used to acylate an aminoalcohol on the
nitrogen, forming the
caxbamate bond, then the phosgene again is added to convert the hydroxyl group
to another
active carbonate derivative. An example of an active carbonate derivative is
compound 39c
shown in Figure 21.
In one embodiment, the active carbonate derivative; is a carbonate ester. The
terms
"carbonate" and "carbonate ester" are used herein in the conventional sense
and relate to
species which comprise the following structure:
0
-o-c-o-R'
wherein R' denotes a carbonate group, such as an organic group having from 1
to 20
carbon atoms. The terms "activated carbonate" and "activated carbonate ester"
are used
herein to refer to carbonates for which R' is an activating group, and for
which the moiety -
O-R' forms a good leaving group. A particularly preferred class of activated
carbonate
CA 02353462 2001-05-31
WO OOI34231 PCT/US99/29339
esters include, but are not limited to para-nitrophenyl carbonate ester
compounds of the
formula:
o _
p IC-O ~ ~ N02
Such "PNP" activated carbonate esters may readily be formed from the
corresponding alcohol, R-OH by reaction with PNP chloro~formate in the
presence of
pyridine (CSHSN) in methylene chloride (CHZC12). '
O _
R-OH + CI-C-p--~~ ~ Npz
pyridine, CH2CI2 O
--~"- R-O_C'p--(~ ~ N02
Examples of other activated carbonate groups include, but are not limited to,
the
following:
0
-o-c~
o
O
-O-C_(~_N
p
0
-o-c-o-cH2
In another step, the activated carbonate ester is converted to the
corresponding
carbamate. The above PNP activated carbonate esters are readily converted to
the
corresponding carbamates by reaction with an amine. The dlendritic structure
may be
extended by employing a primary or secondary amine having j' hydroxy groups.
In this
36
CA 02353462 2001-05-31
WO 00/3423I PCT/US99/29339
way, each original hydroxy group, which led to an activated carbonate ester
group, then
leads to j' hydroxy groups.
Fox example, the PNP activated carbonate ester may be reacted with a primary
or
secondary dihydroxyamine. Examples of primary and secondary amines having two
hydroxyl groups include, but are not limited to:
diethanolamine, ,
/CH2CN2-OH
HN
CN2CH2-OH
bis(diethyleneglycol)amine (compound 7),
(CW2CH20)2-H
HN
~(CH2CH20)2-H
bis(triethyleneglycol)amine,
(CH2CH20)3-H
HN
~(CH2CH20)3-H
bis(tetraethyieneglycol)amine,
(CH2CH20)4-H
HN
~(CH2CHz0)4-H
1 ~ bis(pentaethyleneglycol)amine,
(CH2CH20)5-H
HN
~(CH2CHz0)5-H
bis(hexaethyleneglycol)amine (compound 4),
(CH2CH2O)6-H
HN
~(CH2CH20)6-H
bis(polyethyleneglycol)amine (where n is from I to about 20),
~ (CH2CH20)"-H
HN
~(CH2CH20)"-H
37
CA 02353462 2001-05-31
WO 00/34231 PCTIUS99/29339
diisopropanolamine,
OH'
/CH-CHz-CH3
HN
NCH- i Hz-CH3
OH
serinol (also known as 2-amino-1,3-propanediol),
f H2
HO-CHz-CH-CHz-OH
2-amino-2-methyl-1,3-propanediol,
~ Hz
HO-CHz- ~ H-CHz-OH
CH3
2-amino-2-ethyl-1,3-propanediol,
i Hz
HO-CHz-CH-CHz-OH
CH2CH3
tris(hydroxymethyl)aminomethane,
i H20H
HOCHz- ~ -NHz
CH20H
tris(hydroxyethyl)aminomethane,
i H2CH20H
HOCH2CH2-L-NHz
CH2CH20H
1-amino-1-deoxysorbitol (also referred to as glucamine),
NHz
H OH
HO H
H OH
H OH
CH20H
38
CA 02353462 2001-05-31
WO 00/34231 PCT/US99/29339
N-methyl-D-glucamine,
NHCH3
H OH
HO H
H OH
H OH
CH20H
In one step, the activated carbonate ester is converted to the corresponding
carbamate using a monohydroxyamine to maintain vaiency from one generation to
the next
yet impart unique properties such as arm length, steric bulb;, solubility, or
other physical
properties. Examples of such monohydroxyamines include, but are not limited
to:
3-pyrrolidinol,
OH
NH
2-(hydroxyrnethyl)pyrrolidine,
-CH2-OH
NH
3-hydroxypiperidine (also referred to as 3-piperidinol),
off
i
NH
3-(hydroxymethyl)piperidine,
CH2-OH
NHJ
2-(hydroxymethyl)piperidine,
NH~CH2-OH
39
CA 02353462 2001-05-31
WO 00/34231 PCTlUS99/29339
4-(2-hydroxyethyl)piperidine,
CH2CH20H
NH
4-piperidinol,
OH
J
NH
2,2,6,6,-tetramethyl-4-piperidinol,
OH
CH3 ~CH3
CH3 NH CH3
mono-amino-oligoethylene glycol (where n is from 1 to about 10),
HZN-r-CH2CH20~ H
~n
mono-amino-polyethylene glycol (where n is frorr.~ 1 to about 200),
H2N l CH2CH20-f--H
In one step, the activated carbonate ester is converted to the corresponding
carbamate using a primary or secondary amine which acts as a "terminating"
amine. In one
embodiment, a mono-protected diamine is employed. In a preferred embodiment,
the
I 5 terminating amine is a mono-CBZ protected piperazine, since this compound
provides a
convenient secondary amine handle for adding functionality by acylation with
other
reactive groups such as haloacetyl, maleimidoyl, etc. depending on what is
desired at the N-
terminus. For example, reaction with mono-CBZ-protected piperazine in the
presence of
triethylamine ((CH3CH2)3N) in methylene chlaxide (CHZC:l2) yields the CBZ-
protected
piperazine carbamate, which can then be converted to a haloacetyl group:
CA 02353462 2001-05-31
WO 00/34231 PCT/US99I29339
H-N N-CB;Z
O
_ II (CH3CH2)sN II
O-C O ~ ~ N02 ~'-O C- ~N-CBZ
II ~ o
-O-C- UN-CBZ -----.s ~-O-C-N~N-C-CHZX
Ethylenediamine and other diamines can function ;similarly. Examples of
preferred
terminating amines include, but are not limited to those shown below, as well
as mono-
protected (e.g., mono-CBZ-protected) forms thereof
piperazme,
H-N N-H
ethylenediamine,
H2N-CH2-CH2-NH2
propylenediamine,
H2N-CH2-CH2-CH2-NH2
aikylenediamines (where n is an integer from 1 to about 20),
H2N CH2 NH2
n
N,N'-dimethylethylenediamine,
CH3-NH-CH2-CH2-NH-CH3
a,w-diaminopolyethyleneglycol (where n is an integer from 1 to about 200,
preferably from 1 to about 20),
H2N CHZCH20 CH2CH2-NHZ
n
41
CA 02353462 2001-05-31
WO 00/34231 PCT/US99/Z9339
A particularly preferred terminating amines is mono-CBZ-protected piperazine:
O
H-N\ 'N-C-O-CH2
~/
In principle any primary or secondary amine containing compound which contains
a
reactive conjugating group (such as those described above) or a biologically
active
molecule can be. used to terminate the dendrimer and provide the terminal
functionality that
is desired. For example, amino alcohols would provide terminal hydroxyl
groups, amino
aldehydes would provide terminal aldehyde groups, amino acids would provide
terminal
carboxylic acids, and aminothiols would provide terminal thiols. Methods for
the
introduction of other reactive conjugating functional groups, such as those
described above,
as terminating groups are well known to those of skill in the art.
C. Valenc Platform Con'ugates, Methods of Preparation, and Uses Thereof
In one embodiment, valency platform molecules a~~e provided which act as
scaffolds
to which one yr more molecules may be covalently tethered to form a conjugate.
Thus, in
another aspect, the present invention pertains to valency platform conjugates:
In one embodiment, the valency platform is covalently linked to one or more
biologically active molecules, to form a conjugate. The team "biologically
active
molecule" is used herein to refer to molecules which have biological activity,
preferably in
vivo. In one embodiment, the biologically active molecule; is one which
interacts
specifically with receptor proteins.
In one embodiment, the valency platform is covale,ntly linked to one or more
oligonucleotides, to form a conjugate. In one embodiment, the valency platform
is
covalently linked to one or more peptides, to form a conjul;ate. In one
embodiment, the
valency platform is covalently linked to one or more polypeptides, to form a
conjugate. In
one embodiment, the valency platform is covalently linked to one or more
proteins, to form
42
CA 02353462 2001-05-31
WO 00/34231 PCT/US99129339
a conjugate. In one embodiment, the valency platform is covalently linked to
one or more
antibodies, to form a conjugate. In one embodiment, the valency platform is
covalently
linked to one or more saccharides, to form a conjugate. In one embodiment, the
valency
platform is covalently linked to one or more polysaccharides, to form a
conjugate. In one
embodiment, the valency platform is covalently linked to one or more epitopes,
to form a
conjugate. In one embodiment, the valency platform is covalently linked to one
or more
mimotopes, to form a conjugate. In one embodiment, the valency platform is
covalently
linked to one or more drugs, to form a conjugate.
In one embodiment, the biological molecule is first modified to possess a
functionalized linker arm, to facilitate conjugation. An example of such a
functionalized
linker arm is a polyethylene glycol disulfide, such as, for example:
O S/S, CH3
n I -CH3
CHs
One advantage of the valency platforms of the present invention is the ability
to
introduce enhanced affinity of the tethered biologically active molecules for
their binding
partners. Another advantage of the valency platforms of the present invention
is the ability
to facilitate crosslinking of multiple ligands, as is useful in B cell
tolerance. Another
advantage of the valency platforms of the invention is the ability to include
functionality on
the "core" that can be independently modified to enable the preparation of
conjugates
which can be tailored for specific purposes.
Conjugates of the valency piatfor~rn molecule and one or more biologically
active
molecules may be prepared using known chemical synthetic methods. As discussed
above,
the termini of the valency platform molecule (i. e., the R", R$, and/or
R'°'B of the group -
NR"RB, as discussed above) preferably comprise a reactive conjugating
functional group,
and this reactive functional group may be used to couple fhe valency platform
to the desired
biologically active molecule.
43
CA 02353462 2001-05-31
WO 00/34231 PCT/US99/29339
In one embodiment, the reactive haloacetyl group may be used to couple the
valency platform to a biologically active molecule which possesses one or more
reactive
conjugating functional groups which are reactive towards the haloacetyl group,
and which
react to yield a covalent linkage.
