KR970011150B1 - Starbust conjugates - Google Patents

Abstract

None.

Description

Starburst Conjugate

1 shows various generations of starburst dendrimers.

2a shows dendrimers in which branching connections are asymmetric (not identical).

Figure 2b shows a dendrimer whose branches are symmetrical (identical).

The present invention relates to the use of dense star polymers as carriers for selected materials. Recently, polymers have been developed which are referred to as dense star or starburst polymers. It has been found that the size, shape and properties of these dense star polymers or polymers can be molecularly controlled for particular end use.

Starburst polymers have the significant advantage of being able to provide a means for delivering, controlled delivery, targeted delivery and / or multiple delivery or use of carrier materials at high concentrations per polymer unit.

In a broad aspect, the present invention provides a polymer conjugate material comprising a dense star polymer or starburst polymer in combination with a desired material (hereinafter, these polymer conjugates are referred to as "starburst conjugates" or "conjugates"). Often referred to), methods of making these conjugates, compositions containing semiconductors, and methods of using the conjugates and compositions.

The conjugates of the present invention are suitable for use in various applications where specific delivery is desired. In a preferred embodiment of the invention, the starburst conjugate consists of one or more starburst polymers combined with one or more reagents.

Starburst conjugates offer significant advantages over other carriers known in the art because of the advantageous nature of the starburst polymer.

Starburst polymers exhibit a regular dendritic molecular structure that is branched in radial symmetry. These radially symmetric molecules are said to have a "starburst topology". These polymers are made in such a way that they can provide a concentric dendritic layer around the initiator core. The starburst form is formed by assembling organic repeating units in sequence on the concentric top layer of the initiation center, which is multimodal and self-replication in a geometrical manner (inside each other) through multiple molecular generations. Is introduced. The resulting highly functionalized molecules were called "dendrimers" according to their oligomeric properties as well as branched (resinous) structures. Thus, the terms "starburst oligomer" and "starburst dendrimer" are included in the term "starburst polymer". Morphological polymers having a controlled range of sizes and shapes are dendrimers covalently crosslinked through reactive end groups, which are referred to as starburst "crosslinked dendrimers". The term "crosslinked dendrimer" is also included in the term "starburst polymer".

The following description with reference to the drawings will be helpful in understanding the present invention.

The starburst polymer can be described as first degree, in which, ① denotes the initiation center (the three-functional initiation center shown to the left in this figure) and Z is shown by the second figure from the left. As endgroups, referred to as starbranched oligomers, A, B, C, D and E represent specific molecular generations of starburst dendrimers, (A) _n , (B) _n , (C) _n , (D) _n and (E) _n are starburst crosslinked dendrimers.

Starburst dendrimers bind to three distinct structural features: (a) the initiation center, (b) the inner packing (generation G) and (c) the outermost generation consisting of repeating units radially coupled to the initiation center. Monomolecular collection with the outer surface of a terminal functional group (ie, a terminal functional group). The size and shape of the starburst dendrimer molecule and the functional groups present in the dendrimer molecule are controlled by the choice of initiation center, the number of generations (ie, layers) used to form the dendrimer, and the choice of repeat units used in each generation. Can be. Dendrimers can be separated at any particular generation, so a means for obtaining dendrimers of desired properties is provided.

The choice of starburst dendrimer components affects the properties of the dendrimer. Since the shape of the initiation center influences the shape of the dendrimer, it is possible to obtain, for example, spherical dendrimers, columnar or rod dendrimers, elliptical dendrimers or mushroom dendrimers (depending on the choice of initiation center). The successive formation of generations (ie, the number of generations, and the size and nature of the repeating units) determines the size of the dendrimers and the properties within them.

Starburst dendrimers are branched polymers containing dendritic branches with functional groups distributed at the ends of the branches, so they can be prepared to have a variety of properties. For example, the macromolecule shown in FIG. 2a, and starburst dendrimers as shown in FIG. 2b, may have distinct properties along the length of the branches. The dendrimer morphology shown in FIG. 2a (such as Denk walter, US Pat. No. 4,289,872) is characterized by branching of asymmetric (not identical fragments), outer (i.e., surface) groups (denoted by Z '), (with Z) Internal residues), and much narrower internal spaces. The preferred dendrimer form shown in FIG. 2b is a branched connection of symmetry (identical fragments) with surface groups (denoted by Z '), two different internal residues (X and 2) with varying internal spaces as a function of generation (G). Each represented by Z). As shown in FIG. 2B, the dendrimer can proceed through sufficient generations to encompass and enclose the space as a whole to obtain an object with an almost empty interior and a highly dense surface. In addition, starburst dendrimers exhibit "starburst dense packing" when the surface of the dendrimer contains sufficient terminal residues when progressed through sufficient generation.

The surface chemistry of the dendrimer can be controlled in a predetermined manner by selecting repeating units containing the desired chemical functionality or by modifying all or part of the surface functionalities in an attempt to construct new surface functionality.

In another use of starburst dendrimers, the dendrimers can be joined together to form polydendritic moieties (starburst "crosslinked dendrimers") that are also suitable as carriers.

Dendrimers may also be prepared to deviate from uniform molecularization at a particular generation to provide a means of imparting properties different from discontinuities (ie, deviating from uniform molecularization at specific locations within the dendrimer).

Starburst polymers used in the starburst conjugates of the invention can be prepared by methods known in the art, such as in US Pat. No. 4,587,328.

Dendrimers have a highly uniform form and most importantly can be prepared to allow more functional groups per unit surface area of the dendrimer, which dendrimers have the same molecular weight, the same center and monomer components and the same number of starburst polymers. It may have a larger number of functional groups per unit molecular volume than other polymers having a central branch. Increasing the functional group density in dense starburst polymers allows higher amounts of material to be transported by the dendrimer. Since the number of functional groups present on the dendrimer can be controlled both on the surface and inside, a means of controlling the amount of biologically active agent delivered per dendrimer is also provided.

Similarity may exist between the starburst dendrimer of the early generation (ie generation = 1-7) and the classic spherical Michel. Dendrimer-Michel similarity is derived by comparing features that generally have, such as shape, size, and surface.

TABLE 1

In Table 1, morphology was verified by microscopy by scanning electron microscopy (STEM) and intrinsic viscosity (η) and size exclusion chromatography (SEC) measurements. Surface cohesion was demonstrated by titer and NMR in high fields. Area / surface groups were calculated from SEC hydrodynamic measurements.

The first five generations of Starburst Polyamidoamine (PAMAM) dendrimers are microregions that mimic very similarly to classic spherical micelles in almost every aspect (ie, shape, size, number of surface groups, and area / surface groups).

However, an important difference is that they are already covalently fixed and are stronger than Michelle's mechanical equilibrium. This difference is quite advantageous when using these microregions as an encapsulation device.

If we add more concentric generations after the fifth generation, surface densification occurs. Barrier properties at these surfaces can be increased, which results in a smaller surface area per leading (surface) group, as shown in Table II.

[Characteristics of PAMAM Dendrimer According to Generation II]

* Fluid dynamic diameter

) 폴리에틸렌옥사이드 표준에 대하여 계산한 크기배제 크로마토그라피 측정치로 결정한다.Univariate (

) Determined by size exclusion chromatography measurements calculated against polyethylene oxide standards.

1 Å = 10 ^1- nm; 1 Å ² = 10 ^-2 nm ² ; 1 Å ³ = 10 ^-3 nm ³

For example, amine end generations 5.0, 6.0, 7.0, 8.0 and 9.0 show surface area reductions of 104, 92, 73, 47 and 32 GPa per Z group, respectively. This property corresponds to a transition from a less dense miche-like surface to a more dense bilayer / monolayer barrier layer surface, usually tightly combined with a vesicle (liposomal) or Langmuir-Blodgett type thin film.

If such surface densification occurs, changes in physical properties and morphology should be observed as an increase in generation from the intermediate generation (6-8) to the advanced generation (9 or 10). Scanning electron micrographs (STEM) for generations = 7.0, 8.0 and 9.0 were obtained after removing the methanolol from each sample to give a colorless, pale yellow solid film and coloring with osmium tetraoxide. Morphological changes, for example, occur at generation G = 9.0. The microregions in generation G = 9.0 are estimated to be about 33 mm 3 in diameter, surrounded by a colorless border of about 25 mm 3 in thickness. Methanolic solvent was trapped inside the 25 kPa outer thin film barrier to provide a black colored interior. Thus, at generation = 9.0 Starburst PAMAM morphologically behaves like vesicles (liposomes). However, this starburst is smaller in size and monodisperse than liposomes. Accordingly, the dendrimer of the present invention can be used to molecularly encapsulate a solvent filled in a space having a diameter of about 33 mm 3 (vol. 18,000 m 3) or more.

In addition, the number of functional groups present on the dendrimer can be controlled both at the surface and inside, thus providing a method of controlling the amount of material carried per dendrimer. In one aspect of the invention, the dendrimer is a target carrier of a material capable of delivering the carrier material to a particular location.

Dendrimers suitable for use in the conjugates of the present invention include the dense star polymers or starburst polymers described in US Pat. Nos. 4,507,466, 4,558,120, 4,568,737 and 4,587,329.

