U.S. patent application number 14/160167 was filed with the patent office on 2014-05-15 for multi-arm polyethylene glycol derivatives, conjugates and gels of pharmaceuticals and the same.
This patent application is currently assigned to JENKEM TECHNOLOGY CO., LTD, TIANJIN BRANCH. The applicant listed for this patent is JENKEM TECHNOLOGY CO., LTD, TIANJIN BRANCH. Invention is credited to Meina Lin, Xuan Zhao.
Application Number | 20140135486 14/160167 |
Document ID | / |
Family ID | 44172413 |
Filed Date | 2014-05-15 |
United States Patent
Application |
20140135486 |
Kind Code |
A1 |
Zhao; Xuan ; et al. |
May 15, 2014 |
MULTI-ARM POLYETHYLENE GLYCOL DERIVATIVES, CONJUGATES AND GELS OF
PHARMACEUTICALS AND THE SAME
Abstract
A multi-arm polyethylene glycol (I) having different kinds of
reactive groups and the uses thereof are disclosed, which is formed
by polymerizing ethylene oxide with oligo-pentaerythritol as an
initiator, wherein, PEG is same or different and is --(CH2CH2O)m--,
the average value of m is an integer of 2-250; l is an integer of 1
or more. The method for producing the multi-arm polyethylene glycol
having different kinds of reactive groups, the multi-arm
polyethylene glycol active derivatives comprising linking groups X
attached to PEG and terminal reactive groups F attached to X, the
gels formed by the multi-arm polyethylene glycol active
derivatives, the drug conjugates formed by the multi-arm
polyethylene glycol active derivatives and drug molecules, and the
uses thereof in preparing drugs are also disclosed.
Inventors: |
Zhao; Xuan; (Tianjin,
CN) ; Lin; Meina; (Tianjin, CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
JENKEM TECHNOLOGY CO., LTD, TIANJIN BRANCH |
Tianjin |
|
CN |
|
|
Assignee: |
JENKEM TECHNOLOGY CO., LTD, TIANJIN
BRANCH
Tianjin
CN
|
Family ID: |
44172413 |
Appl. No.: |
14/160167 |
Filed: |
January 21, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13518940 |
Jul 27, 2012 |
|
|
|
PCT/CN2010/002139 |
Dec 23, 2010 |
|
|
|
14160167 |
|
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Current U.S.
Class: |
536/21 ;
536/22.1; 546/48; 548/521; 548/542; 562/564 |
Current CPC
Class: |
C08G 65/3322 20130101;
C08G 65/33337 20130101; C08G 65/2609 20130101; C08L 2203/02
20130101; A61K 47/6903 20170801; A61K 31/727 20130101; A61K 47/60
20170801; C08G 65/329 20130101; C08G 2650/24 20130101 |
Class at
Publication: |
536/21 ; 562/564;
548/542; 548/521; 536/22.1; 546/48 |
International
Class: |
A61K 47/48 20060101
A61K047/48; A61K 31/727 20060101 A61K031/727 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 25, 2009 |
CN |
200910259749.7 |
Claims
1. A multi-arm poly(ethylene glycol) derivative selected from the
group consisting of: a multi-arm poly(ethylene
glycol)amino-single-acetic acid of formula V: ##STR00048## a
multi-arm poly(ethylene glycol)amino-multi-acetic acid of formula
VA: ##STR00049## a multi-arm poly(ethylene
glycol)maleimide-single-N-hydroxysuccinimidyl ester of formula VI:
##STR00050## a multi-arm poly(ethylene
glycol)maleimide-multi-N-hydroxysuccinimidyl ester of formula VIA:
##STR00051## a multi-arm poly(ethylene
glycol)acrylate-single-N-hydroxysuccinimidyl ester of formula VII:
##STR00052## and a multi-arm poly(ethylene
glycol)acrylate-multi-N-hydroxysuccinimidyl ester of formula VIIA:
##STR00053## wherein l is 1, 2 or 3.
2. The multi-arm poly(ethylene glycol) derivative of claim 1,
wherein said multi-arm poly(ethylene glycol) has a molecular weight
of 400-80,000 daltons.
3. The multi-arm poly(ethylene glycol) derivative of claim 2,
wherein said multi-arm poly(ethylene glycol) has a molecular weight
of 1,000-20,000 daltons.
4. A conjugate formed from the active derivative of the multi-arm
poly(ethylene glycol) of claim 3 and a pharmaceutical molecule.
5. A conjugate formed from the active derivative of the multi-arm
poly(ethylene glycol) of claim 1 and a pharmaceutical molecule.
6. The conjugate of claim 5, wherein said pharmaceutical molecule
is selected from the group consisting of proteins, enzymes,
saccharides, organic acids, flavonoids, quinones, terpenoids,
benzene phenols, steroidal, glycosides, alkaloids, and combinations
thereof.
7. The conjugate of claim 6, wherein said organic acids are amino
acids.
8. The conjugate of claim 6, wherein said glycosides are
nucleosides.
9. The conjugate of claim 6, wherein said pharmaceutical molecule
is Irrinitecan.
10. A gel, comprising an active derivative of the multi-arm
poly(ethylene glycol) according to claim 1.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This is a divisional application of copending patent
application Ser. No. 13/518,940, filed Jul. 27, 2012, which is a
.sctn.371 national stage of international patent application
PCT/CN2010/002139, filed Dec. 23, 2010; the application also claims
the priority, under 35 U.S.C. .sctn.119, of Chinese patent
application No. CN 200910259749.7, filed Dec. 25, 2009; the prior
applications are herewith incorporated by reference in their
entirety.
BACKGROUND OF THE INVENTION
Field of the Invention
[0002] The present invention relates to the multi-arm polyethylene
glycol derivatives with different kinds of active groups and their
preparation methods, drug molecules conjugates and the gel
materials. The invention also relates to the role of the described
above new multi-arm polyethylene glycol derivatives with different
types of active groups and its gels in the preparation of
pharmaceutical preparations and medical device materials.
