U.S. patent application number 15/454109 was filed with the patent office on 2017-12-21 for drug delivery conjugates containing unnatural amino acids and methods for using.
The applicant listed for this patent is ENDOCYTE, INC.. Invention is credited to Christopher Paul LEAMON, Iontcho Radoslavov VLAHOV.
Application Number | 20170360950 15/454109 |
Document ID | / |
Family ID | 50488881 |
Filed Date | 2017-12-21 |
United States Patent
Application |
20170360950 |
Kind Code |
A1 |
VLAHOV; Iontcho Radoslavov ;
et al. |
December 21, 2017 |
DRUG DELIVERY CONJUGATES CONTAINING UNNATURAL AMINO ACIDS AND
METHODS FOR USING
Abstract
Described herein are drug delivery conjugates for targeted
therapy. In particular, described herein are drug delivery
conjugates that include polyvalent linkers comprising one or more
unnatural amino acids that are useful for treating cancers and
inflammatory diseases.
Inventors: |
VLAHOV; Iontcho Radoslavov;
(West Lafayette, IN) ; LEAMON; Christopher Paul;
(West Lafayette, IN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ENDOCYTE, INC. |
West Lafayette |
IN |
US |
|
|
Family ID: |
50488881 |
Appl. No.: |
15/454109 |
Filed: |
March 9, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14435919 |
Apr 15, 2015 |
9662402 |
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PCT/US13/65079 |
Oct 15, 2013 |
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15454109 |
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61877317 |
Sep 13, 2013 |
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61865382 |
Aug 13, 2013 |
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61790234 |
Mar 15, 2013 |
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61714565 |
Oct 16, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 47/551 20170801;
A61K 47/549 20170801; A61P 29/00 20180101; A61P 35/00 20180101;
A61K 47/64 20170801; C07K 7/06 20130101 |
International
Class: |
A61K 47/64 20060101
A61K047/64; A61K 47/55 20060101 A61K047/55; A61K 47/54 20060101
A61K047/54; C07K 7/06 20060101 C07K007/06 |
Claims
1.-46. (canceled)
47. A compound of the formula B-L(D).sub.x, or a pharmaceutically
acceptable salt thereof, wherein B is a radical of a cell surface
receptor binding ligand, D is a radical of a drug, x is 1; and L is
a polyvalent releasable linker comprising one or more unnatural
amino acids in the D-configuration; and where B is covalently
attached to L, and L is covalently attached to D, with the proviso
that the compound is not of the formula ##STR00194## ##STR00195##
##STR00196## ##STR00197## ##STR00198## ##STR00199##
##STR00200##
48. The compound of claim 47, or a pharmaceutically acceptable salt
thereof, wherein L further comprises at least one radical selected
from the group consisting of ##STR00201## ##STR00202## ##STR00203##
##STR00204## ##STR00205## ##STR00206## ##STR00207## ##STR00208##
##STR00209## ##STR00210## ##STR00211## ##STR00212## ##STR00213##
##STR00214## ##STR00215## ##STR00216## wherein R is H, alkyl,
cycloalkyl, or arylalkyl; m is an integer from 1 to about 3; n is
an integer from 1 to about 5, p is an integer from 1 to about 5,
and r is an integer from 1 to about 3.
49. The compound of claim 47, or a pharmaceutically acceptable salt
thereof, wherein at least one unnatural amino acid is selected from
the group consisting of D-alanine, D-aspartic acid, D-asparagine,
D-cysteine, D-glutamic acid, D-phenylalanine, D-histidine,
D-isoleucine, D-lysine, D-leucine, D-methionine, D-proline,
D-glutamine, D-arginine, D-serine, D-threonine, D-valine,
D-tryptophan, D-tyrosine, and D-ornithine
50. The compound of claim 47, or a pharmaceutically acceptable salt
thereof, wherein L comprises at least one D-cysteine and at least
two D-glutamic acids.
51. The compound of claim 47, or a pharmaceutically acceptable salt
thereof, wherein L comprises two or more unnatural amino acids.
52. The compound of claim 47, or a pharmaceutically acceptable salt
thereof, wherein L comprises three or more unnatural amino
acids.
53. The compound of claim 47, or a pharmaceutically acceptable salt
thereof, wherein L comprises four or more unnatural amino
acids.
54. The compound of claim 47, or a pharmaceutically acceptable salt
thereof, wherein L further comprises a disulfide bond.
55. The compound of claim 48, or a pharmaceutically acceptable salt
thereof, wherein L further comprises a disulfide bond.
56. The compound of claim 49, or a pharmaceutically acceptable salt
thereof, wherein L further comprises a disulfide bond.
57. The compound of claim 47, or a pharmaceutically acceptable salt
thereof, wherein L further comprises a divalent radical of the
formula ##STR00217## wherein n is an integer selected from 1 to
about 4; R.sup.a and R.sup.b are each independently selected from
the group consisting of hydrogen and alkyl, including lower alkyl
such as C.sub.1-C.sub.4 alkyl that are optionally branched; or
R.sup.a and R.sup.b are taken together with the attached carbon
atom to form a carbocyclic ring; R is an optionally substituted
alkyl group, an optionally substituted acyl group, or a suitably
selected nitrogen protecting group; and each * indicates a covalent
bond to the rest of the compound.
58. The compound of claim 47, or a pharmaceutically acceptable salt
thereof, wherein B is a folate receptor binding ligand.
59. The compound of claim 58, or a pharmaceutically acceptable salt
thereof, wherein B is a folate.
60. The compound of claim 59, or a pharmaceutically acceptable salt
thereof, wherein B is of the formula ##STR00218##
61. The compound of claim 57, or a pharmaceutically acceptable salt
thereof, wherein B is of the formula ##STR00219##
62. The compound of claim 47, or a pharmaceutically acceptable salt
thereof, wherein D is an adrenocorticoid, a corticosteroid, an
alkylating agent, an antiandrogen, an antiestrogen, an androgen, an
aclamycin, an estrogens, an antimetabolite a platinum compound, a
taxane, a plant alkaloid, a mitomycin, a discodermolide, a
microtubule inhibitor, an epothilone, a tubulysin, an antibiotic, a
nitrogen mustard, a nitrosurea, a vinca alkaloid, a dolastatin, an
amanitin, an inflammatory agent, a proinflammatory agent, a
rapamycin, a penicillin, a cephalosporin, an aminoglycoside
antibiotic, a cryptophycin, a maytansine, a cryptophycin,
63. The compound of claim 47, or a pharmaceutically acceptable salt
thereof, wherein D is selected from the group consisting of
methotrexate, busulfan, carboplatin, chlorambucil, cisplatin,
tamoxiphen, taxol, paclitaxel, Taxotere.RTM., cyclophosphamide,
daunomycin, rhizoxin, T2 toxin, prednisone, teniposide, cyclopropyl
benz[e]indolone, seco-cyclopropyl benz[e]indolone,
O-Ac-seco-cyclopropyl benz[e]indolone, bleomycin, vincristine,
vinblastine, vindesine, vinorelbine, deacetylvinblastine
monohydrazide (DAVLBH), colchicine, allocolchicine, thiocolchicine,
trityl cysteine, halicondrin B, dolastatin 10, .alpha.-amanitin,
camptothecin, irinotecan, geldanamycin, estramustine, nocodazole,
colcemid, sirolimus, everolimus, vancomycin, erythromycin,
clindamycin, rifampin, chloramphenicol, gentamicin, amphotericin B,
acyclovir, trifluridine, ganciclovir, zidovudine, amantadine,
ribavirin, bortezomib, thiobortezomib, aminopterin, paclitaxel,
docetaxel, doxorubicin, daunorubicin, verucarin, didemnin B,
purvalanol A, ispinesib, budesonide, dasatinib, bortezomib,
thiobortezomib, and .alpha.-amanatin.
64. The compound of claim 47, or a pharmaceutically acceptable salt
thereof, wherein D is a tubulysin.
65. The compound of claim 61, or a pharmaceutically acceptable salt
thereof, wherein D is a tubulysin.
66. A pharmaceutical composition comprising the compound of claim
47, or a pharmaceutically acceptable salt thereof, in combination
with one or more carriers, diluents, or excipients, or a
combination thereof.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 14/435,919 filed on Apr. 15, 2015, which is a U.S. national
stage application under 35 U.S.C. .sctn.371(b) of International
Application No. PCT/US2013/065079 filed Oct. 15, 2013, and claims
priority under 35 U.S.C. .sctn.119(e) to U.S. Provisional
Application Ser. No. 61/714,565 filed Oct. 16, 2012, U.S.
Provisional Application Ser. No. 61/790,234 filed Mar. 15, 2013,
U.S. Provisional Application Ser. No. 61/865,382 filed Aug. 13,
2013, and U.S. Provisional Application Ser. No. 61/877,317 filed
Sep. 13, 2013. The disclosures of all the above referenced
applications are incorporated herein by reference in their
entirety.
TECHNICAL FIELD
[0002] The invention described herein pertains to drug delivery
conjugates for targeted therapy. In particular, the invention
described herein pertains to drug delivery conjugates that include
polyvalent linkers comprising one or more unnatural amino
acids.
BACKGROUND AND SUMMARY OF THE INVENTION
[0003] The mammalian immune system provides a means for the
recognition and elimination of pathogenic cells, such as tumor
cells, and other invading foreign pathogens. While the immune
system normally provides a strong line of defense, there are many
instances where pathogenic cells, such as cancer cells, and other
infectious agents evade a host immune response and proliferate or
persist with concomitant host pathogenicity. Chemotherapeutic
agents and radiation therapies have been developed to eliminate,
for example, replicating neoplasms. However, many of the currently
available chemotherapeutic agents and radiation therapy regimens
have adverse side effects because they lack sufficient selectivity
to preferentially destroy pathogenic cells, and therefore, may also
harm normal host cells, such as cells of the hematopoietic system,
and other non-pathogenic cells. The adverse side effects of these
anticancer drugs highlight the need for the development of new
therapies selective for pathogenic cell populations and with
reduced host toxicity.
[0004] It has been discovered herein that drug delivery conjugates
that include polyvalent linkers formed from one or more unnatural
amino acids are efficacious in treating pathogenic cell
populations, and exhibit low host animal toxicity.
[0005] In one illustrative and non-limiting embodiment of the
invention, described herein are compounds of the formula
B-L-D.sub.X
wherein each of B, L, D, and x are as defined in the various
embodiments and aspects described herein.
[0006] In another embodiment, pharmaceutical compositions
containing one or more of the compounds are also described herein.
In one aspect, the compositions include a therapeutically effective
amount of the one or more compounds for treating a patient with
cancer, inflammation, and the like. It is to be understood that the
compositions may include other components and/or ingredients,
including, but not limited to, other therapeutically active
compounds, and/or one or more carriers, diluents, excipients, and
the like, and combinations thereof. In another embodiment, methods
for using the compounds and pharmaceutical compositions for
treating patients or host animals with cancer, inflammation, and
the like are also described herein. In one aspect, the methods
include the step of administering one or more of the compounds
and/or compositions described herein to a patient with cancer,
inflammation, and the like. In another aspect, the methods include
administering a therapeutically effective amount of the one or more
compounds and/or compositions described herein for treating
patients with cancer, inflammation, and the like. In another
embodiment, uses of the compounds and compositions in the
manufacture of a medicament for treating patients with cancer,
inflammation, and the like are also described herein. In one
aspect, the medicaments include a therapeutically effective amount
of the one or more compounds and/or compositions for treating a
patient with cancer, inflammation, and the like.
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] FIG. 1A shows the relative affinity of EC1669 in KB cells, 1
h at 37.degree. C. FIG. 1B shows the relative affinity of EC1669 in
CHO-.beta. cells, 1 h at 37.degree. C.
[0008] FIG. 2 shows the cytostatic effect of EC1669 on RAW264.7
cells, as determined by XTT cell viability at 2 h and 72 h.
[0009] FIG. 3A shows in vivo activity of EC1456 against KB tumors
in nu/nu mice dosed at 1 .mu.mol/kg three times per week (M/W/F)
(TIW) for two consecutive weeks ( ), compared to EC1456 co-dosed
with EC0923 at 100 mol/kg (.tangle-solidup.), and untreated (PBS)
controls (.box-solid.). The dotted vertical line represents the day
of the final dose. FIG. 3B shows that EC1456 did not result in any
observable whole animal toxicity as determined by animal body
weight.
[0010] FIG. 4A shows the activity of EC1663 in nu/nu mice bearing
s.c. KB tumors, where EC1663 was administered i.v. starting on Day
7 with 0.5 .mu.mol/kg (.tangle-solidup.), three times per week
(M/W/F) for a 2 week period, and compared to untreated controls
(.box-solid.), N=5 animals per cohort. Dotted vertical line=day of
final dosing day. FIG. 4A shows 4/4 PRs in test animals. FIG. 4B
shows that EC1663 did not exhibit significant host animal
toxicity.
[0011] FIG. 5A shows the activity of EC1456 against established
subcutaneous MDA-MB-231 tumors. Animals bearing s.c. MDA-MB-231
tumors (94-145 mm.sup.3) were treated i.v. starting on Day 17 with
2 .mu.mol/kg (panel A) of EC1456 ( ), three times per week (M/W/F)
for a 2 week period, and compared to untreated animals
(.box-solid.), as shown in FIG. 5A. N=5 animals per cohort. Dotted
vertical line=day of final dose. FIG. 5B shows that EC1456 did not
cause gross whole animal toxicity as determined by % weight
change.
[0012] FIG. 6A shows the activity of EC1456 in animals bearing s.c.
KB-CR2000 (cisplatin resistant) tumors (98-148 mm3), where EC1456
was administered i.v. starting on Day 6 with 2 .mu.mol/kg ( ),
three times per week (M/W/F) for a 2 week period, or with 3 mg/kg
of cisplatin (.tangle-solidup.), twice per week (T/Th) for a 2 week
period, and compared to untreated controls (.box-solid.), N=5
animals per cohort. Dotted vertical line=day of final dosing day.
FIG. 6B shows that EC1456 did not exhibit significant host animal
toxicity. In contrast, cisplatin treatment resulted in substantial
host animal toxicity during the dosing period.
[0013] FIG. 7 shows the in vivo efficacy of EC1496 against
adjuvant-induced arthritis. The arrows indicate the treatment days.
(a) healthy control, (b) untreated control, (c) EC1496, (d)
EC1496+excess EC0923 (comparator/competition compound).
[0014] FIG. 8A shows the in vivo efficacy of EC1669 against
arthritis, (a) healthy control, (a) untreated control, (b) EC1669
(375 nmol/kg), (c) EC1669+500.times. EC0923. FIG. 8B shows that
EC1669 does not exhibit whole animal toxicity, (a) untreated
control, (b) EC1669 (375 nmol/kg), (c) EC1669+500.times. EC0923,
(d) healthy control.
[0015] FIG. 9A shows the in vivo efficacy of EC1669 against
arthritis, as determined by paw swelling. FIG. 9B shows the in vivo
efficacy of EC1669 against arthritis, as determined by bone
radiography.
[0016] FIG. 10A shows the in vivo efficacy of EC1669 alone, and
EC1669 plus CellCept combination co-therapy in AIA rats, where day
0 is 9 days post induction, and the arrows indicate treatment days,
(a) healthy control, (b) untreated control, (c) EC1669 (1000
nmol/kg, siw, sc), (d) CellCept.TM. (30 mg/kg, po, qdx5), (e)
EC1669+CellCept.TM.. FIG. 10B shows the whole animal toxicity
compared to control for each of the administration protocols.
[0017] FIG. 11 shows the in vivo efficacy of EC1669 alone, and
EC1669 plus CellCept combination co-therapy in AIA rats, as
determined by paw swelling.
[0018] FIG. 12A shows the in vivo efficacy of EC1669 against EAU
(total uveitis scores for both eyes). Animals are treated with
EC1669 (.box-solid.), EC1669 plus EC0923 (.quadrature.), and MTX
(.diamond-solid.) every other day starting on day 8 after EAU
induction or from untreated animals (.cndot.). Day 0 is 8 days post
induction, and the arrows indicate treatment days. FIG. 12B shows
that EC1669 does not cause whole animal toxicity.
[0019] FIG. 13A shows the in vivo efficacy of EC1496 against EAU
(total uveitis scores for both eyes), (a) uveitis untreated
control, (b) EC1496 (375 nmol/kg), (c) EC1496+excess EC0923. FIG.
13B shows the in vivo efficacy of EC1496 against EAU, as determined
by histology.
[0020] FIG. 14A shows the in vivo efficacy of EC1669 against EAE,
(a) untreated EAE control, (b) EC1669 (250 nmol/kg), (c)
EC1669+excess EC0923. Animals are treated every other day (as
indicated by arrows) starting on day 8 after EAE induction, and
compared to untreated control. FIG. 14B shows the percent changes
in body weight (B), averaged for each group.
[0021] FIG. 15 shows the in vivo efficacy of EC1496 against EAE.
Individual EAE scores from untreated animals and animals treated
with EC1496 and EC1496 plus EC0923 every other day starting on day
8 after EAE induction are shown, and compared to untreated
control.
[0022] FIG. 16A shows the pharmacokinetics of EC1496 (500 nmol/kg,
s.c.), and in vivo production of aminopterin and aminopterin
hydrazide. FIG. 16B shows the pharmacokinetics of EC0746
(comparator compound, 500 nmol/kg, s.c.), and in vivo production of
aminopterin and aminopterin hydrazide.
[0023] FIGS. 17A, 17C, and 17E show the pharmacokinetic
biodistribution of .sup.3H-EC1669; and FIGS. 17B, 17D, and 17F show
.sup.3H-methotrexate in mice. Test compounds were administered to
Balb/c mice at 500 nmol/kg, s.c.
[0024] FIG. 18 shows the comparison of RBC uptake of .sup.3H-EC1669
(.box-solid.) and .sup.3H-MTX () in mice, as a measure of
radioactivity over time.
[0025] FIG. 19 shows the relative whole animal toxicity between (b)
EC1496 (3 .mu.mol/kg) and (c) EC0746 (comparator compound, 3
.mu.mol/kg)), and compared to vehicle control (a) when dosed BIW
for 2 weeks in folate deficient rats.
[0026] FIG. 20 shows the maximum tolerated dose (MTD) of EC1456
compared to vehicle controls. Vehicle control (.box-solid.), EC1456
at 0.33 .mu.mol/kg (.cndot.), EC1456 at 0.41 .mu.mol/kg
(.tangle-solidup.), EC1456 at 0.51 .mu.mol/kg (), and EC1456 at
0.67 .mu.mol/kg (.diamond-solid.).
DETAILED DESCRIPTION
[0027] Several illustrative embodiments of the invention are
described by the following clauses:
[0028] A compound of the formula B-L(D).sub.X, or a
pharmaceutically acceptable salt thereof, wherein B is a radical of
a cell surface receptor binding and/or targeting ligand, D is in
each instance a radical of an independently selected drug, x is an
integer selected from 1, 2, 3, 4 and 5; and L is a polyvalent
releasable linker comprising one or more unnatural amino acids; and
where B is covalently attached to L, and L is covalently attached
to each D; and
[0029] where the compound is not any one of or any subgroup or
subset of the following formulae
##STR00001## ##STR00002## ##STR00003## ##STR00004## ##STR00005##
##STR00006## ##STR00007##
[0030] and/or where the compound is not of the following
formula
##STR00008##
[0031] and/or where the compound is not any one of or any subgroup
or subset of the following formulae
##STR00009## ##STR00010## ##STR00011## ##STR00012## ##STR00013##
##STR00014##
[0032] and/or where the compound is not any one of or any subgroup
or subset of the following formulae
##STR00015##
[0033] and/or where the compound is not of the following
formula
##STR00016##
[0034] and/or any combination of the foregoing;
[0035] or any pharmaceutically acceptable salt thereof.
[0036] The compound of the preceding clause wherein B-L(D).sub.X is
capable of binding to the cell surface receptor.
[0037] The compound of any one of the preceding clauses wherein the
ligand is a vitamin receptor binding ligand.
[0038] The compound of any one of the preceding clauses wherein the
ligand is a folate receptor binding ligand.
[0039] The compound of any one of the preceding clauses wherein the
ligand is a folate.
[0040] The compound of any one of the preceding clauses wherein the
ligand is a folate comprising D-glutamyl, also referred to herein
as D-folate, or pteroyl-D-glutamic acid. It is to be understood
herein that when B is a radical of D-folate, the included
D-glutamyl portion of B is not part of the linker L.
[0041] The compound of any one of the preceding clauses wherein B
is an unnatural folate radical of the formula
##STR00017##
[0042] The compound of any one of the preceding clauses wherein the
ligand is folic acid.
[0043] The compound of any one of the preceding clauses wherein B
is a radical of the formula
##STR00018##
[0044] The compound of any one of the preceding clauses wherein at
least one unnatural amino acid has the D-configuration.
[0045] The compound of any one of the preceding clauses wherein at
least one unnatural amino acid is selected from D-alanine,
D-aspartic acid, D-asparagine, D-cysteine, D-glutamic acid,
D-phenylalanine, D-histidine, D-isoleucine, D-lysine, D-leucine,
D-methionine, D-proline, D-glutamine, D-arginine, D-serine,
D-threonine, D-valine, D-tryptophan, D-tyrosine, and D-ornithine,
and any amino acid derivatives thereof.
[0046] The compound of any one of the preceding clauses wherein at
least one unnatural amino acid is selected from D-aspartic acid,
D-asparagine, D-cysteine, D-glutamic acid, D-histidine, D-lysine,
D-methionine, D-glutamine, D-arginine, D-serine, D-threonine,
D-tryptophan, D-tyrosine, and D-ornithine, and any amino acid
derivatives thereof.
[0047] The compound of any one of the preceding clauses wherein at
least one unnatural amino acid is selected from D-aspartic acid,
D-asparagine, D-cysteine, D-glutamic acid, D-histidine, D-lysine,
D-glutamine, D-arginine, D-serine, D-threonine, D-tryptophan, and
D-ornithine, and any amino acid derivatives thereof.
[0048] The compound of any one of the preceding clauses wherein at
least one unnatural amino acid is selected from D-aspartic acid,
D-cysteine, D-glutamic acid, D-lysine, D-arginine, D-serine, and
D-ornithine, and any amino acid derivatives thereof.
[0049] The compound of any one of the preceding clauses wherein L
comprises two or more unnatural amino acids.
[0050] The compound of any one of the preceding clauses wherein L
comprises three or more unnatural amino acids.
[0051] The compound of any one of the preceding clauses wherein L
comprises four or more unnatural amino acids.
[0052] The compound of any one of the preceding clauses wherein L
further comprises one or more disulfides.
[0053] The compound of any one of the preceding clauses wherein at
least one disulfide comprises L-cysteinyl.
[0054] The compound of any one of the preceding clauses wherein at
least one disulfide comprises D-cysteinyl.
[0055] The compound of any one of the preceding clauses wherein L
further comprises one or more divalent hydrophilic radicals.
[0056] The compound of any one of the preceding clauses wherein L
further comprises two or more divalent hydrophilic radicals.
[0057] The compound of any one of the preceding clauses wherein L
further comprises three or more divalent hydrophilic radicals.
[0058] The compound of any one of the preceding clauses wherein L
further comprises four or more divalent hydrophilic radicals.
[0059] The compound of any one of the preceding clauses wherein L
further comprises one or more divalent polyoxy radicals.
[0060] The compound of any one of the preceding clauses wherein L
further comprises two or more divalent polyoxy radicals.
[0061] The compound of any one of the preceding clauses wherein L
further comprises three or more divalent polyoxy radicals.
[0062] The compound of any one of the preceding clauses wherein L
further comprises four or more divalent polyoxy radicals.
[0063] The compound of any one of the preceding clauses wherein L
further comprises one or more divalent polyhydroxy radicals.
[0064] The compound of any one of the preceding clauses wherein L
further comprises two or more divalent polyhydroxy radicals.
[0065] The compound of any one of the preceding clauses wherein L
further comprises three or more divalent polyhydroxy radicals.
[0066] The compound of any one of the preceding clauses wherein L
further comprises four or more divalent polyhydroxy radicals.
[0067] The compound of any one of the preceding clauses wherein at
least one unnatural amino acid comprises a polyhydroxy radical.
[0068] The compound of any one of the preceding clauses wherein at
least two unnatural amino acids comprise a polyhydroxy radical.
[0069] The compound of any one of the preceding clauses wherein at
least three unnatural amino acids comprise a polyhydroxy
radical.
[0070] The compound of any one of the preceding clauses wherein at
least four unnatural amino acids comprise a polyhydroxy
radical.
[0071] The compound of any one of the preceding clauses wherein at
least one of the polyhydroxy radicals is of the formula
CH.sub.2--(CH(OH)).sub.n--CH.sub.2--OH
where n is selected from 1, 2, 3, 4, 5, and 6.
[0072] The compound of any one of the preceding clauses wherein n
is selected from 1, 2, 3, and 4.
[0073] The compound of any one of the preceding clauses wherein n
is selected from 3 and 4.
[0074] The compound of any one of the preceding clauses wherein n
is 3.
[0075] The compound of any one of the preceding clauses wherein L
comprises a divalent polyglutamic acid radical, where at least one
glutamic acid forms an amide with an aminopolyhydroxy radical.
