CA1316524C - Starburst conjugates - Google Patents

Abstract

ABSTRACT

Starburst conjugates which are composed of at least one starburst polymer in association with at least one unit of a carried material have been prepared. These conjugates have particularly advantageous properties due to the unique characteristics of the starburst polymer.

35,444-F"B"

Description

1 3 ~ 6~693-4103 STARBURST CONJUGATES
The present invention concerns the use oE dense star polymers as carriers for selected materials (the "carried"
material). In recent years polymers referred to as dense star polymers or Starburst polymers have been developed. It has been found that the size, shape and properties of these dense star polymers or starburst 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 filed on August 18, 1987. Application serial No. 544,734 deals with all cases in which the carried material is a pharmaceutical material, application serial No. 544,736 deals with all cases in which the carried material is an agricultural material and the present application deals with all the remaining cases in which the carried material is neither pharmaceutical nor agricultural.
In its broadest aspect, the present invention is directed to polymer conjugate materials comprising dense star polymers or starburst polymers associated with carried materials which carried materials are other than agricultural or pharma-ceutical carried materials (hereinafter these polymer conjugates will frequently be referred to as "starburst conjugates" or Trade-mark f'~

:. .

-la-1 3 1 ~ 5, 2 ~
"conjugates"), processes for preparing these conjugates, compositions containing the ~3~2~

conjugates, and methods of using the conjugates and compositions.
The conjugates of the present invention are suitable for use in a variety of applications where specific deliver~ is desired. In a preferred embodiment o~ the presen~t invention, the starburst conjugates are comprised of one or more starburst polymers associated with one or more agents.
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 ~- 15 characterized by regular dendritic branching with radial symmetry. These radially symmetrical molecules are referred to as possessing "starburst topology".
These polymers are made in a manner which can provide concentric dendritic tiers around an initiator core.
The starburst topology is achieved by the ordered assembly of uniform (within each tier) organic repeating units in concentric, dendritic tiers around an initiator core; this is accomplished by introducing multiplicity in a geometrically progressive fashion through a number of molecular generations. The resulting highly functionalized molecules generations have been termed "dendrimers" in deference to their branched (tree-like) structure as well as their oligomeric nature. Thus, the terms starburst oligomer.
and starburst dendrimer are encompassed within the term ~tarburst polymer.
Covalent bridging of the starburst dendrimers through their reactive terminal groups produces a class of topological polymers, with size and shape controlled 35,444-F"B" -2-.
..,, ,... ., ~, . .. ,. ,.. ~ ~
. . .

~ 3 ~

domains, which are referred to as "starburst bridged dendrimers", whic~ term is also encompassed within the term starburst polymer.
The following description of the figures aid in understanding the present invention7 Fi~ure 1 depicts various generations of starburst dendrimers.
Fi~ure 2A depicts a dendrimer having un~-ymmetrical tunequal) branch junctures.
Fi~ure 2B depicts a dendrimer having symmetrical (equal) branch junctures.
The starburst polymers are illustrated by Figure 1 wherein ~ represents an initiator core (in thi~ Pigur~ a tri-functional initiator core, shown by the ~ar 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 3tarburst oligomers, called dendrimers; and (A)n~
(B)n~ (C)n, (D)n~ and (E)n represent starburst bridged dendrimers.
The starburst dendrimers are unimolecular assemblage~ 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 core, and (c) an exterior surface of terminal functionality (i.e., terminal functional groups) attached to the outermost generation. The size and shape of the starburst dendrimer and the functional 35,444-F"B" _3_ 13~2~

groups present in the ~endrlmer can be controlled by the cholce of the lnitiator core, the number of generatlons ~i.e., tiers) employed ln creatlng the dendrimer, ~nd the cholce o~ ~he repeat-ing units employed at each generation. Since ~he dendrimers can be isolated at any particular generatlon, a means ls provlded for obtaining dendrlmers havlng desired propertles.
The cholce o~ the starburst dendrlmer components a~fects the propertles of the dendrlmers. The lnltlator core type can affect the dendrlmer shape, produclng (dependlng on the cholce of initiator core)~ for example, spheroid-shaped dendrimers, cylin-drical or rod-shaped dendrimers, ellipsoid-shaped dendrimers, or musllroom-shaped dendrlmers. Sequential bulldlng of generatlons (l.e., generatlon number and the sl~e and nature of the repeatlng unlts) determlnes the dimenslons of the dendrlmers and the nature of their lnterlor.
Because starburst dendrlmers are branched polymers con-talnlng dendrltlc branches having functlonal groups dlstributed on the periphery of the branches, they can be prepared wlth a varlety of propertles. For example, starburst dendrlmers, such as those deplcted ln Flgure 2A and Flgure 2B can have dlstinct propertles due to branch length. The dendrlmer type shown ln Flgure 2A (such as Denkwalter U.S. Patent 4,289,872) possesses unsymmetrlcal (un-equal segment) branch ~unctures, exterlor (l.e., surface) groups (represented by Z'), lnterior moletles (represented by Z) but much less lnternal void space. The dendrlmer type shown ln Figure 2B
possesses symmetrlcal (equal segment) br~nch ~unctures wlth sur-face groups (represented by Z',), two , ' ~5~ ~ 3 1 ~ ~211 different interior moieties (represented respectively by X and Z) with interior void space ~Jhich vaires as a function of the géneration (G). The dendrimers such as those depicted i-n 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 "starburst dense packing" where the surface of the dendrimer contains sufficient terminal moietie~ such that the dendrimer surface becomes congested and encloses void spaces within the interior of the dendrimer. This congestion can provide a f 15 molecular level barrier which can be used to control diffusion of mater-ials into or out of the interior of the dendrimer.
Surface chemistry can be controlled in a predetermined fa~hion by selecting a repeating unit which contains the desired chemical functionality or by chemicall~ modifying all or a portion of the surface functionalities to create new surface functionalities.
In an advantageous use of the dendrimers, the dendrimers can themselves be linked together to create polydendric moieties ("starburst bridged dendrimers") which are also suitable as carriers.
In addition, the dendrimers can be prepared so as to have deviations from uniform branching in 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.

35,444-F"B" -5--6- ~ 3 ~ 2ll The starburst polymers employed in the starburst conjugates of the present invention 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 unlt 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 polymers may allow a greater quantity of material to be carried per dendrimer. Since the number of functional groups on the dendrimers can be controlled on the surface and within the interior, it also provides a means for controlling the amount of agent carried per dendrimer.
I An analogy can be made between early generation starburst dendrimers (i.e. generation -1-7) to clas~ical spherical micelles. The dendrimer-micelles analogy was derived by comparing features which they had in common such as shape, size and surface.

35,444-F"B" -6- _ _7_ ~3~2~

Table I

Parameter Regular Classical Starburst Dendrimers Shape Spherical Spherical Size 20-60A 17~ 7fl (diameter) Sur~ace 4-202 Z=6-192 aggregation number (generation = 2-7) area2/surface group 130-80A2 127-75~2 10 (~ ~
Z ~s the ~umb~r of surface groups; 1A= 10~1 nm;
1A = 10~ nm f 15 In Table I, the shape was verified by scanning transmission electron micrographs (STEM) microscopy and intrinsic viscosity (rl) measurements. The size was verified by intrinsic viscosity (~) and size exclusion chromatography (SEC) measurements. The surface aggregation numbers were verified by titrmetry and high field NMR. The area/surface group was calculated from SEC hydrodynamic measurements.

The first five generations of starbur~t 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 group). 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 advantage when using these microdomains a~
encapsulation devices.