[0003] At present, the polyethylene glycol derivatives are widely
used in combination with proteins, peptides, and other therapeutic
agents to extend the physiological half-life of drugs referred to,
reduce immunogenicity and toxicity. In clinical use, PEG and its
derivatives which act as the carrier in the production of
pharmaceutical preparations have been widely used in lots of
medicines. The attempts to bond PEG to the drug molecules have also
seen significantly development in the past decade, and widely used
in many approved medicines such as PEGasys.RTM., which is a
combination of .alpha.-interferon and polyethylene glycol with a
longer circulation half-life and a better therapeutic effect.
Metabolic processes of polyethylene glycol in the human body have
been quite clear, which is a safe synthetic polymer material
without side effects.
[0004] In terms of drug modification, the latest research direction
is to connect a targeted molecule to the drug molecules through
polyethylene glycol to enrich drug molecules near the focus, so as
to achieve the best therapeutic effect. For example, anticancer
medicine is connected to monoclonal antibodies. According to the
recent research of our company, the combination of two different
medicinal molecules through polyethylene glycol can maintain the
aforementioned increase of physiological half-life and reduce
immunogenicity and toxicity, while giving full play to the two
molecules' synergies. Intermolecular synergy is very important in
the Chinese medicine theory. When two or more different molecules
are connected with polyethylene glycol, a PEG derivative with
metachronous double functional groups is needed. Currently, our
company could be able to produce a variety of PEG derivatives with
metachronous double functional groups, such as MAL-PEG-NHS, acrylic
acid-PEG-NHS, and HO-PEG-COON. However, those PEG derivatives with
metachronous double functional groups are straight-chain
polyethylene glycol derivatives.
[0005] Straight-chain polyethylene glycol derivatives with
metachronous double functional groups are limited to some extent in
applications. The ratio of two molecules connected by the
straight-chain ethylene glycol derivatives with metachronous double
functional groups is basically 1:1. If one molecule is needed more
than another one, for example, if one molecule has with low in vivo
activity, more connections will be required for the low activity
molecules than the high-activity molecules, which is a challenge to
the straight-chain polyethylene glycol derivatives with
metachronous double functional groups. At the same time, in terms
of drug carrying, multi-arm polyethylene glycol is advantageous
over the straight-chain polyethylene glycol. Straight-chain
polyethylene glycol derivatives with metachronous double functional
groups can only carry two molecules, while the multi-arm
polyethylene glycol has several end groups, and thus has more than
one drug connection points, and can carry several drug molecules.
At present, multi-arm polyethylene glycol is widely used in the
PEG-modification of peptides and small molecule drugs. However, the
multi-arm PEG derivatives on the market only have the same active
groups such as 4-arm polyethylene glycol succinate --NHS ester,
(4arm-SS). U.S. Pat. No. 6,046,305 shows a star-shaped polyethylene
glycol derivative formed through polymerization reaction with only
one active group. At the same time, one arm of this star-shaped
polyethylene glycol derivative is connected with the other arms
through the non-ether bond, such as the amide bond or ester bond.
This connection is different in the way, and can reduce the
stability of the derivatives.
SUMMARY OF THE INVENTION
[0006] The purpose of this invention is to increase the kinds of
active groups of the multi-arm polyethylene glycol in the existing
technology by providing a new, simple structure and stable
multi-arm polyethylene glycol derivatives with different types of
active groups. The new multi-arm PEG derivatives with different
types of active groups include metachronous double functional
groups polyethylene glycol derivatives and metachronous three
functional groups polyethylene glycol derivatives. At the same
time, the invention provides a new preparation method for the
multi-arm PEG derivatives with different types of active groups and
conjugates of multi-arm polyethylene glycol active polyethylene
glycol derivatives and pharmaceutical molecules and gels and their
applications.
[0007] The invention provides a novel multi-arm polyethylene glycol
derivative, which has the structure of general formula I:
##STR00001##
wherein:
[0008] F.sub.1, F.sub.2, F.sub.3 and F.sub.4 each has a structure
of --X--Y, and at least two of F.sub.1, F.sub.2, F.sub.3 and
F.sub.4 are different;
[0009] X is a linking moiety, which is selected from a group
consisting of: (CH.sub.2).sub.i, (CH.sub.2).sub.iNH,
(CH.sub.2).sub.iOCOO--, (CH.sub.2).sub.iOCONH--,
(CH.sub.2).sub.iNHCOO--, (CH.sub.2).sub.iNHCONH--,
OC(CH.sub.2).sub.iCOO--, and (CH.sub.2).sub.iCONH--; i is an
integer between 0 and 10;
[0010] Y is a functional end group, which is selected from a group
consisting of: hydroxyl, amino, mercapto, carboxyl, ester group,
aldehyde group, acrylic group and maleimide group;
[0011] PEG is the same or different --(CH.sub.2CH.sub.2O).sub.m--,
the average value m is an integer between 2 and 250; and
[0012] l is an integer.gtoreq.1.