In particular, the present invention relates to starburst conjugates comprising at least one kind of starburst polymer combined with at least one kind of carrier material. Starburst conjugates falling within the scope of the present invention include conjugates represented by the following general formula (I):

(P) _x * (M) _y (I)

Wherein each P is a dendrimer; x is an integer of 1 or more; Each M is a unit (eg, molecule, atom, ion, and / or other basic unit) of a carrier (except pharmaceutical), which may be the same or different from each other; y is an integer of 1 or more; * Indicates that the carrier material is combined with a dendrimer.

Preferred starburst conjugates of formula (I) are those in which M is a signal generator (e.g. a fluorescent substance), a signal reflector (e.g. a paramagnetic object), a signal absorber (e.g. an electron beam impermeable), a fragrance, a pheromone or a dye Phosphorus conjugates, particularly preferred conjugates are those where x = 1 and y = 2 or more.

Starburst conjugation of Formula (I), a starburst crosslinked dendrimer, covalently bonded together, optionally through a bonding group, such that the starburst dendrimer can form polydendritic aggregates (ie, x> 1) Gates are also included.

As used herein, the term "coupled with" means that the carrier (s) is encapsulated or trapped within the center of the dendrimer, partially or fully dispersed throughout the dendrimer, attached to or bound to the dendrimer, or It can be by a combined way. Coupling the carrier material (s) with the dendrimer (s) may optionally use linking groups and / or spacers to facilitate the manufacture or use of starburst conjugates. Appropriate linking groups link the target director to the dendrimer (ie P) with little compromise to the target director (ie T) efficiency or the efficiency of other carrier (s) (ie M) present in the starburst conjugate. It is a group to combine. These linking groups may be separable or non-separable, and they are typically used to avoid steric hindrance between the target director and the dendrimer, and it is preferred that the linking group is stable (ie, inseparable).

Since the size, shape and functional group density of starburst dendrimers can be precisely controlled, there are many ways to link the carrier material with the dendrimer.

For example, (a) there may be a covalent, Cooulomibic, hydrophobic or chelated form of linkage between the carrier (s) and an entity (typically a functional group) located at or near the surface of the dendrimer. There is; (b) there may be a covalent, coulomb, hydrophobic or chelated form of linkage between the carrier material (s) and the electricity located inside the dendrimer; (c) a dendrimer can be prepared to provide a substantially empty space so that the carrier material can be physically confined within (empty volume) and the surface of the dendrimer can be densely packed with diffusion control residues to control the release of the carrier material arbitrarily; Alternatively, (d) various combinations of the above-mentioned phenomena may be used.

Dendrimers denoted herein as "P" include the dense star polymers described in US Pat. Nos. 4,507,466, 4,558,120, 4,568,737 or 4,587,329.

Carriers indicated herein as "M" and suitable for use in the starburst conjugate include any material that can be combined with the starburst dendrimer without compromising the physical inherent properties of the starburst dendrimer, with the exception of pharmaceutical substances. Any material may be included, for example, metal ions (e.g. alkali and alkaline earth metals), signal generating factors (e.g. fluorescent objects), signal reflecting factors (e.g. paramagnetic objects), signal absorbing factors (e.g. electron beam impermeability). Problem), pheromone residue, perfume residue, dye residue and the like. Carriers include chelating agents or scavengers capable of selectively eliminating various agents.

Starburst conjugates of formula (I) are prepared by reacting P with M, which is usually carried out in a suitable solvent at a temperature which promotes the binding of the carrier (M) and the starburst dendrimer (P).

Suitable solvents are those in which P and M can be at least partially miscible and inert to the formation of conjugates. No solvent is required if P and M are at least partially miscible with each other. If desired, a mixture of appropriate solvents may be used. Such suitable solvents are, for example, water, methanol, ethanol, chloroform, acetonitrile, toluene, dimethylsulfoxide and dimethyl formamide.

The reaction conditions for forming the starburst conjugate of general formula (I) depend on the nature of the particular dendrimer (P), the pharmaceutical substance carried (M) and the bond (*) formed. Typically, the temperature can be varied from room temperature to reflux. The choice of particular solvent and temperature is well known to those skilled in the art.

The ratio of M to P depends on the size of the dendrimer and the amount of carrier. For example, the molar ratio of ionic material M to P is usually 0.1 to 1000: 1. Preferably it is 1-50: 1, more preferably 2-6: 1. The weight ratio of the organic substance M contained in P is 0.1-5: 1, Preferably it is 0.5-3: 1.

Particularly preferred starburst conjugates are conjugates which contain a target directing agent (denoted herein as "T") and are represented by the following general formula (II).

(T) e * (P) x * (M) y (II)

Wherein each target director is: e is an integer of at least 1: P, x, *, M and y are as defined above.

Among the starburst conjugates of general formula (II), it is preferable that M is a signal generating factor, a signal reflecting factor or a signal absorption factor. Also preferred are conjugates in which e is 1 or 2, and conjugates in which x is 1 and y is 2 or more. Particular preference is given to conjugates in which x = 1, e = 1, y = 2 or more and M and T are linked with the polymer via the same or different linking groups.

Starburst conjugates of formula (II) can be prepared by forming T * P and then adding M, or by forming P * M and then adding T. These reactions are carried out under temperatures which do not harm certain conjugate components and, if necessary, in the presence of a suitable solvent. To adjust the pH, a buffer or an appropriate acid or base is used. The reaction conditions depend on the binding form formed (*), the starburst dendrimer (P) used, the pharmaceutical substance (M) to be transported, and the target director (T). It is also possible to chelate P and M, usually in water, followed by conjugation with T. Conjugation with T is carried out in a suitable buffer.

The ratio of T: P is preferably 1: 1. The ratio of M: P is as described above.

A target director capable of targeting a starburst conjugate is a substance used in the starburst conjugate of the present invention to deliver at least a portion of the starburst conjugate to a desired target, and including chemical functional groups that exhibit target specificity, and the like. .

In the absence of a target director (or in the presence of a target director in some cases), all or a substantial portion of these functional groups may be anionic, due to the number of functional groups that may be located on or near the dendrimer. Cationic, hydrophobic or hydrophilic can be effectively assisted in delivering the starburst conjugate to the desired target, the hydrophobic or hydrophilic compatible target, before opposition.

Processes for preparing the conjugates of formula (II) using P with protected handles (S) are also regarded as methods for preparing the conjugates of formula (II). The scheme is as follows:

In the above scheme, S * P is a dendrimer: S * P * M is a protected dendrimer conjugated with M: P * M is a dendrimer conjugated with M (starburst conjugate): T * P * M is a starburst conjugate associated with a target director.

In preparing the conjugate, a suitable solvent which does not affect P * M may be used. For example, when S is t-butyloxycarbonyl, S can be removed with an aqueous acid.

Starburst conjugates are also preferred in which the polymer is directly linked or connected via a linker, these starburst conjugates being represented by the following general formula (III):

[(T) _e- (C ') _f ] _g * (P) _x * [(C ") _h- (M)] _y ) _k (III)

Wherein each C 'is the same or different linking group: each C "is the same or different linking group: g and k are each an integer of at least 1: f and h are each an integer of at least 0 and-- If a linking group is present, it represents a covalent bond: P, x, *, M, y, T and e are as defined above.

Starburst conjugates can be used in diagnostic or in vitro diagnostic applications (e.g., radioimmunoassays, electron microscopy, enzyme-linked immunosorbent assays, nuclear magnetic resonance spectroscopy, contrast imaging and immunocytographies), for assays or other useful applications. It can be used as a starting material for preparing a medicament.

The present invention also relates to starburst conjugate compositions wherein the starburst conjugate is formulated with other suitable excipients. The starburst conjugate composition may optionally contain other active ingredients, additives and / or diluents.

Preferred starburst conjugate polymers for use in the starburst conjugates of the present invention are described as starburst polymers having at least one branch extending from the center (hereinafter referred to as the central branch), preferably having at least two branches. Polymers, wherein the branch has at least one or more end groups, provided that (1) the ratio of end groups to the central branch is greater than 1, preferably 2 or more, and (2) per unit volume in the polymer The endgroup density is that of extended conventional star polymers having residues of similar centers and monomers and having the molecular weight and number of the central branches (these branches of the extended conventional starpolymers each contain only one endgroup). At least 1.5-fold and (3) molecular volume using a scaled Corey-Pauling molecular model No more than about 80% of the volume of the extended conventional star polymer molecule as measured by the dimensional study. The term "dense" as used herein to modify "star polymer" or "dendrimer" means that the molecular volume is smaller than that of an extended conventional star polymer of the same molecular weight. Extended conventional star polymers used as base materials for comparison with dense star polymers are the same as those of dense star polymers having a molecular weight, the center and monomer components, and the number of center branches. The term "extended" means that the branches of conventional star polymers are each of their maximum length, elongated or elongated so that such branches can survive if the star polymer is completely dissolved in a solvent ideal for the star polymer, for example. . In addition, although the number of end groups of dense star polymer molecules is higher than that of conventional star polymer molecules, the chemical structure of the end groups is the same.

Dendrimers used in the conjugates of the present invention can be prepared by methods known in the art. The dendrimers, shell reactants and core compounds, and methods for their preparation, are as defined in US Pat. No. 4,587,329.

Dendrimers used in the conjugates of the present invention may contain endgroups that are reactive enough to afford the addition or substitution reaction.