35,444-F"B'~ -7--8- 131~3~

As further concentric generations are added beyond five, congestion of the surface occurs. This congestion can lead to increased barrier characteristics at the surface and manifests itsel~ as a smaller surface area per head ~surface) groups as shown in Table II.

35,444-F7'B" -8-~ 3 ~
_9_ "~ ~ ~ ''C

N
N ~

r1 U~

~` ~ ¢ 1 ~3 C ~ S 0~¢ ' ¦ S

~ o a U~ ~ O NO --H 01 ~
~1 N~ S ~ X

.~
S...... ~ ~I N ~ r r~ g S~
1~ tr~ l _I N N '~ N ~
C O ~y~
. ~
~ . ~ .
o.~l N No~l~ N 0 31 S~ S
CO U~ '7 N 1 " ~
~ O

~ ~ O ~D ~ ~ '~ O
O ~ .
' u~ U U ~ C
a ~ O

Ul U~ U ~ U~ ~ ~ g t E 3 a ~ D. a o o~
35, 444-F "B" -9-~ ~L 3 ~

For example, amine termlnate~ generations 5.0, 6.0, 7.0, .0 and 9.0 have decreased surface areas of 104, 92, 73, 47 and 32A2 per Z group, respec~ively. Thls characterlstic corresponds to a transltlon from a less congested mlcelle-llke surface to a more congested bllayer/monolayer barrier llke surface normally assoclated with vesicles (llposomes3 or Langmuir-Blodgett type membranes.
If this surface congestlon is lndeed occurrlng, the change ln physlcal characterlstlcs and morphology should be observed as the generations increase ~rom the intermediate generatlon (6-8) to the more advanced generatlons (9 or 10). The scannlng transmlsslon electron mlcrographs (ST~M) for generations o 7.0, 8.0 and 9.0 were obtalned after removlng the methanol solvent from each of the samples to provide colorless, llght yellow solld films and staining wlth osmium tetrao~ide. The morphological change preclicted occurred at the generatlon G = 9.0 stage. The interlor microdomalns at generation, G = 9.0, measure about 33A ln dlameter and are surrounded by a colorless rim whlch ls about 25A thlck. Apparently the methanollc solvent has been entrapped wlthln the 25A outer membrane-llke barrler to provlde the dark stalned interlor. Thus, at generation = 9.0, the starburst PAMAM ls behaving topologlcally like a vesicle (llposome). However, thls starburst ls an order of magnltude smaller and very monodlspersed compared to a llposome.
Consequently, the present dendrlmers can be used to molecularly encapsulate solvent fllled vold spaces of as much dlameter as about 33A ~volume about 18,000A3) or more.
Slnce the nurnber of functional groups on the dendrimers can be~controlled on the surface and wlthln . ~ ~
' ., ' 2 ~

the interior, it also provides a means for controlling the amount of carried material to be delivered per dendrimer. In one embodiment of the presen~ invention, the dendrimers are targeted carriers o~ agents capable of delivering the carried agent (material) to a particular locus.
Dendrimers suitable for use in the conjugates of the present invention include the starburst polymers described in U.S. Patents 4,507,466, 4,558,120, 49568,737 and 4,587,329.
In particular, the present invention concerns a starburst conjugate which comprises at least one f 15 starburst polymer associated with at least one carried 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;
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 3 material, said carried material can be the same carried material or a different carried material;
y represents an integer of 1 or greater; and 35,444-F"B" -11-.

-12~ 2 ~

* indicate~ that the carried material i~ associated with the dendrimer.
Preferred starburst conjugates of formula (I) are those in which M is a signal generator such as fluorescing entities9 signal reflector such as paramagnetic entities, signal absorbers such as electron beam opacifiers, fragrance, pheromones, or dye; particularly pre~erred are those in which x-1, and y=2 or more.
Also included are starburst conjugates of formula (I) wherein the dense star dendrimers are covalently linked together, optionally via linking f 15 groups, so as to form polydendric assemblages (i.e., where x~1).
As u~ed herein, "associated with~' means that the carried material(s) can be encapsulated or entrapped within the core of the dendrimer, dispersed partially or fully throughout the dendrimer, or attached or linked to the dendrimer, or any combination thereof. The association of the carried material(s) and the dendrimers may optionally employ connectors and/or spacers to facilitate the preparation or use of the starburst conjugates. Suitable connecting groups are grQups which link a targeting director (i.e., T) to the dendrimer (i.e., P) without significantly impairing the effectiveness of the director or the effectiveness of any other carried material(s) (i.e. 7 M) present in the starburst conjugate. These connecting groups may be cleavable or non-cleavable and are typically used in order to avoid steric hindrance between the target director and the dendrimer, preferably the connecting group3 are stable (i.e., non-cleavable). Since the 35,444-F"B" -12--13- ~ 3~2~
size9 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 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 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 r' 15 entrapment of the carried materials within the interior ~void space) wherein the release of the carried material can optionally be controlled by congesting the sur~ace of the dendrimer with diffusion controlling moietie~; or (d) various combinations of the 2~ aforementioned phenomena can be employed.
Dendrimers, herein represented by "P", include the dense star polymers described in UOS~ Patents 4,507,466, 4,558,120, 4,568,737 or 4,587,329.
Carried materials, herein represented by "M", which are suitable for use in the starburst conjugates include any materials, other than pharmaceutical or agricultural materials, which can be associated with the starburst dendrimer without appreciably disturbing the physical integrity of the dendrimer, for example, metal ions such as the alkali and alkaline-earth metals; signal generators such as fluorescing entities; ~ignal reflectors quch a~ paramagnetic entities; signal absorbers such as an electron beam opacifiers; pheromone moieties; fragrance moieties; dye 35,444-F"B" -13- _~

~1 3 ~
moieties; and the like. Carried materials include scavengin~ agents such as chelants or any moieties capable of selectively scavenging a variety of agents.
The starbursts conjugates of formula (I) are prepared by reacting P with M, usually in a suitable solvent, at a temperature which facilitates the association of the carried material (M) with the starburst dendrimer (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 r` 15 re~uired. When desired, mixtures of suitable solvents can be utilized. Examples of such suitable solvents are water, methanol, ethanol, chloroform, acetonitrile, toluene9 dimethylsulfoxide and dimethylformamide.
The reaction condition for the ~ormation of the starburst conjugate of formula (I) depends upon the particular dendrimer (P), the carried material (M), and the nature of the bond (*) formed. Typically, the temperature can range from room ternperature to reflux.
The selection of the particular solvent and temperature will be apparent to one skilled in the art.
The ratio of M:P will depend on the si~e of the dendrimer and the amount of carried material. For 3 example, the molar ratio (ratio of moles) 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 organic M to P is usually 0.1-5:1, and preferably 0.5-3:1.

35,444-F"B" -14--15- ~3~2~

Other starburst conjugates are those conjugates which contain a target director (herein designated as "T") and which are represented by the formula:

(T)e * (P)x * (M)y (II) wherein each T represents a target director;
e represents an integer of 1 or greater; and P, x, *~ M9 and y are as previously defined herein.
Preferred among the starburst conjugates of formula (~) are those in which M is a signal generator, signal reflector, or signal absorber. Also preferred are those conjugate3 in which e-1; and those in which x=1 and y=2 or more; and particularly preferred are those in which x=1, e=~l, and y=2 or more. Most preferred are those in which M and T are associated with the polymer via the same or different connectors.

The starburst conjugates of Eormula (Il) are prepared either by forming T*P and then adding M or by forming P*M and then adding T. Either reaction scheme is conducted at temperatures which are not detrimental to the particular conjugate component and in the presence of a suitable solvent when required. To control pH, buffers or addition of suitable acid base i~ used. The reaction conditions are dependent on the type of association formed (*), the starburst dendrimer u~ed (P), the carried material (M), ahd the target director (T). Alternatively, P and M can be chelated, 35,444-F"B'i -15-,. ............. .