[0013] In a preferred embodiment, F.sub.1 in the multi-arm
polyethylene glycol derivatives of the invention is --X--COOH;
F.sub.2, F.sub.3 and F.sub.4 each has the structure of --X--Y and
at least one of F.sub.2, F.sub.3 and F.sub.4 is not --X--COOH. The
described active derivatives of multi-arm polyethylene glycol have
the structure of the formula II:
##STR00002##
[0014] In a preferred embodiment, F.sub.1 and F.sub.2 in the
multi-arm polyethylene glycol derivatives of the invention are both
--X--COOH; F.sub.3 and F.sub.4 each has the structure of --X--Y and
at least one of F.sub.3 and F.sub.4 is not --X--COOH. The described
active derivatives of multi-arm polyethylene glycol have the
structure of the formula IIA:
##STR00003##
[0015] In a detailed embodiment, the described multi-arm
polyethylene glycol derivative has the multi-arm polyethylene
glycol hydroxy-single-acetic acid of the structure of the formula
III:
##STR00004##
[0016] In a detailed embodiment, the described multi-arm
polyethylene glycol derivative has the multi-arm polyethylene
glycol hydroxy-multi-acetic acid of the formula IIIA as
follows:
##STR00005##
[0017] In a detailed embodiment, the described multi-arm
polyethylene glycol derivative has the multi-arm polyethylene
glycol hydroxy-single-NHS ester of the formula IV as follows:
##STR00006##
[0018] In a detailed embodiment, the described multi-arm
polyethylene glycol derivative has the multi-arm polyethylene
glycol hydroxy-multi-NHS ester of the structure of the formula IVA
as follows:
##STR00007##
[0019] In a detailed embodiment, the described multi-arm
polyethylene glycol derivative has the multi-arm polyethylene
glycol amino-single-acetic acid of the structure of the formula V
as follows:
##STR00008##
[0020] In a detailed embodiment, the described multi-arm
polyethylene glycol derivative has the multi-arm polyethylene
glycol amino-multi-acetic acid of the structure of the formula VA
as follows:
##STR00009##
[0021] In a detailed embodiment, the described multi-arm
polyethylene glycol derivative has the multi-arm polyethylene
glycol maleimide-single-NHS ester of the structure of the formula
VI as follows:
##STR00010##
[0022] In a detailed embodiment, the described multi-arm
polyethylene glycol derivative has the multi-arm polyethylene
glycol maleimide-multi-NHS ester of the structure of the formula
VIA as follows:
##STR00011##
[0023] In a detailed embodiment, the described multi-arm
polyethylene glycol derivative has the multi-arm polyethylene
glycol acrylate-single-NHS ester of the structure of the formula
VII as follows:
##STR00012##
[0024] In a detailed embodiment, the described multi-arm
polyethylene glycol derivative has the multi-arm polyethylene
glycol acrylate-multi-NHS ester of the structure of the formula
VIIA as follows:
##STR00013##
[0025] In a preferred embodiment, the l in the described multi-arm
polyethylene glycol derivatives is an integer between 1 and 10
(including 1 and 10) and the further preferred l is an integer
between 1 and 3 (including 1 and 3).
[0026] In a preferred embodiment, the molecular weight of the
described multi-arm polyethylene glycol is 400-80,000 daltons,
preferred 1000-20,000 daltons.
[0027] The present invention also provides the conjugates formed
from the active derivatives of the described multi polyethylene
glycol and pharmaceutical molecules through the end group F of the
active derivatives. In some embodiments, the specified
pharmaceutical molecules are selected from the groups consisting
of: amino acids, proteins, enzymes, nucleosides, saccharides,
organic acids, flavonoids, quinones, terpenoids, benzene phenols,
steroidal and glycosides thereof, alkaloids, and the combinations
thereof. Preferably, the present invention provides a conjugate
formed from the eight-arm polyethylene glycol acid and the
Irrinitecan or docetaxel.
[0028] The invention also provides a gel made from the active
derivative of the described active multi-arm polyethylene
glycol.
[0029] The present invention further provides the applications of
the above conjugates in pharmaceutical preparation.
[0030] The present invention further provides the method of
preparing the new, simple and stable multi-arm PEG derivatives with
different types of active groups.
[0031] The described methods include: use the pentaerythritol or
oligomer pentaerythritol as the initiator to polymerize 20 ethylene
oxide and produce multi-arm polyethylene glycol, convert one or
more end hydroxy groups of the multi-arm polyethylene glycol into
carboxylic acid or amine via chemical reaction, then isolate and
purify the single-carboxyl or multi-more carboxyl, single-amino or
multi-amino products with ion exchange column, and then transform
the corresponding hydroxyl, carboxyl and amino groups into the
desired reactive groups through the chemical reaction, and finally
produce the multi-arm polyethylene glycol derivatives with
different types of active groups described in the innovation.
MODE OF CARRYING OUT THE INVENTION
[0032] The method of preparing multi-arm polyethylene glycol
derivatives with different types of active groups is described with
the following examples:
[0033] The structural formula of multi-arm PEG chain is as
follows:
##STR00014##
wherein:
[0034] R is the central molecule or the non-molecular hydroxyl part
of the initiator molecules, which is typically the alkyl,
cycloalkyl or aralkyl;
[0035] n is the number of branches or the number of arms;
[0036] PEG is the same or different --(CH.sub.2CH.sub.2O).sub.m--,
m is any integer, characterizing the single-arm polymerization
degree of polyethylene glycol.
[0037] When R is the non-hydroxyl part of pentaerythritol, the
initiator molecule is pentaerythritol, and the chemical structure
is as follows:
##STR00015##
wherein:
[0038] n is 4, and what is formed is the four-arm polyethylene
glycol.
[0039] When R is the non-hydroxyl part of dimerization of
pentaerythritol, the initiator molecule is dimerization of
pentaerythritol with the following chemical structure:
##STR00016##
wherein:
[0040] n is 6, and what is formed is the six-arm polyethylene
glycol.
[0041] When R is the non-hydroxyl part of trimeric pentaerythritol,
the initiator molecule is trimeric pentaerythritol with the
following chemical structure:
##STR00017##
wherein:
[0042] n is 8, and what is formed is the eight-arm polyethylene
glycol.
[0043] The multi-arm polyethylene glycol used in the invention is
produced by polymerizing 5 ethylene oxide with the above
pentaerythritol or oligomer pentaerythritol as the initiator.