[0076] The compound of any one of the preceding clauses wherein L
comprises a divalent polyglutamic acid radical, where at least two
glutamic acids form an amide with an aminopolyhydroxy radical.
[0077] The compound of any one of the preceding clauses wherein L
comprises a divalent polyglutamic acid radical, where at least
three glutamic acids form an amide with an aminopolyhydroxy
radical.
[0078] The compound of any one of the preceding clauses wherein L
comprises a divalent polyglutamic acid radical, where at least four
glutamic acids form an amide with an aminopolyhydroxy radical.
[0079] The compound of any one of the preceding clauses wherein at
least one of the glutamic acids is D-glutamic acid.
[0080] The compound of any one of the preceding clauses wherein at
least two of the glutamic acids is D-glutamic acid.
[0081] The compound of any one of the preceding clauses wherein at
least three of the glutamic acids is D-glutamic acid.
[0082] The compound of any one of the preceding clauses wherein at
least four of the glutamic acids is D-glutamic acid.
[0083] The compound of any one of the preceding clauses wherein at
least one of the glutamic acids is unsubstituted D-glutamic
acid.
[0084] The compound of any one of the preceding clauses wherein at
least two of the glutamic acids is unsubstituted D-glutamic
acid.
[0085] The compound of any one of the preceding clauses wherein at
least three of the glutamic acids is unsubstituted D-glutamic
acid.
[0086] The compound of any one of the preceding clauses wherein at
least four of the glutamic acids is unsubstituted D-glutamic
acid.
[0087] The compound of any one of the preceding clauses wherein L
comprises a divalent poly(D-glutamic acid) radical, where at least
one glutamic acid forms an amide with an aminopolyhydroxy
radical.
[0088] The compound of any one of the preceding clauses wherein L
comprises a divalent poly(D-glutamic acid) radical, where at least
two glutamic acids form an amide with an aminopolyhydroxy
radical.
[0089] The compound of any one of the preceding clauses wherein L
comprises a divalent poly(D-glutamic acid) radical, where at least
three glutamic acids form an amide with an aminopolyhydroxy
radical.
[0090] The compound of any one of the preceding clauses wherein L
comprises a divalent poly(D-glutamic acid) radical, where at least
four glutamic acids form an amide with an aminopolyhydroxy
radical.
[0091] The compound of any one of the preceding clauses wherein L
comprises a divalent radical of the formula (K-L).sub.d, where K is
a divalent D-glutamic acid radical, L is a divalent L-glutamic acid
radical that forms an amide with an aminopolyhydroxy radical, and d
is 1, 2, 3, or 4.
[0092] The compound of the preceding clause wherein d is 2, 3, or
4.
[0093] The compound of the preceding clause wherein d is 3 or
4.
[0094] The compound of the preceding clause wherein d is 3.
[0095] The compound of any one of the preceding clauses wherein at
least one of the aminopolyhydroxy radicals is of the formula
NH--CH.sub.2--(CH(OH)).sub.m--CH.sub.2--OH
where m is selected from 1, 2, 3, 4, 5, and 6.
[0096] The compound of any one of the preceding clauses wherein at
least one of the aminopolyhydroxy radicals is of the formula
NH--CH.sub.2--(CH(OH)).sub.m--R
where m is selected from 1, 2, 3, 4, 5, and 6; and R is H, alkyl,
cycloalkyl, or arylalkyl.
[0097] The compound of any one of the preceding clauses wherein m
is selected from 1, 2, 3, and 4.
[0098] The compound of any one of the preceding clauses wherein m
is selected from 3 and 4.
[0099] The compound of any one of the preceding clauses wherein L
comprises a divalent radical of the formula
S--CH.sub.2CH.sub.2--O--C(O).
[0100] The compound of any one of the preceding clauses wherein L
comprises a divalent radical of the formula
S--S--CH.sub.2CH.sub.2--O--C(O).
[0101] The compound of any one of the preceding clauses wherein L-D
comprises a radical of the formula
S--CH.sub.2CH.sub.2--O--C(O)-D.
[0102] The compound of any one of the preceding clauses wherein L-D
comprises a radical of the formula
S--S--CH.sub.2CH.sub.2--O--C(O)-D.
[0103] The compound of any one of the preceding clauses wherein x
is 3.
[0104] The compound of any one of the preceding clauses wherein x
is 2.
[0105] The compound of any one of the preceding clauses wherein x
is 1.
[0106] The compound of any one of the preceding clauses wherein at
least one drug is a cytotoxic agent.
[0107] The compound of any one of the preceding clauses wherein at
least one drug is a cancer treating agent.
[0108] The compound of any one of the preceding clauses wherein at
least one drug is a vinca alkaloid.
[0109] The compound of any one of the preceding clauses wherein at
least one drug is desacetylvinblastine monohydrazide.
[0110] The compound of any one of the preceding clauses wherein at
least one drug is a tubulysin.
[0111] The compound of any one of the preceding clauses wherein at
least one drug is tubulysin A.
[0112] The compound of any one of the preceding clauses wherein at
least one drug is tubulysin B.
[0113] The compound of any one of the preceding clauses wherein at
least one drug is tubulysin A hydrazide.
[0114] The compound of any one of the preceding clauses wherein at
least one drug is tubulysin B hydrazide.
[0115] The compound of any one of the preceding clauses wherein at
least one drug is a tubulysin where the Tuv residue includes an
ether aminal.
[0116] The compound of any one of the preceding clauses wherein at
least one drug is a tubulysin hydrazide where the Tuv residue
includes an ether aminal
[0117] The compound of any one of the preceding clauses wherein at
least one drug is a inflammation-treating agent.
[0118] The compound of any one of the preceding clauses wherein at
least one drug is an anti-inflammatory agent.
[0119] The compound of any one of the preceding clauses wherein at
least one drug is a dihydrofolate reductase inhibitor.
[0120] The compound of any one of the preceding clauses wherein at
least one drug is aminopterin or methotrexate.
[0121] The compound of any one of the preceding clauses wherein at
least one drug is an aminopterin.
[0122] The compound of any one of the preceding clauses wherein at
least one drug is an inhibitor of mammalian target of rapamycin
(mTOR).
[0123] The compound of any one of the preceding clauses wherein at
least one drug is sirolimus (rapamycin), temsirolimus, everolimus,
or ridaforolimus.
[0124] The compound of any one of the preceding clauses wherein at
least one drug is not T-2 mycotoxin.
[0125] The compound of any one of the preceding clauses wherein at
least one drug is not a duocarmycin.
[0126] The compound of any one of the preceding clauses wherein at
least one drug is not a mitomycin.
[0127] The compound of any one of the preceding clauses wherein at
least one drug is not desacetylvinblastine monohydrazide.
[0128] The compound of any one of the preceding clauses wherein at
least one D is a radical of the formula
##STR00019##
[0129] The compound of any one of the preceding clauses wherein at
least one D is a radical of the formula
##STR00020##
[0130] The compound of any one of the preceding clauses wherein at
least one D is a radical of the formula
##STR00021##
where n=1, 2, 3, 4, 5, or 6, or alternatively, n=1, 2, or 3, or
alternatively, n=2 or 3.
[0131] The compound of any one of the preceding clauses wherein at
least one D is a radical of the formula
##STR00022##
where n=1, 2, 3, 4, 5, or 6, or alternatively, n=1, 2, or 3, or
alternatively, n=2 or 3.
[0132] The compound of any one of the preceding clauses wherein at
least one drug is a compound capable of binding to or reacting with
a nucleic acid or a DNA transcription factor, or a prodrug
thereof.
[0133] The compound of any one of the preceding clauses wherein B-L
is a radical of the formula
##STR00023##
[0134] The compound of any one of the preceding clauses wherein B-L
is a radical of the formula
##STR00024##
[0135] The compound of any one of the preceding clauses wherein B-L
is a radical of the formula
##STR00025##
[0136] The compound of any one of the preceding clauses wherein B-L
is a radical of the formula
##STR00026##
[0137] The compound of any one of the preceding clauses wherein the
compound is of the formula EC1456
##STR00027##
[0138] or a pharmaceutically acceptable salt thereof.
[0139] The compound of any one of the preceding clauses wherein the
compound is not of the formula EC1456
##STR00028##
or a pharmaceutically acceptable salt thereof.
[0140] The compound of any one of the preceding clauses wherein the
compound is of the formula EC1496
##STR00029##
or a pharmaceutically acceptable salt thereof.
[0141] The compound of any one of the preceding clauses wherein the
compound is not of the formula EC1496
##STR00030##
or a pharmaceutically acceptable salt thereof.
[0142] The compound of any one of the preceding clauses wherein the
compound is of the formula EC1669
##STR00031##
or a pharmaceutically acceptable salt thereof.
[0143] The compound of any one of the preceding clauses wherein the
compound is not of the formula EC1669
##STR00032##
or a pharmaceutically acceptable salt thereof.
[0144] A pharmaceutical composition comprising a compound of any
one of the preceding clauses in combination with one or more
carriers, diluents, or excipients, or a combination thereof.
[0145] A unit dose or unit dosage form composition comprising a
therapeutically effective amount of one or more compounds of any
one of the preceding clauses, optionally in combination with one or
more carriers, diluents, or excipients, or a combination
thereof.
[0146] A composition for treating cancer or inflammation in a host
animal, the composition comprising a therapeutically effective
amount of one or more compounds of any one of the preceding
clauses; or a pharmaceutical composition comprising a
therapeutically effective amount of one or more compounds of any
one of the preceding clauses, optionally further comprising one or
more carriers, diluents, or excipients, or a combination
thereof.
[0147] A method for treating cancer or inflammation in a host
animal, the method comprising the step of administering to the host
animal a composition comprising a therapeutically effective amount
of one or more compounds of any one of the preceding clauses; or a
pharmaceutical composition comprising one or more compounds of any
one of the preceding clauses, optionally further comprising one or
more carriers, diluents, or excipients, or a combination
thereof.
[0148] Use of one or more compounds of any one of the preceding
clauses, optionally in combination with one or more carriers,
diluents, or excipients, or a combination thereof, in the
manufacture of a medicament for treating a cancer or inflammation
in a host animal.
[0149] The method or composition or unit dose or use of any one of
the preceding clauses wherein the cancer is drug resistant
cancer.
[0150] The method or composition or unit dose or use of any one of
the preceding clauses wherein the cancer is a platinum resistant
cancer.
[0151] The method or composition or unit dose or use of any one of
the preceding clauses wherein the cancer is a cisplatin resistant
cancer.
[0152] The method or composition or unit dose or use of any one of
the preceding clauses wherein the cancer is an ovarian cancer.
[0153] The method or composition or unit dose or use of any one of
the preceding clauses wherein the cancer is a drug resistant
ovarian cancer.
[0154] The method or composition or unit dose or use of any one of
the preceding clauses wherein the cancer is a cisplatin resistant
ovarian cancer.
[0155] The method or composition or unit dose or use of any one of
the preceding clauses wherein the cancer is a platinum resistant
ovarian cancer, such as NCI/ADR-RES or NCI/ADR-RES related ovarian
cancer.
[0156] The method or composition or unit dose or use of any one of
the preceding clauses wherein the cancer is a platinum resistant
ovarian cancer, such as IGROVCDDP or IGROVCDDP related ovarian
cancer.
[0157] The method or composition or unit dose or use of any one of
the preceding clauses wherein the cancer is a breast cancer.
[0158] The method or composition or unit dose or use of any one of
the preceding clauses wherein the cancer is a drug resistant breast
cancer.
[0159] The method or composition or unit dose or use of any one of
the preceding clauses wherein the cancer is a triple negative
breast cancer, such as MDA-MB-231 or MDA-MB-231 related breast
cancer.
[0160] The method or composition or unit dose or use of any one of
the preceding clauses wherein the cancer is a non-small cell lung
cancer.
[0161] The method or composition or unit dose or use of any one of
the preceding clauses wherein the cancer is a hepatocellular
carcinoma or cancer.
[0162] An intermediate for preparing a compound of any one of the
preceding clauses of the formula
##STR00033##
or a pharmaceutically acceptable salt thereof, wherein L is a
leaving group.
[0163] An intermediate for preparing a compound of any one of the
preceding clauses of the formula
##STR00034##
or a pharmaceutically acceptable salt thereof, wherein M is
hydrogen or a cation.
[0164] An intermediate for preparing a compound of claim 1 of the
formula
##STR00035##
or a pharmaceutically acceptable salt thereof, wherein L is a
leaving group.
[0165] In another embodiment, the compounds described herein can be
internalized into the targeted pathogenic cells by binding to the
corresponding cell surface receptor. In particular, vitamin
receptors, such as folate receptors, selectively and/or
specifically bind the vitamin, and internalization can occur, for
example, through receptor-mediated endocytosis. Once internalized,
the releasable linker included in the compounds described herein
allows for the delivery of the drug cargo to the interior of the
target cell, thus decreasing toxicity against non-target tissues
because the releasable linker remains substantially or completely
intact until the compounds described herein are delivered to the
target cells. Accordingly, the compounds described herein act
intracellularly by delivering the drug to an intracellular
biochemical process, a decrease the amount of unconjugated drug
exposure to the host animal's healthy cells and tissues.
[0166] In another embodiment, compounds described herein that
include a folate receptor binding ligand exhibit greater
specificity for the folate receptor compared to the corresponding
compounds that do not include at least one unnatural amino acid. In
another embodiment, compounds described herein that include a
folate receptor binding ligand show high activity for folate
receptor expressing cells. In another embodiment, compounds
described herein exhibit potent in vitro and in vivo activity
against pathogenic cells, such as KB cells, including cisplatin
resistant KB cells, NC1/ADR-RES-Cl.sub.2 cells, IGROV1 cells, and
MDA-MB-231 cells. In another embodiment, compounds described herein
that include a folate receptor binding ligand do not show
significant binding to folate receptor negative cells. In another
embodiment, compounds described herein that include a folate
receptor binding ligand enter cells preferentially or exclusively
via the high affinity folate receptors, such as folate receptor
alpha (.alpha.) and/or folate receptor beta (.beta.). In another
embodiment, compounds described herein generally do not
substantially enter cells via passive transport, such as via the
reduced folate carrier (RFC). In another embodiment, compounds
described herein exhibit lower host animal toxicity compared to
compounds that do not include at least one unnatural amino acid. In
another embodiment, compounds described herein exhibit greater
serum stability compared to compounds that do not include at least
one unnatural amino acid. In another embodiment, compounds
described herein are cleared rapidly compared to compounds that do
not include at least one unnatural amino acid. In another
embodiment, compounds described herein are cleared primarily via
renal clearance compared to hepatic clearance.
[0167] The compounds described herein can be used for both human
clinical medicine and veterinary applications. Thus, the host
animal harboring the population of pathogenic cells and treated
with the compounds described herein can be human or, in the case of
veterinary applications, can be a laboratory, agricultural,
domestic, or wild animal. The present invention can be applied to
host animals including, but not limited to, humans, laboratory
animals such rodents (e.g., mice, rats, hamsters, etc.), rabbits,
monkeys, chimpanzees, domestic animals such as dogs, cats, and
rabbits, agricultural animals such as cows, horses, pigs, sheep,
goats, and wild animals in captivity such as bears, pandas, lions,
tigers, leopards, elephants, zebras, giraffes, gorillas, dolphins,
and whales.
[0168] The invention is applicable to populations of pathogenic
cells that cause a variety of pathologies in these host animals. In
accordance with the invention "pathogenic cells" means cancer
cells, infectious agents such as bacteria and viruses, bacteria- or
virus-infected cells, activated macrophages capable of causing a
disease state, other pathogenic cells causing inflammation, any
other type of pathogenic cells that uniquely express,
preferentially express, or overexpress vitamin receptors or
receptors that bind vitamins and/or vitamin receptor binding
ligands, and any other type of pathogenic cells that uniquely
express, preferentially express, or overexpress high affinity
folate receptors or receptors that bind folates and/or folate
receptor binding ligands. Pathogenic cells can also include any
cells causing a disease state for which treatment with the
compounds described herein results in reduction of the symptoms of
the disease. For example, the pathogenic cells can be host cells
that are pathogenic under some circumstances such as cells of the
immune system that are responsible for graft versus host disease,
but not pathogenic under other circumstances.
[0169] Thus, the population of pathogenic cells can be a cancer
cell population that is tumorigenic, including benign tumors and
malignant tumors, or it can be non-tumorigenic. The cancer cell
population can arise spontaneously or by such processes as
mutations present in the germline of the host animal or somatic
mutations, or it can be chemically-, virally-, or
radiation-induced. The invention can be utilized to treat such
cancers as carcinomas, sarcomas, lymphomas, Hodgekin's disease,
melanomas, mesotheliomas, Burkitt's lymphoma, nasopharyngeal
carcinomas, leukemias, and myelomas. The cancer cell population can
include, but is not limited to, oral, thyroid, endocrine, skin,
gastric, esophageal, laryngeal, pancreatic, colon, bladder, bone,
ovarian, cervical, uterine, breast, testicular, prostate, rectal,
kidney, liver, and lung cancers.
[0170] In another embodiment, the method or pharmaceutical
composition of any one of the preceding embodiments wherein the
disease is selected from the group consisting of arthritis,
including rheumatoid arthritis and osteoarthritis,
glomerulonephritis, proliferative retinopathy, restenosis,
ulcerative colitis, Crohn's disease, fibromyalgia, psoriasis and
other inflammations of the skin, osteomyelitis, Sjogren's syndrome,
multiple sclerosis, diabetes, atherosclerosis, pulmonary fibrosis,
lupus erythematosus, sarcoidosis, systemic sclerosis, organ
transplant rejection (GVHD) and chronic inflammations is
described.
[0171] The drug can be any molecule capable of modulating or
otherwise modifying cell function, including pharmaceutically
active compounds. Illustrative drugs include, but are not limited
to, peptides, oligopeptides, retro-inverso oligopeptides, proteins,
protein analogs in which at least one non-peptide linkage replaces
a peptide linkage, apoproteins, glycoproteins, enzymes, coenzymes,
enzyme inhibitors, amino acids and their derivatives, receptors and
other membrane proteins; antigens and antibodies thereto; haptens
and antibodies thereto; hormones, lipids, phospholipids, liposomes;
toxins; antibiotics; analgesics; bronchodilators; beta-blockers;
antimicrobial agents; antihypertensive agents; cardiovascular
agents including antiarrhythmics, cardiac glycosides, antianginals
and vasodilators; central nervous system agents including
stimulants, psychotropics, antimanics, and depressants; antiviral
agents; antihistamines; cancer drugs including chemotherapeutic
agents; tranquilizers; anti-depressants; H-2 antagonists;
anticonvulsants; antinauseants; prostaglandins and prostaglandin
analogs; muscle relaxants; anti-inflammatory substances;
immunosuppressants, stimulants; decongestants; antiemetics;
diuretics; antispasmodics; antiasthmatics; anti-Parkinson agents;
expectorants; cough suppressants; mucolytics; and mineral and
nutritional additives.
[0172] Further, the drug can be one that is cytotoxic, enhances
tumor permeability, inhibits tumor cell proliferation, promotes
apoptosis, decreases anti-apoptotic activity in target cells, is
used to treat diseases caused by infectious agents, enhances an
endogenous immune response directed to the pathogenic cells, or is
useful for treating a disease state caused by any type of
pathogenic cell. Additional illustrative drugs include
adrenocorticoids and corticosteroids, alkylating agents,
antiandrogens, antiestrogens, androgens, aclamycin and aclamycin
derivatives, estrogens, antimetabolites such as cytosine
arabinoside, purine analogs, pyrimidine analogs, and methotrexate,
busulfan, carboplatin, chlorambucil, cisplatin and other platinum
compounds, tamoxiphen, taxol, paclitaxel, paclitaxel derivatives,
Taxotere.RTM., cyclophosphamide, daunomycin, rhizoxin, T2 toxin,
plant alkaloids, prednisone, hydroxyurea, teniposide, mitomycins,
discodermolides, microtubule inhibitors, epothilones, tubulysins,
cyclopropyl benz[e]indolone, seco-cyclopropyl benz[e]indolone,
O-Ac-seco-cyclopropyl benz[e]indolone, bleomycin and any other
antibiotic, nitrogen mustards, nitrosureas, vinca alkaloids, such
as vincristine, vinblastine, vindesine, vinorelbine and analogs and
derivative thereof such as deacetylvinblastine monohydrazide
(DAVLBH), colchicine, colchicine derivatives, allocolchicine,
thiocolchicine, trityl cysteine, halicondrin B, dolastatins such as
dolastatin 10, amanitins such as .alpha.-amanitin, camptothecin,
irinotecan, and other camptothecin derivatives thereof,
geldanamycin and geldanamycin derivatives, estramustine,
nocodazole, MAP4, colcemid, inflammatory and proinflammatory
agents, peptide and peptidomimetic signal transduction inhibitors,
and any other drug or toxin. Other drugs that can be included in
the conjugates described herein include rapamycins, such as
sirolimus or everolimus, penicillins, cephalosporins, vancomycin,
erythromycin, clindamycin, rifampin, chloramphenicol,
aminoglycoside antibiotics, gentamicin, amphotericin B, acyclovir,
trifluridine, ganciclovir, zidovudine, amantadine, ribavirin, and
any other antimicrobial compound.
[0173] In another embodiment, the drug is selected from
cryptophycins, bortezomib, thiobortezomib, tubulysins, aminopterin,
rapamycins, paclitaxel, docetaxel, doxorubicin, daunorubicin,
everolimus, .alpha.-amanatin, verucarin, didemnin B, geldanomycin,
purvalanol A, ispinesib, budesonide, dasatinib, epothilones,
maytansines, and tyrosine kinase inhibitors, including analogs and
derivatives of the foregoing.
[0174] In another embodiment, the compounds described herein
include at least two drugs (D), which are illustratively selected
from vinca alkaloids, cryptophycins, bortezomib, thiobortezomib,
tubulysins, aminopterin, rapamycins, such as everolimus and
sirolimus, paclitaxel, docetaxel, doxorubicin, daunorubicin,
.alpha.-amanatin, verucarin, didemnin B, geldanomycin, purvalanol
A, ispinesib, budesonide, dasatinib, epothilones, maytansines, and
tyrosine kinase inhibitors, including analogs and derivatives of
the foregoing. In one variation, the drugs (D) are the same. In
another variation, the drugs (D) are different.
[0175] The drug delivery conjugate compounds described herein can
be administered in a combination therapy with any other known drug
whether or not the additional drug is targeted. Illustrative
additional drugs include, but are not limited to, peptides,
oligopeptides, retro-inverso oligopeptides, proteins, protein
analogs in which at least one non-peptide linkage replaces a
peptide linkage, apoproteins, glycoproteins, enzymes, coenzymes,
enzyme inhibitors, amino acids and their derivatives, receptors and
other membrane proteins, antigens and antibodies thereto, haptens
and antibodies thereto, hormones, lipids, phospholipids, liposomes,
toxins, antibiotics, analgesics, bronchodilators, beta-blockers,
antimicrobial agents, antihypertensive agents, cardiovascular
agents including antiarrhythmics, cardiac glycosides, antianginals,
vasodilators, central nervous system agents including stimulants,
psychotropics, antimanics, and depressants, antiviral agents,
antihistamines, cancer drugs including chemotherapeutic agents,
tranquilizers, anti-depressants, H-2 antagonists, anticonvulsants,
antinauseants, prostaglandins and prostaglandin analogs, muscle
relaxants, anti-inflammatory substances, stimulants, decongestants,
antiemetics, diuretics, antispasmodics, antiasthmatics,
anti-Parkinson agents, expectorants, cough suppressants,
mucolytics, and mineral and nutritional additives.
[0176] In another embodiment, at least one additional composition
comprising a therapeutic factor can be administered to the host in
combination or as an adjuvant to the above-detailed methodology, to
enhance the drug delivery conjugate-mediated elimination of the
population of pathogenic cells, or more than one additional
therapeutic factor can be administered. The therapeutic factor can
be selected from a compound capable of stimulating an endogenous
immune response, a chemotherapeutic agent, or another therapeutic
factor capable of complementing the efficacy of the administered
drug delivery conjugate. The method of the invention can be
performed by administering to the host, in addition to the
above-described conjugates, compounds or compositions capable of
stimulating an endogenous immune response (e.g. a cytokine)
including, but not limited to, cytokines or immune cell growth
factors such as interleukins 1-18, stem cell factor, basic FGF,
EGF, G-CSF, GM-CSF, FLK-2 ligand, HILDA, MIP-1.alpha., TGF-.alpha.,
TGF-.beta., M-CSF, IFN-.alpha., IFN-.beta., IFN-.gamma., soluble
CD23, LIF, and combinations thereof.
[0177] Therapeutically effective combinations of these factors can
be used. In one embodiment, for example, therapeutically effective
amounts of IL-2, for example, in amounts ranging from about 0.1
MIU/m.sup.2/dose/day to about 15 MIU/m.sup.2/dose/day in a multiple
dose daily regimen, and IFN-.alpha., for example, in amounts
ranging from about 0.1 MIU/m.sup.2/dose/day to about 7.5
MIU/m.sup.2/dose/day in a multiple dose daily regimen, can be used
along with the drug delivery conjugates to eliminate, reduce, or
neutralize pathogenic cells in a host animal harboring the
pathogenic cells (MIU=million international units;
m.sup.2=approximate body surface area of an average human). In
another embodiment IL-12 and IFN-.alpha. are used in the
above-described therapeutically effective amounts for interleukins
and interferons, and in yet another embodiment IL-15 and
IFN-.alpha. are used in the above described therapeutically
effective amounts for interleukins and interferons. In an alternate
embodiment IL-2, IFN-.alpha. or IFN-.gamma., and GM-CSF are used in
combination in the above described therapeutically effective
amounts. The invention also contemplates the use of any other
effective combination of cytokines including combinations of other
interleukins and interferons and colony stimulating factors.