S
For example, if the biologically active molecule is a protein which has one or
more
free amino groups (i.e., -NHZ), the two groups may be used to form the
conjugate:
o
8
vaiency platform-C-CH2X + H2N-protein
C)
II
valency platform-C;-CH2--NH-protein
In another example, if the biologically active molecule is a protein which has
one or
more free thiol groups (i.e., -SH) or sulfide groups {i.e., -SR), the two
groups may be used
to form the conjugate:
o
II
valency platform-C-CHzX + RS-protein
C>
valency platform-C.-GH2--S protein
In another embodiment, a terminal maleimidoyl group may be used to couple the
valency platform to a biologically active molecule which possesses one or more
reactive
conjugating functional groups which are reactive towards i;he maleimidoyl
group, and
which react to yield a covalent linkage.
For example, if the biologically active molecule is a protein which has one or
more
free thiol groups (i.e., -SH) or sulfide groups (i.e., -SR), the two groups
rnay be used to
form the conjugate:
44
CA 02353462 2001-05-31
WO 00/34231 PCT/US99/29339
O
vaiency platform-N~ + RS-protein
O~~~i' O
S-protein
--~~ valency platform--N
O
D. Examples
Several embodiments of the present invention are illustrated in the Examples
below,
which are offered by way of illustration and not by way of limitation.
Example 1
Examples of Synthesis of Amine Diols
IO
A chemical scheme for the preparation of HEGA (bis-hexaethyleneglycolamine) is
shown in Figure I 0. One hydroxy terminus of hexaethylene glycol is f rst
converted to a
tosyl group (compound 1 ), which is then converted to a bromo group (compound
2). The
resulting compound is then reacted with tosylamide to yield tosylated bis-
I S hexaethyleneglycolamine (compound 3). The tosyl group is then removed to
yield the
desired bis-hexaethyleneglycolamine (compound 4).
A chemical scheme for the preparation of DEGA (bis-diethyleneglycolamine) is
also shown in Figure 10. Chlorodiethylene glycol (compound 5) is reacted with
20 aminodiethylene glycol (compound 6) to yield the desired bis-
diethyleneglycolamine
{compound 7).
Compound 1
Hexaethyleneglycol mono-tosylate
25 g (88.5 mmol) of hexaethyleneglycol was stirred at 0°C in 200 mL of
CHZC12,
and 14.3 mL of pyridine ( 177 mmol; 2 eq.) was added to the mixture followed
by 17.4 g
CA 02353462 2001-05-31
WO 00/34231 PCTlUS99/29339
(88.5 mmol) of tosylchloride. The reaction mixture was stirred at room
temperature for 24
hours and partitioned between 400 ml of IN HCl and 200 ml of CHzCl2. The
organic layer
was dried over MgS04, filtered, and concentrated to provide 31 g of a light
yellow oil.
Purification by silica gel chromatography (CHZClZ/MeOH) provided 15.32 g (40%)
of 1 as
S a light yellow oil:'H NMR (CDC13) 8 2.45 (s, 3H), 3.SS- ..75 (m, 22H), 4.1 S
(t, 2H), 7.35
(d, 2H), 7.80 (d, 2H); HRMS (FAB) calculated for C,~H33~O9S (M+H): 437.1845.
Found:
437.1834.
Compound 1
Hexaethyleneglycoi mono-tosyiate
SO g of HEG (177 mmol) was dissolved in 300 ml of CHZCIz and 7.2 ml (8$ mmol)
of pyridine was added at room temperature. 17.4 g (8$ mrnol) of tosylchloride
was added
to the mixture in four batches, each 2 hours apart. After the last addition,
the mixture was
1 S stirred for 16 hours. The reaction mixture was concentrated, 1 S 0 mL of
0.1 M HCI was
added, and the mixture was extracted twice with hexane to remove excess
tosylchloride.
The aqueous layer was washed with three portions of ether to remove di-
tosylate. This was
carefully monitored by TLC to avoid any removal of mono-tosylate. The aqueous
Iayer
was then extracted with portions of CHzGl2 . The combined organic layers were
washed
with 0. I M HCI, dried over MgSOQ, filtered, and concentrated to give 23.6 g
(31 %) of
compound 1.
Compound 2
Hexaethyleneglycol mono-bromide
2S
Compound I (I 8 g, 41.3 mmoI) was dissolved in 12;0 mL of acetone and 10.8. g
of
Liar (124 mmol) was added. The mixture was stirred at 60~°C for 2
hours, the reaction
mixture was allowed to cool to room temperature, and S00 mL of H20 was added.
The
mixture was extracted with 2x 500 mL of CHzCI2. The corxibined organic layers
were dried
over MgS04, filtered, and concentrated to give 13.7 g (96%) of compound 2 as a
light
46
CA 02353462 2001-05-31
WO 00/34231 PCT/US99/29339
yellow oil: 'H NMR {CDCl3) b 3.50 (t, 2H), 3.60-3.75 (m, 20H), 3.83 (t, 2H);
HRMS
(FAB) calculated for C~ZH26BrO6 {M+H): 345.0913. Found: 345.0922.
Compound 3
N,N-his-hexethyleneglycol-tos;ylamide
Compound 2 {3.5 g, 9.8 mmol) and 0.84 g (4.9 mmol) of tosylamide were
dissolved in 35 mL of CH3CN. Potassium carbonate (1.63 g (11.8 mmol), which
had been
dried in the 100°C oven, was added, arid the mixture was refluxed for
18 hours under Nz.
The mixture was allowed to cool to room temperature, and 150 mL of HzO was
added. The
mixture was extracted with 3 X I50 mL of CHzCl2. The combined organic layers
were
washed with HBO, dried (MgS04), f ltered, and concentrated to give 3.3 g of a
light yellow
oil. Purification by silica gel chromatography (CHZCh /MeOH) provided 2.7 g
(79%) of
compound 3 as a light yellow oil:'H NMR (CDC13) b 2.45~ {s, 3H), 3.35 (t, 4H),
3.55-3.8
(m, 44H), 7.27 (d, 2H), 7.69, (d, 2H); HRMS (FAB) calculated for C3,HS,CsNO,4S
(M+Cs):
832.2554. Found: 832.2584.
Compound 4
N,N-bis-hexethyleneglycol-amine
Compound 3, (2.74 g) was dissolved in 4 mL of dry THF and transferred to a
three-
neck flask equipped with a Dewar-condenser. This was sti~~ed at -78°C
as 100 mL of NH3
was condensed into the mixture. Approximately 1-2 g of Nfa was added to the
mixture at
-78°C in small portions until the dark blue color persisted. The
cooling bath was removed,
and the mixture was then stirred at reflux for 30 minutes. Cooling at -
78°C was continued,
and the reaction was carefully quenched with glacial acetic acid until all the
blue color
disappeared. The NH3 was allowed to evaporate, and the white solid was dried
under
vacuum to yield compound 4. This material was used as is in subsequent steps
assuming a
100% yield.
47
CA 02353462 2001-05-31
WO 00/34231 PCTNS99/29339
Compound 7
DEGA {bis-diethyleneglycol~amine)
Compound 7 was prepared according to an existing literature procedure as shown
below (Bondunov et al., J. Org. Chem. I995, Vol. 60, pp. b097-b102). 33.8 g
(32I mmol)
of aminodiethyleneglycol (compound 6), 9.4 g ( 88.3 mmol) of Na2C03, 200 mL of
toluene,
and 10.0 g (80.3 mmol) of chlorotriethyleneglycol (compound 5), were refluxed
with a
Dean-Starke trap to remove water for 48 hours. The mixture was allowed to cool
then
filtered and concentrated. The resulting 45.8 g of material was vacuum
distilled (bp 153-
~ 158°C, 0.1 Torr) to yield 12.0 g {78%) of compound 7.
Example 2
Synthesis of Uctamer of HEGA/TEG Using Segmental Approach
A chemical scheme for the preparation of an octamer of HEGA/TEG is shown in
Figures 11 A and I 1 B. Compound. The bis-hexaethylene~;lycolamine {compound
4) was
reacted with di-tert-butyldicarbonate to yield the N-BOC <;ompound (compound
8), which
was then reacted withpara-nitrophenylchloroformate to yield the para-
nitrophenylcarbonate compound (compound 9). Thepara-nitrophenylcarbonate (PNP)
group was then converted to a carbamate group by reaction with mono-CBZ-
protected
piperazine, yielding compound 10. The BOC group was removed using
trifluoroacetic acid
to yield compound 1 I . Compounds 9 and 11 were then re;~cted together to form
a"one-
sided" dendritic compound (compound I2). Again, the BOC group was removed
using
trifluoroacetic acid to yield compound 13. Compound i 3 was then reacted with
triethyleneglycol bis chlaroformate (from which the "core" is derived) to
yield the "two-
sided" dendritic compound (compound 14). The terminal CBZ-protected amino
groups
were then converted to the hydrobromide salt of amino group, and further
reacted with
bromoacetic anhydride to yield reactive bromaacetyl groups at each of the
termini in
compound 15.
48
CA 02353462 2001-05-31
WO 00/34231 PCT/US99/29339
Compound 8
N-BOC-N,N-bis-hexaethyleneglycol-amine
Compound 4 (797 mg, I .46 mmol) was dissolved in 14 mL of H20, and the mixture
was stirred at room temperature. To the mixture was added 465 mg (4.38 mmol)
of
Na,CO3. The pH was checked to make sure it was basic, and 319 mg (1.46- mmol)
of di-
tert-butyldicarbonate ((BOC)z0)was dissolved in 7 mL of dioxane and the
resulting
solution was added to the reaction mixture. The mixture was stirred at room
temperature
for 6 h and partitioned between 100 ml of H20 and 3 x 100 mL of CHZCIz. The
combined
organic layers were dried (MgS04), f ltered, and concentrated to give 605 mg
(64%) of
compound 8 as a light yellow oil: 'H NMR (CDCl3) 8 I .45 (s, 9H), 3.45 (m,
4H), 3.8-3.5
{m, 44H): MS (ESI) calculated for C29HssNaNO,4 (M+Na): 668. Found: 668.
Compound 9
Compound 8 (52 mg, 0.08 mmol) was dissolved in 3 mL of CHzCl2 and 97 mg
(0.483 mrnol) of p-nitrophenylchloroformate was added to the mixture. The
mixture was
stirred at 0°C, 78 ~,L (0.966 mmol) of pyridine was added, and the
mixture was then stirred
at room temperature for 4 hours. The reaction mixture was cooled to
0°C, acidif ed with 1
N HCI, and partitioned between 10 mL of 1 N HCl and 3 x 10 mL of CHZCI,. The
combined organic layers were dried {MgS04), and concentrated to give I 32 mg
of an oil.
Purif cation was accomplished by silica gel chromatography (98 : 2
CHZCIz/MeOH) to give
57 mg (74.0%) of compound 9 as an oil: 'H NMR (CDC13) 8 1.49 (s, 9H), 3.43 (m,
4H),
3.52-3.77 (rn, 36H), 3.83 (m, 4H), 4.47 (m, 4H), 7.41 {d, 4H), 8.32 (d, 4H);
MS (ESI)
calculated for C43HssNaN3022 (M+Na): 998. Found: 998.