[0044] The polyethylene glycol could be represented with molecular
weight generally as long as one arm's molecular weight of the
polyethylene glycol is between 300 and 60,000 daltons, which is
equivalent to m being about 6 to 1300. More preferably, m is 28,112
and 450, respectively corresponding to a molecular weight of 1,325,
5,000 and 20,000. Due to potential uneven nature of the initial PEG
compounds usually limited by its average molecular weight but not
the repeating units, the molecular weight is preferred to
characterize polyethylene glycol polymer instead of using the
integer m to represent the self-repeating units in the PEG
polymer.
[0045] Active Groups:
[0046] When using the active derivatives of the multi-arm
polyethylene glycol of the invention, different purposes of the
derivatives are determined by the different end functional groups
F. The introduction of these functional groups will determine the
application areas and applicable structures of the derivatives. The
most common functional groups is N-hydroxysuccinimide ester (NHS),
as can be seen in formula IV. The active derivatives with the NHS
ester structure can be linked with the groups with amine.
[0047] Similarly, according to this manual, the technical personnel
in the field can obtain the multi-arm active polyethylene glycol
derivatives with amino functional groups, as can be seen in formula
III.
[0048] Similarly, the technical personnel in the field can obtain
the multi-arm active polyethylene glycol derivatives with carboxyl
functional groups, as can be seen in formula IV.
[0049] Similarly, the technical personnel in the field can obtain
the multi-arm active polyethylene glycol derivatives with Maleimide
functional groups (MAL), as can be seen in formula VI. The active
derivatives with MAL structure can be linked with the thiol
groups.
[0050] Many medicines contain such functional groups as active
amino, carboxyl and hydroxyl, which could combine with 30
monosaccharides, polysaccharides, nucleosides, poly-nucleoside
phosphoryl and other ingredients in vivo to form active
pharmacology structure in organisms.
[0051] Therefore, the PEG derivatives with the modified functional
groups can be combined with these medicine molecules in the same
way to replace bio-organic molecules, so as to overcome such
shortcomings as the short half-life and short-time physiological
efficacy of bio-organic molecules in vivo.
[0052] Active derivatives of multi-arm polyethylene glycol of the
invention could be combined with drug molecules through appropriate
end functional groups (F). The described end functional groups can
connect the free amino groups, hydroxyl, sulfur hydroxyl in
proteins, peptides or other natural medicines with PEG derivatives.
For small molecule medicines, each multi-arm PEG molecule can bond
several medicine molecules. Such PEG derivatives have relatively
high drug load to secure the appropriate concentration of the drug
and enhance the release function, so as to improve the
physiological role of the drug molecules in the body.
[0053] The purpose of all these applications is just to provide a
possible reference model for the medical applications of the PEG
derivatives, and the actual applications and selections shall be
confirmed based on the pharmacological, toxicological and clinical
tests.
[0054] In the combinations of the invention, some of the medicine
molecules are preferred as amino acids, proteins, enzymes,
nucleosides, sugars, organic acids, flavonoids, quinones, terpenes,
phenolic phenylpropanoids, steroidal and its glycosides, alkaloids.
Protein medicine molecules are preferred as interferon drugs, EPO
drugs, growth hormone drugs, antibody drugs, and so on.
[0055] The combinations of the invention can be dosed in the form
of a pure compound or suitable pharmaceutical compositions in any
acceptable dosing method or with the reagents with the similar
purposes. Therefore, the medicine could be taken through oral,
nasal, rectal, transdermal or injection dosing method in the form
of solid, semi-solid, lyophilized powder or liquid, for example,
tablets, suppositories, pills soft and hard gelatin capsules,
powders, solutions, suspensions, or aerosols, etc., and the unit
dosage forms of accurate dose and simple dosing method shall be
preferred. The combinations can include conventional pharmaceutical
carriers or excipients and combinations of the invention, which are
used as active ingredients (one or more). In addition, other drugs,
carriers and auxiliary agents could also be included.
[0056] Typically, according to the desired dosing method, the
pharmaceutically acceptable combinations will include the
combinations of the invention in the weight % from 1 to about 99,
and the suitable pharmaceutical excipient in the weight % from 99
to 1. The preferred combination contains about 5-75% of the
combinations of the invention, and the remaining composition is the
suitable pharmaceutical excipients.
[0057] The preferred dosing method is injection with conventional
daily dose plan which can be adjusted according to the severity of
the disease. Combinations of the invention or their
pharmaceutically acceptable salts can also be made into the
injection, such as dispersing about 0.5 to about 50% active
ingredients in the pharmaceutical auxiliary agents that can be
dosed in liquid, such as water, brine, aqueous glucose, glycerol,
ethanol, etc., so as to form a solution or suspension.
[0058] For the pharmaceutical combinations that can be dosed in
liquid, the combinations of the invention (about 0.5 to about 20%)
and the selectively existing pharmaceutical adjuvants could be
dispersed into carriers, such as water, saline, aqueous glucose,
glycerol, ethanol, etc., to form a solution or suspension.
[0059] If necessary, the pharmaceutical combinations of the
invention may also contain small amounts of auxiliary substances
such as wetting agents or emulsifiers, pH buffers and
anti-oxidants, for example: citric acid, sorbitan monolaurate,
triethanolamine, oleic acidesters, butylated hydroxy toluene.
[0060] The actual preparation method of such formulations is
publicly known by the technical personnel in the field, for
example, it can be found at Remington's Pharmaceutical Sciences,
18th Edition (Mack Publishing Company, Easton, Pa., 1990). In any
case, with the technology of the invention, the combinations used
may contain a therapeutically effective combination for the
treatment of the corresponding disease.
EXAMPLES
[0061] The following examples are offered to illustrate, but not to
limit, the present invention.
[0062] All the reagents referred to in the following examples are
commercially available unless otherwise indicated.