Examples of such endgroups are amino, hydroxy, mercapto, carboxy, alkenyl, allyl, vinyl, amido, halo, urea, oxiranyl, aziridinyl, oxazolinyl, imidazolinyl, sulfonato, Phosphonato, isocyanato and isothiocyanato. Dendrimers differ from conventional star or star-branched polymers in that the concentration of endgroups per unit volume of molecule is higher than conventional extended star polymers having the same number of central branches and the same length of central branches. Accordingly, the terminal group density per unit volume in the dendrimer is usually at least 1.5 times, preferably at least 5 times, more preferably at least 10 times, and preferably 15 to 50 times the conventional extended starpolymer density. The end groups per central branch in the dense polymer are preferably at least 2, more preferably at least 3, most preferably 4 to 1024. Preferably, for a given polymer molecular weight, the molecular volume of the dense star polymer is less than 70 volume percent, more preferably 16 to 60 volume percent, most preferably 7 to 50 volume percent of the syntactic elongated star polymer.

Preferred dendrimers for use in the conjugated cones of the present invention are characterized by having a monovalent or multiatomic center covalently bonded to the dendritic branch. This sequential branching can be explained by the following sequence in which G represents the number of generations.

Mathematically, the relationship between the number of end groups (#) in the dendritic branch and the number of generations in the branch can be expressed as

(Where G is the number of generations and Nr is the degree of redundancy of the repeating units which is at least two or more in the case of amines). The total number of end groups present in the dendrimer is determined as follows:

(Where G and Nr are as defined above, and Nc represents the valence of the central compound (often referred to as central functional group)). Therefore, the dendrimer of the present invention can be represented by its component parts as follows.

Where the center, terminal residue, G and Nc are as defined above and the repeating unit has a valence or functionality of Nr + 1 (where Nr is as defined above)

Dendrimer copolymer dendrimers preferred for the purposes of the present invention are the only compounds composed of multifunctional monomer units in a highly branched (aqueous) arrangement. Dendrimer molecules are prepared from multifunctional initiation units (central compounds), multifunctional repeat units, and terminal units that are the same or different from the repeat units. The central compound is represented by the general formula ① (Z ^c ) N _c (wherein ① represents a center; Z ^c represents a functional group bonded to ①; N _c is a functional group of the center, preferably two or more, and three or more. Is most preferred). Thus, the dendrimer molecule comprises a plurality (N _c) of functional groups is made up of a multifunctional center ① bonded to Z ^c, functional group Z ^c are respectively the first generation repeating unit X ¹ Y ¹ (Z ¹⁾ one of the N ¹ Connected to the functional terminus, the Z groups of one generation of repeating units are each linked to one functional terminus of the repeating units of the next generation until the final generation is reached.

The repeating unit in the dendrimer molecule is the same in a single generation, but may vary with generation. In the repeating unit X ¹ Y ¹ (Z ¹ ) N ¹ , X ¹ is the monofunctional terminal of the first generation repeating unit, Y ¹ is the residue constituting the first generation, and Z ¹ is the first generation repeating unit The functional group of the multifunctional leader of the central compound ① (Z ^c ) N _c or may be the same or different from the functional group of other generations, N ¹ represents the degree of redundancy of the multifunctional leader of the first generation repeating unit 2 In the above, most preferably, it is 2, 3, or 4.

In general, the repeating unit is of general formula XⁱYⁱ(Zⁱ)_Ni(Where "i" represents a specific generation ranging from the first generation to t-1 generations). Therefore, in the preferred dendrimer molecule, each Z of the first generation repeating unit^OneX of the second-generation iteration unit²Repetition unit X in generation "i" connected toⁱYⁱ(Zⁱ)_NiEach ZⁱThe group is the end of the repeating unit in the generation number "i + 1" (X^{i + 1}), Through all generations. The terminal or terminal of the preferred dendrimer molecule is terminal X^tY^t(Z^t)_NtWhere t represents an end generation and X^t, Y^t, Z^t, And N^tZ^tThere is no next generation connected to the group and N^tX except that is less than 2 (for example, 0 or 1)ⁱ, Yⁱ, ZⁱAnd NⁱAnd may be the same as or different from). Accordingly, preferred dendrimers have the following molecular formula:

Where i is from t-1

In the above formula, the symbols are as defined above.

Is a product of all the values within the defined limits.

이고, 이는 하나의 수지상 분지의 i번째 세대를 구성하는 반복단위 XⁱXⁱ(Zⁱ)_Ni의 수이며, i가 1인 경우에는

이다. 공중합체 덴드리머에 있어서, 한 세대의 반복단위는 적어도 하나이상의 다른 세대에 존재하는 반복단위와 상이하다. 바람직한 덴드리머는 이후에 기술되는 구조식에서 알 수 있는 바와 같이 대단히 대칭적이다. 바람직한 덴드리머는 또하나의 시약과 접촉시킴으로써 작용화된 덴드리머로 전환시킬 수 있다.therefore,

, Which is the number of repeating units X ⁱ X ⁱ (Z ⁱ ) _Ni constituting the i th generation of one dendritic branch, where i is 1

to be. In copolymer dendrimers, the repeating unit of one generation is different from the repeating unit present in at least one other generation. Preferred dendrimers are highly symmetric, as can be seen in the structural formulas described below. Preferred dendrimers can be converted to functionalized dendrimers by contacting with another reagent.

For example, when the end generation hydroxyl is converted to an ester by reaction with an acid chloride, an ester terminated dendrimer is obtained. Since such functionalization need not be performed up to the theoretical maximum defined by the number of functional groups available, the functionalized dendrimers may not have a precisely defined molecular formula, such as when using highly symmetrical or preferred dendrimers.

In homopolymer dendrimers, all repeat units X ⁱ X ⁱ (Z ⁱ ) _Ni are the same. Since the values of N ⁱ are all the same (defined as N ^r ), the product of the number of repeat units decreases to a simple exponential form. Therefore, the molecular formula can be represented in a simpler form as follows.

Where i = 1 to t = 1

This form still shows the distinction between different generations i, each consisting of N _c N _r ^(i-1) repeating units X ⁱ X ⁱ (Z ⁱ ) _Ni . Generations, combined in one term:

or

In the above, X ^r X ^r (Z ^r ) _Nr are all repeating units used for generation i.

In conclusion, the polymer is a starburst polymer if the polymer compound meets these general formulas. Conversely, the polymer is not a starburst polymer unless the polymer compound meets these general formulas. In addition, in order to confirm whether or not a polymer is a starburst polymer, it is not necessary to know a manufacturing method, and only to know whether it meets these general formulas. The general formula also demonstrates generation (G) or layered structure of the dendrimer.

There are several ways to measure the ratio of medicine to dendrimer (P), which depends on where and how the binding of P * M occurs. If internal encapsulation is present, the weight ratio of M: P is usually 10: 1, preferably 8: 1, more preferably 5: 1, most preferably 3: 1. The ratio may be as low as 0.5: 1 to 0.1: 1. When internal stoichiometry is used, the weight ratio of M: P is the same as in the case of internal encapsulation. When the external stoichiometry is measured, the mole / mole ratio of M: 9 is given by the following equation.

In the above, N _c means the degree of redundancy of the center: N _t means the degree of redundancy of the end group: N _r means the degree of redundancy of the branched link. N _c N _t N _r ^G-1 represents the number of Z groups. Thus, for example, (A) above may be the result of the presence of proteins, enzymes or highly charged branches on the surface; (B) may be the result of being octinoic acid; (C) may be the result when it is a carboxylate ion or group.

Of course, those skilled in the art will be able to easily produce other structures of various dimensions by appropriately changing the dendrimer component and the number of generations used. Linear polymers of comparable molecular weight (in fully extended form) have a radius of rotation, which is much larger than dendrimers of the same molecular weight.

Binding of the target director to the dendrimer is another aspect of the present invention. In a preferred embodiment of the invention, particularly in the case where it is desired to use an antibody as a target directing agent, reactive functional groups (e.g. carboxyl, sulfhydryl, reactive aldehydes, reactive olefin derivatives, isothiocyanato, isocyanato , Amino, reactive aryl halides or reactive alkyl halides) can be conveniently used on the dendrimer. Reactive functional groups can be introduced into the dendrimer by known techniques, for example:

(1) Use of heterofunctional initiators incorporating different reactive groups (as starting material for the synthesis of dendrimers). In such heterofunctional initiators, at least one of the functional groups serves as an initiation site for dendrimer formation and at least one of the remaining functional groups is used for binding to the target director but cannot initiate dendrimer synthesis. For example, the use of a protected aniline allows for further modification of the NH ₂ group in the molecule without reacting the NH ₂ of the aniline.

The functional groups that can be used for binding to the target director can be part of the initiator molecule in any one of three forms:

(a) The form used for binding to the target director. This is possible when no reaction occurs at the center of any of the synthetic steps involved in the dendrimer synthesis.

(b) if the functional group used for binding to the target director is reactive at the synthesis step involved in the synthesis of the dendrimer, it will be non-reactive to the synthesis process involving the group and the integrity of the remainder of the macromolecule The functional group can be protected by using a protective group that can be easily removed without changing.

(c) If it is not possible to form a simple protecting group for the reactive functional group to be used for binding to the target director, synthetic precursors which are non-reactive may be used in all synthetic processes used for the synthesis of dendrimers. Once the synthesis is complete, this functional group should be readily convertible to the desired linking group using a method that does not change the integrity of the rest of the molecule.

