CA1316364C - Starburst conjugates - Google Patents

Abstract

ABSTRACT

Starburst* conjugates which are composed of at least one dendrimer in association with at least one unit of a carried agricultural material have been prepared. These conjugates have particularly advantageous properties due to the unique characteristics of the dendrimer.
*Trade-mark 35,444-F

Description

- -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 ~' .,;
.

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 ~, . .

.. ..

,~

.. .

1 31 63~

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'~, ~ , . . . ~ , ~ . -, .

, , : , .

1 31 63G!~

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-. .
' '. ~ `

-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 -35,444-F -6-.
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:. , ~ .

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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-~' ; .
.
. . .
. .~ .
, ' ;~

-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.

.

` 35 :

: 35,444-F -8~

:::

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`` 1 3 1 6 3 ,', ''lt a`l 0 r ~ f¢ N ~

0~ ~ N 7~ ~ , N~

~¦ N ~ o~ D N 1¢ 111 q~ ~

Ç ~ ~1 0 f C I O ~ o O ,1 I ~llS ~
¦ N'I O ~ n l I N ~ G~ ~ N~ .¢ ~ ~1 a~ .
.-- ~ ~ N 11 _~ N ~ X ~3 l ~1 X O
C ~ S N N o~:l N .~1 U
N~ N e ~ -I
~ .~1 a ~
", 5 ~ N 01 . C
N O N ~ _I U7 0 0 ~1 11 u ~ a .~ N = ~ U ~ . ~ , ~ .
~ 0 ,~ O ,0~ cn O
~ ~ x ~ e u~ Q
35, 444-F : 9 . ' ~

1 31 63k~1~
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 `~' ~ : 10 " ` .

..... , ~

,:
--- 1 31 63G l "

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 : ' ' ' ~ ' : , ' , . ..

1 3 1 6 3 ~ /lr 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 '~

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 ' -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-.: ~ - . :

~ .

~,, 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-:

- 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~

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~

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 H2N ~ CH2CH

Il .

f ( CH3 ) 3cocNH ~)--CH2CH

O
:

( CH3 ) 3COCNH ~>--CH2CH

:
:

, :

35~ :

` ~ 35, 444-F:: ~ -34 `.'~' `
i~,,~, ,, .:. : , :

1 3 1 6 3 6 /r -35- -.

H2N ~, \~CH2CH

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, 02N /\ ~ CH2CH ; and 1 C f HZCH2NH2 r 1~ 02N~</~)> CH2(~
CH2~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--~

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 (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~

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:

Compound #2 + >

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~

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 >

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:

/--\ 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:

Compound #6 + >

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 -~ >

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~

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 .

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-,

Claims (24)

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 agricultural 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 agricultural materials is a pesticide, radio-nuclide, chelator, chelated metal, toxin, 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 and at least one of which is a bioactive agent.

5. The conjugate of claim 4 wherein the target director is an entity specific for one or more target receptors and the bioactive agent is a radionuclide, pesticide or toxin.

6. The conjugate of claim 2 wherein the dendrimer contains discontinuities.

7. A dense star polymer conjugate of claim 1 having 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 of a carried agricultural material, said carries agricultural material can be the same carried agricultural material or a different carried agricultural material; y represents an integer of 1 or greater;
and * indicates that the carried agricultural material is associated with the dendrimer.

8. The conjugate of claim 7 wherein M is a pesticide, radionuclide, chelant, toxin, signal generator, signal reflector, or signal absorber.

9. The conjugate of claim 7 wherein x=1 and y=2 or more.

10. The conjugate of claim 8 wherein y=2 or more.

11. The conjugate of claim 7 wherein the molar ratio of any ionic M to P is 0.1-1,000:1.

12. The conjugate of claim 9 wherein the weight of the ration of any pesticide or toxin M to P is 0.1-5:1.

13. A process for preparing a dense star polymer conjugate of the formula (P)x * (M)y (I) wherein each P represents a dense star polymer dendrimer; x represents an integer of 1 or greater; each M represents a unit of a carried agricultural material, said carried agricultural material can be the same carried agricultural material or a different carried agricultural material; y represents an integer of 1 or greater; and * indicates that the carried agricultural material is associate with the dendrimer, which comprises reacting a dendrimer P with an agricultural material M, at a temperature which facilitates the association of the carried agricultural material (M) with the dendrimer (P) wherein P and M
are as defined above.

14. A process according to claim 13 wherein the reaction of P with M is effected 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, dimethylsulfoxide or dimethylformamide.

17. A dense star polymer conjugate composition which comprises one or more conjugates of any one of claims 1, 2, 6, 7, 8, 9, 10, 11 or 12 and at least one agriculturally acceptable diluent or carrier.

18. The conjugate composition of claim 17 which also contains other active ingredients.

19. A use for the delivery of at least one carried agricultural material of at least one dense star polymer conjugate, as defined in claims 1, 2, 6, 7, 8, 9, 10, 11 or 12 containing said material.

72a 64693-4102

20. 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.

21. The conjugate of claim 2 wherein the dendrimer is of the formula wherein i is 1 to t-1; the core compound is represented by the formula ? (Zc)Nc where ?
represents the core, Zc represents the functional groups bonded to ?

and Nc 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.

22. A conjugate according to claim 21 wherein the n function is defined as:
i-1 II Nn = (N1) (N2) (N3)...(N1-2) (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 IIo=1 n=1.

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

24. A process for preparing a dense star polymer conjugate as defined in claim 1 wherein the dense star polymer is a dendrimer having reactive moieties which process comprises the reaction of the dendrimer, having reactive moieties, with a compound containing an NH2 group unprotected or protected by a protecting group for an amine which is inert under the conditions used for dense star polymer synthesis and contacting the reactants or the product with said carried agricultural material.