U.S. patent application number 09/308667 was filed with the patent office on 2002-03-14 for bioactive derivatives of camptothecin.
Invention is credited to ANGELUCCI, FRANCESCO, CAIOLFA, VALERIA, FACHIN, GABRIELE, SUARATO, ANTONINO.
Application Number | 20020032331 09/308667 |
Document ID | / |
Family ID | 10820049 |
Filed Date | 2002-03-14 |
United States Patent
Application |
20020032331 |
Kind Code |
A1 |
ANGELUCCI, FRANCESCO ; et
al. |
March 14, 2002 |
BIOACTIVE DERIVATIVES OF CAMPTOTHECIN
Abstract
Water soluble polymeric conjugates of camptothecin consist
essentially of N-(2-hydroxypropyl)methacryloylamide units linked
via a spacer group to a residue of a camptothecin such as
irinotecan or its non-soluble metabolite, 7-ethyl-10-hydroxy
amptothecin. The conjugates possess enhance antitumor activity and
decreased toxicity with respect to the free drug. A process for
their preparation and the pharmaceutical compositions containing
them are also described.
Inventors: |
ANGELUCCI, FRANCESCO;
(MILAN, IT) ; FACHIN, GABRIELE; (PAVIA, IT)
; CAIOLFA, VALERIA; (MILAN, IT) ; SUARATO,
ANTONINO; (MILAN, IT) |
Correspondence
Address: |
OBLON SPIVAK MCCLELLAND
MAIER & NEUSTADT
1755 JEFFERSON DAVIS HIGHWAY
FOURTH FLOOR
ARLINGTON
VA
22202
|
Family ID: |
10820049 |
Appl. No.: |
09/308667 |
Filed: |
June 3, 1999 |
PCT Filed: |
September 25, 1998 |
PCT NO: |
PCT/EP98/06185 |
Current U.S.
Class: |
546/48 ; 546/51;
546/53 |
Current CPC
Class: |
A61K 47/65 20170801;
A61K 47/58 20170801 |
Class at
Publication: |
546/48 ; 546/51;
546/53; 514/19; 514/283 |
International
Class: |
A61K 038/00; C07D
471/22 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 3, 1997 |
GB |
9721070.2 |
Claims
1. A polymeric conjugate which consists essentially of: (i) from 85
to 97 mol % of N-(2-hydroxypropyl)methacryloylamide units
represented by formula (2) 10(ii) from 3 to 15 mol % of units
represented by formula (3) 11wherein [S] is a spacer group; Z is
hydrogen, R.sub.1CO in which R.sub.1 is C.sub.1--C.sub.6 alkyl or
the group 1,4'-bipiperidine and (iii) from 0 to 12 mol % of
N-methacryloyl-glycine or
N-(2-hydroxypropyl)methacryloyl-glycinamide units represented by
formula (4) 12wherein [X] represents hydroxy or a residue of
formula --NH--CH.sub.2--CH(OH)--CH.sub.3.
2. A polymeric conjugate according to claim 1 which contains the
N-(2-hydroxypropyl) methacryloylamide units represented by the
formula (2) in a molar proportion of 90%.
3. A polymeric conjugate according to claim 1 which contains 10 mo%
of the 20-O-(methacryloyl-glycyl-peptidyl)camptothecin units
represented by the formula (3).
4. A polymeric conjugate according to claim 1 wherein the spacer
group [S] in the units of the formula (3) is selected from Ala-Gly,
Phe-Gly, Leu-Gly, Phe-Ala, Leu-Leu, Phe-Leu-Gly, Leu-Leu-Gly and
Phe-Leu-Gly-Gly.
5. A polymeric conjugate according to claim 1 wherein Z in the
units of the formula (3) is H.
6. A polymeric conjugate according to claim 1 in which the content
of active camptothecin derivative of formula (1) 13wherein Z is as
defined in claim 1, is 10% (w/w).
7. A process for preparing a polymeric conjugate as defined in
claim 1, which process comprises reacting a camptothecin derivative
of formula (5): 14wherein [S] and Z are as defined in claim 1, with
a polymer (B) consisting essentially of: (i) from 85 to 97 mol % of
N-(2-hydroxypropyl)methacryloylamide units represented by
formula(2) 15(iv) from 3 to 15 mol % of N-methacryloyl-glycyl units
represented by formula(6) 16wherein R.sub.2is: the residue of an
active ester, or hydroxy; and optionally displacing the remaining
active ester groups with 1-amino-2-propanol.