를 사용한다.(2) The reagent used to bind (covalently) the desired reactive functional group onto a preformed dendrimer, which must contain a functional group that readily reacts with the terminal functional group of the dendrimer. The functional group that is ultimately intended for binding to the target agent may be in its final form as a protected functional group or as a synthetic precursor. The form of use of these binding functionalities depends on their integrity during the synthesis plant to be used and the final macromolecular ability to withstand all the conditions necessary to make this group available for binding. For example, the preferred route for PEI is

Use

Examples of heterofunctional initiators for use in (1) above include the following representative examples.

There are some chemicals that are particularly important:

1) Starburst polyamidoamide ("PAMAM")

2) Starburst Polyethyleneimine ("PEI")

3) Starburst PEI compound with PAMAM surface:

4) Starburst Polyether ("PE") Chemical Properties.

Dendrimer surface functional groups can be modified to provide other useful functional groups, polyethers or other immunosensitive residues such as:

-OPO ₃ H ₂ , -PO ₃ H ₂ , -PO ₃ H ^(-2) , -CO ₂ ^(-1) ,

-SO ₂ H, -SO ₂ ^(-1) , -SO ₃ H, -SO ₃ ^(-1) , -NR ₁ R ₂ ,

-R ⁵ , -OH, -OR ¹ , -NH ² , perfluorinated alkyl,

이고, R²은 알킬, 아릴 또는

이고, R³은 -OH, -SH, -CO₂H, -SI₂H 또는 -SO₃H이고, R⁴은 알킬 아릴, 알콕시, 하이드록실, 머캅토, 카복실, 니트로, 수소, 브로모, 클로로, 요오도 또는 플루오로이고, R⁵은 알킬이고, X는 NR, O 또는 S이고, n은 정수, 1, 2 또는 3이다).(Wherein R is alkyl, aryl or hydrogen, and R ¹ is alkyl, aryl, hydrogen or

And R ² is alkyl, aryl or

R ³ is —OH, —SH, —CO ₂ H, —SI ₂ H or —SO ₃ H, R ⁴ is alkyl aryl, alkoxy, hydroxyl, mercapto, carboxyl, nitro, hydrogen, bromo, Chloro, iodo or fluoro, R ⁵ is alkyl, X is NR, O or S, n is an integer, 1, 2 or 3).

The choice of functional group depends on the particular end use in which the dendrimer is to be used.

The following examples are intended to illustrate the invention and do not limit the scope of the invention. Examples indicated by letters relate to methods of preparing starting materials, and examples indicated by numbers relate to methods of manufacturing products.

Example A

Preparation of 2-carboxamido-3- (4'-nitrophenyl) -propanamide

P-nitrobenzyl malonate diethyl ester (2.4 g, 8.13 mmol) was dissolved in 35 ml of methanol. The solution was heated to 50-55 ° C. with stirring and bubbled in the solution with a stream of anhydrous ammonia for 64 hours. After cooling the solution, the white, furry product was filtered and recrystallized from 225 ml of boiling methanol to yield 1.85 g (7.80 mmol) of bisamide in 96% yield. (Melting point: 235.6 ° C. (d))

The structure was confirmed by MS, ¹ H and ¹³ C NMR spectroscopy.

Elemental Analysis for C ₁₀ H ₁₁ O ₄ N ₃

Theoretic Value (%): C; 50.63, H; 4.69, N; 17.72

Found (%): C; 50.75, H; 4.81, N; 17.94

Example B

Preparation of 1-amino-2- (aminomethyl) -3- (4'-nitrophenyl) propane

2-Carboxamido-3- (4'-nitrophenyl) propanamide (2.0 g, 8.43 mmol) was slurried in 35 ml of anhydrous tetrahydrofuran with stirring under agitation. To the mixture was added a borane / tetra hydrofuran mixture (106ml, 106mmole) by syringe. The reaction mixture was then heated to reflux for 48 hours to dissolve the suspended amide. The solution was cooled and tetrahydrofuran was removed in vacuo using a rotary evaporator.

The crude and borane residues were dissolved in 50 ml of ethanol and the solution was washed with anhydrous hydrogen chloride gas. The solution was refluxed for 1 hour and the solvent was removed in vacuo. The crude hydrochloride was dissolved in 15 ml of deionized water and extracted twice with 50 ml of methylene chloride. The aqueous layer was cooled in an ice bath under an argon blanket and 50% sodium hydroxide was added slowly to a basic pH of 11.7. The basic aqueous layer was extracted four times with 25 ml of methylene chloride and these mixed extracts were evaporated to yield 1.45 g of a tan oil. This oil was treated 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 mixed filtrate was evaporated in vacuo to yield 1.05 g (5.02 mmol) of the titlediamine as a clear oil (melting point: 275-278 ° C. (d) dihydrochloride).

The structure was confirmed by MS, ¹ H and ¹³ C NMR spectroscopy.

Elemental Analysis for C ₁₀ H ₁₇ N ₃ O ₂ Cl ₂

Theoretic Value (%): C; 42.57, H; 6.07, N; 14.89

Found (%): C; 43.00, H; 6.14, N; 15.31

Found (%): C; 43.00, H; 6.14, N; 15.31

Example C

Preparation of 1-amino-2- (aminomethyl) -3- (4'-aminophenyl) propane

Borane / tetrahydrofuran solution (70 ml, 70 mmol) was added to the flask containing 4-amino-benzylmalonamide (1.5 g, 7.24 mmol) with nitrogen using a cannula needle while stirring. The solution was refluxed for 40 hours. The colorless solution was cooled and excess tetrahydrofuran was removed by rotary evaporation to leave a clear gelatinous oil. When the methanol (50 ml) was carefully added to the oil, the gas was released to a noticeable degree.

Dry hydrogen chloride was dissolved with bubbling through the suspension, and then the solution was refluxed for 1 minute. Methane / hydrochloric acid was rotary evaporated and the same dissolution / reflux process was repeated for the resulting hydrochloride. The hydrochloride obtained was dissolved in 10 ml of water and cooled in an ice bath under argon.

Concentrated sodium hydroxide (50%) was added slowly with stirring to pH 11. Thereafter, the aqueous moisture was extracted twice with 100 ml of chloroform, combined, and filtered through a short silica gel plug without drying. The solvent was removed in vacuo (rotary) to give the title compound (0.90 g, 5.02 mmol) in 70% yield (Rf = 0.65-CHCl ₃ / MeOH / conc. NH ₄ OH-2: 2: 1). The structure was confirmed by ¹ H and ¹³ C NMR and used without further purification.

Example D

Preparation of 6- (4-aminobenzyl) -1,4,8,11-tetraaza-5,7-dioxoundecane

4-aminobenzyl malonate dimethyl ester (2.03 g, 8.43 mmol) was dissolved in 10 ml of methanol. To a stirred solution of freshly distilled ethylene diamine (6.00 g, 103.4 mmol) in 10 ml methanol was added dropwise over 2 hours under nitrogen. The clear solution was stirred for 4 days and analyzed by thin layer chromatography (TLC), indicating that all of the diesters (Rf = 0.1) were converted to bisamides (Rf = 0.42-20% concentrated NH ₄ OH / 80% ethanol). . This substance is strong ninhydrin positive. Methanol and excess diamine were removed by rotary evaporator and the resulting white solid was vacuum dried (10 ⁻¹ mm, 50 ° C.) overnight to give crude product (2.54 g, 8.36 mmol). Analytical samples were recrystallized from chloroform / hexanes (melting point = 160-161 ° C.), mass spectrometry, ¹ H and ¹³ C data consistent with the proposed structure.

Example E

Reaction of Mesyl Aziridine with 1-amino-2- (aminomethyl) -3- (4-nitrophenyl) propane

1-amino-2- (aminomethyl) -3- (4-nitrophenyl) -propane (400 mg, 1.91 mmol, at least 96% purity) was dissolved in 10.5 ml of anhydrous ethanol under nitrogen. Mesylaziridine (950 mg, 7.85 mmol) was added to the stirred diamine solution as a solid. The reaction was stirred at 25 ° C. for 14 hours using a magnetic stirrer, resulting in a white rubbery residue on the side of the flask. Ethanol was decanted and the residue was retreated with 15 ml of ethanol to remove all unreacted aziridine. The rubbery product was vacuum dried (10 ⁻¹ mmHg, 25 ° C.) to give tetrakismethyl sulfonamide (1.0 g, 1.44 mmol) in 75% yield. (Rf = 0.74-NH ₄ OH / ethanol = 20: 80). The structure was confirmed by ¹ H and ¹³ C hexa resonance (NMR) spectroscopy.

Example F

Preparation of 2- (4nitrobenzyl) -1,3- (bis-N, N-2-aminoethyl) diaminopropane

Crude methylsulfonamide (650 mg, 0.94 mmol) was dissolved in 5 ml of nitrogen washed concentrated sulfuric acid (98%). The solution was kept under nitrogen and heated to 143-146 ° C. with vigorous stirring for 27 minutes. Slight black light appeared and the cooled solution was poured into ether stirred solution (60 ml). The precipitated white salt cake was filtered and immediately dissolved in 10 ml of deionized water. The solution was adjusted to pH 11 with 50% NaOH while cooling under argon. The resulting solution was mixed with 90 ml of ethanol and the precipitated inorganic salts were filtered off. The solvent was removed from the crude amine under reduced pressure and 190 ml of toluene was added to the light brown oil produced under nitrogen. The mixture was stirred vigorously and water was removed by azeotropic distillation (Dean-Stark trap) until the remaining toluene became light yellow (30-40 ml remained in the pot). Toluene was cooled and decanted from black hard to handle residues and salts. The solution was removed from this solution in vacuo and the resulting pale yellow oil was vacuum dried (0.2 mm, 35 ° C.) overnight to give 21 ml of product (60%) which was confirmed by MS, ¹ H and ¹³ C CMR.