Compound 10
Compound 9 {2.72 g, 2.79 mmoI) was dissolved in :l0 mL of CHZCh and the
mixture was stirred at 0°C. To the mixture was added I.16 ml (8.36
mmol) of Et3N
49
CA 02353462 2001-05-31
WO 00/34231 PCTNS99/29339
followed by I .842g (8.36 mmol) of mono-CBZ-piperazine dissolved in 10 mL of
CHZC12.
The mixture was stirred at room temperature for I 8 hour,;, cooled to
0°C, and acidif ed with
1 N HCI. The mixture was partitioned between I50 mL of I N HCI and 3 x 150 mL
of
CH,CIz. The combined organic layers were washed with :saturated NaHCO~
solution, dried
(MgS04) and concentrated to give 3.64 g of a yellow oil. Purification by
silica geI
chromatography (97/3 CHzCI2JMeOH) gave 3.1 g (98%) of compound _10 as a light
yellow
oil: 'H NMR (CDCI3) 8 1.45 (s, 9H), 3.40-3.55 (m, 12H), 3.55-3.68 (m, 36H},
3.71 (m,
4H), 4.37 {m, 4H), 5.17 (s, 4H), 7.35 {brd s, lOH); MS (ESI) calculated for
CSSHB,NaN502o
(M+Na): 1060. Found: 1060.
Compound 11
Compound I O {3.54 g, 3. I i mmol) was dissolved in 15 mL of CH~CI, and I5 mL
of
trifluoroacetic acid (TFA) was added to the mixture. The nnixture was stirred
at room
temperature for 4 hours and concentrated. The residue was re-dissolved in I O
mL of CHzCIz
and neutralized by shaking with a saturated solution of Na)EiC03 at
0°C. The mixture was
then partitioned between 100 mL of saturated NaHC03 solution and 4 x 100 mL of
CHZCIz.
The combined organic layers were dried (MgS04), f ltered, and concentrated to
give 3.16 g
of compound 11 {98%) as a yellow oil: 'H NMR {CDCl3) C~ 2.88 (t, 4H), 3.50
{brd s, 16H),
3.56-3.69 (m, 36H), 3.7I (m, 4H), 4.28. (m, 4H}, 5.15 (s, 4H), 7.38 (brd s, l
OH); MS (ESI)
calculated for CSOHg°N50'$ (M+H): 1037. Found: 1038.
Compound 12
Compound 9 (647 mg, 0.663 mmol} and 2.065 g ( / .989 mmol) of compound _1 I
were dissolved in 3 mL of CHZC12, and 462 ~cL (3.315 mmol) of Et~N and 40 mg
(0.331
mmol) of DMAP (4-dimethylaminopyridine) was added to the mixture. The reaction
mixture was stirred at room temperature overnight and cooled to 0°C. To
the mixture was
added 5 mL of H20, and the mixture was acidified with I N HCl and partitioned
between
50 mL of H,O and 3 x 50 mL of CH~CIZ. The combined organic layers were dried
{MgSO~}
so
CA 02353462 2001-05-31
WO 00/34231
PCTNS99/29339
and concentrated to give 2.77 g (72.1 %) of a yellow oil. Purification by
silica gel
chromatography (CHZCI2/MeOH) gave 1.307 g (72%) of compound 12 as a yellow
oil: 'H
NMR -(CDCl3) 8 1.46 (s, 9H), 3.49 (brd s, 32H), 3.52-3.67 (m, 92H), 3.69 (m,
8H), 4.22 (m,
12H); 5.15 (s, 8H), 7.3 S (brd s, 20H).
Compound 13
Compound 12 ( 1.3 g, 0.47 mmol) of the starting material was dissolved in i 0
mL of
CHZCh and 10 mL of TFA (trifluoroacetic acid) was added. The mixture was
stirred at
room temperature for 4 hours, concentrated, and re-dissolved in 10 mL of
CHZC12. The
mixture was neutralized shaking with a saturated solution of NaHC03 at
0°C. The mixture
was partitioned between 25 mL of saturated NaHC03 solution and 4 x 2S mL of
CH~C12.
The combined organic layers were dried (MgS04) and concentrated to give 1.23 g
(98%) of
compound 13 as a yellow oil: 'H NMR (CDCl3) 8 3.48 (brd s, 32H), 3.53-3.67 {m,
92H),
3.71 (m, 8H), 4.22 (m, I2H), 5.15 (s, 8H), 7.38 (brd s, 20Ft).
Compound 14
Compound 13 (600 mg, 0.224 mmol) was dissolved in I :5 mL of CHZC12 and the
mixture was stirred at 0°C. To the mixture was added 65 pL {0.373 mmol)
of D1PEA
followed by the slow addition of a solution of 20.5 mg (0.075 mmol) of
triethyleneglycol
bis-chloroformate dissolved in 0.5 mL of CHZCIZ. After 3 hours the reaction
mixture was
cooled to 0°C and acidified with 1 N HCI. The mixture was partitioned
between 25 ml of 1
N HCl and 2 x 25 ml of CHZC12. The combined organic layers were dried (MgS04)
and
concentrated to give 566 mg of alight yellow oil. Purif cati~on by silica gel
chromatography
(95/5 CHZCI2/MeOH) gave 14S mg of compound I4 (35%} as a light yellow oil: 'H
NMR
(CDCl3) 8 3.51 (brd s, 64H}, 3.54-3.77 (m, 272H), 4.23 (m, 28H), 5.17 (s,
I6H}, 7.36 (brd
s, 40H); MS (ESl) calculated for C260H421N22~)06 {M+H): 5549. Found: 5549.
5I
CA 02353462 2001-05-31
WO 00/34231 PCT/US99/29339
Compound 15
Compound 14 (143 mg, 0.026 mmol) was treated with 3 mL of 30% HBr/AcOH far
30 min. The resulting HBr salt was precipitated with ether. The solids were
collected by
S centrifugation and washed three times with ether. The re;>ulting HBr salt
was dried in the
desiccator overnight and dissolved in 1.2 ml of H,O. The mixture was stirred
at 0°C, and
97 mg (1.16 mmol) of sodium bicarbonate was added. A solution of 107 mg (0.412
mmol)
of bromoacetic anhydride in I .2 mL of dioxane was added, the mixture was
stirred at 0°C
for I S-20 min. To the mixture was added 10 mL of HZO, and the mixture was
slowly
acidified with 1 M H~S04 to a pH of 4. The aqueous layer was extracted with 2
x 10 mL of
EtOAc which was discarded. The aqueous layer was then extracted with 6 x 10 mL
of 8/2
CHZCi,/MeOH. The combined organic layers were dried (:MgS04), filtered and
concentrated to give I 17 mg of an oil. Preparative HPLC (C18, gradient, 3S-
SS% B, A =
0.1 % TFA/H,O and B = 0. I % TFAICH,CN) to give 27 ml; (I 9%) of compound _1 S
as a
IS colorless oil: 'H NMR (CDCl3) 3.46-3.75 (m, 336H), 3.90 (m, 16H}, 4.21-4.33
(m, 28H);
MS (ESI) calculated for Cz,2H38,Br$N220~$ (M+H): S43S. hound: 5448.
Example 3
Synthesis of Octamer of DEGA/TEG Using Segmental Approach
A chemical scheme for the preparation of an octamer of DEGA/TEG is shown in
Figures 12A and 12B. The bis-diethyleneglycolamine (compound _7) was reacted
with di-
tert-butyldicarbonate to yield the N-BOC compound {compound _16}, which was
then
reacted with para-nitrophenylchloroformate to yield the para-
nitrophenylcarbonate
2S compound (compound I 7}. The para-nitrophenylcarbonate; (PNP) group was
then
converted to a carbamate group by reaction with mono-CB.Z-protected
piperazine, yielding
compound I 8. The BOC group was removed using trifluoroacetic acid to yield
compound
18a. Compounds 17 and I 8a were then reacted together to form a "one-sided"
dendritic
compound (compound 19}. Again, the BOC group was removed using-
trifluoroacetic acid
to yield compound 19a. Compound 19a was then reacted with triethyleneglycol
bis
chloroformate (from which the "core" is derived) to yield the "two-sided"
dendritic
S2
CA 02353462 2001-05-31
W~ 00/34231 PCT/US99129339
compound (compound 20). The terminal CBZ-protected amino groups were then
converted
to the hydrobromide salt of amino group, and further reacted with bromoacetic
anhydride to
yield reactive bromoacetyl groups at each of the termini in compound 20a.
Compound 16
N-BOC-N,N-bis-diethylenegly~:ol-amine
To a solution of 600 mg ( 3.10 mmol) of bis-diethyleneglycolamine (compound
in 9.9 mL of 10% aqueous Na2C03 was added slowly a solution of 678 mg (3.10
mmol) of
di-tert-butyldicarbonate in S mL of dioxane. The mixture was stirred at roam
temperature
for S hours, and 2S mL of water was added. The mixture was shaken with Et20,
the Et20
layer was discarded, and the mixture was extracted with three 2S mL portions
of CH~CI2.
The CHZC12 extracts were combined, dried {MgS04), filtered and concentrated to
give 717
mg (79%) of compound 16 as a viscous oiI: 'H NMR (CD~Cl3) cS 1.48 (s, 9H},
3.45 (brd s,
1 S 4H), 3.60 {t, 4H}, 3.65 (brd s, 4H), 3.69 (brd s, 4H).
Compound 17
To a solution of 200 mg (0.68 mmol) of compound 16 and 822 mg g (4.08 mmol) of
4-nitrophenyichloroformate in 30 mL of CHZCIz was added 0.66 mL {8.16 mmol) of
pyridine at 0°C. The mixture was stirred at room temperature for 1.S
hours then cooled
back to 0°C, and the mixture was acidified with 1 N HCl and partitioned
between SO mL of
1 N HCl and three SO mL portions of CHzCl2. The combined CHZC12 extracts were
dried
(MgS04), filtered and concentrated to give 1.21 g of yellow oil. The mixture
was partially
purified by silica gel chromatography (CHZC12/MeOH) to ;give S8I mg of
partially purified
compound 17 which still contained 4-nitrophenol: MS (ESl) calculated for
CZ~Ha~NaN3O,4
(M+Na): 646. Found: 646. This material was used directly in the next step.
53
CA 02353462 2001-05-31
WO 00/34231 PCT/US99I29339
Compound I8
To a solution of 42I mg of partially purified compound 17 (0.68 mmol
theoretical)
and 282 ~,L (2.02 mmol) of Et3N in 4 mL of CHzCl2 at 0°C was added a
solution of 446 mg
(2.02 mmol) of mono-CBZ-piperazine in 4 mL of CHZC12. The mixture was stirred
at room
temperature for I.S hours, cooled back to 0°C, acidified with 1 N HCI,
and partitioned
between 50 mL of I N HCl and 3. X 50 mL of CHZCIz. Th.e combined CHzCIz layers
were
washed with saturated NaHC03 solution, dried (MgS04), filtered, and
concentrated to give
500 mg of viscous residue. Purification by silica gel chromatography
(CHZCh/MeOH)
gave 265 mg (50%) of compound 18 as a viscous oil: 'H NMR (CDCl3) 8 I .45 (s,
9H),
3.40-3.70 (m, 28H), 4.28 (t, 4H), S.I6 (s, 4H), 7.37 (brd s, IOH); MS (ESI)
calculated for
C39HSSNaN5~t2 {M+Na): 808. Found: 809.