Example 1
Synthesis of 4-arm poly(ethylene glycol)hydroxy-single-acetic acid
(III-1) and 4-arm poly(ethylene glycol)hydroxy-double-acetic acid
(IIIA-1)
##STR00018##
[0064] Procedure:
[0065] 4-ARM-PEG (10 kDa, 100 g) in 800 ml of THF was
azeotropically dried by distilling off THF (20%) under nitrogen and
the solution was cooled to room temperature. To the solution was
added potassium tert-butoxide (4.48 g) and stirred 2 hours at room
temperature. Tert-butyl bromoacetate (5.17 ml) was added dropwise
and the mixture was stirred at room temperature overnight. The
mixture was filtered and evaporated under vacuum to remove
solvents. The residue was dissolved in a aqueous solution (H.sub.2O
500 mL+sodium hydroxide 8.16 g+sodium phosphate) and stirred 2
hours at 80.degree. C. The PH of the aqueous solution was adjusted
to about 2-3. Sodium chloride (15%) was added and the product was
extracted with dichloromethane. The extract was dried over
anhydrous sodium sulfate, filtered, concentrated under vacuum at
50.degree. C., and then precipitated into diethyl ether. The
filtrate was dried in vacuum and separated with DEAE ion-exchange
chromatography column to afford 4-arm poly(ethylene
glycol)hydroxy-single-acetic acid (III-1) and 4-arm poly(ethylene
glycol)hydroxy-double-acetic acid (IIIA-1).
[0066] NMR (DMSO) .delta.: 4.01 (s, CH.sub.2COOH), 4.54 (t,
CH.sub.2OH).
Example 2
Synthesis of four-arm polyethylene glycol
hydroxy-single-N-hydroxysuccinimidyl ester (IV-1)
##STR00019##
[0068] Procedure:
[0069] 4-arm poly(ethylene glycol)hydroxy-single-acetic acid
(III-1) (10 kDa, 0.5 g) and N-hydroxysiccinimide (0.01439 g) were
dissolved in dichloromethane. DCC (0.01290 g) was added and the
solution was stirred overnight at room temperature, filtered,
concentrated under vacuum at 40.degree. C. The residue was
dissolved in hot iso-propanol and then crystallized by cooling the
solution to 0.degree. C. The resulting precipitate was collected,
washed with iso-propanol and dried to afford the 4-arm
poly(ethylene glycol)hydroxy-single-N-hydroxysuccinimidyl ester
(IV-1).
##STR00020##
Example 3
4-arm poly(ethylene glycol)hydroxy-single-methyl acetate
(IVA-1)
##STR00021##
[0071] Procedure:
[0072] 4-arm poly(ethylene glycol)hydroxyl-single-acetic acid
(III-1) (10 kDa, 3.2 g) was dissolved in methanol (16 ml). The
solution was cooled to 0.degree. C. and the concentrated sulfuric
acid was added drop wise. The solution was stirred 3 hours at room
temperature. The solution was adjusted PH to about 7.0 with 8%
NaHCO.sub.3 aqueous solution. The product was extracted with
dichloromethane. The organic layer was separated, dried over
anhydrous magnesium sulfate, filtered, and concentrated in
40.degree. C., and then precipitated into diethyl ether. The
filtrate was dried over vacuum to afford the four-arm poly(ethylene
glycol)hydroxy-single methyl acetate (IVA-1).
[0073] NMR (DMSO) .delta.: 3.32 (s, CH.sub.2COOCH.sub.3), 4.13 (s,
CH.sub.2COOCH.sub.3), 4.57 (t, CH.sub.2OH).
Example 4
4-arm poly(ethylene glycol)sulfonate-single-methyl acetate
(IVB-1)
##STR00022##
[0075] Procedure:
[0076] Four-arm poly(ethylene glycol)sulfonate-single methyl
acetate (IVB-1) (10000, 3.0 g) in 50 ml of toluene was
azeotropically dried by distilling off 38 ml toluene and the
solution was cooled to room temperature. To the solution was added
5 ml of dichloromethane and 188 .mu.l of triethylamine, 94 .mu.l of
MsCl was added dropwise. The solution was stirred at room
temperature overnight and the reaction was quenched by adding 720
.mu.l of absolute ethanol. The mixture was filtered, evaporated at
60.degree. C., dissolved in hot iso-propanol and then crystallized
by cooling the solution to 0.degree. C. The resulting precipitate
was collected, washed with iso-propanol and dried to afford the
four-arm poly(ethylene glycol)sulfonate-single methyl acetate
(IVB-1).
[0077] NMR (DMSO) .delta.: 3.17 (s, CH.sub.2OSO.sub.2CH.sub.3),
4.13 (s, CH.sub.2COOCH.sub.3), 4.30 (t,
CH.sub.2OSO.sub.2CH.sub.3).
Example 5
4-arm (polyethylene glycol)amino-single-acetic acid (V-1)
##STR00023##
[0079] Procedure:
[0080] 4-arm poly(ethylene glycol)sulfonate-single acetic acid
(V-1) having a molecular weight of 10000 (2.6 g) was dissolved in
7.8 ml of water. The PH of the aqueous solution was adjusted to 12
with 2M sodium hydroxide and the solution was stirred 2-2.5 hours.
Ammonia water (26 ml) and ammonium chloride (1.3 g) was added to
the above aqueous solution. The mixture was stirred 72 hours at
room temperature. Sodium chloride (7 g) was added and the reaction
mixture was exacted with dichloromethane. The organic layer was
collected and concentrated to remove the solvent at 40.degree. C.
Water (30 ml) and sodium chloride was then added and the PH of the
aqueous solution was adjusted to 2-3 with 2M hydrochloric acid. The
product was extracted with dichloromethane. The extract was dried
with anhydrous sodium sulfate, concentrated and added to ethyl
ether. The precipitated product was filtered off and dried under
vacuum to offer the 4-arm poly(ethylene glycol)sulfonate-single
acetic acid (V-1).