Example G

Preparation of 1/2 Generation Starburst Polymer (containing an aniline derivative) by the following reaction scheme

Compound # 2

Methyl acrylate (2.09 g, 24 mmol) was dissolved in methanol (15 ml). Compound 6- (4-aminobenzyl) -1,4,8,11-tetraaza-5,7-dioxoundecane (1.1 g, 3.8 mmol) (ie, compound # 1, as described in Example D) Similarly) was dissolved in methanol (10 ml) and added slowly to the methyl acrylate solution with vigorous stirring over 2 hours. The reaction mixture was stirred at ambient temperature for 48 hours. The solvent was removed with a rotary evaporator while maintaining the temperature below 40 ° C. Ester (Compound # 2) was obtained as yellow oil (2.6 g). No carboxy ethylation of the aniline group was observed.

Example H

Preparation of First Generation Starburst Polymer (Containing Aniline Residue) by the Following Scheme

Ester (Compound # 2) (2.6 g, 3.7 mmol) was dissolved in methanol (100 ml). This was carefully added to a solution in which ethylene diamine (250 g, 4.18 mol) and ethanol (100 ml) were vigorously stirred at a rate such that the temperature did not rise above 40 ° C. After the addition was completed, the reaction mixture was stirred at 35-40 ° C. (mentle heating) for 28 hours. After 28 hours, no ester groups were detected by the IR spectrometer. The solvent was removed at 60 ° C. on a rotary evaporator. Excess ethylenediamine was removed using a ternary azeotrope of toluene-methanol-ethylene diamine. Finally, all residual toluene was azeotropically distilled with methanol. All ethanol was removed to yield 3.01 g of product (Compound # 3) as an orange glassy solid.

Example I

Preparation of 1 1/2 Generation Starburst Polymers with Aniline Residues by the Following Scheme

Amine (Compound # 3) (2.7 g, 3.6 mmol) was dissolved in methanol (7 ml) and slowly added to a stirred solution of methyl acrylate (3.8 g, 44 mmol) in methanol (15 ml) at ambient temperature over 1 hour. It was. A slight increase in the temperature of the solution during the addition was observed. The solution was stirred at ambient temperature for 16 hours. The solvent was removed at 40 ° C. with a rotary evaporator. All solvents and excess methyl acrylate were removed to yield 4.7 g of ester (Compound # 4) as orange oil.

Example J

Preparation of 1/2 Generation Starburst Polymers with Aniline Residues by the Following Scheme

Triamine (Compound # 5, the method for preparing this compound is shown in Example C) (0.42 g, 2.3 mmol) was dissolved in methanol (10 ml) and methyl acrylate (1.98 g, 23 in methanol (10 ml) Millimoles) dropwise over 1 hour. The mixture was stirred at ambient temperature for 48 hours. The solvent was removed on a rotary evaporator while maintaining a temperature below 40 ° C. Excess methylacrylate was removed by repeated azeotropic distillation with methanol. Ester (Compound # 6) was isolated as orange oil (1.24 g).

Example K

Preparation of First Generation Starburst Polymer (Containing Aniline Residue) by the Following Scheme

Ester (Compound # 6) (1.24 g, 2.3 mmol) was dissolved in methanol (50 ml) and added dropwise to ethylenediamine (73.4 g, 1.22 mol) in methanol (10 ml) over 2 hours. A slight exotherm appeared and kept vigorous stirring. The solution was stirred at ambient temperature for 72 hours. The solvent was removed at 60 ° C. on a rotary evaporator. Excess ethylenediamine was removed using a ternary azeotrope of toluene-methanol-ethylenediamine. Finally, all residual toluene was removed using methanol and then vacuum pumped to give amine (Compound # 7) (1.86 g) as a yellow / orange oil.

Example L

Preparation of Starburst Polymers with Aniline Residues by the Next Scheme

The amine (Compound # 7) (1.45 g, trace amount of methanol remaining) was dissolved in methanol (100 ml) and dissolved in methyl acrylate (5.08 g) in methanol (20 ml) between 1 1/2 of the examples. Was added slowly over. This solution was stirred at room temperature for 24 hours. Removal of the solvent followed by repeated azeotropic distillation with methanol may remove all excess methylacrylate. The ester (Compound # 8) was isolated as orange oil (2.50 g, 1.8 mmol) by pumping on a vacuum pump for 48 hours.

Example M

Hydrolysis of 4.5 Generation Dendrimers and Preparation of Calcium Salts

4.5 generation PAMAM (ester terminated, starting from NH ₃ ) (2.11 g, 10.92 meq) was dissolved in 25 ml methanol, to which 10% NaOH (4.37 ml, 10/92 meq) was added (pH = 11.5 to 12). After 24 hours at room temperature, the pH was about 9.5. After an additional 20 hours, the solution was rotovapted and rotavaporized again by addition of 50 ml of toluene.

The resulting oil was dissolved in 25 ml of methanol and 75 ml of diethyl ether was added to precipitate the resulting oil in a white rubbery form. The liquid was decanted and the rubbery material was rotary evaporated to give a very fine gray powder which was further dried to give 2.16 g of product (98% yield).

NMR and IR analysis showed no ester group.

The sodium salt of 4.5 generation PAMAM (ester terminated, initiated from NH ₃ ) was replaced with calcium salt by dialysis. Sodium salt (1.03 g) was taken up in 100 ml of water and passed through hollow fiber dialysis tubing (cut off = 5000) at 3 ml / minute. The outside of the tubing was placed in 5% CaCl ₂ solution. This operation was repeated.

The resulting solution was dialyzed again and this time repeated two more times for water.

The solution was evaporated to produce 0.6 g of a wet solid which was dissolved (but not fully dissolved) in methanol and dried to yield 0.45 g of gray crystals.

C ₃₆₉ H ₅₉₂ O ₁₄₁ N ₉₁ Oa ₂₄ Calcd: 10.10% Ca ⁺⁺

Molecular weight = 9526.3, Content calculation value: C-4432.1, H-601.8, O-2255.9, N-1274.6, Ca-961.9

Theoretical Value (%): C; 46.5, H; 6.32, N; 13.38, Ca; 10.10

Found (%): C; 47.34, H; 7.00, N; 13.38, Ca; 10.10

Example N

Preparation of Dendrimers Containing Terminal Carboxylate Groups

The semi-generation starburst polyamidoamine was hydrolyzed to convert the terminal methylester groups to carboxylates. In this case, spherical molecules of negative charges dispersed around were produced. Hydrolyzed dendrimers ranged from 0.5 generation (3 carboxylates) to 6.5 generation (192 carboxylates).

The product can be produced as Na ⁺ , K ⁺ , Ca ⁺ or Rb ⁺ salts.

Example O

N-t-Butylcarbonyl-4-aminobenzyl dimethyl ester

4-aminobenzyl malonate dimethyl ester (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 was stirred overnight. Butanol was removed on a rotary evaporator to give the product as a yellow suspension in water. Extraction with methylene chloride, drying (MgSO 4) and evaporation gave yellow oil (21.05 g, contaminated as di-t-butoxycarbonate). 2-propanol: recrystallized from water (75:25) to give light yellow crystals (11.1 g, 33 mmol, 67%). The structure was confirmed by ¹³ C NMR and purity was checked by HPLC analysis (Spherisorb ODS-1, 0.05MH ₃ PO ₄ pH 3: CH ₃ CN (55: 45)). This material was used without further purification.

Example P

N-t-butoxycarbonyl-6-4 (4-aminobenzyl) -1,4,8,11-tetraaza-5,7-dioxoundecane

Nt-butoxycarbonyl-4-aminobenzyl malonate dimethyl ester (8.82 g, 26 mmol) prepared in Example O was dissolved in 50 ml of methanol This solution was freshly distilled ethylenediamine (188 g, 3.13 mol) and A solution of 20 ml of methanol was added dropwise (2 hours) under a nitrogen atmosphere The solution was allowed to react for 24 hours The ethylenediamine / methanol solution was removed on a rotary evaporation The product was dissolved in methanol and toluene was added. Removal of solvent on the evaporator yielded the crude product as a white solid (10.70 g, contaminated with ethylenediamine) The sample was divided into two samples for purification Ring-shaped sulfonated to capture ethylenediamine Using a Soxhlet extractor containing ion exchange beads, azeotropic removal of ethylenediamine with toluene was used to remove brown oil from which the product was partially decomposed. The remaining product was separated from toluene on cooling as a white solid (2.3 g, about 50%) 10% solution in methanol was analyzed by gas chromatography (Column, Tenax 60/80). As a result, little ethylenediamine was detected in the sample (<01.%) The second fraction was dissolved in methanol to obtain a 10% by weight solution, and purified from ethylenediamine using methanol as a solvent by reverse osmosis. The resulting membrane is Filmtec FT-30, Amicon TC1R thin channel separator, ethylenediamine crosslinks with membrane, and the product is separated into a white solid (2.7 g), gas chromatograph. the ethylenediamine available amount of detection was not found by the calligraphy ¹³ C NMR data and HPLC analysis (Sperry Zob (Spherisorb (ODS-1, 0.05MH 3 PO 4, pH3:. CH 3 CN = 55: 45) is the proposed structure The product was purified without further purification. Used.