8. A .sup.20-O-peptidyl-camptothecin derivative of formula (5')
17wherein the group [S'] is a peptidyl spacer and Z is as defined
in claim 1, and their salt derivatives.
9. A process for preparing a compound of formula (5') as defined in
claim 8, which process comprises condensing a camptothecin
derivative of formula 1 as defined in claim 6 with a
N-protected-peptidyl derivative of formula (7):R.sub.3--[S']--P
(7)wherein [S'] is as defined in claim 8, R.sub.3 represents an
amino-protecting group and P is a residue of an activated ester, to
give a compound represented by formula (8): 18wherein Z' is
R.sub.3[S'] or R.sub.1CO, wherein R.sub.1, R.sub.3 and [S'] are as
defined above and then deblocking the N-protecting group of the
substituent at position C-20 from the resulting compound.
10. A pharmaceutical composition comprising a pharmaceutically
acceptable diluent or carrier and, as active ingredient, a
polymeric conjugate as defined in any one of claims 1 to 6 or a
compound of formula (5') as defined in claim 8.
Description
[0001] The present invention provides water soluble polymeric
conjugates of camptothecin possessing enhanced antitumor activity
and decreased toxicity with respect to the free drug.
7-Ethyl-10-hydroxy-camptothecin (1a, Z=H), a topoisomerase I
inhibitor belonging to the class of camptothecin, is a non-soluble
compound and it is recognized as the active metabolite of
irinotecan (CPT- 11, 1b, Z=1,4'-bipiperidinecarbonyl- , Cancer
Res.: 50, 1715-20, 1990). 1
[0002] The invention provides a polymeric conjugate which is
denoted herein as (A) and which consists essentially of:
[0003] (i) from 85 to 97 mol % of
N-(2-hydroxypropyl)methacryloylamide units represented by formula
(2) 2
[0004] (ii) from 3 to 15 mol % of units represented by formula (3)
3
[0005] wherein [S] is a spacer group; Z is hydrogen, R.sub.1CO in
which R.sub.1 is C.sub.1-C.sub.6 alkyl or the group
1,4'-bipiperidine and
[0006] (iii) from 0 to 12 mol % of N-methacryloyl-glycine or
N-(2-hydroxypropyl) methacryloyl-glycinamide units represented by
formula (4) 4
[0007] wherein [X] represents hydroxy or a residue of formula
--NH--CH.sub.2--CH(OH)--CH.sub.3. The polymer of the present
invention may also be represented as follows: [(2)].sub.x;
[(3)].sub.y; [(4)].sub.z wherein (2), (3) and (4) are units of the
formula as above defined, and x is from 85 to 97 mol %, y is from 3
to 15 mol % and z is from 0 to 12 mol %. Preferably, the polymeric
conjugate (A) contains the N-(2-hydroxypropyl) methacryloyl amide
units represented by the formula (2) in a proportion of 90 % or
more; more preferably 90%. The conjugate may also contain from 3 to
10 mol% of the 20-O-(methacryloyl-glycyl-pepti- dyl)camptothecin
units represented by the formula (3), more preferably 10 mol % of
such units. Preferably A does not contain residues of formula (4),
i.e.zis0.
[0008] The spacer group is susceptible to intratumoral enzymatic
hydrolysis. The spacer group may be an amino acid residue or a
peptide spacer, for example from 2 to 4 amino acid residues long.
Preferably the spacer group [S] is selected from Ala-Gly, Phe-Gly,
Leu-Gly, Phe-Ala, Leu-Leu, Phe-Leu-Gly, Leu-Leu-Gly and
Phe-Leu-Gly-Gly.
[0009] Preferably, the polymeric conjugate (A) contains the units
represented by formula (3) wherein Z is H.
[0010] Content of active camptothecin derivative of formula (1) in
the conjugate of formula A may be from 2 to 15% (weight/weight),
more preferably 10% (w/w). The invention also provides a process
for preparing a polymeric conjugate as defined above, which process
comprises reacting a camptothecin derivative of formula (5): 5
[0011] wherein [S] and Z are as defined above, with a polymer (B)
consisting essentially of:
[0012] (i) from 85 to 97 mol % of
N-(2-hydroxypropyl)methacryloylamide units represented by formula
(2) 6
[0013] (iv) from 3 to 15 mol % of N-methacryloyl-glycyl units
represented by formula (6) 7
[0014] wherein R.sub.2 is
[0015] the residue of an active ester, or
[0016] hydroxy;
[0017] and optionally displacing the remaining active ester groups
with 1-amino-2-propanol.
