Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
- -1- 131636ll
64693-4~02
STARBURST COMJUGATES
The present invention concerns the use of dense star
polymers as carriers for agricultural materials (the l'carried"
material). In recent years polymers referred to as dense star
polymers or Starburst polymers have been developed. It has
been ~ound that the size, shape and properties of these dense
star polymers or starburst po]ymers can be molecularly tailored
to meet specialized end uses. Starburst polymers have
significant advantages which can provide a means for the delivery
of high concentrations of carried material per unit of polymer,
controlled delivery, targeted delivery and/or multiple species
delivery or use.
This application is one of three closely related
patent applications serial Nos. 544,734, 544,735 and 544,736 all
f~led on August 18, 1987. The present application deals with all
cases in which the carried material is an agricultural material,
application serial No. 544,734 deals with all cases in which the
carried material is a pharmaceutical material and application
serial No. 544,735 deals with all the remaining cases in which
the carried material is neither a pharmaceutical nor an
agricultural material.
In its broadest aspect, the present invention is
directed to polymer conjugate materials comprising dense star
polymers or~starburst polymers associated with agricultural
materials (hereinafter these polymer conjugates will frequently
be referred to as "starburst conjugates" or "conjugates"),
processes for preparing these conjugates, compositions
Trade-mark
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1 31 6~' 1
2 6~693--~lo~
containing the conjugates, and methods of using the conjuyates and
compositions.
The conjugates of the present invention are suitable for
use in a variety of applicakions where specific delivery is
desired, and are particularly suited for khe delivery of
biologically active agents. In a preferred embodiment of the
present invention, the starburst conjugates are comprised of one
or more starburst polymers associaked with one or more bioactive
agenks.
The invention further provides a process for prepariny
starburst polyethyleneimine which comprises reacting a starburst
polyethyleneiminemethane sulfonamide with hydrochloric acid and a
process for purifying a starburst dendrimer having a solvent
present which comprises removing the solvent by ultrafiltration
using a membrane.
The starburst conjugates offer significant benefits over
other carriers known in the art due to the advantageous properties
of the starburst polymers. Starburst polymers exhibit molecular
architecture characterized by reyular dendritic branching with
radical symmetry. These radialIy symmetrical molecules are
referred to as possessing "fstarburst~topology". These polymers
are made in a manner which can provide concankric dendrikic kiers
around an initiator core. ~ The ~starburst topology is achieved by
the ordered assembly of organic repeating units in concenkric,
dendritic tiers around an inikiator core; this is accomplished by
1 3 1 6 3 ~i r
2a 64593-~102
introducing multiplicity and self-replication (within each tier)
in a geometrically progressive fashion throuyh a number of
molecular generations. The resulting highly functionalized
molecules generations have been termed "dendrimers" in deference
to their branched (tree-like) s-tructure as well as their
oligomeric na~ure. Thus, khe terms starburst oIigomer and
starburst dendrimer are encompassed within the term starburst
polymer. Topological polymers, with size and shape controlled
domains, are dendrimers that are covalently bridged
~, . .
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,~
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1 31 63~
-3
through their reactive terminal group which are
referred to as "starburst bridged dendrimers", which is
also encompassed within the term'starburst polymer.
The following description of the figures aids
in understanding the present invention.
Fiaure l depicts various generations of starburst
dendrimers.
Figure 2A depicts a dendrimer having unsymmetrical
(unequal) branch junctures.
Fi~ure 2B depicts a dendrimer having symmetrical
(equal) branch junctures.
Figure 3 shows carbon-13 spin lattice relaxation times
(Tl) for aspirin incorporated into various dendrimer
gsnerations. (E~ample 4)
1~ "
The starburst polymers are illustrated by
Figure 1 wherein ~ represents an initiator core (in
this figure a tri-functional initiator core, shown by
the far left drawing); Z represents a terminal group,
shown in the first instance by the second drawing from
the left, referred to as a starbranched oligomer; A, B,
C, D, and E represent particular molecular generations
of starburst oligomers, called dendrimers; and (~)n~
(B)n~ (C)n, (D)n~ and (E)n represent starburst bridged
dendrimers.
The"starburst dendrimers are unimolecular
assemblages that possess three distinguishing
architectural features, namely, (a) an initiator core,
(b) interior layers (generations, G) composed of
repeating units, radially attached to the initiator
35,444-F -3-
.
,
1 31 63~
646g3-~102
core, and (c) an exterlor surface of termlnal ~unctionallty (l.e.,
termlnal functlonal groups) attache~ to the outermost yeneratlon.
The slze and shape of the starburst dendrlmer molecule and th~
functional groups present ln the dendrlmer molecule can be con-
trolled by the cholce of the lnltlator core, the num~er of genera-
tions (l.e., ~lers) employe~ ln creating the dendrimer, and the
cholce of the repeating unlts employed at each generatlon. Slnce
the dendrimers can be read~ly isolated at any partlcular yenera-
tlon, a means ls provided for obtalning dendrimers having deslred
properties.
The choice of the starburst dendrimer components affects
the propertles of the dendrlmers. The lnltlator core type can
affect the dendrlmer shape, producing ldepending on the cholce of
initiator core)~ for example, spheroid-shaped dendrimers, cylin-
drlcal or rod-shaped dendrlmers, elllpsoid-shaped dendrimers, or
mushroom-shaped dendrlmers. Sequentlal bullding o~ generatlons
(i.e., generatlon number and the size and nature of the repeatin~
units) determlnes the dlmensions of the dendrimers and the na$ure
of thelr interlor.
Because starburst dendrlmers are branched polymers con-
taining dendrltlc branches havlng functlonal groups dlstrlbuted on
the perlphery of the branches, they can be prepared with a varlety
of propertles. For example, starburst dendrimers, such as those
deplcted in Figure 2A and Flgure 2B can have distinct propertles
due to the branch length. The dendrlmer type shown ln Figure 2A
(such as Denkwalter, U.S. Patent 4,289,872) possesses unsymmetri-
cal ~unequal segment) branch ~unctures, exterior ~i.e., surface)
groups (represented by Z'~,
~ , . . . ~ , ~ . -, .
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1 31 63G!~
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interior moieties (represented by Z) but much less
internal no internal void space. The dendrimer type
shown in Figure 2~ possesses symmetrical (equal
segment) branch junctures with surface groups
(represented by Z'), two different interior moieties
(represented respec~tively by X and Z) with interior
void space which varies as a function of the generation
(G). The dendrimers such as those depicted in Figure
2B can be advanced through enough generations to
totally enclose and contain void space, to give an
entity with a predominantly hollow interior and a
highly congested surface. Also, starburst~dendrimers,
when advanced through sufficient generations exhibit
f 15 "starburst dense packing" where the surface of the
dendrimer contains sufficient terminal moieties such
that the dendrimer surface becomes congested and
encloses void spaces within the interior of the
dendrimer. This congestion can provide a molecular
level barrier which can be used to control diffusion of
materials into or out of the interior of the dendrimer.
Surface chemistry can be controlled in a
predetermined fashion by selecting a repeating unit
which contains the desired chemical functionality or by
chemically modifying all or a portion of the surface
functionalities to create new surface functionalities.
These surfaces may either be targeted toward specific
sites or made to resist uptake by cells. In an
3Q alternative use of the dendrimers, the dendrimers can
themselves be linked together to create polydendric
moieties (starburst "bridged dendrimers") which are
also suitable as carriers.
`35 In addition, the dendrimers can be prepared so
as to have deviations from uniform branching in
35,444-F -5-
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-6- 1 3 1 63 ~J~
particular generations, thus providing a means of
adding discontinuities (i.e., deviations from uniform
branching at particular locations within the dendrimer)
and different properties to the dendrimer.
The starburst polymers employed in the
"starburst'~conjugates o~ the present inYention can be
prepared according to methods known in the art, for
example, U~ S. Patent 4,587,329.
Dendrimers can be prepared having highly
uniform size and shape and most importantly allow for a
greater number of functional groups per unit of surface
area of the dendrimer, and can have a greater number of
functional groups per unit of molecular volume as
compared to other polymers which have the same
molecular weight, same core and monomeric components
and same number of core branches as the starburst"
polymers. The increased functional group density of
the starburst'lpolymers may allow a greater quantity of
material to be carried per dendrimer.
An analogy can be made between early generation
~starburstl)dendrimers (i.e., generation = 1-7) to
classical spherical micelles. The dendrimer - micelle
analogy was derived by comparing features which they
had in common such as shape, size and surface.
3o
-
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--7--
TABI.E I
Regular ~Starburst"
Parameter ClassicalDendrimers
Micelles
Shape Spherical Spherical
Size (diameter3 20-60~ 17-~3A
Surface
Aggregation 4-202 Z=6-192
Numbers (Generation = 2-7)
Area/Sur~acOe 130-80~2 127-75~2
Group (A)
Z is the number of surface groups; lA - 10-1 nm;
1A2 = 10-2 nm2.
In Table I, the shape was verified by scanning
transmission electron micrographs (STEM) microscopy and
intrinsic viscosity ~) measurements. The size was
verified by intrinsic viscosity (~) and size exclsion
chromatography (SEC) measurements. The surface
t/ t r,~ Lr ~
aggregation numbers were veri~fied by ~e~y'and high
field NMR. The area/surface group was calculated from
SEC hydrodynamic measurements.
The first five generations of starburstP
polyamidoamine (PAMAM) dendrimers are microdomains
which ~very closely mimic classical spherical micelles
in nearly every respect (i.e shape, size, number of
surface groups, and area/surface group3. A major
difference, however, is that they are covalently fixed
and robust compared to the dynamic equilibration of
nature of micelles. This difference is a significant
35,444-F -7-
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.
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-8- 131 63Gi~
advantage when using these microdomains as controlled
delivery prototypes or encapsulation devices.
As further concentric generations are added
beyond five, congestion of the surface occurs. This
congèstion can lead to increased barrier
characteristics at the surface and manifests itself as
a smaller surface area per head (surface) group as
shown in Table II.
.
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a`l 0 r ~ f¢ N ~
0~ ~ N 7~ ~ , N~
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q~ ~
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f C I O ~ o O ,1
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l I N ~ G~ ~ N~ .¢ ~ ~1 a~ .
.-- ~ ~ N 11 _~ N ~ X ~3
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C ~ S N N o~:l N .~1 U
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~ 0 ,~ O ,0~ cn O
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646g3-4102
For example, amlne terminated generations 5.0, 6.0, 7.0,
8.0 and s.o have decreased surface areas of 104, 92, 73, 47 and
32A2 per Z group, respectlvely. Thls characterlstic corresponds
to a transltlon from a less congèsted mlcelle-llke surface to a
more congested bllayer/monolayer barrier-llke sur~ace normally
assoclated with veslcles ~liposomes) or Langmuir-Blodgett type
membranes.
If thls surface congestlon is lndeed occurrlng, the
change ln physical characterlstlcs and morphology ~hould be
observed as the generations lncrease from the lntermedlate genera-
tlons ~6-8) to the more advanced generatlons (9 or 10). The scan-
nlng transmission electon micrographs (STEM) ~or generatlons =
7.0, 8.0 and 9.0 were obtalned after removlng the methanol solvent
from each of the samples to provlde colorless, llght yellow solid
~llms and stalnlng wlth osmlum tetraoxlde. The morphological
change predlcted occurred at the generation, G = 9.0 stage. The
mlcrodomalns at generatlon = 9.0 measure about 33A ln dlameter and
are surrounded by a colorless rlm which ls about 25~ thick.
Apparently, methanolic solvent has been entrapped wlthin the 25A
outer membrane-like barrler to provide the dark stained lnterior.