Example Q

Preparation of 1/2 Generation Starburst Dendrimer from N-t-Butoxycarbonyl-6- (4-aminobenzyl) -1,4,8,11-tetraaza-5,7-dioxoundecane

Nt-butoxycarbonyl-6- (4-aminobenzyl) -oil prepared in Example P was obtained. The remaining product was separated from toluene as a white solid (2.3 g, about 50%) upon cooling. A 10% solution in methanol was analyzed by gas chromatography (Column, Tenax 60/80), showing little ethylenediamine in the sample (<0.1%). The second fraction was dissolved in methanol to give a 10% by weight solution and purified from ethylenediamine using methanol as a solvent by reverse osmosis (films used were Filmtec FT-30, Amicon TC1R thin-film separator). (thin channel separator) and ethylenediamine passes through the membrane). The product was separated into a white solid (2.7 g) and no detectable amount of ethylenediamine was found by gas chromatography. ¹³ C NMR data and HPLC analysis (Spherisorb (ODS-1, 0.05MH ₃ PO ₄ , pH 3: CH ₃ CN = 55: 45) was consistent with the proposed structure. The product was used without further purification. .

Example Q

Preparation of 1/2 Generation Starburst Dendrimer from N-t-Butoxycarbonyl-6- (4-aminobenzyl) -1,4,8,11-tetraaza-5,7-dioxoundecane

100 ml of Nt-butoxycarbonyl-6- (4-aminobenzyl) -1,4,8,11-tetraaza-5,7-dioxoundecane (5.0 g, 13 mmol) prepared in Example P In methanol. Methyl acrylate (6.12 g, 68 mmol) was added and the solution was stirred at ambient temperature for 72 hours. The reaction was adjusted by HPLC (Sperrizob ODSI, acetonitrile: 0.04 M ammonium acetate = 40: 60) to maximize conversion to the desired product. The solution was concentrated to 30% solids and methylacrylate (3.05 g, 32 mmol) was added. The reaction mixture was stirred at ambient temperature until partially alkylated product was not detected by HPLC (24 hours). The solvent was removed by rotary evaporation at 30 ° C. and pumped at 1 mm Hg for 24 hours to give the product as a yellow viscous oil (yield: 7.81 g). ¹³ C NMR data was consistent with the proposed structure.

The product was used without further purification.

Example R

Preparation of Starburst Dendrimers of Complete Generation from N-t-butoxycarbonyl-6- (4-aminobenzyl) -1,4,8,11-tetraaza-5,7-dioxoundecane

A half generation of product (Example Q) (7.70 g, 10.45 mmol) was dissolved in 75 ml of methanol and added dropwise to a stirred solution of ethylenediamine (400 ml, 7.41 mol) and methanol (50 ml) over 2 hours. The reaction mixture was stirred at ambient temperature for 48 hours. Ethylenediamine and methanol were removed by rotary evaporation to yield a yellow oil (11.8 g, contaminated with ethylenediamine). The product was dissolved in 90 ml of methanol and purified from ethylenediamine by reverse osmosis (Filmtec FT-30 thin film and Amicon TC1R thin film separator, methanol as solvent). After 48 hours, no ethylenediamine was detected by gas chromatography (column, Tenax 60/80). The solvent was removed on a rotary evaporator and then pumped on a vacuum line for 24 hours to give the product as a yellow glassy solid (6.72 g). Analysis with HPLC (PLRP-S column, acetonitrile: 0.015 M NaOH, 10 to 20% gradient for 20 min) and ¹³ C NMR was consistent with the proposed structure.

Example S

Preparation of 1 1/2 Generation Starburst Polymers from N-t-butoxycarbonyl-6- (4-aminobenzyl) -1,4,8,11-tetraaza-5,7-dioxoundecane

The first generation of product (Example R) (2.14 g, 25 mmol) was dissolved in 12.5 ml of methanol and methylacrylate (3.5 g, 39 mmol) in 5 ml of methanol was added. While the solution was stirred at ambient temperature for 48 hours, the progress of the reaction was examined by HPLC (Sperisorb ODS-1, Acetonitrile: 0.04 M ammonium acetate = 60: 40). A second fraction of metal acrylate (3.5 g, 39 mmol) was added and the mixture was stirred for an additional 72 hours at ambient temperature. The solvent was removed on a rotary evaporator to yield the product as yellow oil (3.9 g) which was pumped overnight with a vacuum pump. The product was used without the need for further purification.

Example T

Two complete generations of starburst polymers prepared from N-t-butoxycarbonyl-6- (4-aminobenzyl) -1,4,8,11-tetraaza-5,7-dioxoundecane

1 1/2 generation product (Example S) (3.9 g, 2.5 mmol) was dissolved in 50 ml of methanol and added dropwise to a stirred solution of ethylenediamine (600 g, 10 mol) and methanol (50 ml) over 2 hours. The solution was added dropwise over 9 hours at ambient temperature under a nitrogen atmosphere. Ethylenediamine / methanol was removed on a rotary evaporator to yield a yellow glassy solid (4.4 g, contaminated with ethylenediamine). The product was dissolved in methanol to prepare a 10% solution, and reverse osmosis (use film: Filmtec FT-30, Amicon TC1R thin layer) until ethylenediamine was not detected by gas chromatography (column, Tenax 60/80). Purified from ethylenediamine). Removal of the solvent gave the product as a yellow glassy solid (3.52 g). ¹³ C NMR data and HPLC analysis (PLRP-S column, acetonitrile: 0.015 M NaOH, 10-20% gradient for 20 min) was consistent with the proposed structure.

Example U

Preparation of starburst dendrimers having methylene carboxylate ends by reacting second generation starburst with bromoacetic acid

A second generation of product (Example T) (0.22 g, 0.13 mmol) was dissolved in 15 ml of deionized water and the temperature was equilibrated at 40.5 ° C. 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. Using a pH adjuster (titrated with 0.1N NaOH), the reaction pH was maintained at 40.5 ° C. overnight. When tested by reversed phase HPLC (Sperrizob OSD-1 column, eluent 0.25MH ₃ PO ₄ pH 3 [NaOH]: acetonitrile = 85: 15) it can be seen that the single component predominantly synthesized.

Example V

Preparation of Starburst Dendrimers with Second Generation Methylene-carboxylate Terminations Functionalized with Isothiocyanato

5 ml of a 2.8 mM solution of the second generation of methylenecarboxylate terminated starburst dendrimer (Example U) was diluted with 20 ml of water and the pH was adjusted to 0.5 with concentrated hydrochloric acid. After 1 hour at room temperature, the mixture was analyzed by HPLC to remove butoxycarbonyl groups and treated to 50% sodium hydroxide solution to adjust the pH to 7. The pH was maintained at 7 using a pH controller (titrated with 0.1 N NaOH) and 225 μl of thiophosgen was added. After 15 minutes at room temperature, the pH of the mixture was adjusted to 5 with 1N HC1. The mixture was washed with chloroform (200 ml × 2) and then concentrated under reduced pressure on a rotary evaporator. The 0.91 g recovered residue was a mixture of isothiocyanate and salt.

Example W

Preparation of Second Generation Starburst Polyethylenimine-Methanesulfonamide

25.0 g of tris (2-aminoethyl) amine was added to a solution of 125 g of N-methanesulfonylaziridine in 50 ml of ethanol. The solution was stirred for 4 days at room temperature. Water was added to the reaction mixture to the extent necessary to maintain homogeneity of the solution. The solvent was removed by vacuum distillation to yield the second generation of starburst PEI-methanesulfonamide as yellow glass (161 g).

Example X

Decomposition of Methanesulfonamide to Form Second Generation Starburst Polyethylenimine

A solution of 5.0 g of second generation starburst PEI-methanesulfonamide obtained from Example W dissolved in 20 ml of 38% HCI was sealed in a glass ampoule. The ampoules were heated at 160 ° C. for 16 hours, then cooled in an ice bath and opened. The solvent was removed by vacuum distillation and the residue was dissolved in water. The pH of the solution was adjusted to 10 or more using 50% NaOH, followed by vacuum distillation to remove the solvent. Toluene (150 ml) was added to the residue and the mixture was heated to reflux under a Dean-Stark trap until water could no longer be removed. The solution was filtered to remove salts and the filtrate was concentrated in vacuo to give 1.9 g of second generation Starburst PEI as a yellow oil.

Example Y

Preparation of third generation starburst polyethyleneimine-methanesulfonamide

36.6 g of N-methanesulfonylaziridine was added to a solution of 10.1 g of second generation Starburst PEI prepared in Example X in 100 ml of ethanol. The solution was stirred for 1 week at room temperature. Water was added as needed to maintain the homogeneity of the solution. The solvent was removed with vacuum distilled water to give third generation starburst PEI-methanesulfonamide as yellow glass (45.3 g).

Example Z

Decomposition of Methanesulfonimide to Form Third Generation Starburst Polyethylenimine

2.3 g of agent was removed by removing the methanesulfonamide group of the third generation starburst PEI-methanesulfoamide (5.0 g) prepared from Example Y according to the procedure as described for the second generation material in Example X. Third generation starburst PEI was obtained as a yellow oil.