Compound I9
IS
Compound 18 (77 mg, 0.098 mmol) was dissolved :in I .5 mL of CHZCIZ and I .5
mL
of TFA was added. The mixture was stirred for 6 hours and concentrated. The
residue was
dissolved in 10 mL of CH2Clz and the resulting solution wa.s stirred at
0°C while I5 mL of
saturated NaHC03 solution was added. 'The aqueous layer was extracted with 4 X
10 mL,
of CH,Ch, and the combined organic layers were dried (M~;S04), filtered, and
concentrated
to give 62 mg (92%) of free amine, compound 18a: 'H NMIE~ (CDC13) 8 2.90 (t,
4H), 3.46
(brd s, 16H), 3.66 (m, 8H), 4.25 (t, 4H), 5.16 (s, 4H), 7.36 (brd s, I0H). To
a solution of
compound 17 in CH,CIz is added the free amine, compound I8a (3 eq.) and
pyridine. The
mixture is stirred at room temperature until the reaction appears done by TLC.
The product
is isolated by extractive workup and purification by silica ge;l
chromatography to give
compound 19.
54
CA 02353462 2001-05-31
WO 00/34231 PCTNS99/29339
Compound 20
Compound 19 is dissolved in 1/l CHZCIz/TFA, and stirred at room temperature
for
1 hour. The mixture is concentrated under vacuum to provide an amine
intermediate,
compound 19a. Two equivalents of the intermediate amine is reacted with
triethyleneglycol bis-chloroformate in CHzCIz and pyridine. The product is
isolated by
extractive workup and purif cation by silica gel chromatography to give
compound 20.
Compound 20a
In a process similar to that described above for compound 15, compound 20 is
treated with 30% HBr/AcOH for 30 min. The resulting HBr salt is precipitated
with ether.
The solids are collected by centrifugation and washed with ether. The
resulting HBr salt is
dried in the desiccator overnight and dissolved in HzO. T:he mixture was
stirred at 0°C and
sodium bicarbonate added. A solution of bromoacetic anhydride in dioxane is
added, and
the mixture stirred at 0°C for 15-20 min. To the mixture is added H20,
and the mixture is
slowly acidified with 1 M HZS04 to a pH of 4. The aqueous layer is extracted
with EtOAc
which was discarded. The aqueous layer is then extracted with $/2 CHZC12/MeOH.
The
combined organic layers are dried (MgS04), filtered and concentrated to give
compound
20a.
Example 4
Synthesis of Tetramer of DEGA/TEG Using Core Propagation Approach
A chemical scheme for the preparation of an tetramer of DEGA/TEG is shown in
Figure 13. The bis-diethyleneglycolamine (compound 7) was reacted
triethyleneglycol bis
chloroformate (from which the "core" 'is derived) to yield the tetrahydroxy
compound,
compound 21. Compound 21 was then reacted with para-nitrophenylchioroformate
to
yield the tetrapara-nitrophenylcarbonate compound (com7pound 22). The para-
nitrophenylcarbonate (PNP) group was then converted to a carbarnate group by
reaction
with mono-CBZ-protected piperazine, yielding compound 23. The terminal CBZ-
protected
CA 02353462 2001-05-31
WO 00/34231
PCT/US99/29339
amino groups were then converted to the hydrobromide :;alt of amino group, and
further
reacted with bramoacetic anhydride to yield reactive bromoacetyl groups at
each of the
termini in compound 23a.
S Corapound 21
To a solution of I.94 g (10.0 mmol) of compound 7 and 1.75 mL (1.30 g, 10.0
mmol) of EtjN at 0°C a solution of 980 ~,L (1.31 g, 4.78 nnmol} of
triethyleneglycol bis-
chlorofonnate in 3S mL of CHzCIz. The mixture was stirred for 3 hours at room
temperature and concentrated to give 4.84 g of crude compound 21 which was
used as is in
the next step: 'H NMR (CDCl3) 8 3. I 0 (m, 4H}, 3.45-3.78 (m, 32H), 4.24 (m,
4H).
Compound 22
I S Pyridine (9.3 mL, 114.7 mmol) was added to a stiwed solution of 4.84 g of
crude
compound 21 (4.78 mmol theoretical) and 7.71 g (38.24 mmol) of 4-
nitrophenylchlorofonnate at 0°C, and the mixture was stirred for 4
hours at room
temperature. The mixture was cooled to 0°C and acidified with I N HCI.
The mixture was
partitioned between 2S0 mL of 1 N HCl and 2 X 250 mL of CHZCIZ. The organic
layers
were combined, dried (MgS04), filtered and concentrated to give 9.81 g of
crude product.
Purification by silica gel chromatography (CHZC12/MeOH) gave 4.40 g (74%) of
compound
22 as a sticky viscous oil: 'H NMR (CDCl3) 8 3.56 (m, 8H), 3.61-3.72 (m, 16H),
3.76 (m,
8H), 4.23 (t, 4H), 4.44 (t, 8H), 7.40 (d, 8H), 8.2$, (d, 8H); HRMS (FAB)
calculated for
C52H60CSN~O30 (M+Cs}: 1381.2408. Found: 1381.2476.
2S
Compound 23
A solution of I06 mg (0.48 mrnol) of mono-CBZ-piperazine in O.S mL of CHzClz
was added to a stirred solution of 100 mg (0.08 mmol) of compound _22 and 67
p.L (0.48
mmol) of Et3N at 0°C. The mixture was stirred for 18 hours at room
temperature, cooled to
56
CA 02353462 2001-05-31
WO 00/34231 PCT/US99/29339
0°C, and acidified with 1 N HCI. The mixture was partitiioned between S
mL of 1 N HCl
and 3 X S mL of CHZCI2. The organic layers were combined, washed with
saturated
NaHC03 solution, dried (MgS04), filtered, and concentrated to give 119 mg of
yellow oil.
Purification by silica gel chromatography (CHZCl2/MeOH) gave 81 mg {64%) of
compound
S 23 as a viscous oil: 'H NMR (CDC13) S 3.48 (brd s, 40H), 3.52-3.74 (m, 24H),
4.24 (t,
12H), 5.14 (s, 8H), 7.35 (brd s, 20H); HRMS (FAB) calculated for
C,bH,°4CsN,°026
(M+Cs): 1705.6178. Found: 1705.6269.
Compound 23a
In a process similar to that described above for compound 1 S, compound 23 is
treated with 30% HBrIAcOH for 30 min. The resulting HBr salt is precipitated
with ether.
The solids are collected by centrifugation and washed with ether. The
resulting HBr salt is
dried in the desiccator overnight and dissolved in HZO. The mixture was
stirred at 0°C and
1 S sodium bicarbonate added. A solution of bromoacetic anlhydride in dioxane
is added, and
the mixture stirred at 0°C for 1 S-20 min. To the mixture :is added
H20, and the mixture is
slowly acidified with 1 M HzS04 to a pH of 4. The aqueous layer is extracted
with EtOAc
which was discarded. The aqueous layer is then extracted with 8/2 CHzCI2/MeOH.
The
combined organic layers are dried {MgS04), filtered and concentrated to give
compound,
23a.
Example 5
Synthesis of Tetramer of DEGA/PTH Using Core Propagation Approach
2S A chemical scheme for the preparation of an tetranner of DEGA/PTH is shown
in
Figure 14A. The bis-diethyleneglycolamine (compound i') was reacted with
terephthaloyl
chloride (from which the "core" is derived) to yield the tetTahydroxy
compound, compound
24. Compound 24 was then reacted withpara-nitrophenylchloroformate to yield
the tetra
para-nitrophenylcarbonate compound (compound 2S). The para-
nitrophenylcarbonate
(PNP) group was then converted to a carbamate group by reaction with mono-CBZ-
protected piperazine, yielding compound 26. The terminal CBZ-protected amino
groups
S7
CA 02353462 2001-05-31
WO 00134231 PCT/US99/29339
were then converted to the hydrobromide salt of amino group, and further
reacted with
bromoacetic anhydride to yield reactive bromoacetyl groups at each of the
termini in
compound 26a.
Compound 24
A solution of 300 mg (1.48 mmol) of terephthaloyl chloride in 9 mL of CH2C12
was
slowly added to a 0°C solution of 600 mg (3.10 mmol} of 7 and 540 ~,L
(3.I O mmol} of
diisopropylethylamine in 12 mL of CH2C12, and the mixture was stirred under
nitrogen
I 0 atmosphere for 3 hours at room temperature. The mixture was concentrated
under vacuum
to give I .53 g of a crude mixture which contained 24.
Compound 25
15 The 1.53 g of crude 24 and 2.38 g {I I .8I mmol) of 4-
nitrophenylchloroformate
were dissolved in 30 rnL of pyridine, and the resulting solution was stirred
at 0°C while
1.91 mL (23.62 mmol) of pyridine was added. The mixture was stirred at room
temperature for 4 hours, cooled to 0°C; and acidif ed with I N HCI. The
mixture was
partitioned between 75 mL of 1 N HCl and 2 X 75 mL of CHzCIz. The organic
layers were
20 combined, washed with saturated NaHC03 solution, dried (MgS04), filtered,
and
concentrated to give 2.75 g of an oil. Purification by silica gel
chromatography
(CHzCl2/MeOH) gave 1.33 g (76%) of 25 as a viscous oil: 'H NMR (CDCl3) ~ 3.52
(brd s,
8H), 3.65 {brd s, 4H), 3.81 (brd s, I2H), 4.41 (m, 8H), 7.38 (m, 8H), 7.47 (s,
4H), 8.27 {m,
8H); HRMS {FAB) calculated for CSZH53N6o26 (M+H): 1 I 77.3010. Found: I
177.3062.
Compound 26
A solution of 113 mg (0.51 mmol) of mono-CBZ-piperazine in 0.5 mL of CHZCIz
was added to a stirred solution of 100 mg (0.085 mmol) of compound _25 and 71
pL (0.51
mmol) of Et3N at 0°C. The mixture was stirred for 18 hours at room
temperature, cooled to
0°C, and acidified with 1 N HCl. The mixture was partitioned between 5
mL of 1 N HCI
58
CA 02353462 2001-05-31
WO OoI3423I PCT/US99129339
and 3 X 5 mL of CHZCIZ. The organic layers were combined, washed with
saturated
NaHC03 solution, dried (MgS04), f ltered, and concentrated to give i 25 mg of
yellow oil.