~3~6~
usually in water, before conjugation to T. The conjugation with T is carried out in a suitable buffer.
The ratio of T:P is preferably 1:1. The ratio of M:P will be as before.
Target directors capable of targeting the starburst conjugates are entitie~ which when used in the starburst conjugates of the present invention result in at least a portion-of the starburst conjugates being delivered to a desired target, chemical functionalities exhibiting target specificity, and the like.
In the absence of a target director (or in the presence of a target director if de~ired), due to the number of functional groups which can be located at or near the surface of the dendrimer, all or a sub3tantial portion of such functional groups can be made anionic, cationic, hydrophobic or hydrophilic to effecti~ely aid 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 (II) 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 loadin~ ~ S*P*M deprotection D P*M

T*P*M linking ¦

where 35,444-F"B'1 -16- _ -17- ~ 3~ ~3 2~

S*P represents the protected dendrimer;
S*P*M represents the protected dendrimer conjugated with m;
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 ~tarburst conjugates can be used for a f 15 variety of in vitro applications such as radio-immunoassay~, electron microscopy, enzyme linked immunosorbent assay~, nuclear magnetic resonance spectroscopy, and contrast imaging, and immuno-scintography, in analytical applications; or used as starting materials for making other useful agents.
The present invention is also directed to starburst conjugate compositions in which the ~tarburst conjugates are formulated with other suitable vehicles.
The starburst conjugate compositions may optionally contain other active ingredients, additives and/or diluents.
The preferred ~tarburst polymer for use in the starburst conjugates of the present invention is a polymer that can be described a~ a starburst polymer having at least one branch (hereinafter called a core branch), pre~erably 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 35,444-F"B" -17--18- ~ 3~2~

the core branches is more than one, preferably two or greater, (2) the density oP 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 brancheq of the extended conventional star poly~ler bearing only one terminal group, and t3) 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 is used as the base for comparison with the dense star polymer is one that has the same molecular weight~ same core and monomeric components and same number of core branches as the dense star polymer. By "extended" it i~ meant that the individual branche~ of the conventional star polymer are extended or stretched to their maximum length, e.g.; a~ such branches exist when the star polymer is completely solvated in an ideal solvent for the star polymer. In addition while the number of terminal groups is greater for the dense star polymer molecule than in the conventional star polymer molecule, the chemical structure of the terminal groups is the same.
Dendrimer~ used in the conjugates of the present invention can be prepared by processes known in the art. The above dendrimers, the various coreactants 35,444-F"B" -18-~ 1 9 - .

and core compound~, an~ proces~ for their preparation can be a~ defined in U.S. Patent 4,587,329.
The ~tarburst dendrimers, for use in the starburst conjugate~ 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 9 mercapto, carboxy, alkenyl, allyl, vinyl, amido, halo, urea, oxiranyl, aziridinyl7 oxazolinyl, imidazolinyl, sulfonato, phosphonato, isocyanato and isothiocyanato.
The dendrimers differ from conventional star or 3tar-branched polymers in that the dendrimer~ 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 i~ at least about 1.5 times the density of terminal groups in the conventional extended star polymer, preferably at least 5 times, more preferably at least lQ times, most preferably 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 preferably from 4 tc 1024.
Preferably, for a given polymer molecular weight, the molecular volume of the starburst dendrimer is less than 70 volume percent, more preferably from 16 to 60, 3 most preferably from about 7 to 50 volume percent of the molecular volume of the conventional extended star polymer.
Preferred starburst dendrimers for use in the starburst conjugates of the present invention are characterized as having a univalent or polyvalent core 35,444-F"B" -19- _ --20 1 3 ~ ~ ~ 2 ~

that i9 coYalently bonde~ to dendritic branches. Such ordered branching can be illustrated by the following sequence wherein G indicates the number of generation~:

G = 1 G = 2 N - - N -~ ~

H

H H

G = 3 . N -,~ ~
~ ~ ~

N- f N N N

35,444-F"B" -20--21- ~ 3 2 ~

Mathematically, the relationship between the number (#) of terminal groups on a dendritic branch and the number of generationq of the branch can be represented as follows:
NrG
# of terminal groupq 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 iY determined by the followingo f 15 NcNrG
# of terminal groups per dendrimer =

wherein G and Nr are as defined before and Nc represents the valency (often called core functionality) of the core compound. Accordingly, the dendrimer~ of this invention can be represented in its component parts as follows:

~ Terminal (Core~ (Repeat Unit~N G l Moiety N G
I C
Nr-l /

35,444-F"B" -21--2~- ~ 3 ~

wherein the Core, Terminal Moiety, G and Nc are as de~ined before and the Repeat Unit has a valency or functionality of Nr ~ 1 wherein Nr i~ as defined before.

A copolymeric dendrimer which is a preferred dendrimer for the purposes of this invention i9 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 i~ 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 i~ preferably 2 or more, most pre~erably 3 or more. Thus, the dendrimer molecule comprise3 a polyfunctional core, ~, bonded to a number (Nc) of functional gro~ps, zc, each of which is connected to the monofunctional tail of a repeating unit, X1Y1(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 represent~ the monofunctional tail of the first generation repeating unit, y1 repre~ents the moiety constituting the first generation, z1 represent~
the functional group of the polyfunctional head of the repeating unit of the firYt generation and may be the 35,444-F"B" -22--23- ~3~rj~

same as or different from the functional groups of the core compound, ~ (ZC)Nc~ or other generations; and N1 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 prePerred dendrimer molecule, 0 each zl of the first generation repeating unit is connected to an x2 of a repeating unit of the second generation and so on through the generations such that each zi group ~or a repeating unit XiYi(Zi)Ni in lS generation number "i" is connected to the tail (Xi~1) of the repeating unit of the generation number "i~l".
The final or terminal of a preferred dendrimer molecule comprises terminal units, XtYt(Zt)Nt wherein t represents terminal generation and xt, ytJ 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 ~5 35,444-F"B" -23- _~

-24- l 3 ~

~ n is ~ ~ n LS

1~ where i is 1 to t-1 wherein the symbols are as previously defined. The function is the product of all the values between its defined limits. Thus f 15 n Nn - (N1)(N2)(N3)..~(Ni-2)(Ni-l) 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 IIO = 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 il]ustrated in structural formulas described hereinafter. Preferred dendrimers may be converted to functionalized dendrimers by contact with another reagent. For example, conversion of hydroxyl in the terminal generation to ester by reaction with an aoid chloride gives an ester terminally functionalized dendrimer. This functionalization need not be carried out to the theoretical maximum as defined by the number 35,444-F"B" -24- _~

-25- ~ 2 ~

of available functional groups and, thus, a functionalized dendrimer may not have high symmetry or a precisely defined molecular formula as is the case with the preferred dendrimer.