Thus, at generation = 9.0, the starburst PA~AM ls behavlng topo-
logically like a vesicle (liposome). However, this starburst is
an order of magnitude smaller and very monodispersed compared to a
liposome. Consequently, the present dendrimers can be used to
molecularly encapsulate solvent filled void spaces of as much dla-
meter as about 33A (uolume about 18, oooA3 ) or more. These mlcelle
sized prototypes appear to behave like a covalently fixed liposome
in thls advanced generatlon
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stage. These prototypes may have additional capability
as carriers or as delivery agents.
Since the number of ~unctional groups on the
dendrimers can be controlled on the surface and within
the interior, it also provides a means for controlling
the amount of agricultural material to be delivered per
dendrimer. In a particularly preferred embodiment of
the present invention the dendrimers are targeted
carriers of bioactive agents capable of delivering
bioactive agent to a particular target organism such as
a plant or pest or to a particular determinant or locus
in a target organism. Dendrimers suitable for use in
the conjugates of the present invention include the
dense star polymers or starburst~polymers described in
U. S. Patents 4,507,466, 4,558~120, 4,568,737 and
4,587,329.
In particular, the present invention concerns a
'starburst"conjugate which comprises at least one
"starburst"polymer associated with at least one carried
agricultural material. Starburst~conjugates included
within the scope of the present invention include those
represented by the formula:
(P)x * (M)y (I)
wherein each P represents a dendrimer;
3o
x represents an integer of 1 or greater;
each M represents a unit (for example, a molecule,
atom, ion, and/or other basic unit) of a carried
agricultural material, said carried agricultural
material can be the same carried agricultural material
35,444-F
.
1 3 1 ~3~
646g3-4102
or ~ dlfferent carrled agricultural material, preferably the
c~rrled materlal ls a bloactlve agent;
y represents an integer of l or greater; and
* lndicates that the carried material is associated wlth the
dendrlmer.
Preferred starburst con~ugates of formula (I) are those
in which M is a pestlcide, radionucllde, chelator, chelated metal,
toxin, or slgnal generator, slgnal reflector, or slgnal absorber;
partlcularly preferred are those in which x=l, and y=2 or more.
Also included are starburst con~ugates of formula (I)
wherein the starburst dendrimers are covalently linked together,
optionally via linking groups, so as to ~orm polydendric assem-
blages (i.e., where x~l). Use of these starburst bridged den-
drimers include toplcal controlled release agents.
As used herein, "associated with" means that the carried
material(s) can be encapsulated or entrapped wlthin the core of
the dendrlmer, dispersed partially or fully throughout the den-
drlmer, or attached or linked to the dendrimer, or any comblnation
thereof. The assoclatlon of the carried material(s) and the den-
drlmer(s) may optionally employ connectors and/or spacers tofacllitate the preparatlon or use of the starburst con~ugates.
Suitable connectlng groups represented by C', are groups whlch
link a targetlng dlrector (l.e., T) to the dendrlmer (i.e., P)
wlthout signlflcantly lmpairing the effectlveness of the director
or the effectlveness of any other carried material(s) (i.e., M)
present ln the starburst con~ugate. These connectlng groups may
~ ~ be cleavable or
: '
12
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1 3 1 6 3 ~ /lr
-13-
non-cleavable and are typically used in order to avoid
steric hindrance between the target director and the
dendrimer, preferably the connecting~ groups are stable
(i.e., non-cleavable). Since the size, shape and
functional group density of the dense star dendrimers
can be rigorously controlled, there are many ways in
which the carried material can be associated with the
dendrimer. For example, (a) there can be covalent,
coulombic, hydrophobic, or chelation type association
0 between the carried material(s) and entities, typically
functional groups, located at or near the surface of
the dendrimer; (b) there can be covalent, coulombic,
hydrophobic, or chelation type association between the
f 15 carried material(s) and moieties located within the
interior of the dendrimer; (c) the-dendrimer can be
prepared to have an interior which is predominantly
hollow allowing for physical entrapment of the carried
materials within the interior (void volume), wherein
the release of the carried material can optionally be
controlled by congesting the surface of the dendrimer
with diffusion controlling moieties; or (d) various
combinations of the aforementioned phenomena can be
employed.
Dendrimers, herein represented by "P", include
the dense star polymers described in U. S.
Patents 4,507,466; 4,558,120; 4,568,737 or 4,587,329.
Carried agricultural materials, including the
term "agricultural materials", herein represented by
"M'', which are suitable for use in the starburst
conjugates include any materials for in vivo or in
vivtro treatment, diagnosis, or application to plants
and non-mammals (including microorganisms) which can be
~i associated with the starburst dendrimer without
35,444-F -13-
- t 3 1 636 1,
appreciably disturbing the physical integrity of the
dendrimer. For example, carried materials like toxins
such as diphtheria toxin, gelonin, exotoxin A, abrin,
modeccin, ricin, or toxic fragments thereof; metal ions
such as the alkali and alkaline-earth metals;
radionuclides such as those generated from actinides or
lanthanides or other similar transition elements or
from other elements, such as 67cu~ 90y, 111In, 131I,
186Re, 105Rh, 99mTe, 67Ga, 153Sm~ 159Gd, 175yb, 177LU,
88y, 166Ho, 115mIn~ 109pd, 82Rb, 194Ir~ 140Ba, 149pm~
199AU, 140La, and 188Re; signal generators such as
fluorescing entities; signal reflectors such as
paramagnetic entities; signal absorbers such as
electron beam opacifiers; hormones; biological response
modifiers such as interleukins, interferons, viruses
and viral fragments; pesticides, including
antimicrobials, algicides, arithelmetics, acaricides,
insecticides, attractants, repellants, herbicides
and/or fungicides such as acephate, acifluorfen,
alachlor, atrazine, benomyl bentaz~on, captan,
carbofuran, chloropicrin, ~ chlorsulfuron
c`~ ,e,r h1; ~ rl ~,
cyanazine, cyhexatin, ~ rmctrln, 2,4-dichloro-
phenoxyacetic acid~ dalapon, dicamba, diclofop methyl,
diflubenzuron, dinoseb, endothall, ferbam, fluazifop,
glyphosate, haloxyfop, malathion, naptalam,
pendimethalin, permethrin, picloram, propachlor,
propanil, sethoxydin, temephos, terbufos, trifluralin,
triforine, zineb, and the like. Carried agricultural
materials include scavenging agents such as chelants,
chelated metal (whether or not they are radioactive),
or any moieties capable of selectively scavenging
therapeutic or diagnostic agents.
35,444-F -14-
.
:
:
-15- 131636 ~
Preferably the carried materials are bioactive
agents. As used herein, "bioactive" refers to an
active entity such as a molecule, atom, ion and/or
other entity ~hich is capable of detecting,
identifying, inhibiting, treating, catalyzing,
controlling, killing, enhancing or modifying a targeted
entity such as a protein, glycoprotein, lipoprotein,
lipid, a targeted cell, a targeted organ, a targeted
organism [for example, a microorganism, plant, or animal
(excluding mammals)] or other targeted moiety.
The starbursts~conjugates of formula (I) are
prepared by reactive P with M, usually in a suitable
solvent, at a temperature which facilitates the
association of the carried material (M) with the
'/starburstdendrimer (P).
Suitable solvents are solvents in which P and M
are at least partially miscible and inert to the
formation of the conjugate. If P and M are at least
partially miscible with each other, no solvent may be
required. When desired, mixtures of suitable solvents
can be utilized. ~xamples of such suitable solvents
are water, methanol, ethanol, chloroform, acetonitrile,
toluene, dimethylsulfoxide and dimethylformamide.
The reaction conditlon for the formation of the
starburst~conjugate of formula (I) depends upon the
particular dendrimer (P), the carried agricultural
material (M), and the nature of the bond (*) formed.
For example if P is the PEI (polyethyleneimine)
'starburstdendrimer with a methylene carboxylate
surface, M is a radionuclide, e.g. yttrium, then the
reactlon is conducted at room temperature in water.
Typically, the temperature can range from room
35,444-F -15-
: -,. , :'~ ' ''
. '
' '
-16- 1 31 636 lr
temperature to re~lux. The selection of the particular
solvent and temperature will be apparent to one skilled
in the art.
5 ` The ratio of M:P will depend on the size of the
dendrimer and the amount of carried material. For
example, the molar ratio (ratio of moles) of any ionic
M to P is usually 0.1-1,000:1, preferably 1-50:1, and
more preferably 2-6:1. The weight ratio of any
pesticide or toxin M to P is usually 0.1-5:1, and
preferably 0.5-3:1.
When M is a radionuclide, there are three ways
F the starburst"conjugate can be prepared, namely: (1~ P
can be used as a chelant. For example a
methylenecarboxylate surface PEI or PAMAM will chelate
a metal such as yttrium or indium. (2) A chelate can
be covalently bonded to P. For example, an amine
terminated PEI starburst dendrimer can be reacted with
1-(p-isothiocyanatobenzyl)diethylenetriaminepenta-
acetic acid and then chelated, or a complex such as
rhodium chloride chelated with isothiocyanatobenzyl-
2,3,2-tet can be reacted. (3) A prechelated
radionuclide can be associated with P by hydrophobic or
ionic intereaction.
Other starburst conjugates are those conjugates
~ which contain a target director (herein designated as
"T") and wh1ch are represented by the formula:
(T)e * (P)x * ( )Y (II)
.
wherein
35,444-F -16-
1 3 1 6 3 G '~
64693-41U2
each T represents a target director;
e represents an lnteg~r of 1 or yreater; and
P, x, *, M, and y are as prevlously de~lned herein.
Preferred among the starburst con~ugates o~ formula (II) are those
ln whlch M ls a pestlcide, radlonuclide, chelator, chelated metal,
toxln, slgnal generator, slgnal reflector, or slgnal absorber.
Also preferred are those con~ugates ln whlch e=l or 2; and those
ln whlch x=l and y=2 or more. Par~lcularly preferre~ con~ugates
are those ln which x~l, e=l, y=2 or more and M and T are assocla-
ted with the polymer vla the same or different connectors.
The starburst con~ugates of forrnula (II) are prepared
elther by forming T P and then addlng M or by formlng P M and then
addlng T. Elther reactlon scheme is conducted at temperatures
whlch are not detrlmental to the partlcular con~ugate component
and ln the presence of a sultable solvent when requlred. To con-
trol pH, buffers or addition of sultable acid base ls used. The
reactlon conditions are dependent on the type of associatlon
formed ( )~ the starburst dendrlmer used (P), the carried agrlcul-
tural material (M), and the target director (T). Alternatively, P
and M can be chelated, usually ln water, before con~ugatlon to T.
The con~ugatlon wlth T ls carrled out in a sultable buffer.
The ratio of T,P ls preferably 1:1. The ratlo of M:P
wlll be as before.
Target directors capable of targeting the starburst
con~ugates are entitles whlch when used in the starburst con~u-
gates of the present lnvention
17
'
-18- 13163'~,
F result in at least a portion of the starburst
conjugates being delivered to a desired target (for
example, a protein, glycoprotein, lipoprotein, lipid, a
targeted cell, a targeted organism or other targeted
5 moiety) and include hormones, biological response
modifiers, chemical functionalities exhibiting target
speci~icity, and the like.
In the absence of a target director (or in the
10 presence of a target director if desired), due to the
number of functional groups which can be located at or
near the surface of the dendrimer, all or a substantial
portion of such functional groups can be made anionic,
f cationic, hydrophobic or hydrophilic to effectively aid
5 delivery of the starburst conjugate to a desired target
of the opposite charge or to a hydrophobic or
hydrophilic compatible target.
Preparation of the conjugates of formula (II3
20 using a P with a protected handle (S) is also intended
as a process to prepare the conjugates of formula (II).
The reaction scheme is shown below:
S*P loading ~ S*P*M deprotection ~ P*M
T*P~M linking
~.