Purification by silica gel chromatography (CHZC12/MeOH) gave 59 mg (46%) of 26
as a
viscous oil: 'H NMR (CDC13) 8 3.45 (brd s, 40H), 3.55 (m, 4H), 3.72 (m, 4H),
3.78 (s,
8H), 4.24 (m, 8H), S.I3 (s, 8H), 7.34 (brd s, 20H), 7.42 (s, 4H); HRMS {FAB)
calculated
for C,6H96CsN,flO~ (M+Cs): 1633.5755. Found: 1633.5846.
Compound 26a
Tn a process similar to that described above for compound 15, compound 26 is
treated with 30% HBr/AcOH for 30 min. The resulting FEBr salt is precipitated
with ether.
The solids are collected by centrifugation and washed with ether. The
resulting HBr salt is
dried in the desiccator overnight and dissolved in HZO. The mixture was
stirred at 0°C and
sodium bicarbonate added. A solution of bromoacetic anhydride in dioxane is
added, and
the mixture stirred at 0°C for 1 S-20 min. To the mixture its added
H20, and the mixture is
slowly acidified with 1 M HzSOQ to a pH of 4. The aqueous layer is extracted
with EtOAc
which was discarded. The aqueous layer is then extracted with 8/2 CHzCIz/MeOH.
The
combined organic layers are dried (MgSOd), filtered and concentrated to give
compound
26a.
Example 6
~nthesis of Octamer of DEGAJPTH, Using Core Propagation Approach
A chemical scheme for the preparation of an octarr.~er of DEGA/PTH is shown in
Figure 14B. The bis-diethyleneglycolamine {compound 7;) was reacted with the
tetra para-
nitrophenylcarbonate compound (compound 25) to yield tlae octahydroxy
compound,
compound 27a. Compound 27a was then reacted withpara-nitrophenylchloroformate
to
yield the octapara-nitrophenylcarbonate compound (compound 27). The para-
nitrophenylcarbonate (PNP) group was then converted to a. ca.rbamate group by
reaction
with mono-CBZ-protected piperazine, yielding compound 28. The terminal CBZ-
protected
ammo groups were then converted to the hydrobromide salit of amino group, and
further
59
CA 02353462 2001-05-31
WO 00/34231 PCT/US99/29339
reacted with bromoacetic anhydride to yield reactive bromoacetyl groups at
each of the
termini in compound 28a.
Compound 27a
A solution of 98 mg (0.51 mmol) of compound 7 iin 0.5 mL of CH2C12 was added
to
a stirred solution of 100 mg (0.085 mmol) of compound 25 and 71 ~.L (0.51
mmol) of Et3N
at 0°C, and the mixture was stirred for 18 hours. The mixture was
concentrated to give 250
mg of crude product, compound 27a.
IO
Compound 27
Crude compound 27a (250 mg) was dissolved in 4 mL of CHZC12. The mixture was
cooled to 0°C, and 275 mg (1.36 mrnol) of 4-nitrophenylclllaroformate
was added followed
I S by 220 p.L (2.74 mmol) of pyridine. The mixture was stirred at room
temperature for 7
hours, cooled to 0°C, and acidified with I N HCI. The mixture was
partitioned between 5
mL of 1 N HCl and 2 X 15 mL of CHZClz. The organic layers were combined,
washed with
saturated NaHCO~ solution, dried (MgS04), filtered, and concentrated to give
393 mg of an
oiI. Purification by silica geI chromatography (CHZCIZ/MeOH) gave 123 mg (53%)
of
20 compound 27 as a viscous oil: 'H NMR (CDCl3) 8 3.63-3.136 (m, 72H}, 4.46
{t, 24H), 7.26
(s, 4H), 7.35 (m, 16H), 8.24 (m, 16H}.
Compound 28
25 A solution of 6 eq. of mono-CBZ-piperazine in CHZC12 is added to a stirred
solution
of compound 27 and 6 eq. of Et3N at 0°C. The mixture is shirred for I8
hours at room
temperature, cooled to 0°C, and acidified with 1 N HCI. The mixture is
partitioned
between I N HC1 and CHZCIz. The organic layers are combined, washed with
saturated
NaHC03 solution, dried (MgS04), filtered, and concentrated to give crude
product which
30 can be purified by silica gel chromatography to give compo~.md 28.
CA 02353462 2001-05-31
W4 00/34231 PCT/US99I29339
Compound 28a
In a process similar to that described above for compound 15, compound 28 is
treated with 30% HBr/AcOH fox 30 min. The resulting HBr salt is precipitated
with ether.
The solids are collected by centrifugation and washed witlh ether. The
resulting HBr salt is
dried in the desiccator overnight and dissolved in HzO. The mixture was
stirred at 0°C and
sodium bicarbonate added. A solution of bromoacetic anhydride in dioxane is
added, and
the mixture stirred at 0°C for 15-20 min. To the mixture is added H20,
and the mixture is
slowly acidified with 1 M H~S04 to a pH of 4. The aqueous layer is extracted
with EtOAc
which was discarded. The aqueous layer is then extracted 'with 8/2
CHZCIz/MeOH. The
combined organic layers are dried (MgS04), filtered and concentrated to give
compound
28a.
Example 7
Snthesis of Tetramer of HEGA/TEG Using Core Prop~a~ation Approach
A chemical scheme for the preparation of an tetram~er of HEGA/TEG is shown in
Figure i 5. The bis-hexaethyleneglycolamine (compound ~~) was reacted
triethyleneglycol
bis chloroformate (from which the "core" is derived) to yield the tetrahydroxy
compound,
compound 29. Compound 29 was then reacted withpara-nitrophenylchloroformate to
yield the tetrapara-nitrophenylcarbonate compound (compound 30}. The para-
nitrophenylcarbonate (PNP) group was then converted to a carbamate group by
reaction
with mono-CBZ-protected piperazine, yielding compound 31. The terminal CBZ-
protected
amino groups were then converted to the hydrobrornide salt of amino group, and
further
reacted with bromoacetic anhydride to yield reactive bromoacetyl groups at
each of the
termini in compound 31 a.
6I
CA 02353462 2001-05-31
WO 00/34231 PCT/US99/29339
Compound 29
To a solution of 2. I eq. of compound 4 and 2.1 eq. of Et3N at 0°C is
added a
solution of 1 eq. of triethyleneglycol bis-chloroformate in CHzCIz. The
mixture is stirred at
room temperature until complete by TLC and concentrated to give crude compound
_29
which is used as is in the next step.
Compound 30
Pyridine (12 eq.) is added to a stirred solution of cmde compound _29 and 6
eq. of 4-
nitrophenylchloroformate at 0°C, and the mixture is stirred at room
temperature until the
reaction is complete as evidenced by TLC. The mixture is cooled to 0°C,
and acidified
with 1 N HCI. The mixture is partitioned between 1 N HC'i and CHzCIz. The
organic
layers are combined, dried (MgS04), filtered and concentrated to give crude
product.
Compound 30 is purified by silica geI chromatography.
Compound 3I
A solution of 6 eq. of mono-CBZ-piperazine in CH;zCI2 is added to a stirred
solution
of compound 30 and 6 eq. of Et3N at 0°C. The mixture is stirred for at
room temperature
until the reaction is complete as evidenced by TLC, cooled to 0°C, and
acidified with 1 N
HCI. The mixture is partitioned between 1 N HCI and CHZCIz. The organic layers
are
combined, washed with saturated NaHC03 solution, dried (MgS04), filtered, and
concentrated to give crude compound 31 which is purified by silica gel
chromatography.
Compound 31a
In a process similar to that described above for compound _i 5, compound _31
is
treated with 30% HBr/AcOH for 30 min. The resulting HB:r salt is precipitated
with ether.
The solids are collected by centrifugation and washed with ether. The
resulting HBr salt is
62
CA 02353462 2001-05-31
WO 00/34231 PCT/US99/29339
dried in the desiccator overnight and dissolved in HzO. The mixture was
stirred at 0°C and
sodium bicarbonate added. A solution of bromoacetic anhydride in dioxane is
added, and
the mixture stirred at 0°C for 15-20 min. To the mixture its added H20,
and the mixture is
slowly acidified with 1 M HzS04 to a pH of 4. The aqueous layer is extracted
with EtOAc
which was discarded. The aqueous layer is then extracted with 8/2 CHZC12/MeOH.
The
combined organic layers are dried (MgS04), filtered and concentrated to give
compound
31 a.
Example 8
Synthesis of Tetramer of DEA/PTH Using Core Propagation Approach
A chemical scheme for the preparation of an tetramer of DEGA/PTH is shown in
Figure 16A. Diethanolamine was reacted with terephthaloyl chloride (from which
the
"core" is derived} to yield the tetrahydroxy compound, compound 32. Compound
32 was
then reacted with para-nitrophenylchloroformate to yield the tetra para-
nitrophenyicarbonate compound (compound 33). Thepara-nitrophenylcarbonate
(PNP)
group was then converted to a carbamate group by reaction with mono-CBZ-
protected
piperazine, yielding compound 34. The terminal CBZ-protected amino groups were
then
converted to the hydrobromide salt of amino group, and further reacted with
bromoacetic
anhydride to yield reactive bromoacetyl groups at each of the termini in
compound 34a.
Compound 32
A solution of 2.0 g (9.85 mmol) of terephthaloyl chloride in 25 mL of THF was
added slowly to a 0°C solution of 2.17 g (20.7 mmol) of di.ethanolamine
and 3.6 mL (20.7
mmol} of diisopropylethylamine in 50 mL of THF. The mixture was stirred at
room
temperature for 3 hours and concentrated under vacuum to give 6.7 g of crude
compound
32. A small amount was purified for characterization purposes by preparative
HPLC (1"
C 18 column, gradient 0-15% B, A = 0.1 % TFA/H20 and E~ = 0.1 % TFA/CH;CN): 'H
NMR
(D20) 8 3.52 (m, 4H}, 3.59 (m, 4H), 3.72 {m, 4H}, 3.89 (m, 4H), ?.51 (s, 4H);
MS (EST)
calculated for C16H25N2~6 (M+H): 341. Found: 341.