[0018] The invention also provides 20-O-peptidyl-camptothecin
derivatives of formula (5') 8
[0019] wherein the group [S'] is a peptide spacer, for example from
2 to 4 amino acid residues long, preferably selected from Ala-Gly,
Phe-Gly, Leu-Gly, Phe-Ala, Leu-Leu, Phe-Leu-Gly, Leu-Leu-Gly and
Phe-Leu-Gly-Gly; Z is as defined above, and their salt derivatives.
Such compounds are prepared by condensing camptothecin derivatives
of formula 1 as above defined with a N-protected-peptidyl
derivative of formula (7):
R.sub.3--[S']--P (7)
[0020] wherein [S'] is as defined above and R.sub.3 represents an
amino-protecting group, such as Boc, FMOC, triphenylsilyl,
diphenylmethylene or triphenylmethyl, and P is a residue of an
activated ester, such as p-nitrophenoxy or N-hydroxysuccinimido, to
give a compound represented by formula (8): 9
[0021] wherein Z' is R.sub.3[S'] or R.sub.1CO, wherein R.sub.1,
R.sub.3 and [S'] are as defined above and then deblocking the
N-protecting group of the substituent at position C-20 from the
resulting compound. The compounds of formula (5) as defined above
may be analogously prepared.
[0022] Preparation of compounds of formula (7) follows standard
synthetic procedures that are known from the literature. Suitable
N-protected-peptidyl derivatives of formula 7 include, for example,
N-(tri-phenylmethyl)-L-phenylalanyl-L-leucyl-glycyl
p-nitrophenylester (7a),
N-(t-Butoxycarbonyl)-L-phenyl-alanyl-L-leucyl-glycyl
p-nitrophenylester (7b). Thus, for example,
7-ethyl-10-hydroxy-camptothec- in (1a) may be allowed to react with
a molar excess, for example up to five-fold molar excess or more,
especially 2 mol.equivalent, of a N-protected-peptidyl derivative
of formula (7) in anhydrous solvent such as dry dimethylformamide
in the presence of 4-dimethylaminopyridine. In this manner, the
protected amino acid is introduced at both hydroxylated positions
10--OH and 20--O of compound 1a to give compound of formula (8a:
Z=R.sub.3 [S']).
[0023] The reaction can typically be effected for from 8 to 48
hours. The reaction is typically carried out at a temperature from
15 to 40.degree. C. The substituent group at position 10--OH and
the amino-protected group R.sub.3 of the substituent at 20--OH are
removed by an appropriate deprotecting agent to give the
7-ethyl-10-hydroxy-20-O-peptidyl-camptothe- cin derivative of
formula (5a: Z=H). Deprotection may be therefore achieved by acid
treatment, such as treatment with acetic acid, a mixture of acetic
acid and 1.5N aqueous hydrochloric acid or 90% aqueous
trifluoroacetic acid from one to 6 hours at temperature from 10 to
30.degree. C.; preferably for two hours at room temperature.
[0024] The condensation of derivative of formula (5) with the
polymer of formula (B), is carried out in conditions capable of
preserving the nature of linkage between
7-ethyl-10-(substituted)-camptothecin and spacer [S] as well as
that of the conjugate.
[0025] Polymers consisting essentially of from 85 to 97 mol % of
N-(2-hydroxypropyl)-methacryloylamide units of formula (2) and from
15 to 3 mol % of N-methacryloyl-glycine units of formula (6), are
prepared by copolymerization of N-(2-hydroxypropyl) methacrylamide
with N-methacryloyl-glycine or methacryloyl-glycine active-ester
derivatives, as described in Makromol.Chem. 178, 2159 (1977).
R.sub.2 may represent a phenoxy group which is substituted on the
phenyl ring by one ore more electron-withdrawing groups, such as
nitro or halogen. Preferably R.sub.2 represent p-nitrophenoxy.
Preferably, the reaction between a polymer in which R.sub.2
represents the residue of active ester and a compound of formula
(5) to prepare the polymeric conjugate of the invention is carried
out in an anhydrous polar organic solvent such as
dimethylsulfoxide. The reaction can typically carried out at
temperature from 15 to 30.degree. C., preferably at room
temperature for 15 hours; then the aminolysis of the remaining
active ester groups can be performed in the presence of
1-amino-2-propanol at room temperature, from 0.5 to 1 hour. The
conjugate suitably is precipitate with ethyl acetate, dissolved in
ethanol and reprecipitated with ethyl acetate.