; where
S*P represents the protected dendrimer;
S*P*M represents the protected dendrimer
conJugated wi:th m;
.
~ 35,444-F -18-
~19- 131 63G''t
P~M represents the dendrimer conjugated
with M (starburst conjugate);
T*P*M represents the starburst conjugates
liked to the target director.
Suitable solvents can be employed which do not
effect P*M. For example when S is t- ~ ~ S
can be removed by aqueous acid.
The starburst conjugates can be used for a
variety of in vivo and in vitro diagnostic applications
pertaining to plants and non-mammals, such as
radioimmunoassays, electron microscopy, enzyme linked
immunosorbent assays, nuclear magnetic resonan¢e
spectrosoopy, contrast imaging, and immunoscintography,
in analytical applications; and in biological control
applications as a means of delivering pesticides such
as herbicides, insecticides, fungicides, repellants,
attractants, repellants, attractants, antimicrobials or
other toxins, or used as starting materials for making
other useful agents.
The present invention is also directed to
25 ~starburstJ~conjugate compositions in which the starburst~
conjugates are formulated with other suitable vehicles
useful in agriculture such as on crops, fallow land, or
as pesticides, or in treatment of or in vivo or in
vitro testing of non-mammals. The starburst)conjugate
compositions may optionally contain such other active
ingredients, additives and/or diluents.
An agriculturally acceptable carrier or diluent
which may also be present wi`th one or more"starburst"
conjugates of the present invention includes those
carrie~s or diluents customarily used in granular
35,444-F ~ -19-
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~ .
~,, 1 31 63~1~
formulations, emulsi~ible concentrates, solutions, or
suspensions such as, for example, toluene, xylene,
benzene, phenol, water, methane, hydrocarbons,
naphthalene and others.
The preferred starburst polymer for use in the
starburst conjugates of the present invention is a
polymer that can be described as a 'starburst~having at
least one branch (hereinafter called a core branch),
preferably two or more branches, emanating from a core,
said branch having at least one terminal group provided
that (1) the ratio of terminal groups to the core
branches is more than one, preferably two or greater,
; (2) the density of terminal groups per unit volume in
the polymer is at least 1.5 times that of an extended
conventional star polymer having similar core and
monomeric moieties and a comparable molecular weight
and number of core branches, each of such branches of
the extended conventional star polymer bearing only one
terminal group, and (3) a molecular volume that is no
more than about 80 percent of the molecular volume of
said extended conventional star polymer as determined
by dimensional studies using scaled Corey-Pauling
molecular models. As used herein, the term "dense" as
it modifies "star polymer" or "dendrimer" means that it
has a smaller molecular volume than an extended
conventional star polymer having the same molecular
weight. The extended conventional star polymer which
3 is used as the base for comparison with the starburst')
polymer is one that has the same molecular weight, same
core and monomeric components and same number of core
branches as the starburst polymer. By "extended" it is
meant that the individual branches of the conventional
star~polymer are extended or stretched to their maximum
35,444-F ~20-
'`` `' '`` -` `,
. :. . . .
.
-21- 131 63G~
length, e.g., as such branches exist when the star
polymer is completely solvated in an ideal solvent for
the star polymer. In addition while khe number of
terminal groups is greater ~or the 'starburst polymer
molecule than in the conventional star polymer
molecule, the chemical structure of the terminal grou2s
is the same.
Dendrimers used in the conjugates of the
present invention can be prepared by processes known in
the art.
The above dendrimers, the various coreactants and core
compounds, and process for their preparation can be as
f 15 defined in U. S. Patent 4,587,329.
" 1~
The starburst dendrimers, for use in the
starburst~1conjugates of the present invention, can have
terminal groups which are sufficiently reactive to
undergo addition or substitution reactions. Examples
of such terminal groups include amino, hydroxy,
mercapto, carboxy, alkenyl, allyl, vinyl, amido, halo,
urea, oxiranyl, aziridinyl, oxazolinyl, imidazolinyl,
sulfonato, phosphonato, isocyanato and isothiocyanato.
The terminal groups can be modified to make them
biologically inert. The dendrimers differ from
conventional star or star-branched polymers in that the
dendrimers have a greater concentration of terminal
groups per unit of molecular volume than do
conventional extended star polymers having an
equivalent number of core branches and an equivalent
core branch length. Thus, the density of terminal
groups per unit volume in the dendrimer usually is at
least about 1.5 times the density of terminal groups in
the conventionaI extended star polymer, preferably at
35,444-F -21-
- ~
- , : -- ,.
;
,; ;
:, .
--22- 1 3 1 6 ~Ir
least 5 times, more preferably at least 10 times, most
pre~erably from 15 to 50 times. The ratio of terminal
groups per core branch in the starburst'dendrimer is
preferably at least 2, more preferably at least 3, most
5 preferably from 4 to 1024. Preferably, for a given
polymer molecular weight, the molecular volume of the
"~tarburst' dendrimer is less than 70 volume percent9
more preferably from 16 to 60, most preferably from 7
to 50 volume percent of ~he molecular volume of the
0 conventional extended star polymer.
Preferred starburst dendrimers for use in the
starburst~conjugates of the present invention are
f characterized as having a univalent or polyvalent core
15 that is covalently bonded to dendritic branches. Such
ordered branching can be illustrated by the following
sequence wherein G indicates the number of generations:
.
_.
35,444-F -22-
:
-23- 13163
- G = 1 G = 2
N--
N--~
H H
H EI
f 15 G = 3
~N~
~ ,f ~N
:
:
`~ 35
3 5 ,~ 4:4 4 -F ~ 2 3 -
- ~ ~ ' , . ....
.
-2~- 1 31 636~
Mathematically~ the relationship between the
number (#) of terminal groups on a dendritic branch and
the number of generations of the branch can be
represented as follows:
NrG
# o~ terminal groups per dendritic branch =
wherein G is the number of generations and Nr
is the repeating unit multiplicity which is at least 2
as in the case of amines. The total number of terminal
groups in the dendrimer is determiend by the following:
~ of terminal groups per dendrimer NCNrG
wherein G and Nr are as defined before and Nc
represents the valency (often called core
functionality) of the core compound. Accordingly, the
dendrimers of this invention can be represented in its
component parts as f ollows:
~ Terminal
(Core)~(Repeat Unit! G Moiety Nr
~ Nr~l J
wherein the Core, Terminal Moiety, G and Nc are as
def ined before and the Repeat Unit has a valency or
35,444-F -24-
.
- ,
:~ , , :
i
:
,
-
-25- 1 31 63~ ~
functionality of Nr + 1 wherein Nr is as defined
before.
A copolymeric dendrimer which is a preferred
dendrimer for the purposes of this invention is a
unique compound constructed of polyfunctional monomer
units in a highly branched (dendritic) array. The
dendrimer molecule is prepared from a polyfunctional
initiator unit (core compound), polyfunctional
repeating units and terminal units which may be the
same or different from the repeating units. The core
compound is represented by the formula ~ (ZC)Nc
wherein ~ represents the core, zc represents the
functional groups bonded to I and Nc represents the
core functionality which is preferably 2 or more, most
preferably 3 or more. Thus, the dendrimer molecule
comprises a polyfunctional core, ~, bonded to a number
(Nc) of functional groups, zc, each of which is
connected to the monofunctional tail of a repeating
unit, ~1Y1(Z1)N1~ of the first generation and each of
the Z groups of the repeating unit of one generation is
bonded to a monofunctional tail of a repeating unit of
the next generation until the terminal generation is
reached.
In the dendrimer molecule, the repeating units
are the same within a single generation, but may differ
from generation to generation. In the repeating unit,
X1Y1(Z1)N1, X1 represents the monofunctional tail of
the first generation repeating unit, y1 represents the
moiety constituting the first generation, Z1 represents
the functional group of the polyfunctional head of the
repeating unit of the first generation and may be the
3~ same as or different from the functional groups of the
core compound, ~ (ZC)Nc, or other generations; and N1
35,444-F -25- __
: . .
-26- l 3 1 6 ~
is a number of 2 or more, most preferably 2, 3 or 4,
which represents the multiplicity of the polyfunctional
head of the repeating unit in the first generation.
Generically, the repeating unit is represented by the
formula XiYi(Zi)Ni wherein "i" represents the
particular generation from the first to the t-1
generation. Thus, in the preferred dendrimer molecule,
each Zl o~ the first generatio-n repeating unit is
connected to an x2 of a repeating unit of the second
0 generation and so on through the generations such that
each zi group for a repeating unit XiYi(Zi)Ni in
generation number "i" is connected to the tail (Xi+l)
of the repeating unit of the generation number "i+l".
f 15 The final or terminal of a preferred dendrimer molecule
comprises terminal units, XtYt(Zt)Nt wherein t
represents terminal generation and xt, yt~ zt and Nt
may be the same as or different from xi, yi~ zi and Ni
except that there is no succeeding generation connected
to the zt groups and Nt may be less than two, e.g.,
zero or one. Therefore the preferred dendrimer has a
molecular formula represented by
( ~ ( )No) ~ (X Y Z Ni~ NOn ~ ( N~ NcnN
where i is 1 to t-l
35,444-~ -Z6-
.
-27- 1 31 636~
wherein the symbols are as previously defined. The n
function is the product of all the values between its
defined limits. Thus
~ Nn = (N1)(N2)(N3)... (Ni-2)(Ni-1)
n=1
which is the number of repeat units, XiYi(Zi)Ni,
comprising the ith generation of one dendritic branch
and when i is 1, then
n = 1
n=1
In copolymeric dendrimers, the repeat unit for one
generation differs from the repeat unit in at least one
other generation. The preferred dendrimers are very
symmetrical as illustrated in structural formulas
described hereinafter. Preferreddendrimers may be
converted to functionalized dendrimers by contact with
another reagent. For example, conversion o~ hydroxyl
in the terminal generation to ester by reaction with an
acid chloride gives an ester terminally functionalized
dendrimer. This functionalization need not be carried
out to the theoretical maximum as defined~by the number
of available functional groups and, thus, a
functionalized dendrimer may not~have high symmetry or
a precisely de~ined molecular formula as is the case
with the preferred dendrimer.
In a homoplymer dendrimer, all of the repeat
units,XiYl(Zl)Ni, are identical. Since the values of
35,444-F -27-
;
:
" . ` ~ ' ' ' " . " ' ' ~ : '
,
~` ` ' : . ' ' '': ,
1 31 63k~
-2~-
all Ni are equal (defined as Nr~ the product function
representing the number of repeat units reduces to a
simple exponential form. Therefore, the molecular
formula may be expressed in simpler form as
10 (O(ZC)N ) ~ Y (Z )N) N N i~ N~ N N t-1
where i = 1 to t-l
f 15
This form still shows the distinction between the
different generations i, which each consist of NCNr(i-~)
repeating units, XiYi(Zi)Ni. Combining the
generatlons into one term gives:
~ .
;: :
-~: 35,L~4L~-F -28-
:`:
.
~ ' :
-: '' , . . :
-29- 1 3 1 6 3 ~j ir
(~ (Z )N ) ~X Y (Z )N~ Nr _l~X Y (Z )Nt~ N Nt-l
or
core ~ repeat unit terminal unit
((~)(ZC)N )~_Yr(Zr)Nr) (X Nr(t~ Nc
Nr-1
wherein xryr(zr)Nr is the repeating unit which is used
in all generations i.
Consequently, if a polymer compound will fit
into these above formulae, then the polymer is a
F starburst')polymer. Conversely, if a polymer compound
will not fit into these above formulae, then the
polymer is not a starburst polymer. Also, to determine
whether a polymer is a starburst polymer, it is not
necessary to know the process by which it was prepared,
but only whether it fits the formulae. The formulae
also demonstrate the generations (G) or tiering of
dendrimers.