In a homoplymer dendrimer, all of the repeat units, XiYi~2i)Ni, are identical. Since the values of all Nl 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 ( ~ (Zc)N ) ~ i yi (zi)N~ n ~ (XtY (Z )N~ N Nr where i = 1 to t-1 This form still shows the distinction between the different generations i, which each consist of NcNr (i-1) repeating units, XiYi(Zi)Ni. Combining the generations into one term gives:

3o 35,444-F"B" -25--26- ~3~ ~2~

Z )N )~X y (zi) Nr(t ~(X Y (Z )Nt) NCNr(t-l) or 1C core repeat unitremoval unit, 1' ((~ )N )~XrYr(~r)Nr)( y (Z )Nt) 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 starburst polymer. Conversely, if a polymer compound will not fit into these above formulae, then the polymer is not a starburst polymer. Also r to determine whether à 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 3 also demonstrate the generations (G) or tiering of dendrimers.
~ learly, there are several ways to determine the ratio of agent (M) to dendrimer (P) which depend upon how and where the association of P*M occurs. When there is interior encapsulation, the weight ratio of 35,444-F"B" -26-- 27~

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 i3 determined, the mole/mole ratio of M:P given by the following formulae:

M P

(A) 5 NCNtNr6~l 1 (B) 3 NCNtNr~ 1 ,.
f 15 (C) 1 NCNtNr where Nc means the core multiplicity, Nt means the terminal group multiplicity, and Nr~m,eans branch juncture multiplicity. The NCNtNr~ ~ 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 octanoic acid; (C) above when it is carboxylate ions or groups.
Of course other structures of ~arious dimensions can be readily prepared by one skilled in the art by appropriately varying the dendrimer components and number of generations employed. The dimensions are 3ignificant in that they are small. A
linear polymer of comparable molecular weight would have a radius of gyration, (in its fully extended form), that would be much larger than the same molecular weight dendrimer.

35,444-F"B" -27- _~

: , .

~3~$~

Llnking target directors to dendrlmers ls another aspect of the present lnventlon. In preferred embodlments of the present inventlon, a reactive functional group such as a carboxyl, sulfhydryl, reactive aldehyde, reactlve ole~lnic deriva~ive, l~othlocyanato, lsocyanato, amlno, reactlve aryl halide, or reactive alkyl hallde can convenlently be employed on the dendrlmer. The reactive functional groups can be lntroduced to the dendrimer uslng known technlques, for example (1) Use of a heterofunctlonal lnitiator ~as a star~lng material for syntheslzlng the dendrlmer) which has lncorporated into lt functional groups of different reactivity. In such heterofunctional lnitiator at least one of the functlonal groups will serve as an inltiation si~e for dendrlmer formation and at least one of the other functional groups wlll be avallable for linking to a target director but unable to inltiate dendrimer synthesls. For example, use of protected anlllne allows further modificatlon of NH2 groups within the molecule, without reactlng the NH2 of the anlline.
The functlonal group whlch will be available for linking to a target dlrector may be part of the lnitlator molecule ln any one of three forms, namely:
(a) In the form in which lt will be used for linking with the target dlrectors. Thls ls posslble when none of the synthetic steps lnvolved in the dendrlmer synthesls can result ln reactlon at this center.
(b) When the functlonal group used for linking to the targeting dlrector is reactive in ~T
'~

-29- ~3~ ~5~

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 precursor can be used which is unreactive f 15 in all the synthetic proecedures uqed 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.
(2) 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, as a protected functionality, or as a synthetic precursor. The form in which this linking ~unctionality i~ used depends on itq integrity during the synthetic procedure to be utilized, and the ability of the 35,444-F"B" -29-, : ' , 3 ~ 3 ~

~inal macromolecule to with~tand any conditions necessary to make this group available for linking. For example, the preferred route for PEI uses ~ ~N02 Example~ of heterofunctional initiators ~or use in (1) above, include the following illustrative examples:
f 1~
H2N ~ CH2NH2 H2N~ CH2CH

30 ' 8 35,444-F"B" -30--31~ 2~

(CH3)3 COCNH~ O~> CH2C~H
\ _ ` CNHCH2CH2NH2 ~ ~CH2NH2 ( CH3 ) 3 COCN~ CH2CH

,, lC

H2N ~CH2CH

3C H2N ~ CH2CH2NH2 35, 444-F"B" -31~

, ., .

~3~~ :IL3:1 ~52l~

~2N ~,>--CH2CH

02N--<~(~ CH2CH2NH2 t' 1 _ / \ /
02N <~- CH2CH ; and \
CH .NH2 2~

.
35, 444-F"B" -32- ~

~ 3 ~ 2 ~

f H2CH2NH2 02N C~2CH

There are several chemistrie of particular f 15 importance:

1) Starburst Polyamidoamides ("PAMAM") Chemistry;
2) Starburst Polyethyleneimines ("PEI") Chemistry;
3) Starburst PEI compound with a surface of PAMAM;
4) Starbur~t Polyether ("PE") Chemistry.
ModiYications of the dendrimer surface functionalities may provide other useful functional ~5 groups such as the following:
-OP03H2, -P03H2, -P03H(-~ po3(-2), _C02(-1), -S02H, -S02( 1), -S03H 9 -S03(-1), -NR1R2~ -R5, -OH, -ORl, -NH2, polyetherq, perfluorinated alkyl, -CNHR1, -COH, 1- ,.
O O

35`,444-F"B" -33~

.. ~3~~ ~3~

-(CH2)n ~ -N=C ~

-NHCH2 ~ -(CH2)n ~ ~ -(cH2) wherein R represents alkyl, aryl or hydrogen;
f 15 Rl represents alkyl, aryl, hydrogen, or ~ (CH2)n ~

-N X

(CHz)n R2 represent~ alkyl, aryl, or ~ (CH2)n ~

-N X

~CH2)n , ` 30 - 35,444-F"B" -34- _~

-35~ 2 ~

R3 represents -OH, -SH, -C02H, -S02~, 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 product.

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 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,lH and 13C NMR
spectroscopy.

35,444-F"B" -3 -36- 13~

Anal: Calc. for c1oH1 104N3 C H N
Theo: 50.63 4~69 17.72 Found: 50.75 4.81 17.94 Example B: Preparation of l-Amino-2-(aminomethyl)-3-(4'-nitrophenyl)propane.
2-Carboxamido-3-(4'nitrophenyl)propanamide (2.0 1 g, 8.43 mmole) was slurried in 35 ml of dry tetrahydro-furan under a nitrogen atmosphere with ~tirring. To 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 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 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 chloride. The aqueous layer was cooled in an ice bath under an argon blanket and 50~ sodium hydroxide was slowly added until basic pH-11.7. The basic aqueous layer was extracted with four 25 ml portions of methylene chloride and these combined extracts were evaporated (rotary) to give 1.45 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) o~ the 35,444-F"B" -36-37 ~3~

titled diamine as a clear oil (mp = 275-278C(d) bis HCl salt).
The structure was conflrmed by MS, 1H and 13C NMR
Spectroscopy.
Anal: Calc. for C1oHl7N3o2cl2 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 solution was brought to reflux for 40 hours. The colorle~s solution was cooled and excess tetrahydrofuran was removed by rotary evaporation leaving a clear gelatinous 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 l minute. The methanol/HCl was rotary evaporated and the resulting hydrochloride salt was carried through the same dissolution/reflux procedure again. The hydrochloride silt obtained was dissolved in lO ml of water and cooled in an ice bath under argon. Concentrated sodium hydroxide (50%) was added slowly with stirring to pH=11. The aqueous portion was then extracted with 2 X
100 ml portions o~ chloro~orm which were combined and filtered through a short ~ilica gel plug without drying. The solvent was removed in vacuo (rotary) 35,44~-F':B" -37--38~ J~ 2'1 affording the title compound (0.90 g, 5.02 mmole) in 70% yield (Rf=0.65 - CHC13/MeOH/NH40H conc - 2/2/1).
The structure was confirmed by 1H and 13C NMR and used without further purification.