63
CA 02353462 2001-05-31
WO 00/34231 PCTIUS99/29339
Compound 33
Pyridine (11.4 mL, 14I.2 mmol) was slowly added to a stirred solution of 6.01
g
(8.8 mmol thearetical) of crude compound 32 and 14.2 g (70.6 mmol) of 4-
nitrophenylchloroformate in 88 mL of THF. The mixture was stirred at room
temperature
for I 8 hours and acidified with 1 N HCI. The mixture wars partitioned between
300 mL of
I N HCi and 2 X 300 mL of CH~CI2. The combined organic layers were washed with
saturated NaHC03 solution, dried (MgS04), filtered, and concentrated to give
14.0 g of
sticky orange oil. Purification by silica gel chromatography (CH,C12/MeOH)
provided 3.34
g (38%) of compound 33 as a crystalline solid: mp 77-85°C; 'H NMR
(CDCl3) b 3.76 (brd,
4H), 4.OI (brd, 4H), 4.38 (brd, 4H), 4.64 (brd, 4H), 7.36 (brd, 8H), 7.53 (s,
4H), 8.28 (m,
8H);'3C NMR (CDC13) $ 45.0, 48.5, 65.9, 66.6, 12I.8, 125.0, I25.4, 126.1,
127.2, 137.1,
145.6, 152.4, 155.2, 171.9; HRMS (FAB) calculated for C,,4H36CsN6O2z (M+Cs):
1133.0937. Found: 1133.0988.
Compound 34
A solution of I32 mg (0.6 mmol) of mono-CBZ-pi~rerazine in 0.5 mL of CHzCl2
was added to a 0°C solution of 100 mg (0.1 mmol) of compound 33 and $4
p,L (0.6 mmol)
of Et3N in 0.5 rnL of CHzCIz. The reaction mixture was sti~xed for 18 hours at
room
temperature, cooled to 0°C, and acidified with 1 N HCI. The mixture was
partitioned
between S mL of 1 N HCl and 3 X 5 mL of CHZC12. The cc>mbined organic layers
were
washed with saturated NaHC03 solution, dried (MgS04), filftered, and
concentrated to give
123 mg of white solid. Purification by silica gel chromatography (CH2C12/MeOH)
provided 8d mg (65%) of compound 34 as a crystalline solid: mp 64-67°C;
1 H NMR
(CDCI3) 8 3.35-3.63 (m, 36H), 3.86 (brd, 4H), 4.13 (brd, 4H), 4.41 (brd, 4H),
5.14 (s, 8H),
7.38 (brd s, 20H), 7.43 (s, 4H); '3C NMR (CDC13) S 43.5, 45.1, 48.4, 62.6,
67.4, 126.9,
128.0, 128.2, 128.5, 136.3, 137.4, 155.1, 171.4; HRMS (FAB) calculated for
C68H8°CsN,°O,$ (M+Cs): 1457.4706. Found: 1457.4781.
64
CA 02353462 2001-05-31
WO 00/34231 PCT/US99/29339
Compound 34a
In a process similar to that described above for compound I 5, compound 34 is
treated with 30% HBr/AcOH for 30 min. The resulting I-IBr salt is precipitated
with ether.
The solids are collected by centrifugation and washed wil:h ether. The
resulting HBr salt is
dried in the desiccator overnight and dissolved in HBO. The mixture was
stirred at 0°C and
sodium bicarbonate added. A solution of bromoacetic anihydride in dioxane is
added, and
the mixture: stirred at 0°C for I S-20 min. To the mixture is added
H20, and the mixture is
slowly acidified with I M HZS04 to a pH of 4. The aqueous layer is extracted
with EtOAc
which was discarded. The aqueous layer is then extracted with 8/2 CHZCIz/MeOH.
The
combined organic layers are dried (MgS04), filtered and concentrated to give
compound
34a.
IS Example 9
Synthesis of ~ctamer of DEA/PTH Using Core Fropa~;ation Approach
A chemical scheme for the preparation of an octarner of DEA/PTH is shown in
Figure 16B. Diethanolamine was reacted with the tetrapa~ra-
nitrophenylcarbonate
compound (compound 33) to yield the octahydroxy compound, compound _35a.
Compound
35a was then reacted withpara-nitrophenylchloroformate to yield the octa para-
nitrophenylcarbonate compound (compound 3S). Thepar~a-nitraphenylcarbonate
(PNP)
group was then converted to a carbamate group by reaction with mono-CBZ-
protected
piperazine, yielding compound 36. The terminal CBZ-protected amino groups were
then
converted to the hydrobromide salt of amino group, and further reacted with
bromoacetic
anhydride to yield reactive bromoacetyl groups at each of l;he termini in
compound 36a.
Compound 35a
A solution of 100 mg (0. I mmol) of compound 33 i.n 450 p,L of pyridine was
slowly
added to a 0°C solution of 63 mg (0.6 mmol) of diethanolaa:nine in 150
~L of pyridine. The
CA 02353462 2001-05-31
WO 00/34231 PCT/US99/29339
mixture was stirred for 3 hours at room temperature and cooled back to
0°C, to yield crude
compound 35a, which was used in the next step.
Compound 3S
S
A solution of 443 mg (2.2 mmol) of 4-nitrophenylchloroformate was added to the
reaction mixture above, and the mixture was stirred for 18 hours at room
temperature. The
mixture was then cooled to 0°C, and acidified with 1 N FfCI, and
partitioned between 15
mL of 1 N HCl and 2 X 15 mL of CHzCI2. The combined organic layers were dried
I O (MgS04), filtered, and concentrated to give 462 mg of wlhite sticky solid.
Purification by
silica gel chromatography (CH2C12/MeOH) provided 141 mg (65%) of compound 35
as a
crystalline solid: mp 7S-80°C; 'H NMR (CDCl3) 8 3.52-:3.8I (m, 20H),
3.89 (m, 4H), 4. I2
(m, 4H), 4.36-4.59 (m, 20H), 7.42 (m, 24H), 8.30 (m, 16:H).
15 Compound 36
A solution of 61 rng (0.276 mmol) of mono-CBZ-piperazine in 200 ~,L of CHZCIz
was added to a 0°C solution of 50 mg (0.023 mmol) of compound 35 and 39
~,L (0.276
mmol) of Et3N in 200 ~.L of CHzCl2. The reaction mixtwre was stirred for 7
hours at room
20 temperature, cooled to 0°C, and acidified with 1 N. IICI. The
mixture was partitioned
between 10 mL of 1 N HCl and 3 X 10 mL of CHZC12. The combined organic layers
were
dried (MgS04), filtered, and concentrated to give 85 mg of yellow solid.
Purification by
silica gel chromatography (CHZC12/MeOH) provided 33 rng (51%) of compound 36
as a
sticky low melting solid: 'H NMR (CDCl3) 8 3.37-3.72 (m, 84H), 3.84 (m, 4H),
4.03-4.29
25 (m, 20H), 4.35 (m, 4H}, 5.14 (s, 16H), 7.35 {brd s, 40H}, 7.44 (s, 4H}; MS
(MALDI)
calculated for C,4°H"ZNaNZZO42 {M+Na): 2856. Found: 2,857.
66
CA 02353462 2001-05-31
WO 00/34231 PCT/US99/29339
Compound 36a
In a process similar to that described above for compound _I5, compound _36 is
treated with 30% HBr/AcOH for 30 min. The resulting fl:Br salt is precipitated
with ether.
The solids are collected by centrifizgation and washed with ether. The
resulting HBr salt is
dried in the desiccator overnight and dissolved in H20. Tlhe mixture was
stirred at 0°C and
sodium bicarbonate added. A solution of brornoacetic anhydride in dioxane is
added, and
the mixture stirred at 0°C for 15-20 min. To the mixture is added H20,
and the mixture is
slawly acidified with 1 M H2S04 to a pH of 4. The aqueous Iayer is extracted
with EtOAc
which was discarded. The aqueous layer is then extracted with 8/2 CHzCl2/MeOH.
The
combined organic layers are dried (MgS04), filtered and concentrated to give
compound
36a.
Example 10
Synthesis of Tetramer of DEA/DEG Using Core Propa~zation Approach
A chemical scheme for the preparation of an tetrarner of DEAIDEG is shown in
Figures I7A and 17B. Diethyleneglycol (from which the "core" is derived) was
reacted
withpara-nitrophenylchloroformate to yield the di para-nitrophenylcarbonate
compound
(compound 37). Compound 37 was then reacted with diethanolamine to form the
tetrahydroxy compound, compound 38. Compound _38 was then reacted with para-
nitrophenylchloroformate to yield the tetra para-nitrophenylcarbonate compound
(compound 39}. Thepara-nitrophenylcarbonate (PNP) group was then converted to
a
carbamate group by reaction with mono-CBZ-protected piperazine, yielding
compound _40.
The terminal CBZ-protected amino groups were then converted to the
hydrobromide salt of
amino group (compound 41~, and further reacted with bromoacetic anhydride to
yield
reactive bromoacetyl groups at each of the termini in compound 42.
67
CA 02353462 2001-05-31
WO 00/34231 PCT/US99/29339
Compound 37
Diethyleneglycol bis-4-nitrophenyicarbonate
Pyridine (30.5 mL, 377 mmol) was slowly added 1to a 0°C solution of 5.0
g {47.11
mmol) of diethylene glycol and 23.74 g (118 mmol) of 4-
nitrophenylchloroformate in 500
mL of THF. The cooling bath was removed, and the mixture was stirred for 18
hours at
room temperature. The mixture was cooled back to 0°C, acidified with 6
N HCI, and
partitioned between 400 mL of 1 N HCl and 2 X 400 mL of CHzCl2. The combined
organic layers were dried (MgSOa), filtered, and concentrated to give 24.3 g
of a white
I O solid. Crystallization from hexanes/EtOAc gave I6.0 g ('78%) of compound
37 as a white
powder: mp 110°C; 'H NMR (CDC13) 8 3.89 (t, 4H}, 4.50 (t, 4H), 7.40 (d,
4H), 8.26 (d,
4H).
Compound 38
A solution of 2.5 g {5.73 mmol) of compound 37 in 17 mL of pyridine was added'
to
a 0°C solution of 1.8 g (17.2 mmol) of diethanolamine in :3 mL of
pyridine. The cooling
bath was removed, and the mixture was stirred for 5 hours at room temperature
to yield
compound 38, which was not isolated but was used as is in the next step.
Compound 39
The mixture from the previous step was cooled back to 0°C, 40 mL of
CHZC12 was
added followed by a solution of 11.55 g (57.3 mmol) of 4-
~nitraphenylchloroformate in 60
mL of CHZC12, and the mixture was stirred for 20 hours at room temperature.
The mixture
was cooled back to 0°C, acidified with 1 N HCI, and partitioned between
300 mL of 1 N
HCl and 2 X 200 mL of CHzCIz. The combined organic layers were dried (MgS04),
filtered, and concentrated to give 13.6 g of yellow solid. Purification by
silica gel
chromatography (CHZC12/MeOH and EtOAc/hexanes) provided 4.91 g (83%) of
compound
68
CA 02353462 2001-05-31
WO 00/34231 PCT/US99/29339
39 as a sticky amorphous solid: 'H NMR (CDCI3) 8 3.72 (m, 12H), 4.31 (t, 4H),
4.48 (m,
8H), 7.40 (m, 8H), 8.29 (m, 8H).