Clearly, there are several ways to determine
:the ratio of agent (M) to dendrimer (P) which depend
upon how and where the as:sociation of P*M occurs. When
there is interior encapsulation, the weight ratio of
;.
:
:
~ 35,444-F -29- .
. ~ .
': :
- , - ,, -, . . .
, . . .~ .: .
- . - :
.
- :
.
'~' : ' ' . ' :
-30- 1 31 63~Jil
M:P usually is 10:1, preferably 8:1, more preferably
5:1, most preferably 3:1. The ratio can be as low as
0.5:1 to 0.1:1. When interior stoichiometry is used,
the weight ratio of M:P is the same as for interior
encapsulation. When exterior stoichiometry is
determined, the mole/mole ratio o~ M:P given by the
following formulae:
M : P
(A) 5 NCNtNr
(B) 3 NCNtN~ 1
f 15 (C) 1 NCNtNrG-l 1
where Nc means the core multiplicity, Nt means the
terminal group multiplicity, and N~ means branch
juncture multiplicity. The NCNtNr~-1 term will result
in the number of Z groups. Thus, for example, (A)
above will result when proteins, enzymes or highly
charged molecules are on the surface; (B) above when it
is 2,4-D or octanoic acid; (C) above when it is
carboxylate ions or groups.
Of course other structures of various
dimensions can be readily prepared by one skilled in
the ar~ by appropriately varying the dendrimer
components and l~umber of generations employed. The
dimensions of the dendrimers are significant in that
they are small. A linear polymer o comparable
molecular weight would have a radius of gyration, (in
its fully extended ~orm), that would be much larger
than the same molecular weight dendrimer.
~ .
35,444-F -30-
`~
~` ` ' - :
. :
`` 1 31 63~
64693-4102
Llnklng target dlrectors to dendrlmers ls another aspect of the
present lnventlon. In preferred embodiments of the present
lnvention, a reactlve ~unctlonal group such as a carboxyl,
sulfhydryl, reactlve aldehyde, reactlve olefinlc derivatlve,
lsothlocyanato, isocyanato, amlno, reactlve aryl hallde, or
reac~lve alkyl hallde can convenlently ~e employed on the
dendrimer. The reactlve functional groups can be introduced to
the dendrlmer usln~ known technlques, for e~ample
(1) Use of a hetero~unctlonal lnltlator (as a starting
materlal ~or synthesizlng the dendrlmer) whlch has lncorporated
into lt functlonal groups of dlfferent reactlvity. In such
heterofunctional lnltiator at least one of the functional groups
wlll serve as an lnltlatlon slte for dendrlmer formatlon and at
least one of the other functlonal groups will be available for
linking to a target director but unable to lnltlate dendrlmer
synthesls. For example, use of protected anillne allows further
modlflcatlon o~ NH2 groups wlthin the molecule, without reacting
the NH2 f the anlline.
The function~l group which wlll be available for llnklng
; 20 to a target dlrector may be part of the lnitiator molecule in any
one of three forms namely:
(a~ In the form in which lt wlll be used for llnklng
wlth the target dlrector. Thls is possible when
none of the synthetic steps lnvolved in the
dendrimer synthesis can result ln reactlon at this
center.
(b) When the functlonal group used for linking to the
~` targeting dlrector is reactive in
.
`~ 31
i
:`
~.
: ' ,
-32 1 3 1 6 3 ~J /r
the synthetic steps involved in the
dendrimer synthesis, it can be protected
by use of a protecting group, which
renders the group unreactive to the
synthetic procedures involved, but can
itself be readily removed in a manner
which does not alter the integrity of the
~ remainder of the macromolecule.
(c) In the event that no simple protecting
group can be found for the reactive
functionality to be used for linking with
the targeting director, a synthetic
r` precursor can be used which is unreactive
in all the synthetic proecedures used in
the dendrimer synthesis. On completion of
the synthesis, this functional group must
be readily convertible into the desired
linking group in a manner which does not
alter the integrity of the remainder of
the molecule.
(d) Coupling (covalently) the desired reactive
functional group onto a preformed
dendrimer. The reagent used must contain
a functionality which is readily reacted
with the terminal functional groups of the
dendrimer. The functional group to be
ultimately used to link with the targeting
agent can be in its final form, a~ a
prote¢ted functionality, or as a synthetic
prècursor. The form in which this linking
functionality is used depends on its
`35 integrity during the synthetic procedure
~ to be utilized, and the ability of the
35,444-F -32-
'
- -
:
.
1 3 1 6~
-33-
final macromolecule to withstand any
conditions necessary to m~ke this group
available for linking.
5 - For example, the preferred route ~or PEI uses
~\
. F ~ -NO2
Examples of heterofunctional initiators for use
in (l) above, include the following illus~rative
examples:
- H2N ~/~ CH2NH2
.
~ .
:
' 25 : :::
. , ~
: ,
.
i ,' : ~ ~: ~ -
` ` : : ~ ::
. _.
~ 35,~44-F : ~ _33_
,,
.
: .
: :: : : ~. .
, ~
.
:
.
_34~ 1 3 1 6 3 ù l-lr
NHCH2CH2NH2
H2N ~ CH2CH
CNHCH2CH2NH2
Il .
CNHCH2CH2NH2
f ( CH3 ) 3cocNH ~)--CH2CH
CNHCH2CH2NH2
O
:
O CH2NH2
( CH3 ) 3COCNH ~>--CH2CH
CH2NH2
:
:
, :
35~ :
` ~ 35, 444-F:: ~ -34
`.'~' `
i~,,~, ,, .:. : , :
1 3 1 6 3 6 /r
-35- -.
NH2
H2N ~, \~CH2CH
CH2NH2
1~
H2N~ ~ CH2CH2NH2
1r-
.
` 20 f H2NH2
` H2N = CH2CH
` CH~NH2
02N ~ C3 CHZcH2NH2
:
~ ~ `35
: :~
.~ 3 5, 4 4 4 -F _ 3 5 _
:
:
~ '
"
` -36- 1 3 1 63 ')1l,
CH2NH2
02N /\ ~ CH2CH ; and
CH2NH2
1 C f HZCH2NH2
CH2NCH2CH2NH2
r 1~ 02N~</~)> CH2(~
CH2~CH2CH2NH2
\
CH2CH2NH2
~:~ 2C
: 25 : There are several chemistries of particular
i.mportance:
1) Starburst Polyamidoamides ("PAMAM") Chemistry;
2) Starburst Polyethyleneimines ("PEI") Chemistry;
3) Starburst PEI compound wlth a surface of PAMAM;
4) Starburst polyether ("PE") chemistry.
: Modifications of the dendrimer surface
functionalities may provide other useful functional
:35
:
. : :
:: ~ 35,444-F -36-
` ' ^``': ~ ` : .. ~
.
~. ': . ~ .
: ,
.
_37_ l 3
groups such as the following:
-OP03H2, -P03H2, -P03H(-~ po3(-2), -C02(~ S02H,
_S02( 1), -S03H, -S03(-1), -NR1R2~ -R5, OH, -OR1,
-NH2, polyet'ners, perfluorinated alkyl, -CNHR1, -COH,
" "
O O
- ( CH2 ) n/~ -N= CH--~
R3 R4
N
-NHCH2--((^) ~ 1/ \\
R3~ ' -(CH2)n~\ ~,
:-(CH2)n
'25
~ wherein
: . R represents alkyl, aryl or hydrogen;
~` :
:
35 ~ :
`~: ` : : :
: 35,444-F ~ -37-
.:: , : ,
,
.
, , ,
1 3 1 6 3 6 llr
-38-
(CH2)n
f
Rl represents alkyl, aryl, hydrogen, or -N X;
~(CH2)n J
~ (CH2)n ~
R2 represents alkyl, aryl, or -N X
~ (CH2~n J
R3 represents -OH, -SH, -C02H, -S02H, or -S03H;
R4 represents alkyl, aryl, alkoxy, hydroxyl, mercapto,
carboxyl, nitro, hydrogen, bromo, chloro, iodo, or
fluoro;
R5 represents alkyl;
x represents NR, O or S; and
n represents the integer 1, 2 or 3.
The choice of functional group depends upon the
particular end use for which the dendrimer is designed.
The following examples further illustrate~the
invention but are not to be construed as a limitation
on the scope of the invention. The lettered examples
concern the preparation of starting materials; the
numbered examples concern the preparation of products.
Example A: Preparation of 2-Carboxamido-3-(4~-nitro-
phenyl)-propanamide.
p-Nitrobenzyl malonate diethylester (2.4 grams
(g), 8.13 mmole) was dissolved in 35 ml of methanol.
The solution was heated to 50-55C with stirring~and a
stream of anhydrous ammonia was bubbled through the
solution for 64 hours. The solution was cooled and the
:
~ 35,444-F -38-
.
, . . . . .. ..
-39- 1 31 636 1~
white, flocculant product was filtered and
recrystallized from 225 milliliters (ml) of boiling
methanol to afford 1.85 g (7.80 mmole) of bis amide in
96% yield (mp = 235.6C(d)).
The structure was confirmed by MS~1H and 13C NMR
spectroscopy.
Anal~ Calc. for ClOH1104N3
C H N
Theo: 50.63 4.69 17.72
Found: 50.75 4.81 17.94
f Example B: Preparation of 1-Amino-2-(aminomethyl)-3-
5 (4'-nitrophenyl)propane.
2-Carboxamido-3-(4'nitrophenyl)propanamide (2.0
g9 8.43 mmole) was slurried in 35 ml of dry tetrahydro-
furan under a nitrogen atmosphere with stirring. To
~` 20 this mixture was added borane/tetrahydrofuran complex
(106 ml, 106 mmole) via syringe. The reaction mixture
was then heated to reflux for 48 hours during which
time the suspended amide dissolved. The solution was
25~ cooled and the tetrahydrofuran was removed in vacuo
using a rotary evaporator. The crude product and
borane residue was dissolved in 50 ml of ethanol and
this solution was purged with anhydrous hydrogen
~; chloride gas. The solution was refluxed for 1 hour and
30 the solvent removed in vacuo. The crude hydrochloride
salt was dissolved in 15 ml of deionized water and
extracted with two 50 ml portions of methylene
chlorlde. The aqueous layer was cooled in an ice bath
under an argon blanket and 50% sodium hydroxide was
35 slowly added until basic pH-11.7. The basic aqueous
` layer was extracted with four 25 ml portions of
35,444-F -39-
::
: ~ :
~: ,
: . ,
'
~4~~ 1 31 6 36 ,l
methylene chloride and these combined extracts were
evaporated (rotary) to give 1.~5 g of amber colored
oil. This oil was triturated with diethyl ether (50
ml) and filtered under pressure through a short silica
gel (grade 62 Aldrich) column. The column was washed
with 100 ml of ether and the combined filtrates were
vacuum evaporated giving 1.05 g (5.02 mmole) of the
titled diamine as a clear oil (mp = 275-278C(d) bis HCl
salt).
The structure was confirmed by MS, 1H and 13C NM~
spectroscopy.
Anal: Calc. for C1oHl7N3o2cl2
' 15
C H N
Theo: 42.57 6.07 14.89
Found: 43.00 6.14 15.31
Example C: Preparation of 1-Amino-2-(aminomethyl)-3-
(4'-aminophenyl)propane.
Borane/tetrahydrofuran solution (70 ml, 70
mmole) was added under nitrogen via a cannula needle to
a flask containing 4-amino-benzyl malonamide (1.5 g,
7.24 mmole) with stirring. The soIution was brought to
reflux for 40 hours. The colorless solution was cooled
and excess tetrahydrofuran was removed by rotary
evaporation leaving a clear gelat~inous oil. Methanol
(50 ml) was cautiously added to the oil with notable
gas evolution. Dry hydrogen chloride was bubbled
through the suspension to effect dissolution and the
solution was then refluxed for 1 minute. The
methanoliHCl was rotary evaporated and the resulting
`35 hydrochloride salt was carried through the same
dissolution/reflux procedure again. The hydrochloride
35,444-F -40-
.