[0026] For example, the polymer in which R.sub.2 represents the
residue of an active ester, provided at a concentration of 15%
(w/v) in dry dimethylsulfoxide, is treated with
7-ethyl-10-hydroxy-20-O-peptidyl-campt- othecin derivative of
formula (5a), 3% (w/v), at room temperature for 15 hours. Then
1-amino-2-propanol, 0.1% (w/v) is added and the reaction mixture is
kept at room temperature for 1 hour. The conjugate can be
precipitated with ethyl acetate, collected, washed with ethyl
acetate, then dissolved with absolute ethanol at a concentration of
10% (w/v) and precipitated again with ethyl acetate to give the
conjugate of formula (A) according to the invention.
[0027] The content of active drug (1), in polymeric conjugate of
the invention is determined by HPLC or absorbance spectroscopy
analysis.
[0028] The polymer-bound derivatives of formula (A) are in the
range of 5.000 to 45.000 molecular weight, preferably from 18.000
to 35.000. Polymeric drug derivatives of formula (A) are water
soluble and show enhanced antitumor activity and reduced toxicity
in comparison with the free 7-ethyl-10-hydroxy-camptothecin. They
are useful in the treatment of leukemia and solid tumors, such as
colon, colo-rectal, ovarian, mammary, prostate, lung, kidney and
also melanoma tumors. A human can therefore be treated by a method
comprising administering thereto a therapeutically effective amount
of a polymeric conjugate of the invention. The condition of the
human patient can thus be improved.
[0029] The dosage range adopted will depend on the route of
administration and on the age, weight and condition of the patient
being treated. The polymeric conjugates of formula (A) as well as
soluble salt derivatives of formula (5') are typically administered
by parenteral route, for example intramuscularly, intravenously or
by bolus infusion. A suitable dose range is from 1 to 1000 mg of
equivalent per m.sup.2 body surface area of active drug of formula
(1), for instance from 10 to 100 mg/m.sup.2.
[0030] The polymeric conjugate (A) or soluble salt derivatives of
formula (5') may be formulated into a pharmaceutical composition
together with a pharmaceutically carrier or diluent. Typically they
are formulated for parenteral administration, for example by
dissolution in water for injection or physiological saline.
[0031] The following Examples illustrate the invention.
[0032] Example 1.
[0033] Preparation of: 7-ethyl-10-hydroxy
20-O-(Lmphenylalanyl-L-leucyl-gl- ycyl)camptothecin (5c; H[S]
=H-Phe-Leu-Gly-, Z.apprxeq.H)
[0034] 7-ethyl-10-hydroxy-camptothecin (1a, 0.8g, 2mmol), ),
N-(tert-Butoxycarbonyl)-L-phenyl-alanyl-L-leucyl-glycyl
p-nitrophenylester (7b; 3.3g, 6mmol) and 4-dimethylaminopyridine
(0.5g, 4mmol) were dissolved with dry dimethylsulfoxide (30ml) and
kept at room temperature for three days under stirring. After that
the reaction mixture was poured in 0.1N aqueous hydrochloric acid
(500ml) to give a precipitate which was collected on a sintered
glass funnel. The solid material was dissolved in ethyl acetate
(200ml) and washed with saturated aqueous solution of sodium
hydrogen carbonate (2.times.100ml) and water (2.times.100ml). The
organic phase was separated, dried over anhydrous sodium sulfate
and evaporated under reduced pressure. The residue was treated with
90% aqueous trifluoroacetic acid (40ml) for three hours, then the
solvent was removed under reduced pressure. The crude material was
dissolved in methanol (50ml), diluted with methylene chloride
(200ml), washed with saturated solution of sodium hydrogen
carbonate (3.times.100ml) and water (3.times.100ml). The organic
phase was dried over anhydrous sodium sulfate evaporated under
reduced pressure and flash chromatographed on silica gel using a
mixture of methylene chloride/methanol (95/5 v/v) as eluting system
to give 0.93g of the title compound (5c).
[0035] TLC on Kieselgel plate F.sub.254 (Merck), eluting system
methylene chloride/methanol (95/5 v/v), R.sub.f=0.18.