Example AA

Preparation of Second Generation Starburst Polyamidoamines Having Methylenecarboxylate Terminations (Initiated from Ammonia)

The second generation starburst was dissolved in polyamidoamine (2.71 g, 2.6 mmol) and bromoacetic acid (4.93 g, 31.6 mmol) in 30 ml of deionized water and the pH was adjusted to 9.7 with 5N NaOH using a pH adjuster. It was. The reaction was held at this pH for 30 minutes, the temperature was slowly raised to 60 ° C. and then at constant temperature at 60 ° C. for 3 hours. The pH was raised to 10.3 and the reaction mixture was kept overnight at ambient temperature under the control of a pH regulator. The reaction mixture was refluxed for an additional 4 hours before treatment. The solvent was removed and finally a trace of water was azeotropically distilled with methanol to afford the product as a pale yellow powder (contaminated with 8.7 g sodium bromide). ^{The 13} C NMR spectrum is consistent with the proposed structure (slightly contaminated due to a small amount of impurities as a result of some monoalkylation).

Example BB

Preparation of methylenecarboxylate terminated second generation starburst polyethylenamine (disclosed from ammonia)

The second generation starburst obtained from Example AA was dissolved polyethylenimine (2.73 g.6.7 mmol) and bromoacetic acid (11.29 g, 81 mmol) in 30 ml of deionized water. The pH was slowly raised to 9.5 while maintaining the temperature above 30 ° C. The temperature was slowly raised to 55 ° C. and the pH of the reaction was maintained at 9.5 for 6 hours by pH control (titration with 5N NaOH). The pH was raised to 10.2 and kept at that pH overnight. The solvent was removed on a rotary evaporator and finally azeotropic distillation of the traces of water using methanol yielded the product as a yellow powder (contaminated with 17.9 g · sodium bromide). ^{The 13} C NMR spectrum is consistent with the proposed structure (slightly contaminated due to a small amount of impurities as a result of some monoalkylation).

Example CC

Manufacturing of Starburst PAMAMs of 3.5, 4.5, and 6.5 Generations

2.32 g of methyl acrylate was added to a 10% by weight methanolic solution of 2.46 g of third generation PAMAM starburst. This mixture was left at room temperature for 64 hours. Solvent and excess methyl acrylate were removed and 4.82 g of product was recovered (105% of theory).

Preparation of Higher Generation 1/2 Starburst PAMAM: As described above, generations 4.5, 5.5, and 6.5 were prepared without significant differences in reactant concentration, reactant molar ratio, or reaction time.

Example DD

Manufacturing of 4.5 and 6th Generation Starburst PAMAMs

2000 g of predistilled ethylenediamine was added to a 15 wt% solution of 5.4 g of 1/2 generation Starburst PAMAM in methanol. It was left at room temperature for 48 hours. Methanol and most of the excess ethylenediamine were removed by rotary evaporation under tap water vacuum at temperatures below 60 ° C. The total weight of recovered product was 8.07 g. Gas chromatography showed that the product still contained 34% by weight of ethylenediamine. A 5.94 g fraction of this product was dissolved in 100 ml of methanol and ultrafiltered to remove the residue ethylenediamine. Filter using an Amicon TC1R thin layer recycle separator equipped with an Amicon YM2 membrane. An in-line depressurization valve was used to maintain 55 psig (380 kPa) pressure on the membrane. The solvent was passed through the membrane to initially concentrate 100 ml to 15 ml. After the highest concentration, the flow was switched to constant volume retention recycle for 18 hours. 60 ml of methanol was then passed through the membrane to recover the product into the module and connected tubing. The product was stripped with solvent to recover 2.53 g of fifth generation starburst PAMAM. Analysis by gas chromatography showed that 0.3% of ethylenediamine remained in the product.

The production process of generations 4 and 6 proceeded as described above only by varying the weight ratio of ethylenediamine to starting material. For the fourth generation this ratio was 200: 1 and for the sixth generation this ratio was 730: 1.

Example 1

Preparation of products containing at least one rhodium atom for starburst polymers.

Generation 2.5 PAMAM (ester end, NH ₃ initiation) (0.18 g, 0.087 mmol) and RhCl ₃ .3H ₂ O (0.09 g, 0.3 mmol) were mixed in dimethylformimide (DMF) (15 ml) and then 70 ° C. Heated at for 4 h. When the solution turns magenta, most of the rhodium is dissolved. Unreacted rhodium is removed by filtration and then the solvent is removed on a rotary evaporator. The oil formed was soluble in chloroform. It was washed with water and then dried (MgSO ₄ ) before removing the solvent to give a red oil (0.18 g).

NMR spectra were recorded in CDCl ₃ and only a small difference was observed between chelated and unchelated starbursts.

A portion of this CDCl ₃ solution was diluted with ethanol and then NaBH ₄ was added to precipitate rhodium. RhCl ₃ · 3H ₂ O is insoluble in chloroform and chloroform starburst solution, resulting in chelation.

Example 2

Preparation of Product Containing Starburst Polymer and Chelated pd

Generation 3.5 PAMAM (ester end, NH ₃ initiated) (1.1 g, 0.024 mmol) was dissolved in acetonitrile (50 ml) with stirring. Palladium chloride (0.24 g, 1.4 mmol) was added and the solution was then heated at 70-75 ° C. (water bath) overnight. PdCl ₂ was absorbed into starburst. After removal of the solvent, NMR recording in CDCl _{3 shows} that chelation has already occurred. Diluting the CDCl ₃ solution with ethanol and then adding NaBH ₄ precipitated palladium. Chelated product (1.23 g) was isolated as brown oil.

Example 3

Preparation of Fluorescein-Containing Products Using Starburst Polymers

5-carboxyfluorescein (0.996 g) and starburst polyethylenimine (generation = 2.0, amine termini, NH ₃ initiation) (0.202 g) were mixed in 10 ml of methylene crawl and 5 ml of methanol and then refluxed for 10 minutes. I was. Filtration gave a red insoluble powder (0.37 g) (almost unreacted 5-carboxyfluorescein). 0.4 g of brilliant red solid was isolated from the filtrate. This softened point foamed at 98-103 ° C. to give a brilliant red melt. The NMR spectrum (D ₂ O) of this product was consistent with the dendrimer with fluorescein bound to the surface.

Example 4

Preparation of Fluorescein-Containing Products Using Starburst Polymers

In a similar manner as in Example 3, starburst polyethyleneimine (generation = 2.0, amine termini, NH ₃ initiation) was reacted with fluorescein isothiocyanate to give a brilliant red pearlescent color suitable for use as a fluorescing reagent. A solid was obtained.

Example 5

Hydrolysis of 4.5 Generation Dendrimers and Preparation of Calcium Salts

4.5 generation PAMAM (ester end, NH ₃ initiated) (2.11 g, 10.92 meq) was dissolved in 25 ml of methanol, followed by 10% NaOH (4.37 ml, 10.92 meq) (pH = 11.5-12). After warning for 24 hours at room temperature the pH was 9.5. After a further 20 hours, the solution was rotary evaporated, then 50 ml of toluene was added and then rotary evaporated again.

The resulting oil was dissolved in 25 ml of methanol and then 75 ml of diethyl ether was added to precipitate a white gum. The liquid was decanted and the gum was evaporated to give a very fine gray powder, which was then further dried to give 2.16 g (98% yield) of the product. According to NMR and infrared analysis, no ester group was observed.

The sodium salt of 4.5 generation PAMAM (ester end, NH 3 initiated) was exchanged with calcium salt via dialysis. Sodium salt (1.03 g) was dissolved in 100 ml of water and then passed through a hollow fiber dialysis tube (cut off = 5000) at a rate of 3 ml / min. The outside of the tube was immersed in 5% CaCl ₂ solution. This process was then repeated.

The resulting solution was then dialyzed again against water and then repeated two more times.

Evaporation gave 0.6 g of wet solid, which was absorbed (but not completely dissolved) in methanol and dried to yield 0.45 g of gray crystals.

C ₃₆₉ H ₅₉₂ O ₁₄₁ N ₉₁ Ca ₂₄ calc 10.10% Ca ⁺⁺

Molecular weight = 9526.3 (calculated value = C 44321.1, H 601.8, O-22559, N-1274.6, Ca-961.9)

Theoretical Value (%): C; 46.5, H; 6.32, N1; 3.38, Ca; 10.10

Found (%): C; 47.34, H7.00, N1; 3.55, Ca; 8.83

Example 6

Preparation of Dendrimers Having Terminal Carboxylate Groups

The half generation Starburst polyamidoamines were hydrolyzed to convert their terminal methyl ester groups to carboxylates. The resulting spherical molecules charged with negative charge were dispersed around. Hydrolyzed dendrimers ranged from 0.5 generation (3 carboxylates) to 6.5 generation (192 carboxylates).

The product could be produced as a Na ⁺ , K ⁺ , Cs ⁺ or Rb ⁺ salt.

Example 7

Encapsulation of R (+)-limonene in Polyamidoamine Starburst Dendrimers

Starburst-PAMAM dendrimer (molecular weight = about 175000, generation = 9.0) was added dropwise until a 5-50% by weight solid solution in methanol was saturated with R (+)-lamonene in methanol. This solution was stirred for several hours at room temperature (about 25 ° C.) and then evaporated on a Buchi rotary evaporator at room temperature to give a solid product. Warming at a temperature above 80 ° C. afforded a solvent insoluble product that retained a significant amount of R (+)-limonene in the encapsulated form. These products are a good base for slowly releasing R (+)-limonene as a flavor and deodorant.