:
-
,:
.
~ ' ' ''
-41- 1 3163~)~
- salt obtained was dissolved in 10 ml of water and
cooled in an lce bath under ar~on. Concentrated sodium
hydroxide (50%) I.~as added slowly with stirrina to
pH=ll. The aqueous portion was then extracted wlth 2
5 100 ml portions of chloroform which were combined and
filtered throug~l ~ short silica gel plug without
drying. The solvent was removed ir vacuo (rotary)
affording the title compound (0.90 g, 5.02 mmole) in
70% yield (Rf=0.65 - CHC13/MeOH/NX40H conc - 2/2/1).
0 The structure was confirmed by lH and 13C NMR and used
without further puriflcation.
Example D: Preparation of 6-(4-Aminobenzyl)-1,4,8,11-
f tetraaza-5,7-dioxoundecane.
4-Aminobenzyl malonate dimethylester (2.03 g,
8.43 mmole) was dissolved in lO ml of methanol. This
solution was added dropwise to a stirred solution of
freshly distilled ethylene diamine (6.00 g, 103.4
mmole) in 10 ml of methanol under nitrogen over a 2
hour period. The clear solution was stirred for 4 days
and Thin Layer Chromotography (TLC) analysis indicated
total conversion of diester (Rf = 0.91) to the bis
amide (Rf = 0.42 - 20~ conc NH40H/80% ethanol). This
material was strongly ninhydrin positive. The methanol
and excess diamine were removed on a rotary evaporator
and the resulting white solid was vacuum dried (10~1
mm, 50C) overnight to afford crude product (2.45g, 8.36
mmole) in 99% yield. An analytical sample was
recrystalli~ed from chloroform/hexane, MP - 160-161C.
Thè mass spectral, 1H and 13C NMR data were consistent
with the proposed structure.
.
`' . ~
`- 35,444-F -41-
: ~
-42- l 31~)3~,
Example E: Reaction of Mesyl Aziridine with 1-Amino-2-
(aminomethyl)-3-(4-nitrophenyl)propane.
l-Amino-2-(aminomethyl)-3-(4-nitrophenyl)-
propane (400 mg, 1.91 mmole, ~96% pure) was dissolved
in 10.5 ml of absolute ethanol under nitrogen. Mesyl
aziridine (950 mg, 7.85 mmole) was added to the stirred
diamine solution as a solid. The reaction was stirred
at 25C for 14 hours usi~g a magnetic stirrer and during
this period a white, gummy residue formed on the sides
of the flask. The ethanol was decanted and the residue
was triturated with another 15 ml portion of ethanol to
remove any unreacted aziridine. The gummy product was
vacuum dried (101mm, 25C) to afford the tetrakis methyl
f 15 sulfonamide (1.0 g, 1.44 mmole) in 75~ yield (~ = 0.74
- NH40H/ethanol - 20/80). The structure was confirmed
by 1H and 13C nuclear magnetic resonance (NM~)
spectroscopy.
Example F: Preparation of 2-(4-Nitrobenzyl)-1,3-(bis-
N,N-2-aminoethyl)diaminopropane.
The crude methylsulfonamide (650 mg, 0.94
mmole) was dissolved in 5 ml of nitrogen purged,
concentrated sulfuric acid (98%). This solution was
maintained under nitrogen and heated to 143-146C for 27
minutes with vigorous stirring. A slight darkening was
noted and the cooled solution was poured into a stirred
solution of ether (60 ml). The precipitated white salt
cake was filtered an~ immediately dissolved in 10 ml of
deionized water. The pH of the solution was adjusted
to pH-11 with 50% NaOH under argon with cooling. The
resulting solution was mixed with 90 ml of ethanol and
the precipitated inor~anic salts were filtered. The
solvent was removed from the crude amine under reduced
pressure and to the resulting light brown oil was added
35,444-F -42-
;
.
1 31 63~
64693-4102
190 ml of toluene un~er nltrogen. The mlxture was stirred vigor-
ously and water was removed through azeotropic distlllatlon ~Dean-
Stark trap) until the remaining toluene acqulred a light yellow
color (30-40 ml remainlng in pot). The toluene was cooled and
decanted from the dark, lntrac-table residues and salt. Thls solu-
tion was strippe~ o~ solvent ln vacuo and the resulting llght
yellow oil was vacuum dried (0.2 mm, 35C) overnight affordlng 210
mg of the product (60%) which was characterized by MS, lH and 13C
NMR.
Exa~ ~ Preparatlon of a starburst polymer
~containing an aniline derivative) of one half
generatlon represented by the followlng scheme:
~1
11 H2C=CHCOCH3
H2N ~ C ~ CH2CH(CNHCH2CH2NH2)2
: CH3QE~
Compound #l
O o
H2M ~ H2CH(CNHCH2CH2N/CH2CH2CoCH3)2)2
Compound #2
Methyl acrylate (2.09 g, 24 mmole) was dissolved in
methanol ~15 ml). The compound 6-(4-amlnobenzyl)-1,4,8,11--tetra-
aza-5,7-dloxoundecane ~1.1 g, 3.8 mmole) (l.e., Compound Kl, pre-
paratlon descrlbed ln Example D) was dlssolved in methanol ~10 ml)
and was added slowly over 2 hours wlth rl~orous stlrrlng to the
methyl acrylate solutlon. The
,:
: 43
`: ,i`'`, :
,
:
:
_4L~_ 13163~
reaction mixture was stirred for 48 hours at ambient
temperatures. The solvent was removed on the rotary
evaporator maintaining the temperature below 40C. The
ester (Compound #2) was obtained as a yellow oil (2.6
g). No carboxyethylation of the aniline function was
observed.
~i Example H: Preparation of a starburstl)polymer
(containing an aniline moiety) of one generation;
represented by the ~ollowing scheme:
H2NCH2CH2NH2
Compound #2 + >
CH30H
O O
H2N - ~ CH2CH(CNHCH2CH2N(CH2CH2CNHCH2CH2NH2)2)2
2 Compound #3
The ester (Compound #2) (2.6 g, 3.7 mmole) was
dissolved in methanol (100 ml). this was carefully
added to a vigorously stirring solution of ethylene
diamine (250 g, 4.18 mole) and methanol (100 ml) at
such a rate that the temperature did not rise above
40C. After complete addition the reaction mixture was
stirred ~or 28 hours at 35-40C (heating mantle). After
28 hours no ester groups were detectable by infrared
spectroscopy. The solvent was removed on the rotary
evaporator at 60C. The excess eth~lene diamine was
removed using a ternary azeotrope of toluene~methanol-
ethylene diamine. Finally all remaining toluene was
`35 azeotroped;with~methanol. Removal of all the methanol
35,444-F -44-
`,
. . ; :
~ .:
.,
`-` 1 31 63~
-45-
yielded 3.01 g of the product (Compound #3) as an
orange glassy solid.
Example I: Preparation of a starburst polymer
(containing an aniline moiety) of one and one half
generations represented by the ~ollowing scheme:
O
Compound #3 + H2C=CHCOCH3
>
CH30H
f o o o
H2N--~ CH2CH ( CNE~CH2CH2N ( CH2CH2CNHCE~2CH2N ( CH2CH2COCH3 ) 2 ) 2 ) 2
Compound #4
The amine (Compound #3) (:2.7 g, 3.6 mmole) was
dissolved in methanol (7 ml) and was added slowly over
- one hour to a stirred solution of methyl acrylate (3.8
g, 44 mmole) in methanol (15 ml) at ambient
temperatures.:: A slight warming of the solution was
`` observed during the addition. The solution~ was allowed
to st~ir at ambient temperatures for 16 hours. Th~e ~:
solvent was removed on the rotary evaporator at 40C. :
After:removal of all the solvent and excess methyl
acrylate the e~ster (Compound #4) was obtained in 4.7 g
~:yield as an orange oil.
':
': :
` ~ 35,444~-F~ ~ -45-
, ~ , . :
:
.
` `: : : ` :
:
. . . .
- : , , .
: ,
1 3 1 63fj '',
-46~
Example J: Preparation o~ a starburst polymer
(containing an aniline moiety) of one half generation
represented by the ~ollowing scheme:
0
/--\ E~2C-CHCOCH3
H2 ~ CH2CH(CH2NH2)2 +CH30H
Compound #5
H2l ~ ~ ~ CH2CH(CH2N(CH2CH2COCH3)2)2
Compound #6
The triamine (Compound #5, the preparation of
this compound is shown in Example C) (0.42 g, 2.3
mmole~ was dissolved in methanol (10 ml) and was added
dropwise over one hour to methyl acrylate (1.98 g, 23
mmole) in methanol (lO ml). The~mixture was allowed to
stir at ambient temperatures for 48 hours. The solvent
was removed on the rotary evaporator, maintaining the
temperature at no higher than 40C. The exces$ methyl
acrylate was removed by repeated azeotroping with
methanol. The ester (Compound #6) was~isolated as an
orange oil (1.24 g)~ :
::
-
'
35,444-F ~ -46-
. . ~
- : : ,., ,, ,,. :: . ,
. :: .
.. : , . . . . . . .
- . . .' . ' . ~
-47- l 31 63~
Example K: Preparation of a starburst~polymer
(containing an aniline moiety) o~ one generation;
represented by the following scheme:
H2NCH2CH2NH2
Compound #6 + >
CH30H
1 ' O
H2N ~ CH2CH(CH2N(CH2CH2CNHCH2CH2NH2)2)2
Compound #7
f
The ester (Compound #6) (1.24 g, 2.3 mmole) was
dissolved in methanol (50 ml) and was added dropwise
over two hours to ethylenediamine (73.4 g, 1.22 mole)
in methanol (lO0 ml). A small exotherm was noted,
vigorous stirring was maintained. The solution was
left to stir at ambient temperatures for 72 hours. The
solvent was removed on the rotary evaporator at 60C.
The excess ethylene diamine was removed using a ternary
azeotrope o~ toluene-methanol-ethylenediamine. Finally
all remaining toluene was removed with methanol and
then pumping down with a vaouum~pump for 48 hours gave
the amine (Compound #7) (1.86 g) as a yellow/orange
oil.
'
35,444-F -47-
: ~
- : .
: ' ~. . '
' . ' ,
-48- 1 3 1 63~
Example L: Preparation o~ a starburst)polymer
(containing an aniline moiety) o~ one and one half
generations; represent by the following scheme:
,.
H2C=CHCOCH3
Compound #7 -~ >
CH30H
O O
,. ..
H2N ~ CH2CH(CH2N(CH2cH2CNHcH2CH2N(CH2CH2cocH3)2)2)2
'~ Compound #8
The amine (Compound #7) (1.45 g, trace of
methanol remained) was dissolved in methanol (100 ml)
and was addèd slowly over 1~ hours to a stirred
solution~of methyl acrylate (5.80 g) in methanol (20
; mI). The solution was allowed to~stir for 24 hours at
room temperature. Removal of the solvent followed by
repeated azeotroping with methanol enabled the removal
of all the excess methyl acrylate. After pumping down
on a vacuum pump for 48 hours the ester (Compound #8)
was isolated as an orange oil (2.50 g, 1.8 mmole).
~` :
Example M: Hydrolysis of 4.5 generation dendrimer and
3o preparation of calcium salt.
4.5 Generation PAMAM (ester terminated,
, initiated off NH3) (2.11 g, 10.92 meq) was dissolved in
, 25 ml oP methanol and to it was added 10% NaOH ~(4.37
; 35 ml, 10.92 meq) (pH ~ 11.5-12). After 24 hours at room
temperature, the pH was about 9.5. After an add~itional
:
:
:~ :
.