Example D: Preparation of 6-(4-Aminobenzyl)-1,4~8,11-tetraaza-5,7-dioxoundecane.
4-Aminobenzyl malonate dimethylester (2.03 g, 8.43 mmole) was dissolved in 10 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 o~ methanol under nitrogen over a 2 hour period. The clear solution was stirred for 4 days f 15 and thin layer chromotograph~ (TLC) analysis indicated total conversion of diester (Rf = 0091) 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 (19-1 mm, 50C) overnight to afford crude product (2.45g, 8.36 mmole) in 99% yield. An analytical sample was recrystallized from chloroform/hexane, MP - 160-161C.
The mass spectral, 1H and 13C NMR data were consistent with the proposed structure.

Exam~le E: Reaction of Mesyl Aziridine with 1-Amino-2-(aminomethyl)-3-(4-nitrophenyl)propane.
3 l-Amino-2-(aminomethyl)-3-(4-nitrophenyl)-propane (400 mg, 1.91 mmole, >95% 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 using a magnetic stirrer and during 35,444-F"B" -38- _~

_39_ ~3~

this period a white, gummy re~idue 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 sulfonamide ~1.0 g, 1044 mmole) in 75% yield (Rf = 0.74 - NH40H/ethanol - 20/80). The structure was confirmed by 1H and 13C nuclear magnetic resonance (NMR) spectro~copy.

Example F: Preparation of 2-(4-Nitrobenzyl)-1,3-(bis-N,N-2-aminoethyl)diaminopropane.
The crude methylsulfonamide (650 mg, 0.94 f 15 mmole) wa~ 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 wa~ filtered and immediately dissolved in lO 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 inorganic salts were filtered. The solvent was removed from the crude amine under reduced pressure and to the resulting light brown oil was added 190 ml of toluene under nitrogen. The mixture was stirred vigorously and water wa~ removed through azeotropic distillation (Dean-Stark trap) until the remaining toluene acquired a light yellow color (30~40 ml remaining in pot). The toluene was cooled and decanted from the dark, intractable residues and salt.
This solution wa stripped of solvent in vacuo and the resulting light yellow oil was vacuum dried (0.2 mm, 35,444-F"B" -39- _ `~` 13l6~

35C) overnlght affordlng ~10 mg of the product ~60~) whlch was characterlzed by MS lH and 13C NMR.
E~ample G: Preparatlon of a starburst polymer (containing an aniline derivative) o~ one half generation represented by the ~ollowing scheme:

~ 11 H2c=cHcocH3 --~ O ,>--CH2CH (CNHCH2CH2HN2 ) CH OH
Compound #1 3 2 ~ C~l2cH(cNHcH2cH2N(cH2cocH3)2)2 Compound #2 Methyl acrylate ~2.09 g, 24 mmole) was dissolved in methanol (15 ml). The compound 6-(4-aminobenzyl)-1,4,8,11-tetraaza-5,7-dioxoundecane (1.1 g, 3.8 mmole) (i.e., Compound #1, preparatlon descrlbed ln Example D) was dlssolved ln methanol (10 ml) and was added slowly over 2 hours wlth rlgorous stlrrlng to the methyl acrylate solutlon. The reactlon mlxture was stlrred for 48 hours at ambient temperatures. The solvent was removed on the rotary evaporator malntalnlng the temperature below 40C. The ester (Compound #2) was obtalned as a yellow oll (2.6 g). No carboxyethylatlon of the anlllne functlon was observed.

, -41- ~31~2~

Example H: Preparation of a starburst polymer (containing an aniline moiety) of one generation;
represented by the following scheme:

Compound #2 + H2NCH2CH2NH2 O O
H2N ~ -cH2cH(cNHcH2cH2N(cH2cH2cNHcH2cH2NH2)2)2 Compound #3 .
The ester (Compound #2) (2.6 g, 3.7 mmole) was dissolved in methanol (100 mlS. 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 for 28 hours at 35-40C ~heating mantle). After 28 hours no ester groups were detectable by infrared specroscopy. The solvent was removed on the rot;ary evaporator at 60C. The excess ethylene diamine was removed using a ternary azeotrope of toluene-methanol-ethylene diamine. Finally all remaining toluene was azeotroped with methanol, Removal of all the methanol 3 yielded 3.01 g of the product (Compound #3) as an orange glassy solid.

35,444-F"B" -41-- -~2- ~ 3~6~2~

Example I: Preparation of a ~tarburst polymer (containing an aniline moiety) of one and one half generations represented by the following scheme:
O

Compound #3 + ~2C=CHCOCH3 O O
H2N~ CH2CH ( C'NHC~2C~2N ( Cl~2cl~2cN~lcH2c~l2N ( C}~2C~i2CoC113 ) 2 ) 2 ) 2 Compound #4 The amine (Compound #3) (2.7 g, 3.6 mmole) wa3 dissolved in methanol (7 ml) and was added slowly over one hour to a stirred solution of methyl acrylate t3.8 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 stir at ambient temperatures for 16 hours. The solvent was removed on the rotary evaporator at 40C.
After removal of all the solvent and exces methyl acrylate the ester (Compound #4) was obtained in 4.7 g yield as an orange oil.

35,444-F"B" -42-~ _43_ ~3~

Example J: Preparation of a starburst polymer (contain.ing an aniline moiety) of one half generation -represented by the following scheme:

,.
H2~ ~ -cH2cH(cH2NH2)2 + ~2C-CHCOCH3 Compound #5 H2N- ~ -CH2CH(CH2N(CH2cH2cOcH3)2)2 Compound #6 The triamine (Compound #5, the preparation of this oompound 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 (10 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 waq removed by repeated azeotroping wif,h methanol. The ester (Compound #6) was isolated as an orange oil (1.24 g).

35,444-F"B" -43-~ 3 ~
-~4 Example K: Preparation of a star~urst polymer (containing an aniline moiety) o~ one generation;
represented by the following scheme:

Compound ~,~6 + H2NCH2CH2NH2 H2N ~ CH2cH(cH2N(cH2cH2cNHcH2cH2NH2)2)2 Compound #7 r` 15 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 g9 1~22 mole) in methanol (100 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 of toluene-methanol-ethylenediamine. Finally all remai~ing toluene was removed with methanol and then pumping down with a vacuum pump for 48 hours gave the amine (Compound #7) (1.86 g) as a yellowtorange oil.

3o 35,444-F"B" -44- _ ~45- ~3~6~2~

Example L: Preparation of a starburst polymer (containing an aniline moiety) of one and one half generations; represent by the following scheme:

Compound ~7 + H2C-CHCOCH3 .
O O
H2N- ~ -cH2cH(cH2N(cH2cH2cNHcH2cH2N(cH2cH2cocH3)2)2)~
Compound #8 , 15 The amine (Compound #7) (1.45 g, trace of methanol remained) waq dissolved in methanol (100 ml) and was added slowly over 1~ hours to a stirred solution of methyl acrylate (5.80 g) in methanol (20 ml). 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 exces~ 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 preparation of calcium salt.
3 4.5 Generation PAMAM (ester terminated, initiated off NH3) (2.11 g, 10.92 meq) was dissolved in 25 ml o~ methanol and to it was added 10% NaOH t4.37 ml, 10.92 meq) (pH - 11.5-12). After 24 hours at room temperature, the pH was about 9.5. After an additional 35,444-F"B" -45-, .' -46- ~3~2~

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 for the t- 15 calcium 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.