[0081] NMR (DMSO) .delta.: 2.96 (t, CH.sub.2CH.sub.2NH.sub.2), 4.00
(s, CH.sub.2COOH).
Example 6
4-arm (polyethylene glycol)hydroxy-diethyl methyl ester (IVC-1)
##STR00024##
[0083] The initial material is 4-arm (polyethylene
glycol)hydroxy-acetic acid (IIIA-1) with the molecular weight of
10,000, and its synthetic steps are same as that of Example 3.
[0084] NMR (DMSO) .delta.: 3.32 (s, CH.sub.2COOCH.sub.3), 4.13 (s,
CH.sub.2COOCH.sub.3), 4.57 (t, CH.sub.2OH).
Example 7
4-arm (polyethylene glycol)sulfonate-diethyl methyl ester
(IVD-1)
##STR00025##
[0086] The initial material is 4-arm (polyethylene
glycol)hydroxy-diethyl methyl ester (IVC-1) with the molecular
weight of 10,000, and its synthetic steps are same as that of
Example 4.
[0087] NMR (DMSO) .delta.: 3.17 (s, CH.sub.2OSO.sub.2CH.sub.3),
3.32 (s, CH.sub.2COOCH.sub.3), 4.13 (s, CH.sub.2COOCH.sub.3), 4.30
(t, CH.sub.2OSO.sub.2CH.sub.3).
Example 8
4-arm polyethylene glycol amino-double acetic acid (VA-1)
##STR00026##
[0089] The initial material is 4-arm (polyethylene
glycol)sulfonate-diethyl methyl ester (IVD-1) with the molecular
weight of 10,000, and its synthetic steps is same as that of
Example 5.
[0090] NMR (DMSO) .delta.: 2.96 (t, CH.sub.2CH.sub.2NH.sub.2), 4.00
(s, CH.sub.2COOH).
Example 9
4-arm (polyethylene glycol)-three maleimide-single-acetic acid
(VB-1)
##STR00027##
[0092] Procedure:
[0093] 4-arm poly(ethylene glycol)amino-single acetic acid (V-1)
having a molecular weight of 10000 (1.0 g) was dissolved in 10 ml
of dichloromethane. 42 ul of triethylamine was added and then
MAL-NHS 5 minutes later. The mixture was stirred overnight at room
temperature avoiding light. The reaction solution was concentrated
at 40. The residue was dissolved in hot isopropanol and then
precipitated by cooling in an ice bath. The precipitate was
filtered, washed by isopropanol and dried over vacuum to offer the
4-arm poly(ethylene glycol) three maleimide-single acetic acid
(V-1).
##STR00028##
Example 10
4-arm (polyethylene glycol)-three maleimide-single-NHS ester
(VI-1)
##STR00029##
[0095] Procedure:
[0096] 4-arm poly(ethylene glycol)-three maleimide-single-acetic
acid (VB-1) (10000, 1.0 g) and N-hydroxyl succinimide (0.01496 g)
was dissolved in 10 ml of dichloromethane, DCC (0.02889 g) was
added and the solution was stirred overnight at room temperature
avoid light. The reaction mixture was filtered, concentrated to
remove the solvent. The residue was dissolved in hot isopropanol
and then precipitated by cooling in an ice bath. The precipitate
was filtered, washed by isopropanol and dried over vacuum to offer
4-arm poly(ethylene glycol)-three maleimide-single-succinimide
ester (VI-1).
##STR00030##
Example 11
4-arm (polyethylene glycol)-bimaleimide-acetic acid (VC-1)
##STR00031##
[0098] The initial material is 4-arm (polyethylene
glycol)amino-biacetic acid (VA-1) with the molecular weight of
10,000, and its synthetic steps are same as that of Example 9.
##STR00032##
Example 12
4-arm (polyethylene glycol)-bimaleimide-biNHS ester (VIA-1)
##STR00033##
[0100] The initial material is 4-arm (polyethylene
glycol)-bimaleimide-acetic acid (VC-1) with the molecular weight of
10,000, and its synthetic steps are same as that of Example 10.
##STR00034##
Example 13
4-arm (polyethylene glycol)acrylate-single-acetic acid (VD-1)
##STR00035##
[0102] Procedure:
[0103] In three round-bottomed flask, inlet nitrogen, take 1.0 g
molecular weight 10,000 four-arm polyethylene glycol hydroxy-single
acetic acid (III-1) and 0.0005 gBHT, dissolved in 12 ml
dichloromethane, heating steam 10% solvent, cooling to room
temperature, add 49 ul triethylamine, stir for 5-10 minutes, add
250 ul acryloyl chloride, filling nitrogen, protected from light,
the system is sealed and stirred, overnight reaction, the next day,
concentrated at 30.degree. C. to be thick, add 20 ml water
dissolved to clear, put it aside for 30 minutes, add 15% sodium
chloride, adjusted pH=2-3 with dilute hydrochloric acid, extracted
with dichloromethane three times, combine organic phases, the
organic phase was dried with anhydrous sodium sulfate to clarify,
filter, concentrate filtrate at 30.degree. C. to be thick, ice
bath, add 20 ml isopropanol, thermal dissolution precipitation,
filtration, wash with isopropanol, vacuum drying, get four-arm
polyethylene glycol acrylate-single-acetic acid (VD-1).
[0104] NMR (DMSO) .delta.: 4.00 (s, CH.sub.2COOH), 4.21 (t,
CH.sub.2OCOCH.dbd.CH.sub.2), 5.93 (d, CH.sub.2OCOCH.dbd.CH.sub.2),
6.20 (q, CH.sub.2OCOCH.dbd.CH.sub.2), 6.36 (d,
CH.sub.2OCOCH.dbd.CH.sub.2).