::
~` 35,444-F -48-
:
. ~ . . .
.
. , .; ; , . : ~ . : :
.: :
.. , ~ .
:. - . : . :
. :~;- ~ , ; .
L~g 1 3 1 63 ~
20 hours, the solution was rotovaped, 50 ml of toluene
added, and rotovaped again.
The resulting oil was dissolved in 25 ml of
methanol and precipitated as a white gum upon addition
of 75 ml of diethyl ether. The liquid was decanted and
the gum was rotovaped to give a very fine off-white
powder which upon drying gives 2.16 g of product (98%
yield). No ester groups were found upon NMR and
infrared analysis.
The sodium salt of 4.5 Generation PAMAM (ester
terminated, initiated from NH3) was replaced by the
calcium salt via dialysis. The sodium salt (1.03 g)
was dissolved in 100 ml of water and passed through
hollow fiber dialysis tubing (cut off - 5000) at 3
ml/minute. The exterior of the tubing was bathed in 5
CaC12 solution. This procedure was then repeated.
20The resulting solution was again dialyzed, this
time against water, then repeated two additional times.
Evaporation provided 0.6 g of wet solid, which
was taken up in methanol (not totally soluble) and is
dried to give 0.45 g of off-white crystals.
C36gHsg20l41N9lca24 Calc. - 10.10~ Ca+~
M Wt. - 9526.3 Calc. = C-4432.1, H-601.8,~ 0-2255.9,
30N-1274.6, Ca-961.9)
Theo: C-46.5, H-6.32, N-13.38, Ca-10.10
~~ Found: C-47.34, H-7.00, N-13.55, Ca-8.83
35,444-F ~ -49-
' '. ` ~
, : ' ` ;
~50- 1 31 63 G'l~
Example N: Preparation o~ dendrimers with terminal
carboxylate groups.
Half-~eneration starburst polyamidoamines were
hydrolyzed to convert their terminal methyl ester
groups to carboxylates. Thls generated spheroidal
molecules with negative charges dispersed on the
periphery. The dendrimers hydrolyzed ranged from 0.5
generation (three carboxylates) to 6.5 generation (192
carboxylates).
The products could be generated as Na+, K+, Cs~
or Rb~ salts.
; 15 Example 0: N-t-butoxycarbonyl-4-aminobenzyl malonate
dimethylester
4-Aminobenzyl malonate dimethylester (11.62 g,
49 mmol) was dissolved in 50 ml of t-butanol:water
(60:40 with stirring. Di-t-butoxydicarbonate (19.79 g,
90 mmol) was added and the reaction mixture stirred
overnight. The butanol was removed on the rotary
evaporator, resulting in a yellow suspension of the
pro~duct in water. Extraction into methylene chloride,
drying (MgS04) and evaporation gave a yellow oil (21.05
g, ¢ontaminated by di-t-butoxydicarbonate).
RecrystalIization from 2-propanol:water (75:25) yield
pale~yellow crystals (11.1 g, 33 mmol, 67~). The
structure was confirmed by 13C NMR and purity checked
by hplc analysis (spherisorb ODS-1, 0.05M H3P04 pH 3:
CH3CN 55:45). The material was used without further
purification.
35,444-F -50-
~ '
.
~. ' . , .
,: . . .
.
-51- l 31 63(,il
Example P: N~t-butoxycarbonyl-6-(4-aminobenzyl)-
1,4,8,11-tetraaza-5,7-dioxoundecane
N-t-butoxycarbonyl-4-aminobenzyl malonate
dimethylester (8.82 g 26 mmol), prepared in Example 0,
was dissolved in 50 ml of methanol, This solution was
added dropwise (2 hours) to à solution of freshly
distilled ethylenediamine (188 g 3.13 mole) and 20 ml
of methanol, under a nitrogen atmosphere. The solution
was allowed to stir for 24 hours. The ethylene
diamine/methanol solution was removed on the rotary
evaporator. The product was dissolved in methanol and
toluene added. Solvent removal on the rotary
evaporator gave the crude product as a white solid
r~ l5 (10.70 g contaminated with ethylenediamine). The
sample was divided into two samples for purification.
Azeotropic removal of ethylenediamine with toluene,
using a soxhlet extractor with sulphonated ion exchange
beads in the thimble to trap the ethylenediamine,
resulted in partial decomposition of the product,
giving a brown oil. The remaining product was isolated
as a white solid from the toluene on cooling (2.3 g
approximately 50 percent). Analysis of a 10 percent
? soluti,on in methanol by gas chromatography (Column,
~25 Tenax 60/80) showed no ethylenediamine detectable in
the sample (<O.l percent). The second fraction was
dissolved in methanol to give a 10 percent solution (by
weight) and purified from the ethylened~iamine by
reverse osmosis, using meth~nol as~the solvent. (The
membrane used was a Film,tec FT-30 , in an Ami,,con TC1R
thin channel separator, the ethylenediamine crossing
the membrane.) The product was isolated as a white
solid (2.7 g), in which no detectable amounts of
~; 35 ethylenediamine could be found by gas chromatography.
The`13C NMR data and HLPC analysis (Spherisorb ODS-1,
;~rt~Q~e /`7cl rk
35,444-F ~ ~ -51-
:
:
.
-52- 13163~
0.05M H3P04 pH 3:CH3CN 55:45) were consistent with the
proposed structure. The product was used with no
further purification.
Example Q: Preparation of a'starburst1dendrimer of one
half generation from N-t-butoxycarbonyl-6-(4-
aminobenzyl)-1,4,8,11-tetraaza-5,7-dioxoundecane
N-t-butoxycarbonyl-6-(4-aminobenzyl)-1,4,8,11-
tetraaza-5,7-dioxoundecane (5.0 g 13 mmol), prepared in
Example P, was dissolved in 100 ml of methanol. Methyl
acrylate (6.12 g, 68 mmol) was added and the solution
stirred at ambient temperatures for 72 hours. The
reaction was monitored by HPLC (Spherisorb ODSl,
f 15 Acetonitrile: 0.04M Ammonium acetate 40:60) to optimize
conversion to the desired product. The solution was
concentrated to 30 percent solids, and methyl acrylate
(3.0 g 32 mmol) was added. The reaction mixture was
stirred at ambient temperatures until no partially
alkylated products were detectable by HPLC (24 hours).
Removal of the solvent at 30C by rotary evaporation,
and pumping down at l mm Hg for 24 hours gave the
product as yellow viscous oil, yield 7.81 g. The 13C
NMR data was consistent with the proposed structure.
The product was used without further purification.
Example R: Preparation of a starburst dendrimer of one
full generation from N-t-butoxycarbonyl-6-(4-
aminobenzyl)-1,4,8,11-tetraaza-5,7-dioxoundecane
The half generation product (Example Q) (7.70
g, 10.45 mmol) was dissolved in 75 ml of methanol and
added dropwise over 2 hours to a stirred solution of
ethylenediamine (400 ml, 7.41 mol) and methanol (50
ml). The reaction mixture was stirred at ambient
; temperatures for 48 hours.~ The ethylenediamine and
C /~ rk , ' !
35,444-F ~ -52-
:' :
.
:
- , :
.. . . .
.
.: . ,, ~ ; . .
'. ' ,, ~ ~ ;
,.
:
-53_ 1 3 1 6 ~ ~ ~
methanol were removed by rotary evaporation to give a
yellow oil (11.8 g contaminated with ethylene diamine).
The product was dissolved in 90 ml of methanol, and
purified from the ethylenediamine by reverse osmosis
(Filmtec FT-30 membrane and Amicon TC1R thin channel
sèparator, methanol as solvent). After 48 hours, no
ethylenediamine could be detected by gas chromatography
! (Column, Tenax 60/80). Removal of the solvent on the
rotary evaporator, followed by pumping down on a vacuum
line for 24 hours gave the product as a yellow glassy
solid (6.72 g). Analysis by HPLC, PLRP-S column,
acetonitrile:O.015M NaOH, 10-20 percent gradient in 20
min.) and 13C NMR analysis was consistent with the
proposed structure.
F Example S: Preparation of a starburst'~polymer of one
and one half generation from N-t-butoxycarbonyl-6-(4-
aminobenzyl)-1,4,8,11-tetraaza~5,7-dioxoundecane
The one generation product (Example R) (2.14 g,
25 mmol) was dissolved in 12.5 ml of methanol, and
" methyl acrylate (3.5 g, 39 mmol) in 5 ml of methanol
was added. The solution was stirred at ambient
temperatures for 48 hours, monitoring the progress of
the reaction by HPLC (Spherisorb ODS-l, acetonitrile:
0.04M ammonium acetate, 60:40). A second aliquot of
methyl acrylate was added (3.5 g 39 mmol) and the
reaction mixture stirred at ambient temperatures for a
further 72 hours. Removal of the solvent on the rotary
evaporator gave the product as a yellow oil (3.9 g)
after pumping down overnight with a vacuum pump. The
product was used with no further purification.
~ik Tro~ a r k
35,444-F -53-
,
;~ . -
~54~ 1 31 6 3 Gi~t
Example T: Preparation of a starburst)pol~mer of two
~ full generations from N-t-butoxycarbonyl-6~
aminobenzyl)-1,4,8,11-tetraaza-5,7-dioxoundecane
The one and one half generation product
(Example S) (3.9 g, 2.5 mmol) was dissolved in 50 ml of
methanol, and was added dropwise over 2 hours to a
stirred solution of ethylenediamine (600 g, 10 mol) and
methanol (50 ml). The solution was stirred at ambient
temperatures under an atmosphere of nitrogen for 96
hours. The ethylenediamine/methanol was removed on the
rotary evaporator to give a yellow glassy solid (4.4 g
contaminated with ethylenediamine). A 10 percent
solution of the product was made in methanol, and
purified from the ethylenediamine by reverse osmosis
(membrane used as a Filmtec FT-30, in an Amicon TC1R
thin channel separator) until no ethylenediamine could
be detected by gas chromatography (Column, Tenax 60/oO.
Removal of the solvent gave the product as a yellow
glassy solid (3.52 g). The 13C NMR data and HPLC
analysis (PLRP-S column, acetonitrile:O.015 M ~aOH, 10
to 20 percent gradient in 20 minutes, were consistent
with the proposed structure.
Example U: Reaction of the two generation starburst
with Bromoacetic Acid to give a methylene carboxylate
terminated"starburst'~dendrimer
The second generation product (Example T) ~0.22
g, 0.13 mmol) was dissolved in 15 ml of deionized water
3 and the temperature equilibrated at 40.5C. Bromoacetic
acid (0.48 g, 3.5 mmol) and lithium hydroxide (0.13 g,
3.3 mmol) were dissolved in 5 ml of deionized water,
and added to the reaction mixture. The reaction pH was
carefully maintained at 9, with the use of a pH stat
(titrating with 0.1N NaOH), at 40.5C overnight.
35,444-F -54- _
.: , .
:
-55- 1 3 1 63~il
Monitoring by reverse phase ~IPLC, (Spherisorb O~S-1
column, eluent 0.25 M H3P04 pH 3 [NaOH]; acetonitrile
85:15) confirmed the synthesis of predominantly a
single component.
Example V: Preparation of Isothiocyanato functionalized
second generation methylene-carbox~flate terminated
'~starburst'1dendrimer
Five ml of a 2.8 mM solution of the second
generation methylenecarboxylate terminated~starburst~
dendrimer (Example U) was diluted with 20 ml water and
the pH adjusted to 005 with concentrated hydrochloric
acid. After one hour at room temperature the mixture
f 1 was analyzed by HPLC to verify the removal of the
A butoxycarbonyl group and then treated with 50 percent
sodium hydroxide to ~ the pH to 7. A pH stat
(tltrating with 0.1 N NaOH) was used to maintain the pH
at 7 and 225 ~l thiophosgene was added. After 15
minutes at room temperature the pH of the mixture was
adjusted to 5 with 1N HCl. The mixture washed with
chloroform (20 ml x 2) then concentrated on a rotary
evaporator at reduced pressure. The residue recovered
`;~ 0.91 g is a mixture of the isothiocyanate and salts.