[0178] Chemotherapeutic agents, which are, for example, cytotoxic
themselves or can work to enhance tumor permeability, are also
suitable for use in the method of the invention in combination with
the drug delivery conjugate compounds. Such chemotherapeutic agents
include adrenocorticoids and corticosteroids, alkylating agents,
antiandrogens, antiestrogens, androgens, aclamycin and aclamycin
derivatives, estrogens, antimetabolites such as cytosine
arabinoside, purine analogs, pyrimidine analogs, and methotrexate,
busulfan, carboplatin, chlorambucil, cisplatin and other platinum
compounds, tamoxiphen, taxol, paclitaxel, paclitaxel derivatives,
Taxotere.RTM., cyclophosphamide, daunomycin, rhizoxin, T2 toxin,
plant alkaloids, prednisone, hydroxyurea, teniposide, mitomycins,
discodermolides, microtubule inhibitors, epothilones, tubulysin,
cyclopropyl benz[e]indolone, seco-cyclopropyl benz[e]indolone,
0-Ac-seco-cyclopropyl benz[e]indolone, bleomycin and any other
antibiotic, nitrogen mustards, nitrosureas, vincristine,
vinblastine, and analogs and derivative thereof such as
deacetylvinblastine monohydrazide, colchicine, colchicine
derivatives, allocolchicine, thiocolchicine, trityl cysteine,
Halicondrin B, dolastatins such as dolastatin 10, amanitins such as
.alpha.-amanitin, camptothecin, irinotecan, and other camptothecin
derivatives thereof, geldanamycin and geldanamycin derivatives,
estramustine, nocodazole, MAP4, colcemid, inflammatory and
proinflammatory agents, peptide and peptidomimetic signal
transduction inhibitors, and any other art-recognized drug or
toxin. Other drugs that can be used in accordance with the
invention include penicillins, cephalosporins, vancomycin,
erythromycin, clindamycin, rifampin, chloramphenicol,
aminoglycoside antibiotics, gentamicin, amphotericin B, acyclovir,
trifluridine, ganciclovir, zidovudine, amantadine, ribavirin,
maytansines and analogs and derivatives thereof, gemcitabine, and
any other art-recognized antimicrobial compound.
[0179] As used herein, the term "linker" includes is a chain of
atoms that connects two or more functional parts of a molecule to
form a conjugate. Illustratively, the chain of atoms is selected
from C, N, O, S, Si, and P, or C, N, O, S, and P, C, N, O, and S.
The chain of atoms covalently connects different functional
capabilities of the conjugate, such as binding ligands, drugs,
diagnostic agents, imaging agents, and the like. The linker may
have a wide variety of lengths, such as in the range from about 2
to about 100 atoms in the contiguous backbone. The atoms used in
forming the linker may be combined in all chemically relevant ways,
such as chains of carbon atoms forming alkylene, alkenylene, and
alkynylene groups, and the like; chains of carbon and oxygen atoms
forming ethers, polyoxyalkylene groups, or when combined with
carbonyl groups forming esters and carbonates, and the like; chains
of carbon and nitrogen atoms forming amines, imines, polyamines,
hydrazines, hydrazones, or when combined with carbonyl groups
forming amides, ureas, semicarbazides, carbazides, and the like;
chains of carbon, nitrogen, and oxygen atoms forming alkoxyamines,
alkoxylamines, or when combined with carbonyl groups forming
urethanes, amino acids, acyloxylamines, hydroxamic acids, and the
like; and many others. In addition, it is to be understood that the
atoms forming the chain in each of the foregoing illustrative
embodiments may be either saturated or unsaturated, thus forming
single, double, or triple bonds, such that for example, alkanes,
alkenes, alkynes, imines, and the like may be radicals that are
included in the linker. In addition, it is to be understood that
the atoms forming the linker may also be cyclized upon each other
or be part of cyclic structure to form divalent cyclic structures
that form the linker, including cyclo alkanes, cyclic ethers,
cyclic amines, and other heterocycles, arylenes, heteroarylenes,
and the like in the linker. In this latter arrangement, it is to be
understood that the linker length may be defined by any pathway
through the one or more cyclic structures. Illustratively, the
linker length is defined by the shortest pathway through the each
one of the cyclic structures. It is to be understood that the
linkers may be optionally substituted at any one or more of the
open valences along the chain of atoms, such as optional
substituents on any of the carbon, nitrogen, silicon, or phosphorus
atoms. It is also to be understood that the linker may connect the
two or more functional parts of a molecule to form a conjugate at
any open valence, and it is not necessary that any of the two or
more functional parts of a molecule forming the conjugate are
attached at any apparent end of the linker.
[0180] In another embodiment, a folate-linker radical is described
having the following formula
##STR00036##
[0181] wherein m, n, and q are integers that are independently
selected from the range of 0 to about 8; AA is an amino acid,
R.sup.1 is hydrogen, alkyl, or a nitrogen protecting group, and
drugs are optionally attached at the (*) atoms. In one aspect, AA
is a naturally occurring amino acid of either the natural or
unnatural configuration. In another aspect, one or more of AA is a
hydrophilic amino acid. In another aspect, one or more of AA is Asp
and/or Arg. In another aspect, the integer n is 1 or greater. In
another aspect, the integer n is 2 or greater. In another aspect,
the integer n is 3 or greater. In another aspect, the integer n is
4 or greater. In another aspect, the integer n is 5 or greater. In
another aspect, the integer q is 1 or greater. In another aspect,
the integer q is 1. In another aspect, the integer m is 1 or
greater. In another aspect, the integer m is 1. In another aspect,
R.sup.1 is hydrogen. The drugs and optionally additional linkers
and additional receptor-binding ligands may be connected to the
above formula at the free NH side chains of the
2,.omega.-diaminoalkanoic acid fragments, or at the terminal
carboxylate as indicated by the free valences therein. It is to be
understood that every combination of the foregoing aspects is
described herein as further illustrative embodiments of the
invention. For example, in another embodiment, n is 1 or greater,
and m is one or greater; or n is 1 or greater, m is 1, and q is 1;
and so forth.
[0182] In another embodiment, a folate-linker radical is described
having the following formula
##STR00037##
wherein m, n, q, and p are integers that are independently selected
from the range of 0 to about 8; AA is an amino acid, R.sup.1 is
hydrogen, alkyl, or a nitrogen protecting group, and drugs are
optionally attached at the (*) atoms. In one aspect, AA is as a
naturally occurring amino acid of either the natural or unnatural
configuration. In another aspect, one or more of AA is a
hydrophilic amino acid. In another aspect, one or more of AA is Asp
and/or Arg. In another aspect, the integer n is 1 or greater. In
another aspect, the integer n is 2 or greater. In another aspect,
the integer n is 3 or greater. In another aspect, the integer n is
4 or greater. In another aspect, the integer n is 5 or greater. In
another aspect, the integers q and/or p are 1 or greater. In
another aspect, the integer integers q and/or p are 1. In another
aspect, the integer m is 1 or greater. In another aspect, the
integer m is 1. In another aspect, R.sup.1 is hydrogen. The drugs
and optionally additional linkers and additional receptor-binding
ligands may be connected to the above formula at the free NH side
chains of the 2,.omega.-diaminoalkanoic acid fragments, at the
cysteinyl thiol groups, or at the terminal carboxylate, as
indicated by the free valences therein. It is to be understood that
every combination of the foregoing aspects is described herein as
further illustrative embodiments of the invention. For example, in
another embodiment, n is 1 or greater, and m is one or greater; or
n is 2 or greater, m is 1, and q is 1; or n is 2 or greater, m is
1, q is 1, and p is 1; and so forth.
[0183] In another embodiment, a folate-linker radical is described
having the following formula
##STR00038##
wherein m, n, q, p, and r are integers that are independently
selected from the range of 0 to about 8; AA is an amino acid,
R.sup.1 is hydrogen, alkyl, or a nitrogen protecting group, and
drugs are optionally attached at the (*) atoms. In one aspect, AA
is as a naturally occurring amino acid of either the natural or
unnatural configuration. In another aspect, one or more of AA is a
hydrophilic amino acid. In another aspect, one or more of AA is Asp
and/or Arg. In another aspect, the integer n is 1 or greater. In
another aspect, the integer n is 2 or greater. In another aspect,
the integer n is 3 or greater. In another aspect, the integer n is
4 or greater. In another aspect, the integer n is 5 or greater. In
another aspect, the integers q and/or p and/or r are 1 or greater.
In another aspect, the integers q and/or p and/or r are 1. In
another aspect, the integer m is 1 or greater. In another aspect,
the integer m is 1. In another aspect, R.sup.1 is hydrogen. The
drugs and optionally additional linkers and additional
receptor-binding ligands may be connected to the above formula at
the free NH side chains of the 2,.omega.-diaminoalkanoic acid
fragments, at the cyteinyl thiol groups, at the serinyl hydroxy
groups, or at the terminal carboxylate, as indicated by the free
valences therein. It is to be understood that every combination of
the foregoing aspects is described herein as further illustrative
embodiments of the invention. For example, in another embodiment, n
is 1 or greater, and m is one or greater; or n is 2 or greater, m
is 1, and q is 1; or n is 2 or greater, m is 1, q is 1, and p is 1;
or n is 2 or greater, m is 1, q is 1, and r is 1; or n is 2 or
greater, m is 1, q is 1, p is 1, and r is 1; and so forth.
[0184] In another embodiment, the polyvalent linker includes one or
more divalent hydrophilic radicals, as described herein, also
called linkers or spacer linkers. It is appreciated that the
arrangement and/or orientation of the various hydrophilic linkers
may be in a linear or branched fashion, or both. For example, the
hydrophilic linkers may form the backbone of the linker forming the
conjugate between the ligand and the one or more drugs.
Alternatively, the hydrophilic portion of the linker may be pendant
to or attached to the backbone of the chain of atoms connecting the
binding ligand B to the one or more drugs D. In this latter
arrangement, the hydrophilic portion may be proximal or distal to
the backbone chain of atoms.
[0185] In another embodiment, the linker is more or less linear,
and the hydrophilic groups are arranged largely in a series to form
a chain-like linker in the conjugate. Said another way, the
hydrophilic groups form some or all of the backbone of the linker
in this linear embodiment.
[0186] In another embodiment, the linker is branched with
hydrophilic groups. In this branched embodiment, the hydrophilic
groups may be proximal to the backbone or distal to the backbone.
In each of these arrangements, the linker is more spherical or
cylindrical in shape. In one variation, the linker is shaped like a
bottle-brush. In one aspect, the backbone of the linker is formed
by a linear series of amides, and the hydrophilic portion of the
linker is formed by a parallel arrangement of branching side
chains, such as by connecting monosaccharides, sulfonates, and the
like, and derivatives and analogs thereof.
[0187] It is understood that the linker may be neutral or ionizable
under certain conditions, such as physiological conditions
encountered in vivo. For ionizable linkers, under the selected
conditions, the linker may deprotonate to form a negative ion, or
alternatively become protonated to form a positive ion. It is
appreciated that more than one deprotonation or protonation event
may occur. In addition, it is understood that the same linker may
deprotonate and protonate to form inner salts or zwitterionic
compounds.
[0188] In another embodiment, the hydrophilic spacer linkers are
neutral, an in particular neutral under physiological conditions,
the linkers do not significantly protonate nor deprotonate. In
another embodiment, the hydrophilic spacer linkers may be
protonated to carry one or more positive charges. It is understood
that the protonation capability is condition dependent. In one
aspect, the conditions are physiological conditions, and the linker
is protonated in vivo. In another embodiment, the spacers include
both regions that are neutral and regions that may be protonated to
carry one or more positive charges. In another embodiment, the
spacers include both regions that may be deprotonated to carry one
or more negative charges and regions that may be protonated to
carry one or more positive charges. It is understood that in this
latter embodiment that zwitterions or inner salts may be
formed.
[0189] In one aspect, the regions of the linkers that may be
deprotonated to carry a negative charge include carboxylic acids,
such as aspartic acid, glutamic acid, and longer chain carboxylic
acid groups, and sulfuric acid esters, such as alkyl esters of
sulfuric acid. In another aspect, the regions of the linkers that
may be protonated to carry a positive charge include amino groups,
such as polyaminoalkylenes including ethylene diamines, propylene
diamines, butylene diamines and the like, and/or heterocycles
including pyrollidines, piperidines, piperazines, and other amino
groups, each of which is optionally substituted. In another
embodiment, the regions of the linkers that are neutral include
poly hydroxyl groups, such as sugars, carbohydrates, saccharides,
inositols, and the like, and/or polyether groups, such as
polyoxyalkylene groups including polyoxyethylene, polyoxypropylene,
and the like.
[0190] In one embodiment, the hydrophilic spacer linkers described
herein include are formed primarily from carbon, hydrogen, and
oxygen, and have a carbon/oxygen ratio of about 3:1 or less, or of
about 2:1 or less. In one aspect, the hydrophilic linkers described
herein include a plurality of ether functional groups. In another
aspect, the hydrophilic linkers described herein include a
plurality of hydroxyl functional groups. Illustrative fragments and
radicals that may be used to form such linkers include polyhydroxyl
compounds such as carbohydrates, polyether compounds such as
polyethylene glycol units, and acid groups such as carboxyl and
alkyl sulfuric acids. In one variation, oligoamide spacers, and the
like may also be included in the linker.
[0191] Illustrative divalent hydrophilic linkers include
carbohydrates such as saccharopeptides as described herein that
include both a peptide feature and sugar feature; glucuronides,
which may be incorporated via [2+3] Huisgen cyclization, also known
as click chemistry; .beta.-alkyl glycosides, such as of
2-deoxyhexapyranoses (2-deoxyglucose, 2-deoxyglucuronide, and the
like), and .beta.-alkyl mannopyranosides. Illustrative PEG groups
include those of a specific length range from about 4 to about 20
PEG groups. Illustrative alkyl sulfuric acid esters may also be
introduced with click chemistry directly into the backbone.
Illustrative oligoamide spacers include EDTA and DTPA spacers,
.beta.-amino acids, and the like.
[0192] In another embodiment, the polyvalent linker L comprises one
or more polyethers, such as the linkers of the following
formulae:
##STR00039##
where m is an integer independently selected in each instance from
1 to about 8; p is an integer selected 1 to about 10; and n is an
integer independently selected in each instance from 1 to about 3.
In one aspect, m is independently in each instance 1 to about 3. In
another aspect, n is 1 in each instance. In another aspect, p is
independently in each instance about 4 to about 6. Illustratively,
the corresponding polypropylene polyethers corresponding to the
foregoing are contemplated herein and may be included in the
conjugates as hydrophilic spacer linkers. In addition, it is
appreciated that mixed polyethylene and polypropylene polyethers
may be included in the conjugates as hydrophilic spacer linkers.
Further, cyclic variations of the foregoing polyether compounds,
such as those that include tetrahydrofuranyl, 1,3-dioxanes,
1,4-dioxanes, and the like are contemplated herein.
[0193] In another embodiment, the polyvalent linker L comprises a
plurality of hydroxyl functional groups, such as linkers that
incorporate monosaccharides, oligosaccharides, polysaccharides, and
the like. It is to be understood that the polyhydroxyl containing
spacer linkers comprises a plurality of --(CROH)-- groups, where R
is hydrogen or alkyl.
[0194] In another embodiment, the polyvalent linker L comprises one
or more of the following fragments:
##STR00040##
wherein R is H, alkyl, cycloalkyl, or arylalkyl; m is an integer
from 1 to about 3; n is an integer from 1 to about 5, or from 2 to
about 5, p is an integer from 1 to about 5, and r is an integer
selected from 1 to about 3. In one aspect, the integer n is 3 or 4.
In another aspect, the integer p is 3 or 4. In another aspect, the
integer r is 1.
[0195] In another embodiment, the polyvalent linker L comprises one
or more of the following fragments:
##STR00041##
wherein R is H, alkyl, cycloalkyl, or arylalkyl; m is an integer
from 1 to about 3; n is an integer from 1 to about 5, or from 2 to
about 5, p is an integer from 1 to about 5, and r is an integer
selected from 1 to about 3. In one aspect, the integer n is 3 or 4.
In another aspect, the integer p is 3 or 4. In another aspect, the
integer r is 1.
[0196] In another embodiment, the polyvalent linker L comprises one
or more of the following cyclic polyhydroxyl groups:
##STR00042## ##STR00043## ##STR00044##
wherein n is an integer from 2 to about 5, p is an integer from 1
to about 5, and r is an integer from 1 to about 4. In one aspect,
the integer n is 3 or 4. In another aspect, the integer p is 3 or
4. In another aspect, the integer r is 2 or 3. It is understood
that all stereochemical forms of such sections of the linkers are
contemplated herein. For example, in the above formula, the section
may be derived from ribose, xylose, glucose, mannose, galactose, or
other sugar and retain the stereochemical arrangements of pendant
hydroxyl and alkyl groups present on those molecules. In addition,
it is to be understood that in the foregoing formulae, various
deoxy compounds are also contemplated. Illustratively, compounds of
the following formulae are contemplated:
##STR00045##
wherein n is equal to or less than r, such as when r is 2 or 3, n
is 1 or 2, or 1, 2, or 3, respectively.
[0197] In another embodiment, the polyvalent linker L comprises one
or more polyhydroxyl radicals of the following formula:
##STR00046##
wherein n and r are each an integer selected from 1 to about 3. In
one aspect, the linker includes one or more polyhydroxyl compounds
of the following formulae:
##STR00047##
It is understood that all stereochemical forms of such sections of
the linkers are contemplated herein. For example, in the above
formula, the section may be derived from ribose, xylose, glucose,
mannose, galactose, or other sugar and retain the stereochemical
arrangements of pendant hydroxyl and alkyl groups present on those
molecules.
[0198] In another embodiment, the polyvalent linker L comprises one
or more polyhydroxyl groups that are spaced away from the backbone
of the linker. In one embodiment, such carbohydrate groups or
polyhydroxyl groups are connected to the back bone by a triazole
group, forming triazole-linked hydrophilic spacer linkers.
Illustratively, the linker includes fragments of the following
formulae:
##STR00048##
wherein n, m, and r are integers and are each independently
selected in each instance from 1 to about 5. In one illustrative
aspect, m is independently 2 or 3 in each instance. In another
aspect, r is 1 in each instance. In another aspect, n is 1 in each
instance. In one variation, the group connecting the polyhydroxyl
group to the backbone of the linker is a different heteroaryl
group, including but not limited to, pyrrole, pyrazole,
1,2,4-triazole, furan, oxazole, isoxazole, thienyl, thiazole,
isothiazole, oxadiazole, and the like. Similarly, divalent
6-membered ring heteroaryl groups are contemplated. Other
variations of the foregoing illustrative hydrophilic spacer linkers
include oxyalkylene groups, such as the following formulae:
##STR00049##
wherein n and r are integers and are each independently selected in
each instance from 1 to about 5; and p is an integer selected from
1 to about 4.
[0199] In another embodiment, the polyvalent linker L comprises one
or more carbohydrate groups or polyhydroxyl groups connected to the
back bone by an amide group, forming amide-linked hydrophilic
spacer linkers. Illustratively, such linkers include fragments of
the following formulae:
##STR00050##
wherein n is an integer selected from 1 to about 3, and m is an
integer selected from 1 to about 22. In one illustrative aspect, n
is 1 or 2. In another illustrative aspect, m is selected from about
6 to about 10, illustratively 8. In one variation, the group
connecting the polyhydroxyl group to the backbone of the linker is
a different functional group, including but not limited to, esters,
ureas, carbamates, acylhydrazones, and the like. Similarly, cyclic
variations are contemplated. Other variations of the foregoing
illustrative hydrophilic spacer linkers include oxyalkylene groups,
such as the following formulae:
##STR00051##
wherein n and r are integers and are each independently selected in
each instance from 1 to about 5; and p is an integer selected from
1 to about 4.
[0200] In another embodiment, the polyvalent linker L comprises one
or more of the following fragments:
##STR00052## ##STR00053## ##STR00054##
wherein R is H, alkyl, cycloalkyl, or arylalkyl; m is an
independently selected integer from 1 to about 3; n is an integer
from 1 to about 6, p is an integer from 1 to about 5, and r is an
integer selected from 1 to about 3. In one variation, the integer n
is 3 or 4. In another variation, the integer p is 3 or 4. In
another variation, the integer r is 1.
[0201] In another embodiment, the polyvalent linker L comprises one
or more of the following fragments:
##STR00055##
wherein R is H, alkyl, cycloalkyl, or arylalkyl; m is an
independently selected integer from 1 to about 3; n is an integer
from 2 to about 6, p is an integer from 1 to about 5, and r is an
integer selected from 1 to about 3. In one variation, the integer n
is 3 or 4. In another variation, the integer p is 3 or 4. In
another variation, the integer r is 1.
[0202] In another embodiment, the polyvalent linker L comprises one
or more of the following fragments:
##STR00056## ##STR00057## ##STR00058## ##STR00059##
wherein m is an independently selected integer from 1 to about 3; n
is an integer from 1 to about 6, p is an integer from 1 to about 5,
and r is an integer selected from 1 to about 3. In one variation,
the integer n is 3 or 4. In another variation, the integer p is 3
or 4. In another variation, the integer r is 1.
[0203] In another embodiment, the polyvalent linker L comprises one
or more of the following fragments:
##STR00060##
wherein m is an independently selected integer from 1 to about 3; n
is an integer from 2 to about 6, p is an integer from 1 to about 5,
and r is an integer selected from 1 to about 3. In one variation,
the integer n is 3 or 4. In another variation, the integer p is 3
or 4. In another variation, the integer r is 1.
[0204] In another embodiment, the polyvalent linker L comprises one
or more of the following fragments:
##STR00061## ##STR00062## ##STR00063##
wherein m is an independently selected integer from 1 to about 3; n
is an integer from 1 to about 6, p is an integer from 1 to about 5,
and r is an integer selected from 1 to about 3. In one variation,
the integer n is 3 or 4. In another variation, the integer p is 3
or 4. In another variation, the integer r is 1.
[0205] In another embodiment, the polyvalent linker L comprises a
combination of backbone and branching side motifs such as is
illustrated by the following formulae
##STR00064##
wherein n is an integer independently selected in each instance
from 0 to about 3. The above formula are intended to represent 4,
5, 6, and even larger membered cyclic sugars. In addition, it is to
be understood that the above formula may be modified to represent
deoxy sugars, where one or more of the hydroxy groups present on
the formulae are replaced by hydrogen, alkyl, or amino. In
addition, it is to be understood that the corresponding carbonyl
compounds are contemplated by the above formulae, where one or more
of the hydroxyl groups is oxidized to the corresponding carbonyl.
In addition, in this illustrative embodiment, the pyranose includes
both carboxyl and amino functional groups and (a) can be inserted
into the backbone and (b) can provide synthetic handles for
branching side chains in variations of this embodiment. Any of the
pendant hydroxyl groups may be used to attach other chemical
fragments, including additional sugars to prepare the corresponding
oligosaccharides. Other variations of this embodiment are also
contemplated, including inserting the pyranose or other sugar into
the backbone at a single carbon, i.e. a spiro arrangement, at a
geminal pair of carbons, and like arrangements. For example, one or
two ends of the linker, or the drug D, or the binding ligand B may
be connected to the sugar to be inserted into the backbone in a
1,1; 1,2; 1,3; 1,4; 2,3, or other arrangement.
[0206] In another embodiment, the hydrophilic spacer linkers
described herein include are formed primarily from carbon,
hydrogen, and nitrogen, and have a carbon/nitrogen ratio of about
3:1 or less, or of about 2:1 or less. In one aspect, the
hydrophilic linkers described herein include a plurality of amino
functional groups.
[0207] In another embodiment, the polyvalent linker L comprises one
or more amino groups of the following formulae:
##STR00065##
where n is an integer independently selected in each instance from
1 to about 3. In one aspect, the integer n is independently 1 or 2
in each instance. In another aspect, the integer n is 1 in each
instance.
[0208] In another embodiment, the polyvalent linker L comprises one
or more sulfuric acid esters, such as an alkyl ester of sulfuric
acid. Illustratively, the linker includes the following
formula(e):
##STR00066##
where n is an integer independently selected in each instance from
1 to about 3. Illustratively, n is independently 1 or 2 in each
instance.
[0209] It is understood, that in such polyhydroxyl, polyamino,
carboxylic acid, sulfuric acid, and like linkers that include free
hydrogens bound to heteroatoms, one or more of those free hydrogen
atoms may be protected with the appropriate hydroxyl, amino, or
acid protecting group, respectively, or alternatively may be
blocked as the corresponding pro-drugs, the latter of which are
selected for the particular use, such as pro-drugs that release the
parent drug under general or specific physiological conditions.
[0210] In another embodiment, the polyvalent linker comprises one
or more of the following divalent radicals:
##STR00067##
wherein n is an integer from 2 to about 5, p is an integer from 1
to about 5, and r is an integer from 1 to about 4, as described
above.