[0036] .sup.1H-NMR (200 MHz, DMSO) 0.80 (d, J=5.7Hz, 6H,
.delta.+.delta.'-Leu); 0.90 (t, J=7.3Hz, 3H,
CH.sub.3--CH.sub.2-20); 1.26 (t, J=7.6Hz, 3H,
CH.sub.3--CH.sub.2-7); 1.45 (m, 3H, .beta.+.beta.'+.gamma.-Leu);
2.11 (q, J=7.3Hz, 2H. CH.sub.3--CH.sub.2-20); 2.56 (dd, J=13.2,
8.2Hz, 1H, .beta.-Phe); 2.88 (dd, J=13.2, 4.4 Hz, 1H, .beta.'-Phe);
3.05 (q, J=7.6Hz, 2H, CH.sub.3--CH.sub.2-7); 3.37 (dd, J=8.2,
4.4Hz, 1H, .alpha.-Phe); 3.99 (dd, J=18.0, 5.7Hz, 1H, .alpha.-Gly);
4.14 (dd, J=18.0, 5.7Hz, 1H, .alpha.'-Gly); 4.35 (m, 1H,
.alpha.-Leu); 5.24 (s. 2H, CH.sub.2-5); 5.47 (s, 2H, CH.sub.2-17);
7.02 (s, 1H, H-14); 7.05-7.45 (m, 7H, H-9 +H-11+Ar-Phe); 7,93 (d,
J=8.6Hz, 1H, NH-Leu); 7.99 (d, J=9.8Hz, 1H, H-12); 8.45 (t,
J=5.7Hz, 1H, NH-Gly).
[0037] Example 2.
[0038] Preparation of:7-ethyl-
10-hydroxy-20-O-(L-phenylalanyl-L-leucyl-gl- ycyl)camptothecin
hydrochloride (5d; H[S]=H-Phe-Leu-Gly-, Z=H)
[0039]
7-ethyl-10-hydroxy-20-O-(L-phenylalanyl-L-leucyl-glycyl)camptotheci-
n (5c, 0.5g), prepared as described in Example 1, was dissolved in
methylene chloride (10 ml), cooled at 0.degree. C. and treated with
a 10N methanolic solution of anhydrous hydrochloric (0.2 ml). After
one minute, ethyl ether (100ml) was added to the solution. The
precipitate was collected, washed with ethylene chloride and dried
to give 0.5g of the title compound (5d)
[0040] Example 3.
[0041] Preparation of : copolymer of
N-(2-hydroxypropvlmethacrvlamide {
7-ethyl-10-hydroxy-20-O-[N-methaeryloyl-glycyl-L-phenylalanyl-L-leugyl-gl-
ycyl]camptothecin} and N-(2-hydroxypropyl) methacrvlovlglycinamide
(A1)
[0042] Polymeric precursor (B1) (R.sub.2=p-nitrophenyloxy, 2.58g,
containing 1.16.times.10.sup.-3 eq. of p-nitro phenyl), prepared as
described in Makromol.Chem.178, 2159 (1977), was dissolved with
DMSO dry (15ml) and added with
7-ethyl-10-hydroxy-20-O-(L-phenylalanyl-L-leucyl
glycyl)camptothecin (5c; 0.71g, 1 mmol). The reaction mixture was
kept at room temperature for 22 hours under stirring, then
2-propanolamine (0.05ml) was added and the mixture left under
stirring for one more hour. After that, the reaction mixture was
precipitated with ethyl acetate (200ml) and left under stirring for
30 min. The solid material was collected on a sintered glass
funnel, washed with ethyl acetate (200ml) and ethyl ether (100ml)
and then dissolved with ethanol (30ml). The alcoholic solution was
treated with wet DOWEX-50, sulphonic form, (1.2g) under stirring
for 30min. and, after that, was added dropwise to n-hexane (200ml).
The precipitate was collected on a sintered glass funnel, washed
with ethyl ether and dried to constant weight to give 2.8g of the
title compound (A1).
[0043] Weight-average molecular weight (Mw): 24.300.
[0044] Polydispersity (Mw/Mn): 1.63.
[0045] Content of 7-ethyl-10-hydroxy-camptothecin, determined after
alkaline hydrolysis, 10% w/w.
[0046] Antitumor Activity
[0047] Compound A1 was tested on human colon carcinoma (HT29)
transplanted in nude mice, in comparison with the free drug
7-ethyl-10-hydroxycamptoth- ecin (1a) by i.v. route. A1 was found
non toxic at all tested doses and gave 98% tumor inhibition at the
highest tested dose of 20mg/kg (Table 1).
1TABLE 1 Antitumor Activity of A1 on human colon carcinoma (HT29)
in comparison with 7-ethyl-10-hydroxycamptothecin (1a). treatment
Dose Total Dose TI % compound schedule mg/kg mg/kg 46.sup.th day
Tox A1 iv q4d .times. 8 5 40 77 0/7 10 80 91 0/7 20 160 98 0/7 1a
iv q4d .times. 6 20 120 97 1/7 Reference
[0048] Tumor fragment were implanted sc. Treatment started when
tumor was palpable. TI% (tumor inhibition %) was calculated at day
46.
* * * * *