Compound 40
A solution of 128 mg (0.58 mmol) of mono-CBZ-~piperazine in 1.0 mL of CHZCl2
was added to a 0°C solution of 100 mg (0.10 mmol) of compound 39 and 82
~,L (0.58
mmol) of Et3N in I .0 mL of CHZC12. The reaction mixture was stirred for I 8
hours at room
temperature, cooled to 0°C, and acidified with 1 N HCI. 'The mixture
was partitioned
between I O mL of 1 N HCl and 3 X 10 mL of CHzCl2. Tlle combined organic
layers were
washed with saturated NaHC03 solution, dried (MgS04), filtered, and
concentrated to give
162 mg of yellow oil. Purification by silica gel chromatography (EtOAc/hexanes
followed
by CHZCIZ/MeOH) provided 125 mg {95%) of compound 40 as a glassy amorphous
solid:
'H NMR (CDCl3) 8 3.36-3.59 (m, 40H), 3.68 (t, 4H), 4.2:Z (m, 12H), 5.16, (s,
8H), 7.36
IS (brd s, 20H); MS (ESI) calculated for C66Hz4NaN,o02~ (M-+-Na): 1376. Found:
1376.
Compound 41
Compound 40 (150 mg, 0.1 I mmol) was dissolvedl in 3 mL of 30% HBr/HOAc.
The mixture was stirred for 30 minutes at room temperature, and the HBr salt
was
precipitated by addition of EtzO. The precipitate, compound 4I, was washed
twice with
Et~O and dried under vacuum.
Compound 42
The precipitate, compound 41, was dissolved in 20 mL of HzO. The mixture was
brought to pH 12 by addition of 10 N NaOH and extracted with six 20 mL
portions of 4/1
CHZCIZ/MeOH. The combined organic layers were dried (MgS04), filtered, and
concentrated to provide the free amine., The free amine w~~s dissolved in 8 mL
of CH3CN,
I .5 mL of MeOH was added to improve solubility, and the: solution was stirred
at 0°C
69
CA 02353462 2001-05-31
WO 00/34231 PCT/US99129339
while a solution of 114 mg (0.66 mmol) of chloroacetic anhydride in 2 rnL of
CH3CN was
added. The mixture was stirred at room temperature for 2.5 hours and
concentrated to give
107 mg of an oil. Purification by preparative HPLC (1" C18 column, gradient 25-
45% B,
A = 0.1 % TFA/H,O and B = 0.1 % TFA/CH3CN) provided 44 mg of compound _42 as a
viscous oil: 'H NMR {CDCl3) b 3.41-3.80 (rn, 44H), 4.12 (s, 8H), 4.24 {m,
12H); MS (ESI)
calculated for C4zH65CI4N,o0" (M+H): 1121. Found: 1121.
Example 11
Synthesis of Octamer of DEA/DEG Using Core Propagation Approach
A chemical scheme for the preparation of an octarner of DEA/DEG is shown in
Figures 17C and I7D. The tetrapara-nitraphenylcarbonate compound, compound 39,
was
reacted with diethanolamine to form the octahydroxy compound, compound _43a.
Compound 43a was then reacted withpara-nitrophenylchloroformate to yield the
octa
para-nitrophenylcarbonate compound {compound 43). Thepara-nitrophenylcarbonate
(PNP) group was then converted to a carbonate group by reaction with mono-CBZ-
protected piperazine, yielding compound 44. The terminal CBZ-protected amino
groups
were then converted to the hydrobromide salt of amino group (compound 44a),
and further
reacted with bromoacetic anhydride to yield reactive brom~oacetyl groups at
each of the
termini in compound 45.
Compound 43a
A solution of 100 mg (0.097 minol) of compound 39 in 450 p.L of pyridine was
added to a 0°C solution of 6I mg (0.583 mmol} of dietha.nolamine in 150
p.L of pyridine.
The cooling bath was removed, and the mixture was stirred for 20 hours at room
temperature, to yield the crude compound 43a, which was used as is in the next
step.
CA 02353462 2001-05-31
WO 00134231 PCT/US99/29339
Compound 43
The mixture from the previous step was cooled back to 0°C, and a
solution of 431
mg (2.138 mmol) of 4-nitrophenyichloroformate in 4 mL of THF followed by 2 mL
of
CHzCI, to improve solubility. The mixture was stirred fo:r 18 hours at room
temperature,
cooled back to 0°C, acidified with 1 N HCI, and partitioned between 20
mL of 1 N HCl and
2 X 20 mL of CHZCIz. The combined organic layers were: dried (MgS04), f
ltered, and
concentrated to give 505 mg of yellow solid. Purif cation by silica gel
chromatography
(EtOAc/hexanes) provided 146 mg (68%) of compound 4:3 as a crystalline solid:
mp 67-
69°C; 'H NMR (CDC13) 8 3.50-3.80 (m, 28H), 4.22 (m, 1:2H), 4.43 (m,
1dH}, 7.40 {m,
I6H), 8.30 (m, I6H).
Compound 44
A solution of 476 rng (2.16 mmol) of mono-CBZ-piperazine in 2.0 mL of CHZC12
was added to a 0°C solution of 400 mg (0. I 8 mmol) of compound _43 and
300 p,L (2.16
mmol) of Et3N in 2.0 mL of CHZCh. The reaction mixture was stirred for I 8
hours at room
temperature, cooled to 0°C, and acidified with 1 N HCI. The mixture was
partitioned
between 40 mL of 1 N HCl and 3 X 40.mL of CHzCl2. The combined organic layers
were
washed with 3 X 40 mL of saturated NaHCO~ solution, dried (MgS04), filtered,
and
concentrated to give 483 mg (97%) of compound 44 as a sticky white solid which
was pure
enough for use in the next step: 'H NMR (CDCI,) $ 3.36-3.60 (m, 88H), 3.66 (t,
4I-i~, 4.2I
(brd, 28H), 5. i 5, (s, 16H), 7.36 (brd s, 40H).
Compound 44a
Compound 44 (150 mg, 0.054 mmol) was dissolved in 2 mL of 30% HBrfHOAc.
The mixture was stirred for 30 minutes at room temperature:, and the HBr salt
was
precipitated by addition of EtzO. The precipitate was washed twice with Et,O,
and dried
under vacuum.
71
CA 02353462 2001-05-31
WO 00/34231 PCT/US99/29339
Compound 45
The precipitate, compound 44a, was dissolved in :~0 mL of HzO. The mixture was
brought to pH L2 by addition of 10 N NaOH and extracted with six 20 mL
portions of 4/1
CHZCI,/MeOH. The combined organic layers were dried (MgS04), filtered, and
concentrated to provide the free amine. The free amine was dissolved in 4 mL
of CH3CN,
0.5 mL of MeOH was added to improve solubility, and the solution was stirred
at 0°C
while a solution of 1 I2 mg (0.65 mmol) of chloroacetic aylhydride in I mL of
CH3CN was
added. The mixture was stirred at room temperature for 2 hours and
concentrated to give
1 I I mg of an oil. Purification by preparative HPLC (1" CI8 column, gradient
35-55% B,
A = 0.1% TFA/H,O and B = 0.1% TFA/CH3CN) provided 20 mg (15%) of compound 45
as
a viscous oil: 'H NMR (CDC13) 8 3.40-3.78 (m, 92H), 4.15 (S, 16H), 4.28, (m,
28H).
Example 12
~nthesis of Tetramer and Octamer of ADPIDEG Using Core Propagation Approach
A chemical scheme for the preparation of an octamer of ADP/DEG is shown in
Figures 18A and I 8B. The di pares-nitrophenylcarbonate derivative of
diethyleneglycol,
compound 37 (from which the "core" is derived) was reacted with 2-amino-1,3-
propanediol to yield the tetrahydroxy compound, compound 46. Compound _46 was
reacted
withpares-nitrophenylchloroformate to yield the tetrapara-nitrophenylcarbonate
compound
(compound 47). Compound 47 was then reacted with 2-amino-1,3-propanedial to
yield the
octahydroxy compound, compound 47a. Compound 47a was then reacted with para-
nitrophenylchIoroformate to yield the octapara-nitrophenylcarbonate compound
(compound 48). Thepara-nitrophenylcarbonate (PNP) group was then converted to
a
carbamate group by reaction with mono-CBZ-protected pip~erazine, yielding
compound
48a. The terminal CBZ-protected amino groups were then .converted to the
hydrobromide
salt of amino group (compound 48b), and further reacted with chloroacetic
anhydride to
yield reactive chloroacetyl groups at each of the termini in compound 48c.
72
CA 02353462 2001-05-31
WO 00134231 f'CT/US99I29339
Compound 46
A solution of compound 37 in pyridine is added t:o a 0°C solution of 3
eq. of 2-
amino-1,3-propanediol in pyridine. The cooling bath is removed, and the
mixture is stirred
for 5 hours at room temperature. Compound 46 can be isolated if desired,
however, it is
generally more convenient to isolate after forming the 4-nitrophenylcarbonate
ester.
Compound 47
The mixture above is cooled back to 0°C, a solution of 10 eq. of 4-
nitrophenylchloroformate in 60 mL of CHZC12 is added, and the mixture is
stirred for 20
hours at room temperature. The mixture is cooled back to 0°C, acidified
with 1 N HCi, and
is partitioned between 1 N HCl and CHZCl2. The combined organic layers are
dried
(MgS04), filtered, and concentrated. Purification by silica gel chromatography
provides
compound 47.
Compound 47a
A solution of compound 47 in pyridine is added to a 0°C solution of 6
eq. of 2-
amino-1,3-propanediol in pyridine. The cooling bath is removed, and the
mixture is stirred
for 20 hours at roam temperature to yield compound 47a., which is used in the
next step.
Compound 48
The mixture above is cooled back to 0°C, and a solution of 4-
nitrophenylchloroformate is added. The mixture is stirred for 18 hours at room
temperature, cooled back to 0°C, acidified with 1 N HCI, and
partitioned between 1 N HCI
and CHZCIz. The combined organic layers are dried (Mg;304), filtered, and
concentrated.
Purification by silica gei chromatography provides compound 48.
73
CA 02353462 2001-05-31
WCD 00/34231 PCT/US99/29339
Compound 48a
In a manner similar to that for compound 44 above, a solution of mono-CBZ-
piperazine in CHZCIz is added to a 0°C solution of compound 43 and Et3N
in CHZCIz. The
reaction mixture was stirred for 18 hours at room temper~~ture, cooled to
0°C, and acidified
with 1 N HCI. The mixture was partitioned between 1 N HCl and CHZC12. The
combined
organic layers are washed with saturated NaHC03 solution, dried (MgS04),
filtered, and
concentrated to give compound 48a, for use in the next step.
Compound 48b
In a manner similar to that for compound 44a above, compound _48a is dissolved
in
30% HBr/HOAc. The mixture is stirred for 30 minutes at room temperature, and
the HBr
salt precipitated by addition of Et20. The precipitate is washed with EtzO,
and dried under
vacuum.
Compound 48c
In a manner similar to that for compound 45 above, the precipitate, compound
48b,
is dissolved in H20. The mixture is brought to pH 12 by addition of 10 N NaOH
and '
extracted with 4/1 CHZCIz/MeOH. The combined organic layers are dried (MgS04),
filtered, and concentrated to provide the free amine. The firee amine is
dissolved in CH3CN,
MeOH is added to improve solubility, and the solution is stirred at 0°C
while a solution of
chloroacetic anhydride in CH3CN is added. The mixture was stirred at room
temperature
for 2 hours and concentrated to give crude compound 48c, which is purified by
preparative
HPLC.