The resulting solution was again dialyzed, this time against water, then repeated two additional times.
Evaporation provided 0.6 g o~ wet solid, which was taken up in methanol (not totally soluble) and is dried to give 0.4-5 g of off-white crystals.
c369H592Q14lN91ca24 Calc. 10.10% Ca+~
M Wt. = 9526.3 Calc. - C-4432.1, H-601.8, 0-2255.9, N-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"B" -46-_47_ ~ 2~

Example N: Preparation of dendrimers with terminal carboxylate groups.
Half-generation starburst polyamidoamines were hydrolyzed to convert their terminal methyl ester groups to carboxylates. This generated spheroidal molecules with negative charges dispersed on the periphery. The dendrimers hydrolyzed ranged Prom 0.5 generation (three carboxylates) to 6.5 generation (192 carboxylates).
The products could be generated as Na+, K+, Cs+
or Rb+ salts.

Example 0: N-t-butoxycarbonyl-4-aminobenzyl malonate dimethylester 4-Aminobenzyl malonate dimethylester (11.62 g, 49 mmol) was dissolved in 50 ml of 5-butanol:water 60:40 with stirring. Di-t-butoxydicarbonate (19.79g, 90 mmol) was added and the reaction mixture stirred overnight. The butanol waq removed on the rotary evaporator, resulting in a yellow suspension of the product in water. ~xtraction into methylene chloride, drying (MgS04~ and evaporation gave a yellow oil (21.05 g, contaminated by di-t-butoxydicarbonate).
recrystallization Prom 2-propanol:water (75:25) yield pale yellow crystals (11.1 g, 33 mmol, 67%). The structure was confirmed by 13c NM~ and purity checked by hplc analysis (spherisorb ODS-1, 0.05M H3P04 pH 3:
CH3CN 55:45). The material was used without Purther purification.

35,444-F"B" -47--48~

Example P N-t-butoxycarbonyl-6~(~-aminobenzyl)-1,4,8,11-tetraa~a-5,7-dioxoundecane N-t-butoxycarbonyl-4-aminobenzyl malonate dimethylester ~8.82 g 26 mmol), prepared in Exarnple 0, was dissolved in 50 ml of methanol, This solution was added dropwise (2 hours) to a 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 - 15 (10.70 g contaminated with ethylenediamine). The sample wa3 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 solution in methanol by gas chromatography (Column, Tenax 60/80) showed no ethylenediamne detectable in the sample (<0~1 percent). The second fraction was dis~olved in methanol to give a 10 percent solution (by weight) and purified from the ethylenediamine by reverse osmosis, using methanol as the solvent. (The membrane used was a Filmtec~ FT-30 , in an Amicon TClR
thin channel separator, the ethylenediamine crossing the membrane.) The product waq isolated as a white ~olid (2.7 g), in which no detectable amounts of ethylenediamine could be found by gas chromatography.
The 13C NMR data and hplc analysis (Spheri30rb ODS-1, 35,444-F"B" -48--49- ~31~2~

0.05M H3P04 pH 3:CH3CN 55:45) were consistent with the proposed structure. The product was used with no further puri~ication.

Example Q: Preparation of a starburst dendrimer of one half generation from N~t-butoxycarbonyl-~-(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 wa~ monitored by HPLC (Spherisorb ODSl, 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 1 mm Hg for Z4 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 3o 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 3tirred solution of ethylenediamina (400 ml, 7.41 mol) and methanol (50 ml). The reaction mixture was stirred at ambient temperatures for 48 hours. The ethylenediamine and 35,444-F"B" -49- _~

.. .

-50- ~ 3~ ~2~

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 separator, methanol as solvent). After 48 hours, no ethylenediamine could be detected by gas chromatography (Column, Tena~ 60/80). Removal of the solvent on the rotary evaporator, followed by pumping down on a vacuum 0 line for 24 hours gave the product as a yellow glassy solid (6.72 g). Analysis by HPLC, PL~P-S column, acetonitrile:O.015M NaOH, 10-20 percent gradient in 20 min.) and 13C NMR analysis was consistent with the proposed structure.

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-1, acetonitrile:
0.04M ammonium acetate, 60:40). A second aliquot of methyl aorylate was added (3.5 g 39 mmol) and the reaction mixture stirred at ambient temperatures ~or a further 72 hour~. 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.

i 35,444-F"B" -50- ~

Example T: Preparation of a starburst polymer of two full generations from N-t-butoxycarbonyl-6-(4-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, lO 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 ethylene diamine by reverse osmosis f 15 (membrane used as a Filmtec FT-30, in an Amicon TClR
thin channel separator) until no ethylenediamine could be detected by gas chromatography (Column, Tenax 60/80.
Removal of the solvent gave the product a~ a yellow glassy solid (3.52 g). The 13C NMR data and HPLC
analysis (PLRP-S column, acetonitrile:O.015 M NaOH, 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 O.lN NaOH), at 40.5C overnight.

35,444-F"B"' -51--52- ~3~

Monitoring by reverse phase HPLC, (~pherisorb ODS-1 column, eluent 0.25 M H3P04 pH 3 [NaOH]; acetonitrile 85:15) con~irmed the s~-nthesis of predominantly a single component.

Example V: Preparation of Isothiocyanato functionalized second generation methylene-carboxylate terminated starburst dendrimer 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 0.5 with concentrated hydrochloric acid. After one hour at room temperature the mixture , 15 wa9 analyzed by HPLC to verify the removal of the butoxycarbonyl group and then treated with 50 percent sodium hydroxide to ~ ~g the pH to 7. A pH stat (titrating with 0.1 N NaOH) was used to maintain the pH
at 7 and 225 ~l thiophGsgene 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 o~ second generation starburst polyethyleneimine-methane sulfonamide To a solution of 125 g N-methanesulfonyl-aziridine in 50 ml ethanol was added 25.0 g tris(2-aminoethyl)amine. The solution was stirred at room temperature ~or 4 days. Water was added to the reaction mixture as needed to maintain the homo~eneity of the solution. The solvent was removed by distillation in vacuo to give the 2nd generation 35,444-F"B" -52- _ -53- ~3~

starburst PEI-methane sulfonamide as a yellow glass (161 g).

Example X: Cleavage of methane sulfonamides to form second generat'on starburst polyethyleneimine 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.
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 , 15 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 PEI
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. ~ater was 3 added as needed to maintain the homogeneity of the solution. The solvent was removed by distillation in vacuo to give third generation ~tarbur~qt PEI-methane sulfonamide as a yellow glass (45.3 g).

35~444-F"B" -53- __ -5LI ~3~$2~

Example Z: Cleavage of methane sulfonamides to form 3rd gen starburst polyethyleneimine : The methane sulfonamide groups of third generation starburst PEI-methane sulfonamide (~.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 starburst PEI as a yellow oil.

Example AA: Preparation of a methylenecarboxylate-terminated second generation starburst polyamidoamine (initiated from ammonia) r 15 The second generation starburst polyamidoamine (2.71 g, 2.6 mmmol) and bromoacetic acid (4.39 g, 31.6 mmol) were dissoLved in 30 ml of deionized water and the pH adjusted to 9.7 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 azeotropin~ 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 wa~ consistent with the proposed ~tructure (with some contamination due to a small amount of defected material as a result of some monoalkylation).

35,444-F"B" -54-. ' .

:, . ..... - - , . .

-~55-Example BB: Preparation o~ a methylenecarboxylate terminated second generation starburst polyethyleneimine (initiated from ammonia) The second generation starburst 5 polyethyleneimine (2.73 g, 6.7 mmol), from Example AA, and bromoacetic acid (11.29g, 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 10 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 5 product as a yellow powder (17.9 g, contaminated with sodium bromide). The 13C NMR spectrum was consistent with the proposed structure (with some COntamin~ltiOn due to a small amount of defected material as a result 20 of some monoalkylation).