Example W: Preparation of second generation starburst~
polyethyleneimine-methane sulfonamide
To a solution of 125 g N-methanesulfonyl-
azlridine in 50 ml ethanol was ad~ded 25.0 g tris(2-
aminoethyl)amine. The solution was stirred at room
temperature for 4 days. Water was added to the
reaction mixture as needed to maintain the homogeneity
of the~solution. The solvent was removed by
distillation in vacuo to give the 2nd generation
~: :
35,444-F -55-
, ' : ~. ` ~ ` . , : '
: ' ` -
_5~_ 1 31 63~J'~t
starburst PEI-methane sul~onamide as a yellow glass
(161 g).
Example X: Cleavage of methane sulfonamides to form
second generation"starburst'lpolyethyleneimine
A solution of 5.0 g of second generation
"starburst')PEI-methane sulfonamide, from Example W in 20
ml of 38 percent HCL was sealed in a glass ampoule.
0 The ampoule was heated at 160C for 16 hours, then
cooled in an ice bath and opened. The solvent was
removed by distillation in vacuo and the residue
dissolved in water. After adjusting the pH of the
solution to greater than or equal to 10 with 50 percent
NaOH, the solvent was removed by distillation in vacuo.
Toluene (150 ml) was added to the residue and the
mixture heated at reflux under a Dean-Stark trap until
no more water could be removed. The solution was
filtered to remove salts and the filtrate concentrated
in vacuo to give 1.9 g second generation starburst'1PEI
as a yellow oil.
Example Y: Preparation of third generation starburst
polyethyleneimine-methane sulfonamide
To a solution of 10.1 g second generation
"starburst)PEI, from Example X, in 100 ml ethanol was
added 36.6 g N-methanesulfonylaziridine. The solution
was stirred at room temperature for 1 week. Water was
3 added as needed to maintain the homogeneity of the
solution. The solvent was removed by distillation in
vacuo to give third generation starburst PEI-methane
sulfonamide as a yellow glass (45.3 g).
35,444-F -56-
, '
: ' '
1 31 h3f~
-57-
Example Z: Cleavage o~ methane sulfonamides to form 3rd
gen starburst polyethyleneimine
The methane sulfonamide groups of third
generation starburst~PEI-methane sulfonamide (5.0 g),
from Example Y, were removed by the same procedure as
described for the second generation material in Example
X to give 2.3 g third generation starburstPEI as a
yellow oil.
Example AA: Preparation of a methylenecarboxylate
terminated second generation"starburstnpolyamidoamine
(initiated from ammonia)
The second generation starburst"polyamldoamine
(2.71 g, 2.6 mmol) and bromoacetic acid (4.39 g, 31.6
mmol) were dissolved in 30 ml of deionized water and
the pH adjusted to 907 with 5N NaOH using a pH stat.
The reaction was maintained at this pH for a half hour,
and the temperature was slowly raised to 60C and was
maintained at 60C for three hours at constant pH~ The
pH was raised to 10.3, and the reaction mixture
remained under control of the pH stat at ambient
temperatures overnight. The reaction mixture was
refluxed for a further four hours prior to work up.
Removal of the solvent, and azeotroping the final
traces of water with methanol gave the product as a
pale yellow powder (8.7 g, contaminated with sodium
bromide). The 13C NMR spectrum was consistent with the
propose structure (with some contamination due to a
small amount of defected material as a result of some
monoalkylation).
~ :
35,444-F ~ -57-
:; :
:
.
. -.
~ ~ -
- -, ,
`'`' 1 3 1 6 3 ,~
Example BB: Preparation of a methylenecarboxylate
terminated second generation"starburst''
polyethyleneimine (initiated from ammonia)
: The second generation 'starburst'~
polyethyleneimine (2.73 g, 6.7 mmol), from Example X,
and bromoacetic acid (11.29 g 81 mmol) were dissolved
in 30 ml of deionized water. The pH was slowly raised
to pH 9.5 maintaining the temperature below 30C. The
temperature was raised slowly to 55C, and the reaction
pH maintained at 9.5 for 6 hours with the aid of a pH
stat (titrating with 5N NaOH). The pH was raised to
10.2, and maintained at that pH overnight. Removal of
the solvent on the rotary evaporator, and azeotroping
f the final traces of water using methanol, gave the
15 product as a yellow powder (17.9 g, contaminated with
sodium bromide). The 13C NMR spectrum was consistent
with the proposed structure (with some contamination
due to a small amount of defected material as a result
20 of some monoalkylation).
s, s
Example CC: Preparation of a 3.5,4.5,4.-4 and 6.5
generation'starburst'~PAMAM:
~n ~th Q n~ I, 'c
To a 10 weight percent/solution of 2.46 g 3
generation PAMAM starburst was added 2.32 g of methyl
acrylate. This mixture was allowed to sit at roo~
temperature of 64 hours. After solvent and excess
methyl acrylate removal, 4.82 g of product was
3 recovered (105 percent of theoretical~.
Preparation of higher 1i2 generation starburst~)
` PAMAM's:
; ~ 35 ~ Generations 4.5, 5.5 and 6.5 were prepared as
~ described above with no significant differences in
., :
. '~ :
~ 35,44~4-F -58-
:`:;: : :
- - .
,
. ~ : . - . ; . ,, - ,
., ~
: ' '
_59_ 131 63G~l
~ /e
reactant concentrations, reactant/ratios or reaction
times.
Example DD: Preparation of 4, 5 and 6 generation
starburst PAMAM:
To 2000 g of predistilled ethylenediamine was
added 5.4 g of 4.5 generation starburst'~PAMAM as a 15
weight percent solution in methanol. This was allowed
to sit at room temperature for 48 hours. The methanol
and most of the excess ethylenediarnine were removed by
rotary evaporation under water aspirator vacuum at
temperature less than 60C. The total weight of product
recovered was 8.o7g. Gas chromatography indicated that
'' 15 the product still contained 34 weight percent ethylene-
diamine at this point. A 5.9~ g portion of this
product was dissolved in 100 ml methanol and
ultrafiltered to remove the residual ethylenediamine.
The filtration was run using an Amicon TClR thin
channel recirculating separator equipped with an Amicon
YM2 membrane. An in-line pressure r,elief valve was
used to maintain 55 psig (380 kPa) pressure across the
membrane. The 100 ml was first concentrated to 15 ml
by forcing solvent flow exclusively through the
membrane. After this initial concentration, the flow,
was converted to a constant volume retentate recycle
mode for 18 hours. After this time, 60 mI of methanol
was passed over the membrane to recover product still
in the module and associated tubing. The product was
stripped o~ solvent and 2.53 g of 5 generation
starburst~PAMAM was recovered. Analysis by gas
chromatography indicated 0.3 percent residual ethylene-
diamine remained in the product.
.
35,44~-F -59-
, :
, : : '
,: . .
- \
-60- 1 31 636 J,
Preparation of generation 4 and 6 proceeded as
above with the only difference being the weight ratio
of ethylenediamine to starting material. To prepare
4th generation this ratio was 200:1 and for 6th
~eneration this ratio was 730:1.
Example 1: Preparation of a product containing more
than one rhodium atom per"starburst~polymer.
2.5 Generation PAMAM (ester terminated,
initiated from NH3) (0.18 g, 0.087 mmole) and
RhC13-3H20 (0.09 g, 0.3 mmole) were mixed in
dimethylformamide (DMF) (15 ml) and heated for 4 hours
at 70C. The solution turned crimson and most of the
rhodium was taken up. The unreacted rhodium was
removed by filtration and the solvent removed on the
rotary evaporator. The oil formed was chloroform
soluble This was washed with water and dried (MgS04)
before removal of solvent to yield a red oil (0.18 g).
The NMR spectrum was recorded in CDC13 only minor
differences were noted between the chelated and
unchelated ~tarburst" Dilution of some of this CDC13
solution with ethanol followed by NaBH4 addition
resulted in rhodium precipitation. RhC13-3H20 is
insoluble in chloroform and in chloroform starburst
solution thus confirming chelatlon.
Example 2: Preparation of a product containing Pd
chelated to"starburst'~polymer.
3.5 Generation PAMAM (ester terminated,
i~itiated from NH3) (1.1 g, 0.24 mmole) was dissolved
with stirring into acetonitrile (50 ml). Palladium
chloride (0.24 g, 1.4 mmole) was added and the solution
was heated at 70-75C (water bath) overnight. The PdC12
was taken up into the starburstl) After removal of~the
,
35,444-F ~ -60-
.
J
. ~ :
:. : ~ . . . . ~ ..
-61- 1 31 63~ r
solvent, the ~MR in CDC13 confirmed that chelation had
occurred. Dilution of the CDC13 solution with ethanol
and addition o~ NaBH4 resulted in precipitation of the
palladium. The che]ated product (1~23 g) was isolated
as a brown oil.
Example 3 ~ Attachment of herbicidal molecules (2,4-D)
to the surface of"starburst"dendrimers.
Third generation PAMAM (initiator core-NH3)
(2.0 g, 0.8 mmole) was dissolved in H20 (10 ml) and
combined with toluene (20 ml). The two-phase system
was then stirred and cooled with an ice bath at which
time the acid chloride of 2,4-D [2,4-dichlorophenoxy-
f 15 acetic acid] (2.4 g, 12 equiv) dissolved in toluene (10
ml) was added dropwise over 30 minutes. When the
addition was nearly complete, NaOH (0.5 g, 12.5 mmole,
50% w/w solution) was added and the solution stirred
for an additional two hours. The reaction mixture was
then evaporated to dryness and the re'sulting solid
residue repeatedly taken up in CHC13/MeOH ( 1:1 ) and
filtered. The tan solid was not totally soluble in
CHC13 and appeared to be insoluble in water; however,
the addition of acetone facilitated dissolution. The
tan solid was stirred in CHC13 for 24 hours and the
solution filtered (a sticky tan solid was obtained3.
After drying over MgS04, the filtrate was ooncentrated
to give a viscous orange oil which solidified on
standing. The 13C NMR partial amidation at the surface
by 2,4-D is consistent with the association of the 2,4-
D to starburstl)dendrimer.
'
`
35,41~4-F -61- -
` ` , :
-62- 13167)~'~
Example 4: Incorporation of 2,4-dichlorophenoxyacetic
acid (2,4-D) into"starburst')dendrimers.
A widely accepted method for ascertaining
whether a "probe molecule" is included in the interior
of a micelle is to compare its carhon-13-spin lattice
relaxation times (T1) in a non-micellized versus
micellized mediumO A substantial decrease in Tl for
the micellized medium is indicative of "probe molecule"
inclusion in the micelle. Since starburst'ldendrimers
are "covalently fixed" analogs of micelles, this Tl
relaxation time technique was used to ascertain the
degree/extent to which various herbicide type molecules
were associated with'starburst~)polyamidoamines. In the
f 15 following examples, Tl values for 2,4-dichlorophenoxy-
acetic acid (I) (2,4-D) were determined in solvent
(CDCl3) and then compared to T1 values in CDC13 at
various [I:dendrimer] molar ratios.
Inclusion of 2,4-D into various starburst polyamido-
amine dendrimers as a function of generation.