74
CA 02353462 2001-05-31
WO 00134231 PCTNS99/29339
Example 13
~nthesis of Octamer of DEA/PE Using Core Propagation Approach
A chemical scheme for the preparation of an octa~mer of DEA/PE is shown in
Figure
19. Pentaerythritol (from which the "core" is derived} was reacted with para-
nitrophenylchloroformate to yield the tetrapara-nitrophenylcarbonate compound
(compound 49). Compound 49 was then reacted with die:thanolamine to form the
octahydroxy compound, compound 49a. Compound 49a was then reacted with para-
nitrophenylchloroforrnate to yield the octapara-nitrophenylcarbonate compound
(compound 50). Thepara-nitrophenylcarbonate (PNP) group was then converted to
a
carbamate group by reaction with mono-N-BOC-ethylene;diamine, yielding
compound 51.
Compound 49
Pentaerythritol tetrakis-4-nitrophenylcarbonate
Pyridine (950 p,L, 11.74 mmol) was slowly added to a 0°C solution of
100 mg
(0.734 mmol) of pentaerythrital and 1.18 g (5.88 mmol) of 4-
nitrophenylchloroformate in
I 0 mL of CHzCIz. The cooling bath was removed, and the mixture was stirred
for 24 hours
at room temperature. The mixtuxe was cooled back to 0°C, acidified with
1 N HCI, and
partitioned between 50 mL of 1 N HCl and 2 X 50 mL of CHZC12. The combined
organic
layers were dried (MgS04), filtered, and concentrated to give 1.123 g of a
white solid.
Purification by silica gel chromatography (EtOAc/hexane;s followed by
CH,CI,/MeOH)
provided 128 mg (22%} of compound 49 as a white crystalline solid: mp
175°C; : 'H NMR
(CDCl3) b 4.61 (s, 8H), 7.40, (m, 8H), 8.30 (m, 8H).
Compound 49a
To solution of 120 mg (0.150 mmol) of compound 49 in 1.6 mL of pyridine at
0°C
was added 87 p,L (95 mg, 0.90 mmol) of diethanolamine. The cooling bath was
removed,
and the mixture was stirred for 7 hours,at room temperature, and cooled back
to 0°C, to
yield compound 49a, which was used as is in the next step.
CA 02353462 2001-05-31
WO 00/34231 PCT/US99129339
Compound 50
A solution of 756 mg {3.75 mmol) of 4-nitrophenylchloroformate in 3 mL of
CH~C12 was added the mixture above. The mixture was stirred for 18 hours at
room
temperature, cooled back to 0°C, acidified with 1 N HCI, and
partitioned between 20 mL of
1 N HCl and 2 X 20 mL of CHZC12. The combined organic layers were dried
(MgS04),
filtered, and concentrated to give 819 mg of sticky yellow solid. Purification
by silica gel
chromatography {EtOAc/hexanes and CHZCIz/MeOH) provided 134 mg (47%) of
compound 50 as a sticky viscous oil with some impurities: 'H NMR (CDCl3) 8
3.69 (m,
16H), 4.31 (s, 8H), 4.4I (m, 16H), 7.39 (m, 16H), 8.25 (m, 16H).
Compound 51
A solution of compound SO is treated with 10 eq, ofmono-N-BOC-ethylenediamine
in pyridine and CHZCI2. The mixture is stirred at room terr.~perature until
complete as'
evidenced by TLC, and partitioned between I N HCl and C:HzCl2. The combined
organic
layers is dried (MgS04), filtered, and concentrated to give crude product.
Purification by
silica gel chromatography (EtOAc/hexanes and CHZCIz/Me:OH) provides compound
51.
Example I4
Solid Phase Synthesis of Tetramer of DEA/DEG Usin :segmental Approach
A chemical scheme for the solid phase synthesis of .a tetramer of octamer of
DEA/DEG is shown in Figures 20A and 20B. Wang resin, having terminal hydroxy
groups, was reacted withpara-nitrophenylchloroformate to yield the para-
nitrophenylcarbonate compound (compound 52). Compound _52 was then reacted
with
diethanolamine to form the dihydroxy compound, compound _52a. Compound _52a
was then
reacted withpara-nitrophenylchioroformate to yield the dipara-
nitrophenylcarbonate
compound (compound 52b). Thepara-nitrophenylcarbonate (PNP) group was then
converted to a carbamate group by reaction with mono-CB2:-protected
piperazine, yielding
76
CA 02353462 2001-05-31
WO 00134231 PCTJUS99/29339
compound 53. The CBZ-protected compound was then cleaved from the resin, and
reacted
with diethyleneglycol bis chloroformate (from which the "core" is derived), to
yield the
tetra-CBZ-protected amino compound, compound 40. The terminal CBZ-protected
amino
groups may be converted to the hydrobromide salt of amino group, and further
reacted with
S chloroacetic anhydride to yield reactive chloroacetyl groups at each of the
termini.
Compound 52
Wang resin (25 mg, subst. 0.58 mmol/g, 0.0145 mrnol) was washed with CHZCIz.
The resin was suspended in S80 ~,L of CHzCl2, and IS mg (0.145 mmol} of 4-
nitrophenylchloroformate was added followed by 97 p,L of pyridine. After
gentle agitation
of the mixture for 4 hours, the resin was washed with CHzCI2 and dried, to
yield compound
S2.
1 S Compound 52a
The resin was then suspended in 410 pL of CHZCIz, and 71 mg (0.673 mmol) of
diethanolamine {410 p.L of a solution of 82.5 mg of diethanolamine dissolved
in 493 p.L of
pyridine}. After gentle agitation of the mixture for 16 hours, the resin was
washed with
CH~C12 and dried, to yield compound S2a.
Compound 52b
To the resin was added S80 p.L of CHZC12, and to the mixture was added 15.2 mg
2S (0.145 mmol) of 4-nitrophenylchloroformate followed by f7 ~.L of pyridine.
After gentle
agitation of the mixture for 4 hours, the resin was washed vrith CHZCl2 and
dried, to yield
compound S2b.
77
CA 02353462 2001-05-31
WO 00/34231 PCT/US99/29339
Compound 53
To the resin was added 410 p.L of CHZCl2, and to the mixture was added 130 p.L
of
mono-CBZ-piperazine followed by 410 pL of pyridine. After gentle agitation of
the
mixture for 18 hours, the resin was washed with CHzCI2 and dried, to yield
compound 53.
Compound 54
To the resin was added 1 mL of 10% TFA in CHZC;12, the mixture was agitated
for
10 min, and the mixture was f ltered. The TFA treatment 'was repeated twice,
and the
combined filtrates were combined and concentrated to give 3 mg {35%) of
compound _54;
'H NMR (CDCl3) 8 3.13 (m, 4H), 3.48 (m, 8H), 3.80 {m, 4~H), 4.50 (m, 1H), 5.18
{s, 4H),
7.37 (brd s, lOH); MS (ESI) calculated for C3°H4°NSO$ (M-a-H):
598. Found: 598.
Compound 40
To a solution of 2.1 eq. of compound 54 and 2.1 eq. of Et3N in CHZCIz at
0°C is
added a solution of 1 eq. of diethyleneglycol bis-chloroforrnate in CHZCI2.
The mixture is
stirred fox at room temperature and concentrated to give cnide compound _40
which can be
purified by silica gel chromatography.
Example 15
Compound 39b
To a solution of 3. I 7 g (3.08 rnmol) of compound 3!3 in 35 mL of CHZCIZ at
0°C was
added 2.6 mL of Et3N followed by a solution of 3.26 g ( 18.49 mmol) of mono-N-
Boc-
ethylenediamine (also referred to as tert-butyl N-(2-aminoethyl)carbamate,
Aldrich
Chemical Co.) in 30 mL of CHZC12. The mixture was stirred at room temperature
for 18
hours, cooled to 0°, and acidified with 1 N HCI, The mixture was then
partitioned between
150 mL of 1 N HCl and three 100 mL portions of CHzCl2. 7Che organic layers
were
combined and washed with three portions of saturated sodium bicarbonate
solution, dried
(MgS04), f Itered and concentrated to provide 3.17 g of yellow solid.
Purification by silica
78
CA 02353462 2001-05-31
WO 00/34231 PCT/US99/29339
gel chromatography (step gradient 98/2 to 95/5 to 90/10 CHzCIz/MeOH) provided
2.76 g
(80%) of compound 39b as a white solid. 'H NMR (CDCI~) ~ 1.45 {s, 36H), 3.23
(s, I6H),
3.50 (m, 8H), 3.72 (t, 4H), 4.I9 (m, 8H), 4.26 (t, 4H), 5.38 (brd s, 4H), 5.80
(brd s, 2H),
6.00 (brd s, 2H); mass spectrum (ES) m/z calculated for C;46H84NfoOzt (M+Na):
1135.
Found:ll35.
Compound 39c
A solution of 1 g (9.42 mmol) of diethylene glycol, in 20 mL of EtOAc was
added to
a solution of 3.82 g (23.5 mmol) of carbonyldiimidazole in 80 mL EtOAc and the
resulting
mixture was stirred for 2 hours at room temperature. The :mixture was
concentrated to an
oily solid, and the product was purified by silica gel chromatography (97/2
CHZCIZ/MeOH)
to give 2.04 g (73%) of the bis-imidazolide of diethylene glycol as a white
solid: 'H NMR
(CDCI~) 8 3.87 {t, 4H), 4.58 (t, 4H), 7.08 (s, 2H), 7.41 (s, 2H), 8.16 (s,
2H). A solution of
50 mg (0.17 mmol) of the bis-imidazolide of diethylene glycol in 1 mL of
CHZCIz was
added to a solution of 54 mg (O.SI) mmol) of diethanoIamine and 82 N.L (80.6
mg, 1.02
mmol) of pyridine in 0.5 mL of CHZC12. The mixture was ;stirred at room
temperature for
four hours, and to the mixture was added a solution of 248 mg (1.53 mmol) of
carbonyldiimidazole in 5 mL of CHzCIz. The mixture was .stirred at room
temperature for
1.5 hours and concentrated to an oily solid. Purification by silica gel
chromatography (98/2
CHZCh/MeOH) provided 103 mg (82%) of the multivalent activated carbonate
derivative
compound 39c, including minor impurities. The resulting ail crystallized when
placed in
the freezer: 'H NMR (CDC13) 8 3.71 (m, 12H), 4.32 (m, 4H), 4.59 (t, 8H), 7.10
(s, 4H),
7.43 (s, 4H), $.18 (s, 4H); mass spectrum (ES) m/z (relative intensity) 380
(100), 443 (18),
S I2 (2ti), no parent ion observed.
79