Example CC: Preparation of a 3.5, 4O5~ 5.5 and 6.5 generation starburst PAMAM
~tef~arJol/c 2 To a 10 wt%~solution of 2.46 g 3 generation 5 PAMAM starburst was added 2.32 g of methyl acrylate.
This mixture was allowed to sit at room temeprature for 64 hr. After solvent and excess methyl acrylate removal, 4.82 g of product was recovered (105~ of 30 theoretical).

Preparation of higher 1/2 generation starburst PAMAM'S:
' , .
Generations 4.5, 5.5 and 6.5 were prepared as described above with no significant 35,444-F"B" -55- _ --56- ~316~

differences in reactant concentrations, reactant ratios or reaction times.

Example DD: Preparation of a ~, 5 and 6 generation starburst PAMAM:
To 2000 g of predistilled ethylenediamine was added 5.4 g of 4 1/2 generation starburst PAMAM as a 15 wt% solution in methanol. This was allowed to sit at room temperature for 48 hrs. The methanol and most of the excess ethylenediamine were removed by rotary evaporation under water aspirator vacuum at temperature less than 60C. The total wt o~ product recovered was ô.07 g. Gas chromatography indicated that the product still contained 34 wt% ethylenediamine at this point.
A 5.94 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 relief 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 hrs. After this time~ 60 ml of methanol was passed over the membrane to recover product still in the module and associated tubing. The product was stripped of solvent and 2.53 g of 5 generation starburst PAMAM was recovered. Analysis by gas chromatography indicated 0.3% residual ethylenediamine remained in the product.

35,444-F"B" -56- __ . . .
.

~57~ 13~2~

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 generation this ratio wa~ 730:1.

~xample 1: Preparation of a product containing more than one rhodium atom per starburst polymer.

2.5 Gen PAMAM (ester termlnated, initiated off 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 almost all of the rhodium was taken up.
f 15 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 ~ield a red oil (0.18 g). The NMR spectrum was recorded in CDC13 only minor differences were noted between the chelated and unchelated starburst.
Dilution o~ some of this CDC13 solution with ethanol followed by NaBH4 addition resulted in rhodium precipitation. RhC13~3H~0 is insoluble in chloroform and in chloroform starburst solution thus confirming chelation.
Example 2: Preparation of a product containing chelated with a starburst polymer 3o 3.5 Generation PAMAM (ester terminated, initiated o~f NH3) (1.1 g, 0.24 mmole) was dissolved with stirring into acetonitrile (50 ml)O Palladium chloride (0.24 g, 1.4 mmole) was added and the solution was heated at 70-75C (water bath) overnight. All the PdC12 was taken up into the starburst. Removal of the 35,444-F"B" -57- _ , - _5~_ ~3~

solvent and recording the NMR in CDC13 confirmed that chelation had occurred. Dilution of the CDCl~ solution with ethanol and addition of NaBH~ resulted in precipitation of the palladium. The chelated product (1.23 g) was isolated as a brown oil.
Example 3: Preparation of a product containing ~ffi~seee~ with a starburst polymer ~J~ r~ScL~
~ A sample of 5-carboxyfluorescein (0.996 g) and starburst polyethyleneimine (Gen-2.0; amine terminated, initiated off NH3) (0.202 g) were mixed in 10 ml of methylene chloride and 5 ml of methanol and allowed to reflux for 10 minutes. Upon filtering, an insoluble red powder (0.37 g) was obtained (mostly unreacted 5-oarboxy fluorescein). From the filterate was isolated0.4 g o~ a brilliant-red solid which exhibited a softening point of 98-103C and foamed to a brilliant red melt at 175-180C; NMR spectra (D20~hof thi~ product were consistent with dendrimer having ~ ~s~ bound to the surface Example 4: Preparation of a product containing fluoro~oei-n with a starburst polymer ol~ ~s~e/~7 In a procedure similar to that described in Example 3, starburst polyethyleneimine (Gen-2.0; amine terminated, initiated off NH3) was reacted with fluorescein isothiocyanate to give a brilliant-red iridescent solid which was suitable for use as a fluorescent labelling reagent.

35,444-F"B" -58-.

~ 3 ~

Example 5: Hydrolysi~ of 4.5 generation dendrimers and preparation of calcium salt.
4.5 Generation PAMAM (ester terminated, initiated off NH3~ (2.11 g, 10.92 meq) was dissolved in 25 ml of methanol and to it was added 10% NaOH (4.37 ml, 10.92 meq) (pH = 11.5-12). After 24 hours at room temperature, the pH was about 9.5. A~ter an additional 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 off, and the gum was rotovaped extensively to give a very fine off-white powder which upon further drying gives 2.16 g of product (98% yield). No ester ~sroups were found upon NM~ and infrared analysis.
The sodium salt of 4.5 Generation PAMAM (ester terminated, initiated from NH3) was exchanged for 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%
CaCl2 solution. This procedure was then repeated.
The resulting solution wa~ again dialyzed, this time against water, then repeated two additional times.
3 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.
.
c369H592ol41N9lca24 Calc. - 10.10% Ca++

35,444-F"B" -59- _~
, M Wt. - 9526.3 Calc. - C-4432.1, H-601.~, 0-2255.9, N-1274.6, Ca-961.9) Theo: C-46.5, H~6.32, N-13.38, Ca-10.l0 Found: C-47.34~ H-7.00, N-13.55, Ca~8.83 Example 6: Preparation of dendrimers with terminal carboxylate groups.
Hal~-generation starburst polyamidoamines were hydrolyzed to convert their terminal methyl ester groups to carboxylates. Thi3 generated spheroidal molecules with negative charges dispersed on the periphery. The dendrimers hydrolyzed ranged ~rom 0.5 generation (three carboxylateq) to 6.5 generation (192 carboxylates).
The products could be generated as Na+, K+, Cs+
or Rb~ salts.
Example 7- Encapsulation of R(+) - Limonene in Polyamidoamine Starburst Dendrimers A 5-50 weight percent solids solution in methanol of ~tarburst - PAM~M dendrimer (M.W. about 175,000; generation - 9.0) is added dropwise to (R(+) limonene in methanol until saturated. The solu~ion is stirred at room temperature (about 25C) for several hours and then devolatized on a Buchi rotovap at room temperature to give a solid product. Warming at temperatures greater than 80C gives solvent insoluble products which retain substantial amounts of (R~)-limonene in an encapsulated form. Theqe product3 are excellent prototypes for slow release of (R~)-limonene as a fragrance and deodorizer product.

35,444-F"B" -60-~3~24 Example ~: Encapsulation of Heavy Metal Salts in Polyamindoamlne Starburst Dendrimers A 5-50 weight percent solids solutlon in water of starburst PAMAM dendrimer (M.W. about 350,000; gen~eration = 10.0) ls stirred as a saturated solutlon of lead acetate ~Pb(C2~302)2]
is added dropwise. The solution ls stirred at room temperature (about 25C) for several hours and then ~evolatilized on a B~chi rotorap to give solld products. Scanning transmls~ion electronmicrograph of these products showed that these heavy m0tal l~ salts are encapsulated ln the lnterlor of the dendrlmers. These fllms contalnlng heavy metal salts are useful as s~lelds for absorblng electromagnetlc radlatlon.

ExamPle 9: Encapsulatlon of Fluoresceln (water soluble) Dye ln Polyamldoamlne Starburst Dendrlmers A 50-50 welght percent sollds solutlon (H20/CH30H) of starburst-PAMAM dendrlmer (M.W. about 175,000; generation = 9.0) is stirred as fluoresceln, dlsodlum salt (Acld Yellow 73, Cl.
45350; Uranine; ; avallable from Aldrlch Chemical Co. (Milwaukee, WI) is added untll saturated. The solutlon ls stlrred at room temperature (about 25C) for several hours and then devolatlllzed at room temperature to give a colored solld product. These dye encapsulated dendrlmers are excellent reference probes for calibrating ultrafiltration membranes.