Example 14
4-arm (polyethylene glycol)acrylate-single-NHS ester (VII-1)
##STR00036##
[0106] Procedure:
[0107] In three round-bottomed flask, inlet nitrogen, dark, weighed
0.8 g molecular weight 10,000 four-arm polyethylene glycol
acrylate-single-acetic acid (VD-1) and 0.011 gNHS, dissolved with 8
ml dichloromethane, add 0.0206 g DCC to system, dark airtight
stirred overnight reaction, the next day, filter, concentrate the
filtrate at 30.degree. C. to be thick, stirring 30 minutes, in the
ice bath after the heat dissolution with 16 ml isopropanol,
precipitation, filtration, wash with isopropanol, vacuum drying,
obtain four-arm polyethylene glycol acrylate-single-NHS ester
(VII-1).
##STR00037##
Example 15
4-arm polyethylene glycol acrylic acid-acetic acid (VE-1)
##STR00038##
[0109] The initial material is 4-arm (polyethylene
glycol)hydroxy-biacetic acid (IIIA-1) with the molecular weight of
10,000, and its synthetic steps are same as that of Example 13.
[0110] NMR (DMSO) .delta.: 4.00 (s, CH.sub.2COOH), 4.21 (t,
CH.sub.2OCOCH.dbd.CH.sub.2), 5.93 (b, CH.sub.2OCOCH.dbd.CH.sub.2),
6.20 (4, CH.sub.2OCOCH.dbd.CH.sub.2), 6.36 (b,
CH.sub.2OCOCH.dbd.CH.sub.2).
Example 16
4-arm polyethylene glycol acrylic acid-bi NHS ester (VIIA-1)
##STR00039##
[0112] The initial material is 4-arm (polyethylene glycol)acrylic
acid-biacetic acid (VE-1) with the molecular weight of 10,000, and
its synthetic steps are same as that of Example 14.
##STR00040##
Example 17
Synthesis of 8-arm (polyethylene glycol)hydroxy-single acetate
(III-2) and 8-arm (polyethylene glycol)hydroxy-diacetate
(IIIA-2)
##STR00041##
[0114] Procedure:
[0115] In three round-bottomed flask, inlet nitrogen, add 100 g
8ARM-PEG-10K and 800 mlTHF, heating dissolved, and steam about 20%
solvents, cooling, add 8.96 g potassium tert-butyl alcohol, at room
temperature for 2 hours, add dropping 10.34 ml bromine tert-butyl
acetate, at room temperature overnight reaction, the next day
filtration, the reaction solution was concentrated to be thick, add
1000 ml alkaline hydrolyzate (1000 ml water, add 16.32 g sodium
hydroxide and the 155.04 g sodium), 80.degree. C. alkaline
hydrolysis for 2 hours, 2N hydrochloric acid solution to adjust the
pH to 2-3, add 15% sodium chloride solution, extracted with
dichloromethane three times, combined organic extracts, dry with
anhydrous sodium sulfate, filtered, 50.degree. C., concentrated to
be viscous, precipitate with ether and vacuum drying. 40 g crude
product dubbed aqueous solution, DEAE anion column separation,
respectively collected sodium chloride aqueous solution eluent,
adjusted aqueous phase to pH 2-3 with 2N hydrochloric acid,
extracted with dichloromethane, combined organic extracts, dried
with anhydrous sodium sulfate, filtered, concentrated, precipitated
with diethyl ether, get molecular weight 10,000 eight-arm
polyethylene glycol hydroxy-single-acetic acid (III-2) and
eight-arm polyethylene glycol hydroxyl groups-acetic acid (IIIA-2),
respectively.
[0116] NMR (DMSO) .delta.: 4.01 (s, CH.sub.2COOH), 4.54 (t,
CH.sub.2OH).
Example 18
8-arm polyethylene glycol hydroxy-single methyl acetate
(IIIB-2)
##STR00042##
[0118] Procedure:
[0119] In single-port round-bottomed flask, add 4.0 g molecular
weight 10,000 eight-arm polyethylene glycol hydroxy-single acid
(111-2), dissolved in 20 ml anhydrous methanol, ice water bath,
added 0.8 ml concentrated sulfuric acid at room temperature for 3
hours, with 8% aqueous solution of sodium bicarbonate to adjust the
pH value of 7.0, extracted with dichloromethane three times,
combined organic phases, the organic phase was dried with anhydrous
magnesium sulfate, filtered, 40.degree. C., concentrated to be
viscous, precipitated with ether and vacuum drying, get the
eight-arm polyethylene glycol hydroxy-single methyl acetate
(IIIB-2).
[0120] NMR (DMSO) .delta.: 3.32 (s, CH.sub.2COOCH.sub.3), 4.13 (s,
CH.sub.2COOCH.sub.3), 4.57 (t, CH.sub.2OH).
Example 19
8-arm polyethylene glycol sulfonate-single methyl acetate
(IIIC-2)
##STR00043##
[0122] Procedure:
[0123] In three round-bottomed flask, inlet nitrogen, add 3.0 g
molecular weight 10,000 eight-arm polyethylene glycol
hydroxy-single methyl acetate, dissolved in 50 ml toluene, heat it
to stream out 38 ml toluene, distillate to be clear, cool down to
room temperature, add 5 ml dichloromethane, stirred for 10 minutes,
add 439 ul triethylamine, stirred 5 minutes, add dropping 220 ul
methyl chloride, sealed reaction overnight, the next day, add 1.44
ml ethanol, stirring for 15 minutes, filtered, concentrated to be
viscous, heat dissolved with 60 ml isopropanol, ice water bath,
sedimentate, filtrate, wash cake with isopropanol one time, vacuum
drying, get eight-arm polyethylene glycol sulfonate-single methyl
acetate (IIIC-2).
[0124] NMR (DMSO) .delta.: 3.17 (s, CH.sub.2OSO.sub.2CH.sub.3),
4.13 (s, CH.sub.2COOCH.sub.3), 4.30 (t,
CH.sub.2OSO.sub.2CH.sub.3).