Various half generation~(ester terminated,
initiated from~NH3)'starburst'polyamidoamine dendrimers
(Generation (Gen) = 0.5, 1.5, 2.5, 3.5, 4.5 and 5~.5)
were combined with 2,4-dichlorophenoxyacetic acid (I)
in CDC13 to give an acid:tertiary amine ratio of 1:3.5
and molar ratios of acid:dendrime~r of 1:86 as shown in
Table III. The;relaxation times~(Tl) obtained for the
various carbon atoms in 2,4-dichlorophenoxyacetic acid
and a generation - 3.5 starburst"PAMAM dendrimers are
shown in Table IV, both for 1:1 acid/amine ratios;and
for saturated solutions of 2,4-D.
:
,
: , : ~
35,444-F ~ -62-
~ -
.. . . .. .
,, , .:
. ~ . ,,: . , :
, . , : ~ ; . .
, , . .~ : . '
-63- 1 3 1 6 3 ~) r
Table III
(A) (B) (C) Molar Ratio
Gen Acid/Amine Acid/Amine Acid/Total burst~
0.5 1 -- 1 1
1.5 1 1.33 0.57 6
1t 2.5 1 (3.5)* 1.11 (3.8~k 0.53 (1.8)* 9 (34~
: 3.5 1 (3.0)* 1.05 (3.2)* 0.51 (1.6)* 20 (67)*
4.5 1 1.02 0.51 42
5.5 1 1.01 0.50 86
c* represents examples of 2,4-D inclusion into the i~terior a the
dendrimer in amounts greater than stoichiometric.
Tl's for 2 4-D/G - 3.5 PAMAM Starburst
Inclusion complex: Concentration Effects
(A) (B3
Carbon 1:1 Acid/~nine Saturated with 2~4-D
Tl 13C** Tl l3C*
1 3~19+ol2 ~152.73)3O08+.09 (152.30)
~; 3 0.34~.01 (128.64)0.29+.01 (129.623
0.38_.01 (127.41)0.32_.0l (127 D 34)
2 3.28+.08 (125.79)2.72+.68 (125.99)
4 4.58+.16 (123.2733.95+.07 (123.16)
6 0.31~.01 (114.66)0.28~.01 (114~4~)
CH2 0.16+.01 (67.29j0.146i.003 (66.79)
C-O 1.24+.07 (170.12) - -
:
** represents 13C chemical shifts referenced to chloroform
:at 76.9 ppm..
35,444-F -63-
: .~ ~ , . . .
.
. . ~ .
: . : . ~ ,
'
`\
-64- 1 3 1 6 ~
These data show that larger than stoichiometric amounts
of 2,4-dichlorophenoxyacetic acid (i.e., [(I):Gen=3.5
dendrimer)] = 67 can be used without increasing ~he T1
in any case in the saturated state (see Columns (A) and
(B) in Table IV). In fact, the relaxation times T1
(Column (B) are decreased slightly, thus indicating
that larger than stoichiometric amounts of 2,4-
dichlorophenoxyacetic acid can be included into the
interior of the dendrimer. For example, a molar ratio
of
[(I):Gen=2.5 dendrimer]= 34 whereas [(I):Gen=3.5
dendrimer]= 67,
(see Column D in Table III).
Figure 3 is a plot of T1 values for carbons-3,
5 and 6 in 2,4-dichlorophenoxyacetic acid as a function
of dendrimer generation (i.e., 0.5 ~ 5.5). A minimum
in T1 is reached in all cases of generation 2.5 ~ 5.5,
thus indicating incorporation in that dendrimer
: generation range is occurrin~ Figure 3 also includes
T1 values for 2,4-D in the presence of triethylamine
~; [N(Et)3] and N(Et)3 + N-methylacetamide. It can be
seen that these values are much larger than for
dendrimers G - 1.5 ~ 5.5, thus further supporting
molecular incorporation into the dendrimer molecule.
Example 5: Demonstration of multiple chelation of
yttrium by a methylene carboxylate terminated second
F generation"starburstpolyethyleneimine by trans
chelation from yttrium acetate
The starburst polyethyleneimine methylene
oarboxylate terminated material (0.46 g 52. 5 percent
active, remainder sodium bromide, 0.18 mmal active
"s~tarburst~)dendrimer), from Example BB, was dissolved in
35,444-F -64-
. ~ .
,
.
;
65- 131 63G l
4.5 ml of deuterium oxide. The resuLtant pH was 11.5-
12. A solution of yttrium acetate was prepared by
dissolving yttrium chloride (0.15 g, 0.5 mmol) and
sodium acetate (0.41 g, 0.5 mmol) in 1.5 ml of
deuterium oxide (2.9 moles of yttrium per mole of
dendrimer). Aliquots of 0.5 ml of the yttrium acetate
solution were added to the dendrimer solution and the
13C NMR spectra recorded at 75. 5 MHz .
13
The C NMR spectrum of yttrium acetate shows
two resonances, 184.7 ppm for the carboxyl carbon and
23.7 ppm for the methyl carbon, compared with 182.1 and
24.1 ppm for sodium acetate, and 177.7 and 20.7 ppm for
acetic acid (Sadtler 13C NMR Standard Spectra).
Monitoring the positions of these bands indicates
degree of chelation with the starburstJ'dendrimer. The
most informative signal for the starburs~'dendrimer
which is indicative of chelation is the a-CH2 (of the
methylene carboxylate group involved in chelation),
which appears at 58.4 ppm in the unchelated dendrimer,
and 63.8 ppm in the chelated dendrimer. Upon chelation
with yttrium, the spin lattice relaxation times of the
time a-CH2 shortens as expected from 0.24 + O.Ols to
0.14 + 0.01s, indicative of chelation.
Following the addition of 0.5 ml of the yttrium
acetate solution to the 'starburst dendrimer, all the
3 yttrium appeared to be chelated by the dendrimer,
confirmed by the signals for the acetate being that of
sodium acetate. The same observation was noted for the
addition of a second 0.5 ml aliquot of the yttrium
acetate solution. Upon addition of the third aliquot
of yttrium acetate, not all of the yttrium was observed
-
~ ~ 35,444-F -65-
.
-
'
~66- 1 3163r~
to be taken up as the starburst chelate, the acetate
carboxyl resonance was observed to shi~t to 183.8 ppm
indicating that some o~ the yttrium was associated with
the acetate. The integrated area of the chelated -CH2
groups on the dendrimer increased, indicatin~ that some
of the third mole equivalent of yttrium added was
indeed chelated with the dendrimer. These results
`indicate that the dendrimer can chelate from 2-3
yttrium ions per dendrimer molecule.
Example 6: Demonstration of Multiple Chelation of
Yttrium by a methylene carboxylate terminated second
generationl~starburst~lpolyamidoamine by trans chelation
from yttrium acetate.
The same experimental methods were used for
this study as were used for Example 5. The"starburst~)
polyamidoamine methylene-carboxylate terminated
material (0.40g 62.5% active, remainder sodium bromide,
0.12 mmol.) was dissolved in 4-5 ml of deuterium oxide~
The resultan~ pH was 11.5-12,`which was lowered to 9.4
with 6N HCl prior to the experiment. A solution of
yttrium acetate was prepared by dissolving yttrium
- chloride (0.1125g, .37 mmol.) and sodium acetate
(0.0915g, 1.1 mmol.) in 1.5 ml of deuterium oxide, thus
every 0.5 ml of solution contains one mole equivalent
of metal.
The first two mole equivalents of yttrium
acetate added were fully chelated by the starburst)~
` polyamidoamine. On addition of a third mole equivalent
of yttrium, precipitation of the product occurred and
as such no NMR data could be obtained. The signals
which gave the most information about chelation by the
~35 starburst'dendrimer were those of the two carbons
`~adjacent to the chelating nitrogen. The chemical
~: :
_"
35,444-F -66-
:: `
`:
~ - .
~: ~
.` ~ ' , ~ '`'' ' " ' ' ,
, ',' " , ' ' . ' -' ~ ~ : . ~
': . ` : ' . '
-67- l 31 63~i~
shifts of these carbons in the unchelated dendrimer
occurred at 59.1 ppm for the a-CH2 and 53.7 ppm for
the first methylene carbon of the backbone. Upon
chelation these two resonance~ were observed to shift
downfield to 60.8 and 55.1 ppm respectively. The trans
chelation shows that two metal ions can be readily
chelated per dendrimer molecule however upon chelation
of some unknown ~raction of a third mole equivalent
the product precipitates out o~ solution.
Example 7: Demonstration of Multiple Chelation of 90f
by a methylenecarboxylate terminated second generation
F '(starburst))polyethyleneimine.
~- 15 Standard solution of yttrium chloride (3x10-2
M, spiked with non-carrier added 9Y) and
methylenecarboxylate terminated second generation
'/starburst')polyethyleneimine (6x10-2 M) were prepared.
These were reacted together at various metal:starburst'
ratios in HEPES buf~er.- The complex yield was
determined by ion exchange chromatography using
Sephadex G50 ion exchange beads, eluting with 10
NaCl:NH40H, 4:1 at pH lO. Noncomplexed metal is
removed on the column complexed metal elutes. Yields
were obtained by comparing the radioactivity eluted
with that on the column using a well counter.
.
35,444-F -67-
,
.
. ' .
`. : . , ,
~ 1 31 63' '1
Table V
Chelation of 2.5 Gen. PEI Acetate with 9Y
Vol. Y+3 Vol. PEI ~lol HEPES M:L Theor. 96 Coml~lex M:L ACt.
~ 30 370 o .1 110 o .1
360 0.2 101 o .2
0 20 30 350 0.4 95 4
340 0.5 97 0.5
340 0.5 102 0.5
310 1.0 99 1.0
120 3Q 250 2.0 100 2.0
180 30 lB0 3.0 94 2.8
250 30 120 4.1 80 3.3
300 20 80 7.5 44 3.3
300 20 70 . 5.0 40 2.0
300 20 70 5.0 41 2.0
All volumes in Table V are in microlitres
Within the accuracy of the experiments, these results
F 25 indicate that the 2.5 Gen. starburst~PEI acetate can
chelate between 2 and 3 metals per polymer giving a
soluble complex.
Example 8: Demonstration of multiple chelation of iron
by a sodium propionate terminated sixth generation
~starburs~1polyamidoamine.
The sodium propionate terminated sixth
generation`polyamidoamine (initiated from ammonia)
(97.1 mg, 2.45 mol.) was dissolved in 1.5 ml of
deionized water. Addition of 0.5 ml of 0.5N HCl
35, 444-F -68-
`` ```' `~ ' ' ~ ' ` ,' '
.
.
-69- 1 3 1 6 ) ~ 1
reduced the pH to 6.3. Ferric chloride was added (0.5
ml of 0.1.2M solution, 0.051 mmol) producin~ a light
brown gelatinous precipitate. On heating a~ 60C ~or
0.5 hours, the gelatinous precipitate became soluble,
resulting in a homogeneous orange solution. The
solution was filtered through Biogel P2 acrylamide gel
(10 g, twice) isolating the orange band ~free of halide
contamination). Removal of the solvent in vacuo gave
the product as an orange film (30 mg). Analysis was
consistent with chelation of approximately 20 moles of
F ferric ions per mole of starburst~7dendrimer.
Table IV
Theoretical
Found ~ -
Na4Fe20H128SNasFe2oHl27sBNa6Fe20Hl26s
Na 0.39,0.240.25 0.31 0.38
(0.31 0.1%)
20 Fe 3.14,3.113,05 3,05 3.04
(3.12 0.02~)
C 47.11 49.87 49.84 49.81
H 7.33 7.31 7.30 7.29
N 14.81 14.49 14.48 14.47
25 0 ____ 25.03 25.02 25.01
Mwt.36632.23 36654.21 36375.18
SB = C1521H2467N379573 "
3 These results confirm chelation of 20~2 moles
of ferric ions per mole of starburst dendrimer.
35,444-~ 69-
,