ExamPle lO: Preparation of dendrlmers wlth termlnal fluorescent groups A. Reactlon of Amine Terminated Dendrlmer wlth N-Dansyl A~lrldlne Trade-mark 61 ~f~

~3~ 2l~

A sample (1.5 g, 1.6 x 10-3 mole) of starburst polyethylenelmlne (LPEI), G = 3.0, terminal groups (Z) = 12, M.W.
= 920) ls dissolved ln 20 ml of methanol. The solutlon was stlrred and 0.884 g (3.84 ~ 10-2 mole) of a solutlon of N-dansyl azirldine (ICN Biomedlcals, Costa Mesa, CA) is added dropwise over a period of 20 mlnu~es. The reactlon mlxture ls allowed to stlr at room temperature overnlght. Removal of solvent under vacuum glves a solld product. NMR and lnfrared analysls lndlcates that the product ls covalently bonded dansyl groups ln the surface of the dendrlmer.
B. Reactlon of Amlne Termlnated Dendrlmers wlth Dansyl Chlorlde A solutlon of starburst pol~amldoamine (?..0 g, 1.9 x 10-4 mole) (lnitiated from ammonla, G = 4.0, termlnal groups (Z) =
24, M.W. = 5,147) in 30 ml of water ls stlrred ln a 3-neck flask wlth 80 ml of toluene whlle a solution of dansyl chlorlde (1.23 g, 4.5 x 10-3 mole) (5-dlmethyl-amino-1-naphthalenesulfonyl chloride, from Aldrlch Chemlcal Co., Mllwaukee WI) ~n 40 ml of toluene is added dropwise whlle coollng wlth lce. Concurrently, a solutlon of 10~ NaOH (13.3 mole, 10% excess) is added to the reactlon mixture to glve an olly ball. The product ls washed wlth water, dlssolved ln methanol, and preclpltated wlth dlethyl ether to glve a solld product. NMR and infrared analysls ls consistent with covalently bonded dansyl groups in the dendrimer surface.

-63- 13~6~2ll Example 11: Demonstration of multiple chelation of iron by a sodium propionate tsrminated sixth generation starburst polyamidoamine.
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 HC1 reduced the pH to 6.3. Ferric chloride was added (0.5 ml of 0.1.2M solution, 0.051 mmol) producing a light brown gelatinous precipitate. On heating at 60C for 0.5 hours, the gelatinous precipitate became soluble, resulting in a homogeneous orange solution. The solution wa~ filtered through Biogel P2 acrylamide gel tlO 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 ferric ions per mole of starburst dendrimer.

35,444-F"B" -63-~31~2~

Table III

Theoretical Found Na4Fe20H 1 28SB NasFe20H 1 27SB Na6Fe20H 1 26sg Na0.39,0.24 0.25 0.31 0.3 (0.31 0.1%) Fe3.14,3.11 3.05 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 o ~ 25.03 25.02 25.01 Mwt. 36632.23 36654.21 36375.18 SB = C1s21H2467N379573 These results confirm chelation of 20~2 moles of ferric ions per mole of starburst dendrimer.

35,444-F"B" ~64- _~

Claims (23)

1. A dense star polymer conjugate which comprises at least one dense star polymer associated with at least one unit of at least one carried material wherein the carried material is other than an agricultural or pharmaceutical carried material.

2. The conjugate of claim 1 wherein the dense star polymer is a dense star polymer dendrimer.

3. The conjugate of claim 1 or 2 wherein at least one of the carried materials is a signal generator, signal reflector, or signal absorber.

4. The conjugate of claim 2 wherein there are at least two different carried materials, at least one of which is a target director.

5. The conjugate of claim 1 wherein the dendrimer contains discontinuities.

6. A dense star polymer conjugate of claim 1 of the formula:

(P)x * (M)y wherein each P represents a dendrimer; x represents an integer of 1 or greater; each M represents a unit of a carried material wherein the carried material is other than an agricultural or pharmaceutical carried material, said carried material can be the same carried material or a different carried material; y represents an integer of 1 or greater; and * indicates that the carried material is associated with the dendrimer.

7. The conjugate of claim 6 wherein M is signal reflector, or signal absorber.

8. The conjugate of claim 6 wherein x=1 and y=2 or more.

9. The conjugate of claim 7 wherein y=2 or more.

10. The dense star polymer conjugate of claim 6 wherein the molar ratio of any ionic M to P is 0.1-1,000:1.

11. A dense star polymer conjugate composition which comprises one or more dense star polymer conjugates of any one of claims 1, 2, or 4 to 11 and at least one suitable diluent or carrier.

12. A use of a dense star polymer conjugate of any one of claims 1, 2 or 4 to 11 as a carrier for a dye, fragrance, fluorescing entity, paramagnetic entity, pheromone or election beam opacifier.

13. A process for preparing (P)x * (M)y (I) wherein each P represents a dendrimer; x represents an integer of 1 or greater; each M represents a unit of a carried material wherein the carried material is other than an agricultural or pharmaceutical carried material, said carried material can be the same carried material or a different carried material; y represents an integer of 1 or greater; and * indicates that the carried material is associated with the dendrimer, which comprises reacting P with M at a temperature which facilitates the associa-tion of the carried material (M) with the dendrimer (P).

14. A process according to claim 13 wherein P is reacted with M in a solvent.

15. The process of claim 13 wherein the temperature is from room temperature to reflux.

16. The process of claim 14 wherein the solvent is water, methanol, ethanol, chloroform, acetonitrile, toluene, dimethyl-sulfoxide or dimethylformamide.

17. The conjugate of claim 2 wherein the dense star polymer dendrimer is of the formula wherein the core is # of terminal groups per dendritic branch =

;

G is the number of generations; Nr is the repeating unit multiplicity which is at least 2; Nc is the valency of the core compound; the terminal moiety is determined by the following:
# of terminal moieties per dendrimer =

wherein Nr, G and Nc are as defined above, and the Repeat Unit has a valency or functionality of Nr + 1 wherein Nr is as defined above.

18. The conjugate of claim 2 wherein the dense star polymer dendrimer is of the formula wherein i is 1 to t-1; the core compound is represented by the formula ? (Z)Nc where ?

represents the core Zc represents the functional groups bonded to ?
and N represents the core valency; the Repeat Unit is represented by the formula XiYi(Zi)Ni wherein "i" is defined as above; the final or terminal units are represented by 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; the n function is the product of all the values between its defined limits, such as i-1 H 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 .

19. A process for preparing a dense star polymer conjugate as defined in claim 1 which comprises the reaction of a dense star polymer, having reactive moieties, with an aniline moiety, unprotected or in which the NH2 group is protected by an N-phthalimide of the formula and contacting the reactants or the product with said carried material.

20. A process for preparing a dense star polymer conjugate as defined in claim 1 which comprises the reaction of a dense star polymer, having reactive moieties including NH2 groups which are unprotected or protected by a protecting group for an amine which is inert under the conditions of the dense star polymer synthesis and contacting the dense star polymer with said carried material.

21. A process for preparing a dense star polymer polyethyl-eneimine which comprises reacting a dense star polymer polyethyl-eneiminemethane sulfonamide with hydrochloric acid and contacting the reactants or product with a carried material wherein the carried material is other than an agricultural or pharmaceutical carried material.

22. A process for purifying a dense star polymer dendrimer conjugate according to claim 2 having a solvent present which comprises removing the solvent by ultrafiltration using a membrane.

23. The process of claim 22 wherein the solvent is ethylenediamine.