[0211] It is to be further understood that in the foregoing
embodiments, open positions, such as (*) atoms are locations for
attachment of the binding ligand (B) or any drug (D) to be
delivered. In addition, it is to be understood that such attachment
of either or both of B and any D may be direct or through an
intervening linker comprising one or more of the radicals described
herein. In addition, (*) atoms may form releasable linkers with any
drug D, or other portion of the linker L.
[0212] In another embodiment, the hydrophilic spacer linker
comprises one or more carbohydrate containing or polyhydroxyl group
containing linkers. In another embodiment, the hydrophilic spacer
linker comprises at least three carbohydrate containing or
polyhydroxyl group containing linkers. In another embodiment, the
hydrophilic spacer linker comprises one or more carbohydrate
containing or polyhydroxyl group containing linkers, and one or
more aspartic acids. In another embodiment, the hydrophilic spacer
linker comprises one or more carbohydrate containing or
polyhydroxyl group containing linkers, and one or more glutamic
acids. In another embodiment, the hydrophilic spacer linker
comprises one or more carbohydrate containing or polyhydroxyl group
containing linkers, one or more glutamic acids, one or more
aspartic acids, and one or more beta amino alanines. In a series of
variations, in each of the foregoing embodiments, the hydrophilic
spacer linker also includes one or more cysteines. In another
series of variations, in each of the foregoing embodiments, the
hydrophilic spacer linker also includes at least one arginine.
[0213] In another embodiment, the polyvalent linker L includes a
hydrophilic spacer linker comprising one or more divalent
1,4-piperazines that are included in the chain of atoms connecting
at least one of the binding ligands (L) with at least one of the
drugs (D). In one variation, the hydrophilic spacer linker includes
one or more carbohydrate containing or polyhydroxyl group
containing linkers. In another variation, the hydrophilic spacer
linker includes one or more carbohydrate containing or polyhydroxyl
group containing linkers and one or more aspartic acids. In another
variation, the hydrophilic spacer linker includes one or more
carbohydrate containing or polyhydroxyl group containing linkers
and one or more glutamic acids. In a series of variations, in each
of the foregoing embodiments, the hydrophilic spacer linker also
includes one or more cysteines. In another series of variations, in
each of the foregoing embodiments, the hydrophilic spacer linker
also includes at least one arginine.
[0214] In another embodiment, the hydrophilic spacer linker
comprises one or more oligoamide hydrophilic spacers, such as but
not limited to aminoethylpiperazinylacetamide.
[0215] In another embodiment, the polyvalent linker L includes a
hydrophilic spacer linker comprising one or more triazole linked
carbohydrate containing or polyhydroxyl group containing linkers.
In another embodiment, the hydrophilic spacer linker comprises one
or more amide linked carbohydrate containing or polyhydroxyl group
containing linkers. In another embodiment, the hydrophilic spacer
linker comprises one or more PEG groups and one or more cysteines.
In another embodiment, the hydrophilic spacer linker comprises one
or more EDTE derivatives.
[0216] In another embodiment, the polyvalent linker L includes a
divalent radical of the formula
##STR00068##
wherein * indicates the point of attachment to a folate and **
indicates the point of attachment to a drug; and F and G are each
independently 1, 2, 3 or 4 are described.
[0217] In another embodiment, the polyvalent linker L includes a
trivalent radical of the formula
##STR00069##
wherein *, **, *** each indicate points of attachment to the folate
receptor binding moiety B, and the one or more drugs D. It is to be
understood that when there are fewer drugs, *, **, *** are
substituted with hydrogen or a heteroatom. F and G are each
independently 1, 2, 3 or 4; and W.sup.1 is NH or O is described. In
another aspect, m.sup.1 is 0 or 1.
[0218] In any of the embodiments described herein heteroatom
linkers can also be included in the polyvalent linker L, such as
--NR.sup.1R.sup.2--, oxygen, sulfur, and the formulae
--(NHR.sup.1NHR.sup.2)--, --SO--, --(SO.sub.2)--, and
--N(R.sup.3)O--, wherein R.sup.1, R.sup.2, and R.sup.3 are each
independently selected from hydrogen, alkyl, aryl, arylalkyl,
substituted aryl, substituted arylalkyl, heteroaryl, substituted
heteroaryl, and alkoxyalkyl. It is to be understood that the
heteroatom linkers may be used to covalently attach any of the
radicals described herein, including drug radicals D to the
polyvalent linker, ligand radicals B to the polyvalent linker, or
various di and polyvalent radicals that from the polyvalent linker
L
Illustrative additional bivalent radicals that can be used to form
part of the linker are as follows.
##STR00070## ##STR00071## ##STR00072## ##STR00073## ##STR00074##
##STR00075##
[0219] The polyvalent linker L is a releasable linker.
[0220] As used herein, the term "releasable linker" refers to a
linker that includes at least one bond that can be broken under
physiological conditions when the compounds described herein are
delivered to or inside of the target cell. Accordingly, the term
releasable linker does not generally refer simply to a bond that is
labile in vivo, such as in serum, plasma, the gastrointestinal
tract, or liver, unless those systems are the target for the cell
surface receptor binding ligand. However, after delivery and/or
selective targeting, releasable linkers may be cleaved by any
process that includes at least one bond being broken in the linker
or at the covalent attachment of the linker to B or any D under
physiological conditions, such as by having one or more pH-labile,
acid-labile, base-labile, oxidatively labile, metabolically labile,
biochemically labile, and/or enzyme-labile bonds. It is appreciated
that such physiological conditions resulting in bond breaking do
not necessarily include a biological or metabolic process, and
instead may include a standard chemical reaction, such as a
hydrolysis reaction, for example, at physiological pH, or as a
result of compartmentalization into a cellular organelle such as an
endosome having a lower pH than cytosolic pH.
[0221] It is understood that a cleavable bond can connect two
adjacent atoms within the releasable linker, and/or connect other
linkers with B, and/or any D, as described herein, at any ends of
the releasable linker. In the case where a cleavable bond connects
two adjacent atoms within the releasable linker, following breakage
of the bond, the releasable linker is broken into two or more
fragments. Alternatively, in the case where a cleavable bond is
between the releasable linker and another moiety, such as an
additional heteroatom, a spacer linker, another releasable portion
of the linker, any D, or B, following breakage of the bond, the
releasable linker is separated from the other moiety.
[0222] Illustrative radicals that themselves include a cleavable
bond, or form a cleavable bond with B and/or any D hemiacetals and
sulfur variations thereof, acetals and sulfur variations thereof,
hemiaminals, aminals, and the like, or which can be formed from
methylene fragments substituted with at least one heteroatom, such
as 1-alkoxyalkylene, 1-alkoxycycloalkylene,
1-alkoxyalkylenecarbonyl, 1-alkoxycycloalkylenecarbonyl, and the
like. Illustrative releasable linkers described herein include
polyvalent linkers that include carbonylarylcarbonyl,
carbonyl(carboxyaryl)carbonyl, carbonyl(biscarboxyaryl)carbonyl,
haloalkylenecarbonyl, and the like. Illustrative releasable linkers
described herein include polyvalent linkers that include
alkylene(dialkylsilyl), alkylene(alkylarylsilyl),
alkylene(diarylsilyl), (dialkylsilyl)aryl, (alkylarylsilyl)aryl,
(diarylsilyl)aryl, and the like. Illustrative releasable linkers
described herein include oxycarbonyloxy, oxycarbonyloxyalkyl,
sulfonyloxy, oxysulfonylalkyl, and the like. Illustrative
releasable linkers described herein include polyvalent linkers that
include iminoalkylidenyl, carbonylalkylideniminyl,
iminocycloalkylidenyl, carbonylcycloalkylideniminyl, and the like.
Illustrative releasable linkers described herein include polyvalent
linkers that include alkylenethio, alkylenearylthio, and
carbonylalkylthio, and the like. Each of the foregoing fragments is
optionally substituted with a substituent X.sup.2, as defined
herein.
[0223] The substituents X.sup.2 can be alkyl, alkoxy, alkoxyalkyl,
hydroxy, hydroxyalkyl, amino, aminoalkyl, alkylaminoalkyl,
dialkylaminoalkyl, halo, haloalkyl, sulfhydrylalkyl,
alkylthioalkyl, aryl, substituted aryl, arylalkyl, substituted
arylalkyl, heteroaryl, substituted heteroaryl, carboxy,
carboxyalkyl, alkyl carboxylate, alkyl alkanoate, guanidinoalkyl,
R.sup.4-carbonyl, R.sup.5-carbonylalkyl, R.sup.6-acylamino, and
R.sup.7-acylaminoalkyl, wherein R.sup.4 and R.sup.5 are each
independently selected from amino acids, amino acid derivatives,
and peptides, and wherein R.sup.6 and R.sup.7 are each
independently selected from amino acids, amino acid derivatives,
and peptides. In this embodiment the heteroatom linker can be
nitrogen, and the substituent X.sup.2 and the heteroatom linker can
be taken together with the releasable linker to which they are
bound to form an heterocycle.
[0224] The heterocycles can be pyrrolidines, piperidines,
oxazolidines, isoxazolidines, thiazolidines, isothiazolidines,
pyrrolidinones, piperidinones, oxazolidinones, isoxazolidinones,
thiazolidinones, isothiazolidinones, and succinimides.
[0225] In any of the embodiments described herein, the releasable
linker may include oxygen bonded to methylene, 1-alkoxyalkylene,
1-alkoxycycloalkylene, 1-alkoxyalkylenecarbonyl, and
1-alkoxycycloalkylenecarbonyl to form an acetal or ketal, wherein
each of the fragments is optionally substituted with a substituent
X.sup.2, as defined herein. Alternatively, the methylene or
alkylene is substituted with an optionally-substituted aryl.
[0226] In any of the embodiments described herein, the releasable
linker may include oxygen bonded to sulfonylalkyl to form an
alkylsulfonate.
[0227] In any of the embodiments described herein, the releasable
linker may include nitrogen bonded to iminoalkylidenyl,
carbonylalkylideniminyl, iminocycloalkylidenyl, and
carbonylcycloalkylideniminyl to form an hydrazone, each of which is
optionally substituted with a substituent X.sup.2, as defined
herein. In an alternate configuration, the hydrazone may be
acylated with a carboxylic acid derivative, an orthoformate
derivative, or a carbamoyl derivative to form releasable linkers
containing various acylhydrazones.
[0228] In any of the embodiments described herein, the releasable
linker may include oxygen bonded to alkylene(dialkylsilyl),
alkylene(alkylarylsilyl), alkylene(diarylsilyl),
(dialkylsilyl)aryl, (alkylarylsilyl)aryl, and (diarylsilyl)aryl to
form a silanol, each of which is optionally substituted with a
substituent X.sup.2, as defined herein.
[0229] In any of the embodiments described herein, the releasable
linker may include nitrogen bonded to carbonylarylcarbonyl,
carbonyl(carboxyaryl)carbonyl, carbonyl(biscarboxyaryl)carbonyl to
form an amide, or alternatively an amide with a drug nitrogen.
[0230] In any of the embodiments described herein, the releasable
linker may include oxygen bonded to carbonylarylcarbonyl,
carbonyl(carboxyaryl)carbonyl, carbonyl(biscarboxyaryl)carbonyl to
form an ester, or alternatively an ester with drug oxygen.
[0231] It is to be understood that the bivalent spacer linkers may
be combined in any chemically relevant way, either directly or via
an intervening heteroatom to construct the releasable linkers
described herein. It is further understood that the nature of the
arrangement of spacer and heteroatom linkers defines where the
releasable linker will cleave in vivo. For example, two spacer
linkers that terminate in a sulfur atom when combined form a
disulfide, which is the cleavable bond in the releasable linker
formed thereby.
[0232] For example, in another embodiment, the polyvalent linker
comprises a 3-thiosuccinimid-1-ylalkyloxymethyloxy moiety, where
the methyl is optionally substituted with alkyl or substituted
aryl.
[0233] In another embodiment, the polyvalent linker comprises a
3-thiosuccinimid-1-ylalkylcarbonyl, where the carbonyl forms an
acylaziridine with the drug.
[0234] In another embodiment, the polyvalent linker comprises a
1-alkoxycycloalkylenoxy moiety.
[0235] In another embodiment, the polyvalent linker comprises an
alkyleneaminocarbonyl(dicarboxylarylene)carboxylate.
[0236] In another embodiment, the polyvalent linker comprises a
dithioalkylcarbonylhydrazide, where the hydrazide forms an
hydrazone with the drug. In another embodiment, the polyvalent
linker comprises a 3-thiosuccinimid-1-ylalkylcarbonylhydrazide,
where the hydrazide forms a hydrazone with the drug.
[0237] In another embodiment, the polyvalent linker comprises a
3-thioalkylsulfonylalkyl(disubstituted silyl)oxy, where the
disubstituted silyl is substituted with alkyl or optionally
substituted aryl.
[0238] In another embodiment, the polyvalent linker comprises a
plurality of spacer linkers selected from the group consisting of
the naturally occurring amino acids and stereoisomers thereof.
[0239] In another embodiment, the polyvalent linker comprises a
2-dithioalkyloxycarbonyl, where the carbonyl forms a carbonate with
the drug.
[0240] In another embodiment, the polyvalent linker comprises a
2-dithioarylalkyloxycarbonyl, where the carbonyl forms a carbonate
with the drug and the aryl is optionally substituted.
[0241] In another embodiment, the polyvalent linker comprises a
4-dithioarylalkyloxycarbonyl, where the carbonyl forms a carbonate
with the drug, and the aryl is optionally substituted.
[0242] In another embodiment, the polyvalent linker comprises a
3-thiosuccinimid-1-ylalkyloxyalkyloxyalkylidene, where the
alkylidene forms an hydrazone with the drug, each alkyl is
independently selected, and the oxyalkyloxy is optionally
substituted with alkyl or optionally substituted aryl.
[0243] In another embodiment, the polyvalent linker comprises a
2-dithioalkyloxycarbonylhydrazide.
[0244] In another embodiment, the polyvalent linker comprises a 2-
or 3-dithioalkylamino, where the amino forms a vinylogous amide
with the drug.
[0245] In another embodiment, the polyvalent linker comprises a
2-dithioalkylamino, where the amino forms a vinylogous amide with
the drug, and the alkyl is ethyl.
[0246] In another embodiment, the polyvalent linker comprises a 2-
or 3-dithioalkylaminocarbonyl, where the carbonyl forms a carbamate
with the drug.
[0247] In another embodiment, the polyvalent linker comprises a
2-dithioalkylaminocarbonyl, where the carbonyl forms a carbamate
with the drug. In another aspect, the alkyl is ethyl.
[0248] In another embodiment, the polyvalent linker comprises a
2-dithioalkyloxycarbonyl, where the carbonyl forms a carbamate with
the drug. In another aspect, the alkyl is ethyl.
[0249] In another embodiment, the polyvalent linker comprises a
2-dithioarylalkyloxycarbonyl, where the carbonyl forms a carbamate
or a carbamoylaziridine with the drug.
[0250] In another embodiment, the polyvalent linker comprises a
4-dithioarylalkyloxycarbonyl, where the carbonyl forms a carbamate
or a carbamoylaziridine with the drug.
[0251] In another embodiment, the polyvalent linkers described
herein comprise divalent radicals of formulae (II)
##STR00076##
where n is an integer selected from 1 to about 4; R.sup.a and
R.sup.b are each independently selected from the group consisting
of hydrogen and alkyl, including lower alkyl such as
C.sub.1-C.sub.4 alkyl that are optionally branched; or R.sup.a and
R.sup.b are taken together with the attached carbon atom to form a
carbocyclic ring; R is an optionally substituted alkyl group, an
optionally substituted acyl group, or a suitably selected nitrogen
protecting group; and (*) indicates points of attachment for the
drug, vitamin, imaging agent, diagnostic agent, other bivalent
linkers, or other parts of the conjugate.
[0252] In another embodiment, the polyvalent linkers described
herein comprise divalent radicals of formulae (III)
##STR00077##
where m is an integer selected from 1 to about 4; R is an
optionally substituted alkyl group, an optionally substituted acyl
group, or a suitably selected nitrogen protecting group; and (*)
indicates points of attachment for the drug, vitamin, imaging
agent, diagnostic agent, other bivalent linkers, or other parts of
the conjugate.
[0253] In another embodiment, the polyvalent linkers described
herein comprise divalent radicals of formulae (IV)
##STR00078##
where m is an integer selected from 1 to about 4; R is an
optionally substituted alkyl group, an optionally substituted acyl
group, or a suitably selected nitrogen protecting group; and (*)
indicates points of attachment for the drug, vitamin, imaging
agent, diagnostic agent, other divalent linkers, or other parts of
the conjugate.
[0254] In another embodiment, the compounds described herein
comprise one or more radicals selected from the formulae:
##STR00079##
wherein X is NH, O, or S.
[0255] In another embodiment, the polyvalent linkers herein
described comprise a radical having the formula:
##STR00080##
[0256] Another embodiment, the polyvalent linkers described herein
comprise a radical of having the formula:
##STR00081##
where X is an heteroatom, such as nitrogen, oxygen, or sulfur, n is
an integer selected from 0, 1, 2, and 3, R is hydrogen, or a
substituent, including a substituent capable of stabilizing a
positive charge inductively or by resonance on the aryl ring, such
as alkoxy, and the like, and the symbol (*) indicates points of
attachment. It is appreciated that other substituents may be
present on the aryl ring, the benzyl carbon, the alkanoic acid, or
the methylene bridge, including but not limited to hydroxy, alkyl,
alkoxy, alkylthio, halo, and the like.
[0257] In another embodiment, the polyvalent linkers described
herein comprise radicals selected from carbonyl, thionocarbonyl,
alkylene, cycloalkylene, alkylenecycloalkyl, alkylenecarbonyl,
cycloalkylenecarbonyl, carbonylalkylcarbonyl,
1-alkylenesuccinimid-3-yl, 1-(carbonylalkyl)succinimid-3-yl,
alkylenesulfoxyl, sulfonylalkyl, alkylenesulfoxylalkyl,
alkylenesulfonylalkyl, carbonyltetrahydro-2H-pyranyl,
carbonyltetrahydrofuranyl,
1-(carbonyltetrahydro-2H-pyranyl)succinimid-3-yl, and
1-(carbonyltetrahydrofuranyl)succinimid-3-yl, wherein each of said
spacer linkers is optionally substituted with one or more
substituents X.sup.1;
[0258] wherein each substituent X.sup.1 is independently selected
from the group consisting of alkyl, alkoxy, alkoxyalkyl, hydroxy,
hydroxyalkyl, amino, aminoalkyl, alkylaminoalkyl,
dialkylaminoalkyl, halo, haloalkyl, sulfhydrylalkyl,
alkylthioalkyl, aryl, substituted aryl, arylalkyl, substituted
arylalkyl, heteroaryl, substituted heteroaryl, carboxy,
carboxyalkyl, alkyl carboxylate, alkyl alkanoate, guanidinoalkyl,
R.sup.4-carbonyl, R.sup.5-carbonylalkyl, R.sup.6-acylamino, and
R.sup.7-acylaminoalkyl, wherein R.sup.4 and R.sup.5 are each
independently selected from the group consisting of an amino acid,
an amino acid derivative, and a peptide, and wherein R.sup.6 and
R.sup.7 are each independently selected from the group consisting
of an amino acid, an amino acid derivative, and a peptide.
[0259] The compounds described herein may contain one or more
chiral centers, or may otherwise be capable of existing as multiple
stereoisomers. It is to be understood that in one embodiment, the
invention described herein is not limited to any particular
stereochemical requirement, except where specifically indicated,
and that the compounds, and compositions, methods, uses, and
medicaments that include them may be optically pure, or may be any
of a variety of stereoisomeric mixtures, including racemic and
other mixtures of enantiomers, other mixtures of diastereomers, and
the like. It is also to be understood that such mixtures of
stereoisomers may include a single stereochemical configuration at
one or more chiral centers, while including mixtures of
stereochemical configuration at one or more other chiral
centers.
[0260] Similarly, the compounds described herein may include
geometric centers, such as cis, trans, E, and Z double bonds. It is
to be understood that in another embodiment, the invention
described herein is not limited to any particular geometric isomer
requirement, and that the compounds, and compositions, methods,
uses, and medicaments that include them may be pure, or may be any
of a variety of geometric isomer mixtures. It is also to be
understood that such mixtures of geometric isomers may include a
single configuration at one or more double bonds, while including
mixtures of geometry at one or more other double bonds.
[0261] As used herein, the term "cell surface receptor binding or
targeting ligand" generally refers to compounds that bind to and/or
target receptors that are found on cell surfaces, and in particular
those that are found on, over-expressed by, and/or preferentially
expressed on the surface of pathogenic cells. Illustrative ligands
include, but are not limited to, vitamins and vitamin receptor
binding compounds.
[0262] Illustrative vitamin moieties include carnitine, inositol,
lipoic acid, pyridoxal, ascorbic acid, niacin, pantothenic acid,
folic acid, riboflavin, thiamine, biotin, vitamin B.sub.12, and the
lipid soluble vitamins A, D, E and K. These vitamins, and their
receptor-binding analogs and derivatives, constitute the targeting
entity from which a radical can be formed for covalent attachment
to the polyvalent linker L. Illustrative biotin analogs that bind
to biotin receptors include, but are not limited to, biocytin,
biotin sulfoxide, oxybiotin, and the like.
[0263] Illustrative folic acid analogs that bind to folate
receptors include, but are not limited to folinic acid,
pteropolyglutamic acid, and folate receptor-binding pteridines such
as tetrahydropterins, dihydrofolates, tetrahydrofolates, and their
deaza and dideaza analogs. The terms "deaza" and "dideaza" analogs
refer to the art-recognized analogs having a carbon atom
substituted for one or two nitrogen atoms in the naturally
occurring folic acid structure, or analog or derivative thereof.
For example, the deaza analogs include the 1-deaza, 3-deaza,
5-deaza, 8-deaza, and 10-deaza analogs of folate, folinic acid,
pteropolyglutamic acid, and folate receptor-binding pteridines such
as tetrahydropterins, dihydrofolates, and tetrahydrofolates. The
dideaza analogs include, for example, 1,5-dideaza, 5,10-dideaza,
8,10-dideaza, and 5,8-dideaza analogs of folate, folinic acid,
pteropolyglutamic acid, and folate receptor-binding pteridines such
as tetrahydropterins, dihydrofolates, and tetrahydrofolates. Other
folates useful as complex forming ligands for this invention are
the folate receptor-binding analogs aminopterin, amethopterin (also
known as methotrexate), N.sup.10-methylfolate,
2-deamino-hydroxyfolate, deaza analogs such as 1-deazamethopterin
or 3-deazamethopterin, and
3',5'-dichloro-4-amino-4-deoxy-N.sup.10-methylpteroylglutamic acid
(dichloromethotrexate). The foregoing folic acid analogs and/or
derivatives are conventionally termed "folates," reflecting their
ability to bind with folate-receptors, and such ligands when
conjugated with exogenous molecules are effective to enhance
transmembrane transport, such as via folate-mediated endocytosis as
described herein.
[0264] Additional analogs of folic acid that bind to folic acid
receptors are described in US Patent Application Publication Serial
Nos. 2005/0227985 and 2004/0242582, the disclosures of which are
incorporated herein by reference. Illustratively, such folate
analogs have the general formula:
##STR00082##
wherein X and Y are each-independently selected from the group
consisting of halo, R.sup.2, OR.sup.2, SR.sup.3, and
NR.sup.4R.sup.5;
[0265] U, V, and W represent divalent moieties each independently
selected from the group consisting of --(R.sup.6a)C.dbd., --N.dbd.,
--(R.sup.6a)C(R.sup.7a)--, and --N(R.sup.4a)--; Q is selected from
the group consisting of C and CH; T is selected from the group
consisting of S, O, N, and --C.dbd.C--;
[0266] A.sup.1 and A.sup.2 are each independently selected from the
group consisting of oxygen, sulfur, --C(Z)--, --C(Z)O--, --OC(Z)--,
--N(R.sup.4b)--, --C(Z)N(R.sup.4b)--, --N(R.sup.4b)C(Z)--,
--OC(Z)N(R.sup.4b)--N(R.sup.4b)C(Z)O--,
--N(R.sup.4b)C(Z)N(R.sup.5b)--, --S(O)--, --S(O).sub.2--,
--N(R.sup.4a)S(O).sub.2--, --C(R.sup.6b)(R.sup.7b)--,
--N(C.ident.CH)--, --N(CH.sub.2C.ident.CH)--, C.sub.1-C.sub.12
alkylene, and C.sub.1-C.sub.12 alkyeneoxy, where Z is oxygen or
sulfur;
[0267] R.sup.1 is selected-from the group consisting of hydrogen,
halo, C.sub.1-C.sub.12 alkyl, and C.sub.1-C.sub.12 alkoxy; R.sup.2,
R.sup.3, R.sup.4, R.sup.4a, R.sup.4b, R.sup.5, R.sup.5b, R.sup.6b,
and R.sup.7b are each independently selected from the group
consisting of hydrogen, halo, C.sub.1-C.sub.12 alkyl,
C.sub.1-C.sub.12 alkoxy, alkanoyl, alkenyl, alkynyl,
alkoxy)carbonyl, and (C.sub.1-C.sub.12 alkylamino)carbonyl;
[0268] R.sup.6 and R.sup.7 are each independently selected from the
group consisting of hydrogen, halo, C.sub.1-C.sub.12 alkyl, and
C.sub.1-C.sub.12 alkoxy; or, R.sup.6 and R.sup.7 are taken together
to form a carbonyl group; R.sup.6a and R.sup.7a are each
independently selected from the group consisting of hydrogen, halo,
C.sub.1-C.sub.12 alkyl, and C.sub.1-C.sub.12 alkoxy; or R.sup.6a
and R.sup.7a are taken together to form a carbonyl group;
[0269] L is a divalent linker as described herein; and
[0270] n, p, r, s and t are each independently either 0 or 1.