Example 8

Encapsulation of Heavy Metal Salts in Polyamidoamine Starburst Dendrimers

A 5-50% by weight solid aqueous solution of Starburst-PAMAM dendrimer (molecular weight = about 350000, generation = 10.0) was stirred with dropwise addition of a saturated leadacetate [Pb (C ₂ H ₃ O ₂ ) ₂ ] solution. The solution was stirred for several hours at room temperature (about 25 ° C.) and then evaporated on a Buchner rotary evaporator to give a solid product. Scanning transmission micrographs of these products show that these heavy metal salts are encapsulated inside the dendrimer. These films containing heavy metal salts are useful as shields for absorbing electromagnetic waves.

Example 9

Encapsulation of Fluorescein (Water-Soluble) Dye in Polyamidoamine Starburst Dendrimers

Fluorescein disodium salt (acid yellow 73, Cl. 45350; uranine) in a 5 to 50% by weight solid solution (H ₂ O / CH ₃ OH) of Starburst-PAMAM dendrimer (molecular weight = about 175000, generation = 9.0) (Aldrich Chemical Co., Milwaukee, Wisconsin) was added until saturation and stirred. The solution was stirred for several hours at room temperature (about 25 ° C.) and then evaporated at room temperature to give a colored solid product. These dye encapsulated dendrimers are excellent control probes that scale the ultrafiltration thin films.

Example 10

Preparation of Dendrimers Having Terminal Fluorescent Groups

A. Reaction of Amine Terminated Dendrimers with N-Single Acrylziridine

A sample of starburst polyethyleneimine (LPEI, gen = 3.0, terminal group (Z) = 12, molecular weight = 920) (1.5 g, 1.6 × 10 ⁻³ mol) was dissolved in 20 ml of methanol.

After stirring the solution, 0.884 g (3.84 × 10 ⁻² mol) of N-silyl aziridine (ICN Biomedicals, Costa Mesa, Canada) was added dropwise over 20 minutes. The reaction mixture was stirred at rt overnight. Removal of solvent in vacuo gave a solid product. According to NMR and infrared analysis, the product is a covalently bound single group present on the surface of the dendrimer.

B. Reaction of Amine Terminated Dendrimer with Single Chloride

Starburst polyamidoamine (1.0 g, 1.9 x 10 ^-4 mol) in water (30 ml) (initiated from ammonia, generation = 4.0, terminal group (Z) = 24, molecular weight = 5147) solution containing 80 ml of toluene Solution of single-chloride chloride (1.23 g, 4.5 × 10 ⁻³ mol) (5-dimethylamino-1-naphthalenesulfonyl chloride; sold by Aldrich Chemical Company) in toluene (4 ml) with stirring in a three neck flask Was added dropwise under ice-cooling. At the same time, 10% NaOH solution (13.3 mol, 10% excess) was added to the reaction mixture to give an oily ball. The product was washed with water, then methanol was dissolved and then precipitated with diethyl ether to give a solid product. According to NMR and infrared analysis, it coincides with covalently bonded groups present on the dendrimer surface.

Example 11

Demonstrating Complex Chelation of Iron by Sodium Propionate-terminated Sixth Generation Starburst Polyamidoamine

Sodium propionate terminated sixth generation polyamidoamine (ammonia initiated) (97.1 mg, 2.45 mol) was dissolved in 1.5 ml of demineralized water. 0.5 ml of 0.5N HCI was added to reduce the pH to 6.3. Ferrous chloride (0.5 ml of 0, 1 and 2M solutions, 0.051 mmol) was added to produce a light brown glue precipitate. Heated at 60 ° C. for 0.5 h, the glue precipitate dissolved to make a uniform orange solution. The solution was filtered through a Biogel P2 acrylamide gel (10 g, twice) to separate the orange band (without halide contaminants). The solvent was removed in vacuo to give the product as an orange film (30 mg). The analysis was consistent with chelation of about 20 mloe of iron ion per mole of starburst dendrimer.

TABLE III

SB = C ₁₅₂₁ H ₂₄₆₇ B ₃₇₉ O ₅₇₃

These results demonstrate that 20 ± 2 mole of iron ions are chelated per mole of starburst dendrimer.

Claims (22)

a star having (a) an initiation center, (b) at least three generations of inner layers consisting of repeating units (generations) radially coupled to the initiation center, and (c) an outer surface of the terminal functional groups bonded to the outermost generation Burst dendrimers; And a starburst conjugate comprising a volatile substance (except for a pharmaceutical substance) that is transported in combination with the starburst dendrimer. The conjugate of claim 1 wherein the carrier material is a signaling factor, a signal reflecting factor or a signal absorbing factor. 2. The conjugate of claim 1, wherein at least two different carriers are present, at least one of which is a target agonist. The conjugate of claim 1, wherein the dendrimer has discontinuity. The conjugate of claim 1 wherein the conjugate of the general formula: (P) _x * (M) _y Wherein P is a dendrimer; x is an integer of 1 or more; Each M is a unit of carrier (except for pharmaceuticals), which may be the same or different from each other; y is an integer of 1 or more; * Indicates that the carrier material is associated with the dendrimer. 6. The conjugate of claim 5, wherein M is a signal reflector or a signal absorption factor. The conjugate of claim 6, wherein y is 1 and y is 2 or more. The conjugate of claim 6, wherein y is 2 or more. The conjugate of claim 6 wherein the molar ratio of ionic M to P is from 0.1 to 1000: 1. The conjugate of claim 5 wherein the weight ratio of the pesticide or toxin M to P is from 0.1 to 5: 1. A star used as a carrier for dyes, perfumes, phosphors, analogs, pheromones or electron beam impermeables, comprising at least one starburst conjugate according to any one of claims 1 and 2 to 10 and a diluent or carrier. Burst conjugate composition. A process for preparing a starburst conjugate of formula (I), characterized by reacting P (as defined below) with M (as defined below): (P) _x * (X) _y (I) Wherein each P is a dendrimer; x is an integer of 1 or more; Each M is a unit of carrier (except for pharmaceuticals), which may be the same or different from each other; y is an integer of 1 or more; * Indicates that the carrier material is associated with the dendrimer. The process of claim 12, wherein the reaction is carried out at room temperature to reflux. The process of claim 12 wherein the reaction is performed in a solvent selected from the group consisting of water, methanol, ethanol, chloroform, acetonitrile, toluene, dimethylsulfoxide and dimethylformamide. The conjugate of claim 1 wherein the starburst dendrimer has the formula:

Wherein the center is bonded to the dendritic branch of the dendrimer; The number of terminal residues per dendritic branch is Nr ^G / 2, where G is the number of generations and Nr is the degree of redundancy of the repeating units above; Nc is the valence of the central compound; The terminal residue is determined by NcNr ^G / 2, where Nr, G and Nc are as defined above, the number of terminal residues per dendrimer; The repeating unit has a valence or functionality of Nr + 1, where Nr is as defined above. The conjugate of claim 1 wherein the starburst dendrimer has the formula:

Wherein i is 1 to t-1; The central compound is represented by the general formula ① (Z ^c ) _Nc where ① represents a center, Z ^c represents a functional group bonded to ①, and Nc is a central valence; The repeating unit is represented by the general formula X ⁱ X ⁱ (Z ⁱ ) _Ni , where i is as defined above; The final or terminal unit is of the general formula X ^t Y ^t (Z ^t ) _N ^t , where t is the terminal generation and Z ^t , Y ^t , Z ^t , and N ^t have no continuous generation associated with the Z ^t group, N ^t is 2 less than the number being itneungeot represented by and may be the same or different from X ^i, Y ^i, Z ⁱ and N ^i), except for the; as the product of all the values contained in the Π function is the range before, a Number of repeat units X ⁱ Y ⁱ (Z ⁱ ) _Ni including the i th generation of dendritic branches

= (N ₁ ), (N ₂ ), (N ₃ ). (N ^i-2 ), (N ^i-1 ), where i is 1

to be. A method for producing starburst polyethyleneimine, characterized by reacting starburst polyethyleneimine metal sulfonamide with hydrochloric acid. A method for purifying a starburst dendrimer in which a solvent is present, characterized in that the solvent is removed by ultrafiltration using a membrane. The method of claim 18, wherein the solvent is ethylenediamine. Starburst conjugate of general formula (III) [(T) _e- (C ') _f ] _g * (P) _x * [(C ") _h- (M) _y ] | _k (III) Wherein each C 'is the same or different linking group; Each C ″ is the same or different linking group; g and k are each independently an integer of 1 or more; f and h are each independently an integer of 0 or more;-represents a covalent bond, if a linking group is present; P Is a dendrimer; x is an integer of at least 1; T is a target director; each M is a unit of carrier (except pharmaceutical), which may be the same or different from each other; y is an integer of at least 1 * Indicates that the carrier material is associated with a dendrimer. Reacting a starburst dendrimer (P) having a reactive moiety with a linking group which may have an NH 2 group protected by a protecting group which is inert under the conditions used for starburst synthesis as a protecting group used for amines The manufacturing method of the starburst conjugate of Claim 20 to perform. The method of claim 21, wherein the linking group is an aniline moiety that may have an NH ₂ group protected by N-phthalimide of the formula:

Images