Example 20
Synthesis of 8-arm polyethylene glycol amino-single-acetic acid
(V-2)
##STR00044##
[0126] Procedure:
[0127] In single-port round-bottomed flask, add 2.6 g molecular
weight 10,000 eight-arm polyethylene glycol sulfonate-single methyl
acetate, dissolved in 7.8 ml degassed water, use 2N aqueous sodium
hydroxide to adjust the solution pH 12.0, at room temperature for
2-2.5 hours, add 26 ml ammonia solution dissolved 1.3 g ammonium
chloride to the system, at room temperature stirred for 72 hours,
after the reaction, add 7 g sodium chloride, dissolved, extracted
with methylene chloride reaction mixture three times, combine
organic phase, concentrated at 40.degree. C. to dryness, add 30 ml
water, stirring to dissolve to clear, adjust the pH to 2-3 with 2N
hydrochloric acid, add 5 g sodium chloride, again extracted with
dichloromethane three times, and combined organic phases, the
organic phase with anhydrous sodium sulfate to dry to clarify,
filter, concentrated at 40.degree. C. to be viscous, sedimentation
with 50 ml ether, filtration, vacuum drying, get eight-arm
polyethylene glycol amino-single-acetic acid (V-2).
[0128] NMR (DMSO) .delta.: 2.96 (t, CH.sub.2CH.sub.2NH.sub.2), 4.00
(s, CH.sub.2COOH).
Example 21
Synthesis of 8-arm polyethylene glycol acrylate-single-acetic acid
(VI-2)
##STR00045##
[0130] Procedure:
[0131] In three round-bottomed flask, inlet nitrogen, take 1.0 g
molecular weight 10,000 eight-arm polyethylene glycol
hydroxy-single acetic acid (III-2) and 0.0005 gBHT, dissolved in 12
ml dichloromethane, heating to steam out 10% solvent, cooling to
room temperature, add 115 ul triethylamine, stirring for 5-10
minutes, adding 59 ul acryloyl chloride, filling nitrogen, protect
from light, confined system, overnight reaction with stirring, the
next day, concentrated at 30.degree. C. to be thick, add 20 ml,
dissolved to clear, put it aside for 30 minutes, adding 15% sodium
chloride, to adjust pH=2-3 with dilute hydrochloric acid, extracted
with dichloromethane three times, combine organic phases, the
organic phase was dried with anhydrous sodium sulfate to clarify,
filter, concentrated filtrate to be thick at 30.degree. C., ice
bath add 20 ml isopropanol, thermal dissolution and precipitation,
filtration, wash with isopropanol, vacuum drying, get eight-arm
polyethylene glycol acrylate-single-acetic acid (VI-2).
[0132] NMR (DMSO) .delta.: 4.00 (s, CH.sub.2COOH), 4.21 (t,
CH.sub.2OCOCH.dbd.CH.sub.2), 5.93 (d, CH.sub.2OCOCH.dbd.CH.sub.2),
6.20 (q, CH.sub.2OCOCH.dbd.CH.sub.2), 6.36 (d,
CH.sub.2OCOCH.dbd.CH.sub.2).
Example 22
The synthesis of 8-arm polyethylene glycol acrylate-single-NHS
ester (VII-2)
##STR00046##
[0134] Procedure:
[0135] 8-arm poly(ethylene glycol)acrylate-single-acetic acid
(VI-2) (10 kDa, 0.8 g) and N-hydroxysiccinimide (0.011 g) were
dissolved in 8 ml of dichloromethane. DCC (0.0206 g) was added and
the solution was stirred overnight at room temperature, filtered,
concentrated under vacuum at 40.degree. C. The residue was
dissolved in hot iso-propanol and then crystallized by cooling the
solution to 0.degree. C. The resulting precipitate was collected,
washed with iso-propanol and dried to afford the 4-arm
poly(ethylene glycol)acrylate-single-N-hydroxysuccinimidyl ester
(VII-2).
##STR00047##
Example 23
Conjugates Combined by eight-arm polyethylene glycol
acrylate-single-acetic acid (VI-2) and enoxaparin derivatives
[0136] 1 gram molecular weight 10,000 eight-arm polyethylene glycol
acrylate--single-acetic acid (VI-2) (produced in Implementation
Example 21) was dissolved in 10 ml dichloromethane, and then add
0.12 g enoxaparin topotecan glycine cool (Glycine-Irrinitecan)
(Irrinitecan purchased from Chengdu Furunde Industrial Co., Ltd.),
and 50 mg dimethyl-aminopyridine, and 95 mg
dicyclohexylcarbodiimide two sub-15 amine. This solution was
stirred at room temperature for 6 hours, vacuum to recover solvent,
and add 20 ml 1,4-dioxane to the residue to dissolve. Filter to
remove the precipitate, the solution is concentrated, the residue
by adding 20 ml ether and filtered to collect precipitation and
then vacuum drying with ether.
[0137] Yield: 0.8 g (80%), melting point: 46-50.degree. C.
Example 24
[0138] Synthesis of Stable Eight-arm polyethylene glycol acrylate
with drug gel Eight-arm polyethylene glycol acrylate-single-acetic
acid (VI-2) (10 k Da, 0.5 g) and enoxaparin derivatives (prepared
in Example 23) were dissolved in 10 ml phosphate buffer (pH 7.4).
4-arm polyethylene glycol SH (5 k Da, 0.4 g) was dissolved in 10 ml
phosphate buffer (pH 7.4). The two solutions were mixed quickly,
and the eight-arm polyethylene glycol gel formed in 2 minutes. The
formed gel was placed in 100 ml phosphate buffer (pH 7.4), stored
at 37.degree. C. The gel will be stable in 360 days, and will
witness and degeneration or melting, but the enoxaparin will
release slowly.
* * * * *