[0271] As used herein, the term "amino acid" refers generally to
beta, gamma, and longer amino acids, such as amino acids of the
formula:
--N(R)--(CR'R'').sub.q--C(O)--
where R is hydrogen, alkyl, acyl, or a suitable nitrogen protecting
group, R' and R'' are hydrogen or a substituent, each of which is
independently selected in each occurrence, and q is an integer such
as 1, 2, 3, 4, or 5. Illustratively, R' and/or R'' independently
correspond to, but are not limited to, hydrogen or the side chains
present on naturally occurring amino acids, such as methyl, benzyl,
hydroxymethyl, thiomethyl, carboxyl, carboxylmethyl,
guanidinopropyl, and the like, and derivatives and protected
derivatives thereof. The above described formula includes all
stereoisomeric variations. For example, the amino acid may be
selected from asparagine, aspartic acid, cysteine, glutamic acid,
lysine, glutamine, arginine, serine, ornitine, threonine, and the
like.
[0272] As used herein, the term "amino acid derivative" generally
refers to an amino acid as defined herein where either, or both,
the amino group and/or the side chain is substituted. Illustrative
amino acid derivatives include prodrugs and protecting groups of
the amino group and/or the side chain, such as amine, amide,
hydroxy, carboxylic acid, and thio prodrugs and protecting groups.
Additional Illustrative amino acid derivatives include substituted
variations of the amino acid as described herein, such as, but not
limited to, ethers and esters of hydroxy groups, amides,
carbamates, and ureas of amino groups, esters, amides, and cyano
derivatives of carboxylic acid groups, and the like.
[0273] As used herein, the terms "tubulysin" and "tubulysins" refer
generally to tetrapeptide compounds of the formula
##STR00083##
and pharmaceutical salts thereof, where
[0274] n is 1-3;
[0275] V is H, OR.sup.2, or halo, and W is H, OR.sup.2, or alkyl,
where R.sup.2 is independently selected in each instance from H,
alkyl, and C(O)R.sup.3, where R.sup.3 is alkyl, cycloalkyl,
alkenyl, aryl, or arylalkyl, each of which is optionally
substituted; providing that R.sup.2 is not H when both V and W are
OR.sup.2; or V and W are taken together with the attached carbon to
form a carbonyl;
[0276] X=H, C.sub.1 alkyl, alkenyl, each of which is optionally
substituted, or CH.sub.2QR.sup.9; where Q is --O--, or --S--;
R.sup.9=H, C.sub.1 alkyl, alkenyl, aryl, or C(O)R.sup.10; and
R.sup.10=C.sub.1-6 alkyl, alkenyl, aryl, or heteroaryl, each of
which is optionally substituted;
[0277] Z is alkyl and Y is O; or Z is alkyl or C(O)R.sup.4, and Y
is absent, where R.sup.4 is alkyl, CF.sub.3, or aryl;
[0278] R.sup.1 is H, or R.sup.1 represents 1 to 3 substituents
selected from halo, nitro, carboxylate or a derivative thereof,
cyano, hydroxyl, alkyl, haloalkyl, alkoxy, haloalkoxy, and
OR.sup.6, where R.sup.6 is hydrogen or optionally substituted aryl,
a phenol protecting group, a prodrug moiety, alkyl, arylalkyl,
C(O)R.sup.7, P(O)(OR.sup.8).sub.2, or SO.sub.3R.sup.8, where
R.sup.7 and R.sup.8 are independently selected in each instance
from H, alkyl, alkenyl, cycloalkyl, heterocyclyl, aryl, and
arylalkyl, each of which is optionally substituted, or R.sup.8 is a
metal cation; and
[0279] R is OH or a leaving group, or R forms a carboxylic acid
derivative, such as an acylhydrazide.
[0280] Conjugates of each of the foregoing tubulysins are described
herein. In one variation, Z is methyl. In another variation,
R.sup.1 is H. In another variation, R.sup.1 is OR.sup.6 at C(4),
where R.sup.6 is H, alkyl, or COR.sup.7. In another variation, V is
H, and W is OC(O)R.sup.3. In another variation, X=CH.sub.2QR.sup.9.
In another variation, X=CH.sub.2OR.sup.9. In another variation,
R.sup.9 is alkyl or alkenyl. In another variation, R.sup.9 is
C(O)R.sup.10. In another variation, R.sup.10=optionally substituted
C.sub.1-6 alkyl. In another variation, R.sup.10=C.sub.1-6 alkyl. In
another variation, R forms an acylhydrazide. It is to be understood
that the foregoing description is an explicit description of each
chemically possible combination of variations of the general
tubulysin structure. For example, it is to be understood that the
foregoing description is a description of the variation where Z is
methyl, and R.sup.1 is H; where R.sup.1 is OR.sup.6 at C(4), and
R.sup.6 is H; where Z is methyl, R.sup.1 is OR.sup.6 at C(4),
R.sup.6 is H, and X=CH.sub.2OR.sup.9; and the like.
[0281] Natural tubulysins are generally linear tetrapeptides
consisting of N-methyl pipecolic acid (Mep), isoleucine (Ile), an
unnatural aminoacid called tubuvaline (Tuv), and either an
unnatural aminoacid called tubutyrosine (Tut, an analog of
tyrosine) or an unnatural aminoacid called tubuphenylalanine (Tup,
an analog of phenylalanine). In another embodiment, naturally
occurring tubulysins, and analogs and derivatives thereof, of the
following general formula are described
##STR00084##
and pharmaceutical salts thereof, where R, R', and R.sup.10 are as
described in the various embodiments herein. Conjugates of each of
the foregoing tubulysins are described herein.
[0282] In another embodiment, conjugates of naturally occurring
tubulysins of the following general formula are described
##STR00085##
TABLE-US-00001 Factor R.sup.10 R.sup.1 A (CH.sub.3).sub.2CHCH.sub.2
OH B CH.sub.3(CH.sub.2).sub.2 OH C CH.sub.3CH.sub.2 OH D
(CH.sub.3).sub.2CHCH.sub.2 H E CH.sub.3(CH.sub.2).sub.2 H F
CH.sub.2CH.sub.3 H G (CH.sub.3).sub.2C.dbd.CH OH H CH.sub.3 H I
CH.sub.3 OH
and pharmaceutical salts thereof.
[0283] In another embodiment, compounds are described herein where
the conjugate is formed at the terminal carboxylic acid group or
the terminal acylhydrazine group of each of the tybulysins
described herein.
[0284] As used herein, the term "a rapamycin" is understood to
include sirolimus (rapamycin), temsirolimus, everolimus, and
ridaforolimus, and related compounds, and compounds of the
formula
##STR00086##
[0285] and pharmaceutically acceptable salts thereof, wherein
[0286] Y.sup.A is OR.sup.C or OCH.sub.2CH.sub.2OR.sup.C;
[0287] one of R.sup.A, R.sup.B, or R.sup.C is a bond connected to
L; and
[0288] the other two of R.sup.A, R.sup.B, and R.sup.C are
independently selected in each case from the group consisting of
hydrogen, optionally substituted heteroalkyl, prodrug forming
group, and C(O)R.sup.D, where R.sup.D is in each instance
independently selected from the group consisting of hydrogen, and
alkyl, alkenyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl,
arylalkyl, heteroaryl, and heteroarylalkyl, each of which is
optionally substituted is described.
[0289] As used herein, the term "alkyl" includes a chain of carbon
atoms, which is optionally branched. As used herein, the term
"alkenyl" and "alkynyl" includes a chain of carbon atoms, which is
optionally branched, and includes at least one double bond or
triple bond, respectively. It is to be understood that alkynyl may
also include one or more double bonds. It is to be further
understood that in certain embodiments, alkyl is advantageously of
limited length, including C.sub.1-C.sub.24, C.sub.1-C.sub.12,
C.sub.1-C.sub.8, C.sub.1-C.sub.6, and C.sub.1-C.sub.4.
Illustratively, such particularly limited length alkyl groups,
including C.sub.1-C.sub.8, C.sub.1-C.sub.6, and C.sub.1-C.sub.4 may
be referred to as lower alkyl. It is to be further understood that
in certain embodiments alkenyl and/or alkynyl may each be
advantageously of limited length, including C.sub.2-C.sub.24,
C.sub.2-C.sub.12, C.sub.2-C.sub.8, C.sub.2-C.sub.6, and
C.sub.2-C.sub.4. Illustratively, such particularly limited length
alkenyl and/or alkynyl groups, including C.sub.2-C.sub.8,
C.sub.2-C.sub.6, and C.sub.2-C.sub.4 may be referred to as lower
alkenyl and/or alkynyl. It is appreciated herein that shorter
alkyl, alkenyl, and/or alkynyl groups may add less lipophilicity to
the compound and accordingly will have different pharmacokinetic
behavior. In embodiments of the invention described herein, it is
to be understood, in each case, that the recitation of alkyl refers
to alkyl as defined herein, and optionally lower alkyl. In
embodiments of the invention described herein, it is to be
understood, in each case, that the recitation of alkenyl refers to
alkenyl as defined herein, and optionally lower alkenyl. In
embodiments of the invention described herein, it is to be
understood, in each case, that the recitation of alkynyl refers to
alkynyl as defined herein, and optionally lower alkynyl.
Illustrative alkyl, alkenyl, and alkynyl groups are, but not
limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,
sec-butyl, tert-butyl, pentyl, 2-pentyl, 3-pentyl, neopentyl,
hexyl, heptyl, octyl, and the like, and the corresponding groups
containing one or more double and/or triple bonds, or a combination
thereof.
[0290] As used herein, the term "alkylene" includes a divalent
chain of carbon atoms, which is optionally branched. As used
herein, the term "alkenylene" and "alkynylene" includes a divalent
chain of carbon atoms, which is optionally branched, and includes
at least one double bond or triple bond, respectively. It is to be
understood that alkynylene may also include one or more double
bonds. It is to be further understood that in certain embodiments,
alkylene is advantageously of limited length, including
C.sub.1-C.sub.24, C.sub.1-C.sub.12, C.sub.1-C.sub.8,
C.sub.1-C.sub.6, and C.sub.1-C.sub.4. Illustratively, such
particularly limited length alkylene groups, including
C.sub.1-C.sub.8, C.sub.1-C.sub.6, and C.sub.1-C.sub.4 may be
referred to as lower alkylene. It is to be further understood that
in certain embodiments alkenylene and/or alkynylene may each be
advantageously of limited length, including C.sub.2-C.sub.24,
C.sub.2-C.sub.12, C.sub.2-C.sub.8, C.sub.2-C.sub.6, and
C.sub.2-C.sub.4. Illustratively, such particularly limited length
alkenylene and/or alkynylene groups, including C.sub.2-C.sub.8,
C.sub.2-C.sub.6, and C.sub.2-C.sub.4 may be referred to as lower
alkenylene and/or alkynylene. It is appreciated herein that shorter
alkylene, alkenylene, and/or alkynylene groups may add less
lipophilicity to the compound and accordingly will have different
pharmacokinetic behavior. In embodiments of the invention described
herein, it is to be understood, in each case, that the recitation
of alkylene, alkenylene, and alkynylene refers to alkylene,
alkenylene, and alkynylene as defined herein, and optionally lower
alkylene, alkenylene, and alkynylene. Illustrative alkyl groups
are, but not limited to, methylene, ethylene, n-propylene,
isopropylene, n-butylene, isobutylene, sec-butylene, pentylene,
1,2-pentylene, 1,3-pentylene, hexylene, heptylene, octylene, and
the like.
[0291] As used herein, the term "cycloalkyl" includes a chain of
carbon atoms, which is optionally branched, where at least a
portion of the chain is cyclic. It is to be understood that
cycloalkylalkyl is a subset of cycloalkyl. It is to be understood
that cycloalkyl may be polycyclic. Illustrative cycloalkyl include,
but are not limited to, cyclopropyl, cyclopentyl, cyclohexyl,
2-methylcyclopropyl, cyclopentyleth-2-yl, adamantyl, and the like.
As used herein, the term "cycloalkenyl" includes a chain of carbon
atoms, which is optionally branched, and includes at least one
double bond, where at least a portion of the chain in cyclic. It is
to be understood that the one or more double bonds may be in the
cyclic portion of cycloalkenyl and/or the non-cyclic portion of
cycloalkenyl. It is to be understood that cycloalkenylalkyl and
cycloalkylalkenyl are each subsets of cycloalkenyl. It is to be
understood that cycloalkyl may be polycyclic. Illustrative
cycloalkenyl include, but are not limited to, cyclopentenyl,
cyclohexylethen-2-yl, cycloheptenylpropenyl, and the like. It is to
be further understood that chain forming cycloalkyl and/or
cycloalkenyl is advantageously of limited length, including
C.sub.3-C.sub.24, C.sub.3-C.sub.12, C.sub.3-C.sub.8,
C.sub.3-C.sub.6, and C.sub.5-C.sub.6. It is appreciated herein that
shorter alkyl and/or alkenyl chains forming cycloalkyl and/or
cycloalkenyl, respectively, may add less lipophilicity to the
compound and accordingly will have different pharmacokinetic
behavior.
[0292] As used herein, the term "heteroalkyl" includes a chain of
atoms that includes both carbon and at least one heteroatom, and is
optionally branched. Illustrative heteroatoms include nitrogen,
oxygen, and sulfur. In certain variations, illustrative heteroatoms
also include phosphorus, and selenium. As used herein, the term
"cycloheteroalkyl" including heterocyclyl and heterocycle, includes
a chain of atoms that includes both carbon and at least one
heteroatom, such as heteroalkyl, and is optionally branched, where
at least a portion of the chain is cyclic. Illustrative heteroatoms
include nitrogen, oxygen, and sulfur. In certain variations,
illustrative heteroatoms also include phosphorus, and selenium.
Illustrative cycloheteroalkyl include, but are not limited to,
tetrahydrofuryl, pyrrolidinyl, tetrahydropyranyl, piperidinyl,
morpholinyl, piperazinyl, homopiperazinyl, quinuclidinyl, and the
like.
[0293] As used herein, the term "aryl" includes monocyclic and
polycyclic aromatic carbocyclic groups, each of which may be
optionally substituted. Illustrative aromatic carbocyclic groups
described herein include, but are not limited to, phenyl, naphthyl,
and the like. As used herein, the term "heteroaryl" includes
aromatic heterocyclic groups, each of which may be optionally
substituted. Illustrative aromatic heterocyclic groups include, but
are not limited to, pyridinyl, pyrimidinyl, pyrazinyl, triazinyl,
tetrazinyl, quinolinyl, quinazolinyl, quinoxalinyl, thienyl,
pyrazolyl, imidazolyl, oxazolyl, thiazolyl, isoxazolyl,
isothiazolyl, oxadiazolyl, thiadiazolyl, triazolyl, benzimidazolyl,
benzoxazolyl, benzthiazolyl, benzisoxazolyl, benzisothiazolyl, and
the like.
[0294] As used herein, the term "amino" includes the group
NH.sub.2, alkylamino, and dialkylamino, where the two alkyl groups
in dialkylamino may be the same or different, i.e. alkylalkylamino
Illustratively, amino includes methylamino, ethylamino,
dimethylamino, methylethylamino, and the like. In addition, it is
to be understood that when amino modifies or is modified by another
term, such as aminoalkyl, or acylamino, the above variations of the
term amino are included therein. Illustratively, aminoalkyl
includes H.sub.2N-alkyl, methylaminoalkyl, ethylaminoalkyl,
dimethylaminoalkyl, methylethylaminoalkyl, and the like.
Illustratively, acylamino includes acylmethylamino, acylethylamino,
and the like.
[0295] As used herein, the term "amino and derivatives thereof"
includes amino as described herein, and alkylamino, alkenylamino,
alkynylamino, heteroalkylamino, heteroalkenylamino,
heteroalkynylamino, cycloalkylamino, cycloalkenylamino,
cycloheteroalkylamino, cycloheteroalkenylamino, arylamino,
arylalkylamino, arylalkenylamino, arylalkynylamino,
heteroarylamino, heteroarylalkylamino, heteroarylalkenylamino,
heteroarylalkynylamino, acylamino, and the like, each of which is
optionally substituted. The term "amino derivative" also includes
urea, carbamate, and the like.
[0296] As used herein, the term "hydroxy and derivatives thereof"
includes OH, and alkyloxy, alkenyloxy, alkynyloxy, heteroalkyloxy,
heteroalkenyloxy, heteroalkynyloxy, cycloalkyloxy, cycloalkenyloxy,
cycloheteroalkyloxy, cycloheteroalkenyloxy, aryloxy, arylalkyloxy,
arylalkenyloxy, arylalkynyloxy, heteroaryloxy, heteroarylalkyloxy,
heteroarylalkenyloxy, heteroarylalkynyloxy, acyloxy, and the like,
each of which is optionally substituted. The term "hydroxy
derivative" also includes carbamate, and the like.
[0297] As used herein, the term "thio and derivatives thereof"
includes SH, and alkylthio, alkenylthio, alkynylthio,
heteroalkylthio, heteroalkenylthio, heteroalkynylthio,
cycloalkylthio, cycloalkenylthio, cycloheteroalkylthio,
cycloheteroalkenylthio, arylthio, arylalkylthio, arylalkenylthio,
arylalkynylthio, heteroarylthio, heteroarylalkylthio,
heteroarylalkenylthio, heteroarylalkynylthio, acylthio, and the
like, each of which is optionally substituted. The term "thio
derivative" also includes thiocarbamate, and the like.
[0298] As used herein, the term "acyl" includes formyl, and
alkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl,
heteroalkylcarbonyl, heteroalkenylcarbonyl, heteroalkynylcarbonyl,
cycloalkylcarbonyl, cycloalkenylcarbonyl, cycloheteroalkylcarbonyl,
cycloheteroalkenylcarbonyl, arylcarbonyl, arylalkylcarbonyl,
arylalkenylcarbonyl, arylalkynylcarbonyl, heteroarylcarbonyl,
heteroarylalkylcarbonyl, heteroarylalkenylcarbonyl,
heteroarylalkynylcarbonyl, acylcarbonyl, and the like, each of
which is optionally substituted.
[0299] As used herein, the term "carbonyl and derivatives thereof"
includes the group C(O), C(S), C(NH) and substituted amino
derivatives thereof.
[0300] As used herein, the term "carboxylic acid and derivatives
thereof" includes the group CO.sub.2H and salts thereof, and esters
and amides thereof, and CN.
[0301] The term "optionally substituted" as used herein includes
the replacement of hydrogen atoms with other functional groups on
the radical that is optionally substituted. Such other functional
groups illustratively include, but are not limited to, amino,
hydroxyl, halo, thiol, alkyl, haloalkyl, heteroalkyl, aryl,
arylalkyl, arylheteroalkyl, heteroaryl, heteroarylalkyl,
heteroarylheteroalkyl, nitro, sulfonic acids and derivatives
thereof, carboxylic acids and derivatives thereof, and the like.
Illustratively, any of amino, hydroxyl, thiol, alkyl, haloalkyl,
heteroalkyl, aryl, arylalkyl, arylheteroalkyl, heteroaryl,
heteroarylalkyl, heteroarylheteroalkyl, and/or sulfonic acid is
optionally substituted.
[0302] As used herein, the terms "optionally substituted aryl" and
"optionally substituted heteroaryl" include the replacement of
hydrogen atoms with other functional groups on the aryl or
heteroaryl that is optionally substituted. Such other functional
groups illustratively include, but are not limited to, amino,
hydroxy, halo, thio, alkyl, haloalkyl, heteroalkyl, aryl,
arylalkyl, arylheteroalkyl, heteroaryl, heteroarylalkyl,
heteroarylheteroalkyl, nitro, sulfonic acids and derivatives
thereof, carboxylic acids and derivatives thereof, and the like.
Illustratively, any of amino, hydroxy, thio, alkyl, haloalkyl,
heteroalkyl, aryl, arylalkyl, arylheteroalkyl, heteroaryl,
heteroarylalkyl, heteroarylheteroalkyl, and/or sulfonic acid is
optionally substituted.
[0303] Illustrative substituents include, but are not limited to, a
radical --(CH.sub.2).sub.xZ.sup.X, where x is an integer from 0-6
and Z.sup.X is selected from halogen, hydroxy, alkanoyloxy,
including C.sub.1-C.sub.6 alkanoyloxy, optionally substituted
aroyloxy, alkyl, including C.sub.1-C.sub.6 alkyl, alkoxy, including
C.sub.1-C.sub.6 alkoxy, cycloalkyl, including C.sub.3-C.sub.8
cycloalkyl, cycloalkoxy, including C.sub.3-C.sub.8 cycloalkoxy,
alkenyl, including C.sub.2-C.sub.6 alkenyl, alkynyl, including
C.sub.2-C.sub.6 alkynyl, haloalkyl, including C.sub.1-C.sub.6
haloalkyl, haloalkoxy, including C.sub.1-C.sub.6 haloalkoxy,
halocycloalkyl, including C.sub.3-C.sub.8 halocycloalkyl,
halocycloalkoxy, including C.sub.3-C.sub.8 halocycloalkoxy, amino,
C.sub.1-C.sub.6 alkylamino, (C.sub.1-C.sub.6 alkyl)(C.sub.1-C.sub.6
alkyl)amino, alkylcarbonylamino, N--(C.sub.1-C.sub.6
alkyl)alkylcarbonylamino, aminoalkyl, C.sub.1-C.sub.6
alkylaminoalkyl, (C.sub.1-C.sub.6 alkyl)(C.sub.1-C.sub.6
alkyl)aminoalkyl, alkylcarbonylaminoalkyl, N--(C.sub.1-C.sub.6
alkyl)alkylcarbonylaminoalkyl, cyano, and nitro; or Z.sup.X is
selected from --CO.sub.2R.sup.4 and --CONR.sup.5R.sup.6, where
R.sup.4, R.sup.5, and R.sup.6 are each independently selected in
each occurrence from hydrogen, C.sub.1-C.sub.6 alkyl,
aryl-C.sub.1-C.sub.6 alkyl, and heteroaryl-C.sub.1-C.sub.6
alkyl.
[0304] As used herein the term "radical" with reference to, for
example, the cell surface receptor binding and/or targeting ligand,
and/or the independently selected drug, refers to a cell surface
receptor binding and/or targeting ligand, and/or an independently
selected drug, as described herein, where one or more atoms or
groups, such as a hydrogen atom, or an alkyl group on a heteroatom,
and the like, is removed to provide a radical for conjugation to
the polyvalent linker L. Such ligand radicals and drug radicals may
also be referred herein as ligand analogs and drug analogs,
respectively.
[0305] As used herein, the term "leaving group" refers to a
reactive functional group that generates an electrophilic site on
the atom to which it is attached such that nucleophiles may be
added to the electrophilic site on the atom. Illustrative leaving
groups include, but are not limited to, halogens, optionally
substituted phenols, acyloxy groups, sulfonoxy groups, and the
like. It is to be understood that such leaving groups may be on
alkyl, acyl, and the like. Such leaving groups may also be referred
to herein as activating groups, such as when the leaving group is
present on acyl. In addition, conventional peptide, amide, and
ester coupling agents, such as but not limited to PyBop, BOP-Cl,
BOP, pentafluorophenol, isobutylchloroformate, and the like, form
various intermediates that include a leaving group, as defined
herein, on a carbonyl group.
[0306] It is to be understood that in every instance disclosed
herein, the recitation of a range of integers for any variable
describes the recited range, every individual member in the range,
and every possible subrange for that variable. For example, the
recitation that n is an integer from 0 to 8, describes that range,
the individual and selectable values of 0, 1, 2, 3, 4, 5, 6, 7, and
8, such as n is 0, or n is 1, or n is 2, etc. In addition, the
recitation that n is an integer from 0 to 8 also describes each and
every subrange, each of which may for the basis of a further
embodiment, such as n is an integer from 1 to 8, from 1 to 7, from
1 to 6, from 2 to 8, from 2 to 7, from 1 to 3, from 2 to 4,
etc.
[0307] As used herein, the term "composition" generally refers to
any product comprising the specified ingredients in the specified
amounts, as well as any product which results, directly or
indirectly, from combinations of the specified ingredients in the
specified amounts. It is to be understood that the compositions
described herein may be prepared from isolated compounds described
herein or from salts, solutions, hydrates, solvates, and other
forms of the compounds described herein. It is appreciated that
certain functional groups, such as the hydroxy, amino, and like
groups form complexes and/or coordination compounds with water
and/or various solvents, in the various physical forms of the
compounds. It is also to be understood that the compositions may be
prepared from various amorphous, non-amorphous, partially
crystalline, crystalline, and/or other morphological forms of the
compounds described herein. It is also to be understood that the
compositions may be prepared from various hydrates and/or solvates
of the compounds described herein. Accordingly, such pharmaceutical
compositions that recite compounds described herein are to be
understood to include each of, or any combination of, the various
morphological forms and/or solvate or hydrate forms of the
compounds described herein. In addition, it is to be understood
that the compositions may be prepared from various co-crystals of
the compounds described herein.
[0308] Illustratively, compositions may include one or more
carriers, diluents, and/or excipients. The compounds described
herein, or compositions containing them, may be formulated in a
therapeutically effective amount in any conventional dosage forms
appropriate for the methods described herein. The compounds
described herein, or compositions containing them, including such
formulations, may be administered by a wide variety of conventional
routes for the methods described herein, and in a wide variety of
dosage formats, utilizing known procedures (see generally,
Remington: The Science and Practice of Pharmacy, (21.sup.st ed.,
2005)).
[0309] The term "therapeutically effective amount" as used herein,
refers to that amount of active compound or pharmaceutical agent
that elicits the biological or medicinal response in a tissue
system, animal or human that is being sought by a researcher,
veterinarian, medical doctor or other clinician, which includes
alleviation of the symptoms of the disease or disorder being
treated. In one aspect, the therapeutically effective amount is
that which may treat or alleviate the disease or symptoms of the
disease at a reasonable benefit/risk ratio applicable to any
medical treatment. However, it is to be understood that the total
daily usage of the compounds and compositions described herein may
be decided by the attending physician within the scope of sound
medical judgment. The specific therapeutically-effective dose level
for any particular patient will depend upon a variety of factors,
including the disorder being treated and the severity of the
disorder; activity of the specific compound employed; the specific
composition employed; the age, body weight, general health, gender
and diet of the patient: the time of administration, route of
administration, and rate of excretion of the specific compound
employed; the duration of the treatment; drugs used in combination
or coincidentally with the specific compound employed; and like
factors well known to the researcher, veterinarian, medical doctor
or other clinician of ordinary skill.
[0310] The term "administering" as used herein includes all means
of introducing the compounds and compositions described herein to
the patient, including, but are not limited to, oral (po),
intravenous (iv), intramuscular (im), subcutaneous (sc),
transdermal, inhalation, buccal, ocular, sublingual, vaginal,
rectal, and the like. The compounds and compositions described
herein may be administered in unit dosage forms and/or formulations
containing conventional nontoxic pharmaceutically-acceptable
carriers, adjuvants, and vehicles.
[0311] Illustrative formats for oral administration include
tablets, capsules, elixirs, syrups, and the like.
[0312] Illustrative routes for parenteral administration include
intravenous, intraarterial, intraperitoneal, epidurial,
intraurethral, intrasternal, intramuscular and subcutaneous, as
well as any other art recognized route of parenteral
administration.
[0313] Depending upon the disease as described herein, the route of
administration and/or whether the compounds and/or compositions are
administered locally or systemically, a wide range of permissible
dosages are contemplated herein, including doses falling in the
range from about 1 .mu.g/kg to about 1 g/kg. The dosages may be
single or divided, and may administered according to a wide variety
of protocols, including q.d., b.i.d., t.i.d., or even every other
day, once a week, once a month, once a quarter, and the like. In
each of these cases it is understood that the therapeutically
effective amounts described herein correspond to the instance of
administration, or alternatively to the total daily, weekly, month,
or quarterly dose, as determined by the dosing protocol.
[0314] The term "prodrug" as used herein generally refers to any
compound that when administered to a biological system generates a
biologically active compound as a result of one or more spontaneous
chemical reaction(s), enzyme-catalyzed chemical reaction(s), and/or
metabolic chemical reaction(s), or a combination thereof. In vivo,
the prodrug is typically acted upon by an enzyme (such as
esterases, amidases, phosphatases, and the like), simple biological
chemistry, or other process in vivo to liberate or regenerate the
more pharmacologically active drug. This activation may occur
through the action of an endogenous host enzyme or a non-endogenous
enzyme that is administered to the host preceding, following, or
during administration of the prodrug. Additional details of prodrug
use are described in U.S. Pat. No. 5,627,165; and Pathalk et al.,
Enzymic protecting group techniques in organic synthesis,
Stereosel. Biocatal. 775-797 (2000). It is appreciated that the
prodrug is advantageously converted to the original drug as soon as
the goal, such as targeted delivery, safety, stability, and the
like is achieved, followed by the subsequent rapid elimination of
the released remains of the group forming the prodrug.
[0315] Prodrugs may be prepared from the compounds described herein
by attaching groups that ultimately cleave in vivo to one or more
functional groups present on the compound, such as --OH--, --SH,
--CO.sub.2H, --NR.sub.2. Illustrative prodrugs include but are not
limited to carboxylate esters where the group is alkyl, aryl,
arylalkyl, heteroaryl, heteroarylalkyl, acyloxyalkyl,
alkoxycarbonyloxyalkyl as well as esters of hydroxyl, thiol and
amines where the group attached is an acyl group, an
alkoxycarbonyl, aminocarbonyl, phosphate or sulfate. Illustrative
esters, also referred to as active esters, include but are not
limited to 1-indanyl, N-oxysuccinimide; acyloxyalkyl groups such as
acetoxymethyl, pivaloyloxymethyl, .beta.-acetoxyethyl,
.beta.-pivaloyloxyethyl, 1-(cyclohexylcarbonyloxy)prop-1-yl,
(1-aminoethyl)carbonyloxymethyl, and the like;
alkoxycarbonyloxyalkyl groups, such as ethoxycarbonyloxymethyl,
.alpha.-ethoxycarbonyloxyethyl, .beta.-ethoxycarbonyloxyethyl, and
the like; dialkylaminoalkyl groups, including di-lower alkylamino
alkyl groups, such as dimethylaminomethyl, dimethylaminoethyl,
diethylaminomethyl, diethylaminoethyl, and the like;
2-(alkoxycarbonyl)-2-alkenyl groups such as 2-(isobutoxycarbonyl)
pent-2-enyl, 2-(ethoxycarbonyl)but-2-enyl, and the like; and
lactone groups such as phthalidyl, dimethoxyphthalidyl, and the
like.
[0316] Further illustrative prodrugs contain a chemical moiety,
such as an amide or phosphorus group functioning to increase
solubility and/or stability of the compounds described herein.
Further illustrative prodrugs for amino groups include, but are not
limited to, (C.sub.3-C.sub.20)alkanoyl;
halo-(C.sub.3-C.sub.20)alkanoyl; (C.sub.3-C.sub.20)alkenoyl;
(C.sub.4-C.sub.7)cycloalkanoyl;
(C.sub.3-C.sub.6)-cycloalkyl(C.sub.2-C.sub.16)alkanoyl; optionally
substituted aroyl, such as unsubstituted aroyl or aroyl substituted
by 1 to 3 substituents selected from the group consisting of
halogen, cyano, trifluoromethanesulphonyloxy,
(C.sub.1-C.sub.3)alkyl and (C.sub.1-C.sub.3)alkoxy, each of which
is optionally further substituted with one or more of 1 to 3
halogen atoms; optionally substituted
aryl(C.sub.2-C.sub.16)alkanoyl and optionally substituted
heteroaryl(C.sub.2-C.sub.16)alkanoyl, such as the aryl or
heteroaryl radical being unsubstituted or substituted by 1 to 3
substituents selected from the group consisting of halogen,
(C.sub.1-C.sub.3)alkyl and (C.sub.1-C.sub.3)alkoxy, each of which
is optionally further substituted with 1 to 3 halogen atoms; and
optionally substituted heteroarylalkanoyl having one to three
heteroatoms selected from O, S and N in the heteroaryl moiety and 2
to 10 carbon atoms in the alkanoyl moiety, such as the heteroaryl
radical being unsubstituted or substituted by 1 to 3 substituents
selected from the group consisting of halogen, cyano,
trifluoromethanesulphonyloxy, (C.sub.1-C.sub.3)alkyl, and
(C.sub.1-C.sub.3)alkoxy, each of which is optionally further
substituted with 1 to 3 halogen atoms. The groups illustrated are
exemplary, not exhaustive, and may be prepared by conventional
processes.
[0317] It is understood that the prodrugs themselves may not
possess significant biological activity, but instead undergo one or
more spontaneous chemical reaction(s), enzyme-catalyzed chemical
reaction(s), and/or metabolic chemical reaction(s), or a
combination thereof after administration in vivo to produce the
compound described herein that is biologically active or is a
precursor of the biologically active compound. However, it is
appreciated that in some cases, the prodrug is biologically active.
It is also appreciated that prodrugs may often serves to improve
drug efficacy or safety through improved oral bioavailability,
pharmacodynamic half-life, and the like. Prodrugs also refer to
derivatives of the compounds described herein that include groups
that simply mask undesirable drug properties or improve drug
delivery. For example, one or more compounds described herein may
exhibit an undesirable property that is advantageously blocked or
minimized may become pharmacological, pharmaceutical, or
pharmacokinetic barriers in clinical drug application, such as low
oral drug absorption, lack of site specificity, chemical
instability, toxicity, and poor patient acceptance (bad taste,
odor, pain at injection site, and the like), and others. It is
appreciated herein that a prodrug, or other strategy using
reversible derivatives, can be useful in the optimization of the
clinical application of a drug.
[0318] The compounds, linkers, intermediates, and conjugates
described herein may be prepared using conventional processes,
including those described in International Patent Publication Nos.
WO 2009/002993, WO 2004/069159, WO 2007/022494, and WO 2006/012527,
and U.S. patent application Ser. No. 13/837,539 (filed Mar. 15,
2013). The disclosures of each of the foregoing are herein
incorporated by reference in their entirety.
[0319] Each publications cited herein is incorporated herein by
reference.
[0320] The following examples further illustrate specific
embodiments of the invention; however, the following illustrative
examples should not be interpreted in any way to limit the
invention.
EXAMPLES
Compound Examples
[0321] The compounds described herein may be prepared using the
process and syntheses described herein, as well as using general
organic synthetic methods. In particular, methods for preparing the
compounds are described in U.S. patent application publication
2005/0002942, the disclosure of which is incorporated herein by
reference.
[0322] EXAMPLE. General formation of folate-peptides. The
folate-containing peptidyl fragment
Pte-Glu-(AA).sub.n-NH(CHR.sub.2)CO.sub.2H (3) is prepared by a
polymer-supported sequential approach using standard methods, such
as the Fmoc-strategy on an acid-sensitive Fmoc-AA-Wang resin (1),
as shown in the following Scheme:
##STR00087##
[0323] It is to be understood that unnatural amino acids may be
included in the foregoing process using the appropriate starting
materials.
[0324] In this illustrative embodiment of the processes described
herein, R.sub.1 is Fmoc, R.sub.2 is the desired
appropriately-protected amino acid side chain, and DIPEA is
diisopropylethylamine Standard coupling procedures, such as PyBOP
and others described herein or known in the art are used, where the
coupling agent is illustratively applied as the activating reagent
to ensure efficient coupling. Fmoc protecting groups are removed
after each coupling step under standard conditions, such as upon
treatment with piperidine, tetrabutylammonium fluoride (TBAF), and
the like. Appropriately protected amino acid building blocks, such
as Fmoc-Glu-OtBu, Fmoc-D-Glu-OtBu, N.sup.10-TFA-Pte-OH, and the
like, are used, as described in the Scheme, and represented in step
(b) by Fmoc-AA-OH. Thus, AA refers to any amino acid starting
material, that is appropriately protected. It is to be understood
that the term amino acid as used herein is intended to refer to any
reagent having both an amine and a carboxylic acid functional group
separated by one or more carbons, and includes the naturally
occurring alpha and beta amino acids, as well as amino acid
derivatives and analogs of these amino acids. In particular, amino
acids having side chains that are protected, such as protected
serine, threonine, cysteine, aspartate, and the like may also be
used in the folate-peptide synthesis described herein. Further,
gamma, delta, or longer homologous amino acids may also be included
as starting materials in the folate-peptide synthesis described
herein. Further, amino acid analogs having homologous side chains,
or alternate branching structures, such as norleucine, isovaline,
.beta.-methyl threonine, .beta.-methyl cysteine,
.beta.,.beta.-dimethyl cysteine, and the like, may also be included
as starting materials in the folate-peptide synthesis described
herein.
[0325] The coupling sequence (steps (a) & (b)) involving
Fmoc-AA-OH is performed "n" times to prepare solid-support peptide
(2), where n is an integer and may equal 0 to about 100. Following
the last coupling step, the remaining Fmoc group is removed (step
(a)), and the peptide is sequentially coupled to a glutamate
derivative (step (c)), deprotected, and coupled to TFA-protected
pteroic acid (step (d)). Subsequently, the peptide is cleaved from
the polymeric support upon treatment with trifluoroacetic acid,
ethanedithiol, and triisopropylsilane (step (e)). These reaction
conditions result in the simultaneous removal of the t-Bu, t-Boc,
and Trt protecting groups that may form part of the
appropriately-protected amino acid side chain. The TFA protecting
group is removed upon treatment with base (step (f)) to provide the
folate-containing peptidyl fragment (3).
##STR00088##
[0326] LCMS [ESI [M+H].sup.+: 1046; Partial .sup.1H NMR (D.sub.2O,
300 MHz): .delta. 8.68 (s, 1H, FA H-7), 7.57 (d, 2H, J=8.4 Hz, FA
H-12 &16), 6.67 (d, 2H, J=9 Hz, FA H-13 &15), 4.40-4.75
(series of m, 5H), 4.35 (m, 2H), 4.16 (m, 1H), 3.02 (m, 2H),
2.55-2.95 (series of m, 8H), 2.42 (m, 2H), 2.00-2.30 (m, 2H),
1.55-1.90 (m, 2H), 1.48 (m, 2H) ppm.
[0327] EXAMPLE. The corresponding compounds containing one or more
D-amino acids may also be prepared, such as the following:
##STR00089##
[0328] LCMS [ESI, [M+H].sup.+1) 1046. Partial 1H-NMR (DMSO) .delta.
(ppm): 8.6 (s), 7.5 (d), 6.6 (d), 3.8-4.6 (m), 2.8-3.2 (m), 2.2-2.8
(m), 1-2.2 (m)
##STR00090##
[0329] MS (ESI, [M+H].sup.+1)=1046.5. Partial 1H-NMR (DMSO) .delta.
(ppm): 8.6 (s), 7.5 (d), 6.6 (d), 3.8-4.6 (m), 2.8-3.2 (m), 2.2-2.8
(m), 1-2.2 (m)
##STR00091##
[0330] MS (ESI, [M+H].sup.+1)=1046.4. Partial 1H-NMR (DMSO) .delta.
(ppm): 8.6 (s), 7.6 (d), 6.6 (d), 4-4.6 (m), 3.4-3.8 (m), 3-3.15
(m), 1-2.8 (m)
##STR00092##
[0331] [M+H].sup.+=1047.52. Partial 1H NMR (D.sub.2O): 8.6 (s, 1H),
7.5 (d, 2H), 6.65 (d, 2H), 4.4 (dd, 2H), 4.18 (m, 4H), 2.9 (t, 2H),
2.75 (t, 2H), 2.6-2.15 (m, 10H), 2.1-1.8 (m, 3H), 1.7-1.4 (m, 3H),
1.3 (m, 3H).
##STR00093##
[0332] MS (ESI [M+H].sup.+): 1046. Partial .sup.1H NMR data
(D.sub.2O, 300 MHz): .delta. (ppm) 8.68 (s, 1H, FA H-7), 7.57 (d,
2H, J=8.4 Hz, FA H-12 &16), 6.67 (d, 2H, J=9 Hz, FA H-13
&15).
##STR00094##
[0333] MS (ESI, [M+H].sup.+)=1046.7. Partial 1H-NMR (D2O) .delta.
(ppm): 8.6 (s), 7.5 (d), 6.6 (d), 4.4-4.8 (m), 4-4.2 (m) 2.2-3 (m),
1.8-2.2 (m), 1.3-1.7 (m)
##STR00095##
[0334] EXAMPLE. Preparation of tubulysin hydrazides. Illustrated by
preparing EC0347 (TubB-H).
##STR00096##
N,N-Diisopropylethylamine (DIPEA, 6.1 .mu.L) and isobutyl
chloroformate (3.0 .mu.L) were added with via syringe in tandem
into a solution of tubulysin B (0.15 mg) in anhydrous EtOAc (2.0
mL) at -15.degree. C. After stirring for 45 minutes at -15.degree.
C. under argon, the reaction mixture was cooled down to -20.degree.
C. and to which was added anhydrous hydrazine (5.0 .mu.L). The
reaction mixture was stirred under argon at -20.degree. C. for 3
hours, quenched with 1.0 mM sodium phosphate buffer (pH 7.0, 1.0
mL), and injected into a preparative HPLC for purification. Column:
Waters XTerra Prep MS C.sub.18 10 .mu.m, 19.times.250 mm; Mobile
phase A: 1.0 mM sodium phosphate buffer, pH 7.0; Mobile phase B:
acetonitrile; Method: 10% B to 80% B over 20 minutes, flow rate=25
mL/min. Fractions from 15.14-15.54 minutes were collected and
lyophilized to produce EC0347 as a white solid (2.7 mg). The
foregoing method is equally applicable for preparing other
tubulysin hydrazides by the appropriate selection of the tubulysin
starting compound.
Example. Synthesis of Coupling Reagent EC0311
##STR00097##
[0335] DIPEA (0.60 mL) was added to a suspension of
HOBt-OCO.sub.2--(CH.sub.2).sub.2--SS-2-pyridine HCl (685 mg, 91%)
in anhydrous DCM (5.0 mL) at 0.degree. C., stirred under argon for
2 minutes, and to which was added anhydrous hydrazine (0.10 mL).
The reaction mixture was stirred under argon at 0.degree. C. for 10
minutes and room temperature for an additional 30 minutes,
filtered, and the filtrate was purified by flash chromatography
(silica gel, 2% MeOH in DCM) to afford EC0311 as a clear thick oil
(371 mg), solidified upon standing.
Example. Preparation of Tubulysin Disulfides (Stepwise Process)
##STR00098##
[0336] Illustrated for EC0312. DIPEA (36 .mu.L) and isobutyl
chloroformate (13 .mu.L) were added by syringe in tandem into a
solution of tubulysin B (82 mg) in anhydrous EtOAc (2.0 mL) at
-15.degree. C. After stirring for 45 minutes at -15.degree. C.
under argon, to the reaction mixture was added a solution of EC0311
in anhydrous EtOAc (1.0 mL). The resulting solution was stirred
under argon at -15.degree. C. for 15 minutes and room temperature
for an additional 45 minutes, concentrated, and the residue was
purified by flash chromatography (silica gel, 2 to 8% MeOH in DCM)
to give EC0312 as a white solid (98 mg). The foregoing method is
equally applicable for preparing other tubulysin derivatives by the
appropriate selection of the tubulysin starting compound.
Example
##STR00099##
[0337] To a solution of doxorubicin (100 mg, 0.184 mmol) and
2-[benzotriazole-1-yl-(oxycarbonyloxy)-ethyldisulfanyl]-pyridine
(77.8 mg, 0.184 mmol) in DCM (4 ml) was added DIPEA (0.064 ml,
0.368 mmol.). The reaction was allowed to stir for 2 hours. TLC
(10% MeOH in DCM) indicated that the reaction was complete. DCM was
removed under reduced pressure and purified on SiO.sub.2 column
(10% MeOH in DCM) to yield pure product (90 mg, 65%). LCMS (ESI):
(M+H).sup.+ Calculated for C.sub.35H.sub.36N.sub.2O.sub.13S.sub.2,
757.17; found 757.30, .sup.1H NMR (300 MHz, CDCl.sub.3/CD.sub.3OD):
.delta. 8.44 (br s, 1H), 8.00 (d, 1H), 7.65-7.82 (m, 3H), 7.38 (d,
1H), 7.18 (br s, 1H), 5.45 (s, 1H), 5.25 (s, 3H), 4.70 (m, 2H), 4.3
(m, 1H), 4.22-3.90 (m, 2H), 3.75 (s, 1H), 3.62 (s, 1H), 3.35-2.90
(m, 2H), 2.45-2.10 (m, 2H), 1.85 (m, 5H), 1.32 (d, 3H).
Example. Tubulysin B Pyridyldisulfide
##STR00100##
[0338] Similarly, Tubulysin B pyridyldisulfide is prepared as
described herein.
[0339] EXAMPLE. EC1663 and EC1664 The following additional
compounds are preparable using the methods and processed described
herein:
##STR00101##
[0340] EXAMPLE. EC1426 is prepared according to the following
process.
##STR00102## ##STR00103##
[0341] EXAMPLE. EC1456 is prepared according to the following
process.
##STR00104##
[0342] EXAMPLE. N.sup.10-TFA Protected EC1454 is prepared according
to the following process.
##STR00105##
[0343] EXAMPLE. EC1454 is prepared according to the following
process.
##STR00106##
[0344] EC1454: MS (ESI, [M+2H].sup.2+)=840.90, [M+H].sup.+=1681.3.
Partial 1H-NMR (DMSO) .delta. (ppm): 8.6 (s), 7.6 (d), 6.6 (d),
4.45 (s), 4.35 (t), 4.15-4.3 (m), 3.3-3.6 (m), 3.25 (m), 3.0 (m),
2.7-2.9 (m), 2-2.3 (m), 1.6-2 (m).
##STR00107##
[0345] EC1415: [M+H].sup.+=1709.69, [M+2H].sup.2+=855.22. Partial
.sup.1H NMR (D.sub.2O, 300 MHz) .delta. (ppm): 8.6 (s, 1H), 7.45
(d, 2H), 6.5 (d, 2H), 4.5 (s, 2H), 4.3-4.1 (m, 6H), 3.95 (t, 1H),
3.8-3.4 (m, 19H), 3.4-2.95 (m, 7H), 2.4-1.7 (m, 26H), 1.6 (m, 1H),
1.25 (s, 2H), 1.05 (s, 3H).
[0346] EXAMPLE. EC1004 is prepared according to the following
process.
##STR00108##
Into a round bottomed flask equipped with magnetic stir bar and
temperature probe dipeptide EC1458, imidazole, and methylene
chloride is added. Once all the solids have dissolved, the solution
is cooled using an ice bath. Chlorotriethylsilane (TESCl) is added
drop wise and the ice bath is removed. The reaction is monitored
for completion. A second portion of chlorotriethylsilane and/or
imidazole is added if necessary. The imidazole HCl salt is removed
by filtration and methylene chloride is added. The organics are
washed with a saturated solution of sodium chloride (brine), the
aqueous layer is back extracted once with methylene chloride, and
the combined organic layers are washed with brine. The organic
layer is dried over sodium sulfate and concentrated on a rotary
evaporator. The residue is dissolved in tetrahydrofuran (THF) and
cooled to approximately -45.degree. C. A solution of potassium
bis(trimethylsilyl)amide (KHMDS) in toluene is added drop wise.
With stirring, chloromethyl butyrate is added and the reaction is
monitored. The reaction is quenched with methanol and then ethyl
acetate and brine are added. The aqueous layer is discarded and the
organics are washed once with brine. The organic layer is
concentrated on a rotary evaporator and the oily residue is passed
through a short plug of silica gel. The plug is washed with a 20%
solution of ethyl acetate in petroleum ether. The combined organics
are concentrated on a rotary evaporator until distillation ceases.
The crude EC1004 oil is analyzed by LC and NMR and stored in a
freezer until use.
[0347] EXAMPLE. EC1005 is prepared according to the following
process.
##STR00109##
[0348] Into an appropriately sized hydrogenation flask place
R--N-methyl pipecolinate (MEP), pentafluorophenol, N-methyl
pyrrolidinone (NMP), and ethyl dimethylaminopropyl carbodiimide
(EDC). The mixture is stirred for at least 16 h. EC1004 dissolved
in N-methyl pyrrolidinone (NMP) and 10 wt % Pd/C are added. The
reaction mixture is stirred/shaken under hydrogen pressure until
the reaction is complete by LC analysis. The Pd/C is removed by
filtration through celite. The celite is washed with ethyl acetate
and the combined organics are washed three times with a 1% sodium
bicarbonate/10% sodium chloride solution. The organic layer is
dried over sodium sulfate and concentrated on a rotary evaporator.
The residue is dissolved in DCM and purified by silica gel
chromatography using ethyl acetate and petroleum ether as eluents.
Fractions are collected, checked for purity, combined and dried on
a rotary evaporator. The EC1005 oil is assayed by LC and stored in
a freezer until use.
[0349] EXAMPLE. EC1008 is prepared according to the following
process.
##STR00110##
[0350] EC1005 is dissolved in 1,2-dichloroethane (DCE) and
trimethyltin hydroxide is added. The reaction mixture is heated and
reaction is monitored by LC. On completion, the mixture is cooled
with an ice bath and filtered. The solids are then washed with DCE.
The organic layer is washed once with water and dried over sodium
sulfate. The solution is concentrated on a rotary evaporator and
the residue dissolved in tetrahydrofuran (THF). Triethylamine
trihydrofluoride is added and the mixture stirred while monitoring
with LC. Pyridine, dimethylaminopyridine (DMAP), and acetic
anhydride are added. The reaction is stirred and monitored by LC.
The reaction mixture is concentrated to a residue and the product
is purified by C18 column chromatography with acetonitrile and
water as eluents. Product fractions are collected, concentrated,
and lyophilized to yield a white to off-white powder.
[0351] EXAMPLE. EC1426 is prepared according to the following
process.
##STR00111##
[0352] EC1422 is dissolved in tetrahydrofuran (THF) and
(Benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate
(PyBop) and diisopropylethylamine (DIPEA) are added. Once all the
solids have dissolved hydrazine is added and the reaction is
stirred and monitored for completion. EC0607 is added and the
mixture stirred and monitored for completion by LC. Ethyl acetate
is added and the organics are washed once with saturated ammonium
chloride, twice with saturated sodium bicarbonate, and once with
saturated sodium chloride. The organics are dried over sodium
sulfate and concentrated on a rotary evaporator. The crude EC1426
is purified by silica column chromatography with dichloromethane
and methanol as eluents. Fractions are collected and the combined
product fractions are concentrated on a rotary evaporator to yield
a yellow solid.
[0353] EXAMPLE. EC1428 is prepared according to the following
process.
##STR00112##
[0354] EC1008 is dissolved in dichloromethane and pentafluorophenol
dissolved in DCM along with N-cyclohexylcarbodiimide,N'-methyl
polystyrene (DCC-resin) are added. The mixture is stirred and
reaction completion is monitored by LC. The mixture is filtered to
remove the resin and the organic layer is concentrated on a rotary
evaporator to yield activated EC1008. In a separate flask, EC1426
is dissolved in dichloromethane and trifluoroacetic acid is added.
The reaction mixture is stirred and monitored for completion by LC.
The reaction mixture is concentrated on a rotary evaporator to
yield deprotected EC1426. The activated EC1008 is dissolved in DMF
and diisopropylethylamine (DIPEA) is added. The deprotected EC1426
is dissolved in DMF and added to the reaction mixture. The reaction
is stirred and monitored for completion by LC. Ethyl acetate is
added and the organics are washed three times with saturated
aqueous sodium chloride. The organic layer is dried over sodium
sulfate and the volatiles removed by rotary evaporation. The crude
EC1428 is purified by silica column chromatography using
dichloromethane and methanol as eluents. Fractions are collected,
checked for purity, and the combined product fractions are
concentrated by rotary evaporation to yield a yellow solid. The
EC1428 is stored in a freezer.
[0355] EXAMPLE. Additional tubulsyins and tubulysin intermediates
may be prepared according to the processes described in WO
2012/019123, WO 2009/055562, PCT International Application Serial
No. US2013/034672, and U.S. Provisional application Ser. No.
61/793,082, the disclosures of each of which are incorporated
herein by reference in their entirety.
[0356] EXAMPLE. Illustrative tubulysins are as follows:
##STR00113##
TABLE-US-00002 Compound 100a 100b 100c Tub B R allyl n-butyl
n-pentyl IC50 1.2 0.7 0.8 1.2 on FR + KB cell (nM)
[0357] EXAMPLE. EC1454 is prepared according to the following
process.
##STR00114##
[0358] The solid phase synthesis of N.sup.10-TFA protected EC1454
starts with resin bound trityl protected D-cysteine. The resin is
suspended in dimethylformamide (DMF) and washed twice with DMF.
EC0475 (glucamine modified L-glutamic acid),
(Benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate
(PyBOP), and diisopropylethylamine (DIPEA) are added to reaction
mixture. After at least 1 hour, a Kaiser test is performed to
ensure the coupling is complete. The resin is washed three times
with DMF, three times with IPA, and three times with DMF. The resin
is slowly washed three times with piperidine in DMF, three times
with DMF, and three times with IPA. A Kaiser test is performed to
confirm deprotection. The resin is washed three times with DMF and
the next amino acid in the sequence is coupled following the same
process. Monomers are coupled in the following order: 1) EC0475, 2)
Fmoc-D-Glu(OtBu)--OH, 3) EC0475, 4) Fmoc-D-Glu(OtBu)--OH, 5)
EC0475, 6) Fmoc-D-Glu-OtBu, and 7) N.sup.10-TFA-Pte-OH.
[0359] Once the final coupling is complete, the resin is washed
three times with methanol and dried by passing argon through the
resin at room temperature. The dried resin is suspended in a
mixture of TFA, water, ethanedithiol, and triisopropylsilane. After
1 hour the resin is removed by filtration and washed with TFA. The
product is precipitated by addition to cold ethyl ether, filtered,
and washed with ether. The solids are dried under vacuum at room
temperature and stored in a freezer.
##STR00115##
[0360] N.sup.10-TFA EC1454 is dissolved in argon sparged water.
Sodium carbonate (1M in water, argon sparged) is added to achieve a
pH of 9.4-10.1. The reaction mixture is stirred for at least 20
minutes. Once the reaction is complete as determined by LC, it is
quenched by adjusting the pH to 1.9-2.3 with 2M HCl. The product is
purified by C18 column chromatography using acetonitrile and pH 5
ammonium acetate buffer as eluents. Fractions are collected and
checked for purity by HPLC. The combined product fractions are
concentrated on a rotary evaporator and then lyophilized to yield
EC1454 as a yellow solid. MS (ESI, [M+2H].sup.2+)=840.90,
[M+H1].sup.+=1681.3. Selected 1H-NMR (DMSO, 300 MHz) .delta. (ppm):
8.6 (s), 7.6 (d), 6.6 (d), 4.45 (s), 4.35 (t), 4.15-4.3 (m),
3.3-3.6 (m), 3.25 (m), 3.0 (m), 2.7-2.9 (m), 2-2.3 (m), 1.6-2 (m).
The product is stored at -20.degree. C.
[0361] EXAMPLE. EC1456 is prepared according to the following
process.
##STR00116##
[0362] EC1428 is dissolved in acetonitrile and a solution of EC1454
in pH 7.4 Sodium phosphate buffer is added. The solutions are
sparged with argon before and after addition. The reaction mixture
is stirred for at least 15 minutes and then checked for completion.
The desired product is purified by C18 column chromatography using
acetonitrile and pH 7.4 phosphate buffer as eluents. The product
fractions are collected, checked for purity, combined and
concentrated by ultra-filtration to yield an aqueous solution that
is 10-20 mg/mL EC1456. The final product solution is sampled for
assay and then stored in a freezer.
[0363] The positive electrospray mass spectrum of EC1456 was
obtained on a high resolution Waters Acquity UPLC Xevo Gs-S QTOF
mass spectrometer. The spectrum was obtained following separation
of the major component on a UPLC inlet system, the resolving power
was approximately 35,000. The accurate mass measurement of the M+H
monoisotopic peak was 2625.0598, which is 1.1 ppm error difference
from the theoretical value of 2625.0570 for an ion of formula
C.sub.110H.sub.166N.sub.23O.sub.45S.sub.3. The isotopic
distribution is also consistent with that formula.
Mass spectral features of the ES+ spectrum for EC1456
TABLE-US-00003 Observed Ion Interpretation 2626.06 .sup.13C isotope
of the (M + H).sup.+ ion for the MW 2624 drug substance 1313.54
.sup.13C isotope of the (M + 2H).sup.++ ion for the MW 2624 drug
substance 1150.43 .sup.13C isotope of the (M + 2H - 326).sup.++
fragment, corresponding to the cleavage of the peptide bond at the
tertiary nitrogen and the loss of the butyric acid moiety. 876.03
.sup.13C isotope of the (M + 3H).sup.+++ ion for the MW 2624 drug
substance 657.27 .sup.13C isotope of the (M + 4H).sup.++++ ion for
the MW 2624 drug substance
[0364] A sample of .about.30 mg EC1456 was dissolved in 665 .mu.L
of a 9:1 mixture of deuterated dimethylsulfoxide and deuterated
water. The .sup.1H NMR spectrum was obtained at 500 MHz at 26 deg.
C. on an Agilent model DD2 spectrometer fitted with a 2 channel
probe containing both broadband and proton observe coils. The
.sup.13C NMR spectrum was obtained at 125 MHz on the same
instrument under identical conditions. All spectra were referenced
to the DMSO solvent residual signals at 2.5 ppm (.sup.1H) and 39.50
ppm (.sup.13C).
[0365] All spectral features are assigned for both NMR spectra in
the tables below (.sup.1H and .sup.13C) using the atom numbering in
the following figure, where the * symbols indicate the connection
for the disulfide bond.
##STR00117##
Assignments were made on the basis of both 1D and 2D NMR
experiments, including through bond H--H connectivity using the
COSY and TCSY 2D experiments, through space H--H proximity using 2D
NOESY, carbon multiplicity measurement using the 1D DEPT experiment
and through bond C--H connectivity using the proton detected 2D
experiments HSQC and HMBC. In most cases of overlap in the 1D
spectra (different protons or carbons resonating at the same
chemical shift) could be resolved in the 2D spectra, in these cases
the tables reflect the chemical shifts measured from the 2D spectra
but summed integrations for the group of co-resonating species. In
some cases of 1D overlap (such as the nearly identical glutamic
acid and glucamine subunits) there was also overlap in the 2D
correlation spectra which precludes unambiguous assignment of
single or multiple resonances between multiple atom numbers, in
these cases there are multiple entries for chemical shift and/or
atom number assignments in a single table row.
[0366] NH and OH protons were exchanged by the D.sub.2O deuterium
atoms and are mostly absent from the spectrum, except weak broad
peaks in the 5-10 ppm region. The .sup.1H peaks in the spectrum
that are not listed in the table include a broad HOD peak at 3.75
ppm, and a DMSO peak at 2.50 ppm. The HOD peak does not obscure any
resonances, but elevates the integrations for nearby resonances at
4.2 and 3.4-3.7 ppm due to the broad baseline rise. The DMSO peak
obscures the resonance for H129, which is not integrated for this
reason. The .sup.13C peaks in spectrum not listed in the table
include the very large DMSO solvent at 39.50 ppm. The DMSO peak
obscures both the signals from C91 and C93. The C116 peak is not
observable in the .sup.13C spectrum due to extensive broadening due
to conformational changes around the nearby amide group. All three
chemical shifts (C91, C93, C116) are visible in and measured in the
proton detected 2D correlation spectra.
Proton NMR Assignments for EC1456
TABLE-US-00004 [0367] Proton Chemical Shift (ppm) Assignment #
protons 8.61 5 1 8.16 103 1 7.58 15, 17 2 6.96 95, 99 2 6.62 14, 18
4 6.59 96, 98 6.18 116 Ha 1 5.7 107 1 5.24 116 Hb 1 4.47 11 2 4.39
111, 122 2 4.21 78 10 4.21 65 4.18 84 4.15 46 4.15 59 4.13 21 4.13
40 4.09 27 4.09 92 3.61 33, 52, 71 3 3.56 34, 53, 72 6 3.54 37Ha,
56Ha, 75Ha 3.46 36, 55, 74 3 3.4 35, 54, 73 6 3.38 37Hb, 56Hb, 75Hb
3.21 80Ha, 32Ha, 51 Ha, 70 Ha 4 3.05 32Hb, 51Hb, 70Hb 3 2.93 80 Hb
3 2.91 83 2.8 133Ha 1 2.68 93 2 2.49 (see text) 129 1 2.35 89 2
2.33 110Ha 2.8 133Hb 37 2.17 118 2.14-2.08 24, 29, 42, 48, 61, 67
2.09 110Hb 2.08 109 2.02 135 1.97-1.70 28, 41, 47, 60, 66 1.92 23Ha
1.88 123 1.8 91Ha 1.79 23Hb 1.77 112 1.6 131Ha 9 1.56 130Ha 1.5
132Ha 1.5 91Hb 1.45 125Ha 1.42 119 1.4 132Hb 1.33 130Hb 1.14 131Hb
2 1.07 125Hb 1 90 3 0.94 114 3 0.79 124 3 0.77 126 3 0.75 120 3
0.64 113 3
Carbon NMR Assignments for EC1456
TABLE-US-00005 [0368] Carbon Chemical shift (ppm) Assignment
176.77, 176.32 43, 62 175.74 88 175.42 22 174.75 121 173.87,
172.68, 25, 38, 44, 57, 63 172.15, 171.94, 171.84 173.43 79 173.3
128 172.79 (2x), 30, 49, 68 172.72 172.46 117 170.87 76 170.39 108
169.3 105 166.09 19 162.4 9 160.7 101 156.4 85 156.09 3 155.71 97
154.59 1 150.84 13 149.63 102 149.11 6 148.99 5 130.44 95, 99
128.99 15, 17 128.89 94 127.99 8 124.97 103 122.24 16 115.25 96, 98
111.86 14, 18 72.17 (3x) 35, 54, 73 71.78, 71.74, 71.71 33, 52, 71
71.62, 71.59 (2x) 36, 55, 74 69.65, 69.57 (2x) 34, 53, 72 69.45 107
69.34 116 68.51 129 63.42 (3x) 37, 56, 75 63.03 84 55.08 133 54.05
40 53.88 78 53.46 (2x) 46, 59 53.33 27 52.96 (2x) 122, 111 52.89 21
52.55 65 49.77 92 46.07 11 44.02 135 42.85 80 42.34 (2x), 42.29 32,
51, 70 39.52 93 38.95 91 37.43 83 35.95 118 35.43 123 35.38 89
34.86 110 32.56, 32.36, 24, 29, 42, 48, 61, 67 32.16, 32.09 (2x),
31.81 30.5 112 29.95 130 28.60, 28.04, 27.78 28, 41, 47, 60, 66
(2x), 27.66 27 23 25.01 132 24.43 125 23.04 131 20.86 109 20.56 114
19.64 113 18.36 90 18.04 119 15.64 124 13.72 120 10.28 126
[0369] The IR spectrum of EC1456 was acquired on a Nexus 6700.RTM.
Fourier transform infrared (FT-IR) spectrophotometer (Thermo
Nicolet) equipped with an Ever-Glo mid/far IR source, an extended
range potassium bromide (KBr) beam splitter, and a deuterated
triglycine sulfate (DTGS) detector. An attenuated total reflectance
(ATR) accessory (Thunderdome.TM. Thermo Spectra-Tech), with a
germanium (Ge) crystal was used for data acquisition. The spectrum
represents 256 co-added scans collected at a spectral resolution of
4 cm-1. A background data set was acquired with a clean Ge crystal.
A Log 1/R (R=reflectance) spectrum was acquired by taking a ratio
of these two data sets against each other. Wavelength calibration
was performed using polystyrene.
Infrared Band Assignments for EC1456 Reference Substance
TABLE-US-00006 [0370] Characteristic Absorption(s) (cm.sup.-1)
Functional Group 1700-1500 (m, m) Aromatic C.dbd.C Bending
2950-2850 (m or s) Alkyl C--H Stretch ~3030 (v) Aromatic C--H
Stretch 3550-3200 (broad, s) Alcohol/Phenol O--H Stretch 3700-3500
(m) Amide C.dbd.O Stretch
[0371] The ultraviolet spectrum EC1456 acquired on a Perkin-Elmer
Lambda 25 UV/Vis spectrometer. The spectrum was recorded at 40.7 uM
in 0.1M NaOH solvent on a 1 cm path-length cell at 25 deg. C. The
local maxima at 366 nm, 288 nm and 243 nm are due primarily to the
Pteroic acid, benzamide/phenol and thiazole-amide substructures,
respectively, although the molecule contains dozens of chromaphores
with overlapping absorption in the UV region.
[0372] EXAMPLE. N.sup.10-TFA Protected EC1579 is prepared according
to the following process.
##STR00118##
[0373] EXAMPLE. EC1579 is prepared according to the following
process.
##STR00119##
[0374] EC1579 MS (ESI, [M+2H].sup.2+)=840.89 (M+1H)1+=1681.0.
Partial 1H-NMR (D.sub.2O) .delta. (ppm): 8.6 (s), 7.5 (d), 6.65
(d), 4.4-4.8 (m), 4-4.2 (m), 3.4-3.8 (m) 3-3.3 (m) 2.75 (s),
1.6-2.4 (m).
[0375] EXAMPLE. EC0948 is made by the processes described
herein.
##STR00120##
[0376] EC0848: MS (ESI, [M+2H]2+)=840.8. [M+H]+=1681.1. Selected
1H-NMR (DMSO) .delta. (ppm): s, 8.6; d, 7.6; d, 6.6; s, 4.45; m,
4-4.2; m, 3.3-3.8; m, 3.1-3.3; m, 3-3.1; m, 2.7-2.9; m, 1.7-2.3; s,
1.15
##STR00121##
[0377] EXAMPLE. EC1669 is prepared according to the processes
described herein from EC1579 and EC0469 as follows:
##STR00122##
EC1579 (200 mg, 1.0 eq) is dissolved in deoxygenated (bubbling
argon) 20 mM PO.sub.4 (pH=7) buffer (4.0 mL) and added dropwise to
a stirring solution of EC0469 (80 mg, 1.0 eq) in dry
dimethylsulfoxide (4.0 mL) at room temperature with argon bubbling.
After 30 min, EC1669 (132 mg) is purified by preparative HPLC in
0-30% acetonitrile/50 mM NH.sub.4HCO.sub.3 pH7 buffer and
lyophilized (49% yield). Chemical Formula:
C.sub.87H.sub.122N.sub.26O.sub.40S2; Exact Mass: 2234.78; MW
2236.18. MS (ESI, [M+2H].sup.2+) Predicted 1118.39, Found 1119.52.
Partial .sup.1H NMR (DMSO w/10% D.sub.2O) .delta. (ppm) 8.67 (s),
8.59 (2), 7.61 (d), 7.56 (d), 6.71 (d), 6.61 (d), 3.34-3.39
(m)'
[0378] EXAMPLE. The following additional compounds are described
and are prepared according to the general processes described
herein.
##STR00123## ##STR00124##
EC1454 (8.5 mg, 1.5 eq) was dissolved in degassed (Ar bubbling) 20
mM phosphate pH7 buffer (2.0 mL) and added dropwise to a stirring
solution of EC1717 (3.8 mg, 1.0 eq) in dry dimethylsulfoxide (2.0
mL, Aldrich) at room temperature with Ar bubbling. After 30 min,
EC1739 (5.3 mg, 59%) was purified by preparative HPLC in 10-100%
acetonitrile/50 mM NH.sub.4HCO.sub.3 pH7 buffer and lyophilized. MS
(ESI, [M+2H].sup.2+)=1327.06, Found 1327.73
##STR00125##
EC1454 (5.5 mg, 1.0 eq) was dissolved in degassed (Ar bubbling) 20
mM phosphate pH7 buffer (2.0 mL) and added dropwise to a stirring
solution of EC1662 (3.6 mg, 1.0 eq) in dry dimethylsulfoxide (2.0
mL, Aldrich) at room temperature with Ar bubbling. After 30 min,
EC1664 (4.6 mg, 54%) was purified by preparative HPLC in 10-100%
acetonitrile/50 mM NH.sub.4HCO.sub.3 pH7 buffer and lyophilized. MS
(ESI, [M+2H].sup.2+) Predicted 1313.05, Found 1313.37. Partial
.sup.1H NMR (DMSO w/10% D.sub.2O, 300 MHz) .delta. (ppm) 8.61 (s),
8.15 (s), 7.58 (d), 6.94 (d), 6.60 (m), 5.78 (d), 5.22 (d), 4.47
(m), 4.09-4.33 (m), 0.99 (d), 0.93 (d), 0.76 (t), 0.71 (t), 0.61
(d).
##STR00126##
EC1454 (16.1 mg, 1.2 eq) was dissolved in degassed (Ar bubbling) 20
mM phosphate pH7 buffer (2.0 mL) and added dropwise to a stirring
solution of EC1661 (8.7 mg, 1.0 eq) in dry dimethylsulfoxide (2.0
mL, Aldrich) at room temperature with Ar bubbling. After 30 min,
EC1663 (15.8 mg, 76%) was purified by preparative HPLC in 10-100%
acetonitrile/50 mM NH.sub.4HCO.sub.3 pH7 buffer and lyophilized. MS
(ESI, [M+2H].sup.2+) Predicted 1306.04, Found 1306.82.
##STR00127##
EC1415 (20 mg) was dissolved in pH7 phosphate (pH 7.75, purged with
argon). To this solution was added a suspension of EC0312 (14 mg)
in equal volume of MeOH. The reaction mixture was stirred at
ambient temperature under argon for 45 min, and then loaded onto a
preparatory HPLC (Mobile phase A=50 mM NH.sub.4HCO.sub.3 buffer,
pH=7.0. B=ACN. Method: 5-80% B in 20 min.) for purification.
Fractions containing the desired product were collected, combined,
and freeze-dried to afford the product (18 mg) as a pale yellow
solid. MS(ESI, [M+2H].sup.2+) 1328, 1H NMR (DMSO-d6, D.sub.2O, 300
MHz): 8.6 (s, 1H), 8.15 (s, 1H), 7.85 (bd, 1H), 7.55 (d, 2H), 6.95
(d, 2H), 6.6 (m, 4H), 6.2 (d, 1H), 5.68 (d, 1H), 5.2 (d, 1H), 4.5
(bs, 3H), 4.5-4.3 (m, 4H), 4.3-4.0 (m, 10H), 3.5-3.3 (m, 13H), 3.2
(bd, 5H), 3.1-2.8 (m, 8H), 2.75 (bs, 5H), 2.6-1.6 (m, 50H), 1.4 (m,
9H), 1.2 (m, 9H), 1.0 (dd, 9H), 0.7 (m, 11H), 0.6 (d, 3H).
##STR00128##
[0379] A solution of EC0259 (35 mg) in 20 mM pH7 phosphate buffer
(3.0 mL) and a saturated NaHCO.sub.3 solution (1.5 mL) were added
to a solution of EC0312 (39 mg) in MeOH (5.5 mL) in tandem. The
resulting homogeneous solution was stirred at ambient temperature
under argon for 20 min. and then loaded directly onto a preparatory
HPLC (Mobile phase A=50 mM NH.sub.4HCO.sub.3 buffer, pH=7.0. B=ACN.
Method: 5-80% B in 20 min.) for purification. Fractions containing
the desired product were collected, combined, and freeze-dried to
afford the product (25 mg) as a pale yellow solid. MS (ESI,
[M+H].sup.+) 1993.
##STR00129##
A solution of EC0259 (35 mg) in 20 mM pH7 phosphate buffer (3.0 mL)
and a saturated NaHCO.sub.3 solution (1.5 mL) were added to a
solution of EC0312 (39 mg) in MeOH (5.5 mL) in tandem. The
resulting homogeneous solution was stirred at ambient temperature
under argon for 20 min. and then loaded directly onto a preparatory
HPLC (Mobile phase A=50 mM NH.sub.4HCO.sub.3 buffer, pH=7.0. B=ACN.
Method: 5-80% B in 20 min.) for purification. Fractions containing
the desired product were collected, combined, and freeze-dried to
afford the product (25 mg) as a pale yellow solid. MS (ESI,
[M+H].sup.+) 1993.
##STR00130##
A solution of EC1544 (55.1 mg) in 20 mM pH7 phosphate buffer (1.95
mL) and a saturated NaHCO.sub.3 solution (0.30 mL) were added to a
solution of EC1248 (58.0 mg) in MeOH (2.30 mL) in tandem. The
resulting homogeneous solution was stirred at ambient temperature
under argon for 20 min and then loaded directly onto a preparatory
HPLC (Mobile phase A=50 mM NH.sub.4HCO.sub.3 buffer, pH=7.0. B=ACN.
Method: 5-80% B in 20 min.) for purification. Fractions containing
the desired product were collected, combined, and freeze-dried to
afford the product (61.5 mg) as a pale yellow solid. MS (ESI,
[M+H].sup.+) 1993.
##STR00131##
The pH of a solution of EC1392 (20 mg) in 40 mM pH7 phosphate
buffer was adjusted to 8 with a saturated NaHCO.sub.3 solution. To
the solution was added a suspension of EC0312 (20 mg) in equal
volume of MeOH. The reaction mixture was stirred at ambient
temperature under argon for 30 min, and then loaded onto a
preparatory HPLC (Mobile phase A=50 mM NH.sub.4HCO.sub.3 buffer,
pH=7.0. B=ACN. Method: 5-80% B in 20 min.) for purification.
Fractions containing the desired product were collected, combined,
and freeze-dried to afford the product (15 mg) as a pale yellow
solid. MS(ESI, [M+2H].sup.2+) 1011.39. .sup.1H NMR (DMSO-d6,
D.sub.2O, 300 MHz): 8.6 (s, 1H), 8.15 (s, 1H), 7.85 (bd, 1H), 7.55
(d, 2H), 6.95 (d, 2H), 6.6 (m, 4H), 6.2 (d, 1H), 5.68 (d, 1H), 5.2
(d, 1H), 4.6 (t, 1H), 4.5 (m, 3H), 4.5-4.0 (m, 11H), 3.2-2.8 (m,
6H), 2.8-2.5 (m, 8H), 2.4 (m, 5H), 2.2-2.0 (m, 14H), 2.0-1.7 (m,
7H), 1.6-1.3 (m, 13H), 1.25 (d, 8H), 1.1-0.95 (dd, 8H), 0.75 (m,
10H), 0.6 (d, 2H).
[0380] EXAMPLE. The compounds described herein can also be prepared
by following two methods:
[0381] Method A: Folate spacer is dissolved in water by adjusting
the pH of the solution with NaHCO.sub.3 solution to a pH=7 with
argon purging. The thiophilic agent in organic solvent (MeOH, ACN,
THF or DMSO) is then added. The reaction mixture is stirred at room
temperature with argon purging. The progress of reaction is
monitored by analytical HPLC (Mobile phase A=50 mM
NH.sub.4HCO.sub.3 buffer, pH=7.0; B=ACN). After the reaction is
complete, the organic solvent is evaporated and the resulted
solution is then purified by prep-HPLC with C18 column (Mobile
phase A=50 mM NH.sub.4HCO.sub.3 buffer or 2 mM phosphate buffer,
pH=7.0; B=ACN).
[0382] Method B: Folate spacer is dissolved in water and the pH is
adjusted to 2 with acid (AcOH or dilute HCl). The resulting pH
adjusted spacer is lyophilized, and then redissolved in DMSO. The
reaction mixture is purged with argon, and 10 molar equivalents of
Et.sub.3N (or DIPEA) are added. To this solution is added the
thiophilic agent in organic solvent (DMSO, THF, ACN, etc.). The
progress of the reaction is monitored by HPLC (Mobile phase A=50 mM
NH.sub.4HCO.sub.3 buffer or 2 mM phosphate buffer, pH=7.0. B=ACN).
After the reaction is complete, the reaction mixture is purified by
prep-HPLC with C18 column (Mobile phase A=50 mM NH.sub.4HCO.sub.3
buffer or 2 mM phosphate buffer, pH=7.0; B=ACN).
[0383] EXAMPLE. Additional illustrative linker intermediates (also
referred as folate spacers) are described herein:
##STR00132##
EC0014: .sup.1H NMR (D.sub.2O, 500 MHz) .delta. (ppm) 8.73 (s, 1H,
FA H-7), 7.56 (d, 2H, FA H-12&H16), 6.73 (d, 2H, FA
H-13&H15), 4.45 (m, 2H), 4.1 (m, 2H), 3.61 (d, 2H), 2.82 (m,
3H), 2.74 (dd, 1H), 2.37 (m, 2H), 2.18 (m, 1H), 2.09 (m, 3H), 1.74
(m, 1H)
Example
##STR00133##
[0384] EC0020: MS (ESI, [M+H].sup.+) 746. .sup.1H NMR (D.sub.2O,
500 MHz) .delta. (ppm) 8.76 (s, 1H, FA H-7), 7.68 (d, 2H, FA
H-12&H16), 6.8 (d, 2H, FA H-13&H15), 4.71 (dd, 1H, Asp
H-2), 4.64 (s, 2H FA H-9), 4.41 (dd, 1H, D-Glu H-2), 4.3 (dd, 1H,
Cys H-2), 4.1 (dd, Dpr H-2), 3.72 (dd, 1H, Dpr H-3A), 3.52 (dd, 1H,
Dpr H-3B), 2.89 (dd, 1H, Cys H-3A), 2.85 (dd, 1H, Cys H-3B), 2.81
(dd, 1H, Asp H-3A), 2.62 (dd, 1H, Asp H-3B), 2.44 (dd, 2H, D-Glu
H-4), 2.27 (m, 1H, D-Glu H-3A), 2.08 (m, 1H, D-Glu H-3B). .sup.13C
NMR (DMSO-d6+D.sub.2O, 75 MHz): .quadrature. 174.78, 174.42, 172.68
(2C), 170.45, 168.25, 167.08, 162.24, 156.24, 154.38, 151.24,
149.41 (2C), 129.52, 128.14, 121.74, 111.98, 55.76, 53.02 (2C),
52.77, 50.89, 46.16, 36.61, 32.26, 27.32, 26.60
EXAMPLES
##STR00134## ##STR00135## ##STR00136##
[0385] EC0149: [M+H].sup.+=631. .sup.1H NMR (D.sub.2O): 8.55 (s,
1H), 7.5 (d, 2H), 6.61 (d, 2H), 4.42 (s, 2H), 4.35 (dd, 1H), 4.25
(m, 2H), 4.1 (s, 1H), 3.68 (m, 1H), 3.5 (m, 1H), 3.35-3.2 (m, 3H),
3.1 (dd, 1H), 2.4-2.1 (m, 3H), 2.1-1.9 (m, 4H).
##STR00137##
EC0150: MS (ESI, [M+H].sup.+) 631. Selected .sup.1H NMR (D.sub.2O)
.delta. (ppm) 8.42 (s, 1H, FA H-7), 7.50 (d, 2H, FA H-12&16),
6.65 (d, 2H, FA H-13&15), 4.42 (s, 2H), 4.3-4.1 (m, 2H),
4.0-3.85 (m, 1H), 3.35-3.30 (m, 1H), 3.30-3.10 (m, 2H), 3.10-2.90
(m, 2H), 2.80-2.70 (m, 1H), 2.65-2.50 (m, 2H), 2.30-2.10 (m, 3H),
2.10-1.85 (m, 2H), 1.95-1.80 (m, 2H).
##STR00138##
EC0151: MS (ESI, [M+H].sup.+) 630. Selected .sup.1H NMR (D.sub.2O)
.delta. (ppm) 8.42 (s, 1H, FA H-7), 7.50 (d, 2H, FA H-12&16),
6.65 (d, 2H, FA H-13&15), 4.42 (s, 2H), 4.3-4.1 (m, 2H),
4.0-3.85 (m, 1H), 3.35-3.30 (m, 1H), 3.30-3.10 (m, 2H), 3.10-2.90
(m, 2H), 2.80-2.70 (m, 1H), 2.65-2.50 (m, 2H), 2.30-2.10 (m, 3H),
2.10-1.85 (m, 2H), 1.95-1.80 (m, 2H).
##STR00139##
EC0232: MS (ESI, [M+H].sup.+) 774. .sup.1H NMR (D.sub.2O): 8.56
(s), 7.50 (d), 6.65 (d), 4.48-4.41 (m), 4.21 (dd), 4.08 (dd),
3.48-3.42 (m), 3.28-3.09 (m), 2.61-2.35 (m), 2.28-2.18 (m),
2.16-2.02 (m), 1.97-1.62 (m).
##STR00140##
EC0252: [M+H].sup.+=1046.83. 1H NMR (D.sub.2O): 8.58 (s, 1H), 7.5
(d, 2H), 6.6 (d, 2H), 3.05-2.6 (m, 5H), 2.3-1.9 (m, 4H), 1.8-1.2
(m, 7H).
##STR00141##
EC0259: [M+H].sup.+=1047.52. 1H NMR (D.sub.2O): 8.6 (s, 1H), 7.5
(d, 2H), 6.65 (d, 2H), 4.4 (dd, 2H), 4.18 (m, 4H), 2.9 (t, 2H),
2.75 (t, 2H), 2.6-2.15 (m, 10H), 2.1-1.8 (m, 3H), 1.7-1.4 (m, 3H),
1.3 (m, 3H).
##STR00142##
EC1213: LCMS (ESI [M+H].sup.+): 1046. Selected .sup.1H NMR data
(D.sub.2O, 300 MHz): .delta. 8.68 (s, 1H, FA H-7), 7.57 (d, 2H,
J=8.4 Hz, FA H-12 &16), 6.67 (d, 2H, J=9 Hz, FA H-13
&15).
##STR00143##
EC1214: LCMS (ESI [M+H].sup.+): 1046. Selected .sup.1H NMR data
(D.sub.2O, 300 MHz): .delta. 8.68 (s, 1H, FA H-7), 7.57 (d, 2H,
J=8.4 Hz, FA H-12 &16), 6.67 (d, 2H, J=9 Hz, FA H-13
&15).
##STR00144##
EC1215: LCMS (ESI [M+H].sup.+): 1046. Selected .sup.1H NMR data
(D.sub.2O, 300 MHz): .delta. 8.68 (s, 1H, FA H-7), 7.57 (d, 2H,
J=8.4 Hz, FA H-12 &16), 6.67 (d, 2H, J=9 Hz, FA H-13
&15).
##STR00145## [0386] EC1216: LCMS (ESI [M+H].sup.+): 1046 Selected
.sup.1H NMR data (D.sub.2O, 300 MHz): .delta. 8.68 (s, 1H, FA H-7),
7.57 (d, 2H, J=8.4 Hz, FA H-12 &16), 6.67 (d, 2H, J=9 Hz, FA
H-13 &15).
##STR00146##
[0386] EC1217: LCMS (ESI [M+H].sup.+): 1046. Selected .sup.1H NMR
data (D.sub.2O, 300 MHz): .delta. 8.68 (s, 1H, FA H-7), 7.57 (d,
2H, J=8.4 Hz, FA H-12 &16), 6.67 (d, 2H, J=9 Hz, FA H-13
&15).
##STR00147##
EC1392: [M+H].sup.+=1074.85. 1H NMR (D.sub.2O, 300 MHz) .delta.
(ppm): 8.55 (s, 1H), 7.45 (d, 2H), 6.5 (d, 2H), 4.6 (m, 2H), 4.45
(t, 1H), 4.35 (bs, 2H), 4.2 (m, 1H), 4.1 (s, 1H), 4.05 (m, 1H), 2.9
(t, 2H), 2.75-2.4 (m, 6H), 2.3 (m, 2H), 2.2-1.9 (m, 2H), 1.8-1.4
(m, 2H), 1.2 (m, 2H), 1.3 (s, 3H), 1.2 (s, 3H).
##STR00148##
EC1347: MS (ESI, [M+H].sup.+)=701.57. Selected 1H-NMR (DMSO, 300
MHz) .delta. (ppm): 8.65 (s), 7.6 (d), 6.6 (d), 4.2-4.6 (m),
2.6-3.2 (m), 1.8-2.6 (m), 1.1-1.7 (m)
##STR00149##
EC0589: MS (ESI, [M+H].sup.+) 746. Selected .sup.1H NMR
(DMSO-d6+D.sub.2O, 300 MHz): .quadrature. 8.46 (s, 1H), 7.45 (d,
J=8.4 Hz, 2H), 6.47 (d, J=8.4 Hz, 2H), 4.39 (t, J=6.6 Hz, 1H).
##STR00150##
EC0819: MS (ESI, [M+H].sup.+)=1046.4. Selected 1H-NMR (DMSO)
.delta. (ppm): 8.6 (s), 7.6 (d), 6.6 (d), 4-4.6 (m), 3.4-3.8 (m),
3-3.15 (m), 1-2.8 (m).
##STR00151##
EC0823: MS (ESI, [M+H].sup.+)=672.3. Selected 1H-NMR (DMSO) .delta.
(ppm): 8.8 (s), 7.6 (d), 6.6 (d), 4.4-4.6 (m), 4.2-4.4 (m), 3.4-3.8
(m), 1.8-2.8 (m), 1.15 (s)
##STR00152##
EC0835: MS (ESI, [M+H]+)=1046.5. Selected 1H-NMR (DMSO) .delta.
(ppm): 8.6 (s), 7.5 (d), 6.6 (d), 3.8-4.6 (m), 2.8-3.2 (m), 2.2-2.8
(m), 1-2.2 (m)
##STR00153##
EC0923: MS (ESI, [M+H].sup.+)=672.3. Selected 1H-NMR (D.sub.2O)
.delta. (ppm): 8.8 (s), 7.75 (d), 6.85 (d), 4.4-5 (m), 2.6-2.9 (m),
2.4-2.6 (m), 2-2.6 (m)
##STR00154##
EC0879: MS (ESI, [M+H].sup.+)=442.3. Selected 1H-NMR (DMSO) .delta.
(ppm): 8.7 (s), 7.6 (d), 6.6 (d), 4.55 (s), 4.3 (m), 2.2-2.6 (m),
1.8-2.2 (m), 1-1.2 (m)
##STR00155##
EC0306: [M+H].sup.+=1100.51. 1H NMR (D.sub.2O): .delta. 8.75 (s,
1H), 7.6 (d, 2H), 6.75 (d, 2H), 4.7-4.5 (m, 5H), 4.38 (m, 2H), 4.2
(m, 2H), 4.1 (d, 1H), 3.85-3.5 (m, 10H), 2.95-2.6 (m, 4H), 2.45 (m,
2H), 2.3-2.0 (m, 2H).
##STR00156##
EC0368: [M+H]+=2175.5. 1H NMR (D.sub.2O): 8.6 (s, 1H), 7.5 (d, 2H),
6.6 (d, 2H), 4.45 (bs, 3H), 4.35-4.2 (m, 4H), 4.05 (t, 1H),
3.6-3.35 (bs, 114H), 3.2 (s, 6H), 2.77 (t, 2H), 2.65 (dd, 1H),
2.55-2.45 (m, 3H), 2.4-2.2 (m, 6H), 2.1-1.8 (m, 2H).
##STR00157##
[0387] EC0373: [M+H].sup.+=1346.0. 1H NMR (D.sub.2O): 8.55 (s, 1H),
7.5 (d, 2H), 6.6 (d, 2H), 4.4 (s, 2H), 4.25 (m, 2H), 4.05 (t, 1H),
3.7 (dd, 1H), 3.6-3.3 (m, 50H), 3.25 (dd, 3H), 3.05 (dd, 3H), 2.8
(t, 2H), 2.7 (dd, 2H), 2.6 (dd, 1H), 2.4 (t, 2H), 2.2-1.9 (m,
4H).
##STR00158##
EC0536: [M+2H].sup.2+=941.2. 1H NMR (D.sub.2O): 8.55 (s, 1H), 7.5
(d, 2H), 6.6 (d, 2H), 4.4 (s, 2H), 4.25 (m, 2H), 4.1 (m, 5H), 3.85
(t, 1H), 3.8-3.4 (m, 21H), 3.4-2.95 (m, 7H), 2.8 (s, 2H), 2.7-2.4
(ddd, 2H), 2.4-1.7 (m, 22H), 1.55 (m, 1H).
[0388] EXAMPLE. Additional illustrative compounds and processes for
preparing the compounds are described herein:
Conjugates of EC1579
##STR00159## ##STR00160##
[0389] A solution of EC1579 (acidified, 13.0 mg, 0.0077 mmole) in
DMSO (0.4 mL) and 12 .mu.L of DIPEA (0.070 mmole, 13.5 eq.) were
added to a solution of EC1822 (5.6 mg, 0.0052 mmole) in DMSO (0.2
mL) in tandem. The resulting homogeneous solution was stirred at
ambient temperature under argon for 20 min. and then loaded
directly onto a preparatory HPLC (Mobile phase A=50 mM
NH.sub.4HCO.sub.3 buffer, pH=7.0. B=ACN. Method: 5-80% B in 20
min.) for purification. Fractions containing the desired product
were collected, combined, and freeze-dried to afford the product
(12.4 mg) as a pale yellow solid. Selected .sup.1H NMR (DMSO-d6)
.delta. (ppm) 8.62 (s, 1H), 8.20 (s, 1H), 7.60 (d, 2H), 7.56 (d),
6.93 (d, 2H), 6.61 (m, 3H), 5.25 (d, 1H), 4.51 (d, 1H), 4.50-4.40
(m, 3H), 4.32-4.10 (m, 10H), 3.65-3.50 (m, 10H), 3.40-3.30 (m,
10H), 3.30-3.10 (m, 7H), 3.10-2.95 (m, 3H), 2.95-2.80 (m, 3H),
2.75-2.60 (br, 3H), 2.40-2.00 (m, 14H), 2.0-1.3 (m, 24H), 1.30-1.05
(m, 6H), 0.99 (d, 3H), 0.88 (d, 3H), 0.86 (d, 3H), 0.79 (t, 6H),
0.73 (t, 3H), 0.64 (br, 3H)
##STR00161## ##STR00162##
Example. Synthesis of EC1746
##STR00163##
[0390] A solution of EC1579 (30.9 mg) in 20 mM pH7 phosphate buffer
(4.2 mL) and a saturated NaHCO.sub.3 solution (0.30 mL) were added
to a solution of EC1662 (16.9 mg) in MeOH (4.8 mL) in tandem. The
resulting homogeneous solution was stirred at ambient temperature
under argon for 20 min. and then loaded directly onto a preparatory
HPLC (Mobile phase A=50 mM NH.sub.4HCO.sub.3 buffer, pH=7.0. B=ACN.
Method: 5-80% B in 20 min.) for purification. Fractions containing
the desired product were collected, combined, and freeze-dried to
give the product (33.1 mg) as a fluffy yellow solid. MS (ESI,
M+H)=2627. EC1746 .sup.1H NMR (D.sub.2O): 8.66 (s), 8.10 (s), 7.62
(b), 6.99 (b), 6.69 (b), 5.81 (b), 5.18 (b), 4.60-4.18 (m),
3.91-0.57 (m).
Example. Synthesis of EC1669
##STR00164##
[0391] EC1579 (200 mg, 1.0 eq) was dissolved in degassed (Ar
bubbling) 20 mM PO.sub.4 pH7 buffer (4.0 mL) and added dropwise to
a stirring solution of crude EC0469 (80 mg, 1.0 eq) in dry
dimethylsulfoxide (4.0 mL, Aldrich) at room temperature with Ar
bubbling. After 30 min, EC1669 (132 mg, 49%) was purified by
preparative HPLC in 0-30% acetonitrile/50 mM NH.sub.4HCO.sub.3 pH7
buffer and lyophilized. MS (ESI, [M+H].sup.2+) Predicted 1118.39,
Found 1119.52. Partial .sup.1H NMR (DMSO w/10% D.sub.2O) d (ppm)
8.67 (s), 8.59 (2), 7.61 (d), 7.56 (d), 6.71 (d), 6.61 (d),
3.34-3.39 (m).
Example. Synthesis of EC1665
##STR00165##
[0392] EC1579 (15 mg, 1.0 eq) was dissolved in degassed (Ar
bubbling) 20 mM PO.sub.4 pH7 buffer (2.0 mL) and added dropwise to
a stirring solution of EC0564 (10.5 mg, 1.0 eq) in dry
dimethylsulfoxide (4.0 mL, Aldrich) at room temperature with Ar
bubbling. After 30 min, EC1665 (13.4 mg, 55%) was purified by
preparative HPLC in 10-100% acetonitrile/10 mM NH.sub.4OAc pH5
buffer and lyophilized. MS (ESI, [M+2H].sup.2+) Predicted 1368.09,
Found 1368.30
Conjugates of EC1454
Example. Synthesis of EC1751
##STR00166##
[0393] EC1454 (21.1 mg, 1.3 eq) was dissolved in degassed (Ar
bubbling) 20 mM PO.sub.4 pH7 buffer (2.0 mL) and added dropwise to
a stirring solution of EC1716 (10.8 mg, 1.0 eq) in dry
dimethylsulfoxide (2.0 mL, Aldrich) at room temperature with Ar
bubbling. After 30 min, EC1751 (8.5 mg, 33%) was purified by
preparative HPLC in 10-100% acetonitrile/50 mM NH.sub.4HCO.sub.3
pH7 buffer and lyophilized. MS (ESI, [M+2H].sup.2+) Predicted
1320.05, Found 1320.72. Selected .sup.1H NMR (DMSO w/10% D.sub.2O)
.delta. (ppm) 8.61 (s), 8.15 (s), 7.58 (d), 6.94 (d), 6.60 (m),
5.79 (d), 5.22 (d), 4.47 (m), 4.09-4.33 (m), 0.98 (d), 0.93 (d),
0.75 (m), 0.61 (d)
Example. Synthesis of EC1750
##STR00167##
[0394] EC1454 (31.1 mg, 1.3 eq) was dissolved in degassed (Ar
bubbling) 20 mM PO.sub.4 pH7 buffer (2.0 mL) and added dropwise to
a stirring solution of EC1715 (15.3 mg, 1.0 eq) in dry
dimethylsulfoxide (2.0 mL, Aldrich) at room temperature with Ar
bubbling. After 30 min, EC1750 (18.0 mg, 97%) was purified by
preparative HPLC in 10-100% acetonitrile/50 mM NH.sub.4HCO.sub.3
pH7 buffer and lyophilized. MS (ESI, [M+2H].sup.2+) Predicted
1299.03, Found 1299.19. Partial .sup.1H NMR (DMSO w/10% D.sub.2O)
.delta. (ppm) 8.61 (s), 8.14 (s), 7.57 (d), 6.93 (d), 6.60 (m),
5.77 (d), 5.23 (d), 4.47 (m), 0.98 (d), 0.92 (d), 0.76 (m), 0.71
(t), 0.61 (d)
Example. Synthesis of EC1739
##STR00168##
[0395] EC1454 (8.5 mg, 1.5 eq) was dissolved in degassed (Ar
bubbling) 20 mM PO.sub.4 pH7 buffer (2.0 mL) and added dropwise to
a stirring solution of EC1717 (3.8 mg, 1.0 eq) in dry
dimethylsulfoxide (2.0 mL, Aldrich) at room temperature with Ar
bubbling. After 30 min, EC1739 (5.3 mg, 59%) was purified by
preparative HPLC in 10-100% acetonitrile/50 mM NH.sub.4HCO.sub.3
pH7 buffer and lyophilized. MS (ESI, [M+H].sup.2+) predicted
1327.06, Found 1327.73
##STR00169##
[0396] MS (ESI, [M+H].sup.2+) Predicted 1313.05, Found 1313.37.
Selected .sup.1H NMR (DMSO w/10% D.sub.2O) .delta. (ppm) 8.61 (s),
8.15 (s), 7.58 (d), 6.94 (d), 6.60 (m), 5.78 (d), 5.22 (d), 4.47
(m), 4.09-4.33 (m), 0.99 (d), 0.93 (d), 0.76 (t), 0.71 (t), 0.61
(d)
Example. Synthesis of EC1664
[0397] EC1454 (5.5 mg, 1.0 eq) was dissolved in degassed (Ar
bubbling) 20 mM PO.sub.4 pH7 buffer (2.0 mL) and added dropwise to
a stirring solution of EC1662 (3.6 mg, 1.0 eq) in dry
dimethylsulfoxide (2.0 mL, Aldrich) at room temperature with Ar
bubbling. After 30 min, EC1664 (4.6 mg, 54%) was purified by
preparative HPLC in 10-100% acetonitrile/50 mM NH.sub.4HCO.sub.3
pH7 buffer and lyophilized.
Example. Synthesis of EC1663
##STR00170##
[0398] EC1454 (16.1 mg, 1.2 eq) was dissolved in degassed (Ar
bubbling) 20 mM PO.sub.4 pH7 buffer (2.0 mL) and added dropwise to
a stirring solution of EC1661 (8.7 mg, 1.0 eq) in dry
dimethylsulfoxide (2.0 mL, Aldrich) at room temperature with Ar
bubbling. After 30 min, EC1663 (15.8 mg, 76%) was purified by
preparative HPLC in 10-100% acetonitrile/50 mM NH.sub.4HCO.sub.3
pH7 buffer and lyophilized. MS (ESI, [M+2H].sup.2+) Predicted
1306.04, Found 1306.82
Example. Synthesis of EC1653
##STR00171##
[0399] EC1454 (8.3 mg, 1.0 eq) was dissolved in degassed (Ar
bubbling) 20 mM PO.sub.4 pH7 buffer (2.0 mL) and added dropwise to
a stirring solution of EC0564 (5.8 mg, 1.0 eq) in dry
dimethylsulfoxide (4.0 mL, Aldrich) at room temperature with Ar
bubbling. After 30 min, EC1653 (6.3 mg, 46%) was purified by
preparative HPLC in 10-100% acetonitrile/10 mM NH.sub.4OAc pH5
buffer and lyophilized. MS (ESI, ((M-2)/2)) Predicted 1368.09,
Found 1368.74
##STR00172##
Example. Synthesis of EC1496
##STR00173##
[0400] EC1454 (324 mg, 1.0 eq) was dissolved in degassed (Ar
bubbling) 20 mM PO.sub.4 pH7 buffer (4.0 mL) and added dropwise to
a stirring solution of crude EC0469 (142 mg, 1.1 eq) in dry
dimethylsulfoxide (4.0 mL, Aldrich) at room temperature with Ar
bubbling. After 30 min, EC1496 (221 mg, 51%) was purified by
preparative HPLC in 0-30% acetonitrile/50 mM NH.sub.4HCO.sub.3 pH7
buffer and lyophilized. MS (ESI, ((M+2)/2)) Predicted 1118.39,
Found 1119.02
##STR00174##
Examples. Conjugates of EC1415
##STR00175##
[0401] Example. Synthesis of EC1416
##STR00176##
[0402] EC1415 (20 mg) was dissolved in pH7 phosphate (pH 7.75,
purged with argon). To this solution was added a suspension of
EC0312 (14 mg) in equal volume of MeOH. The reaction mixture was
stirred at ambient temperature under argon for 45 min, and then
loaded onto a preparatory HPLC (Mobile phase A=50 mM
NH.sub.4HCO.sub.3 buffer, pH=7.0. B=ACN. Method: 5-80% B in 20
min.) for purification. Fractions containing the desired product
were collected, combined, and freeze-dried to afford the product
(18 mg) as a pale yellow solid. MS(ESI, [M+2H].sup.2+) 1328. 1H NMR
(DMSO-d6, D.sub.2O, 300 MHz): 8.6 (s, 1H), 8.15 (s, 1H), 7.85 (bd,
1H), 7.55 (d, 2H), 6.95 (d, 2H), 6.6 (m, 4H), 6.2 (d, 1H), 5.68 (d,
1H), 5.2 (d, 1H), 4.5 (bs, 3H), 4.5-4.3 (m, 4H), 4.3-4.0 (m, 10H),
3.5-3.3 (m, 13H), 3.2 (bd, 5H), 3.1-2.8 (m, 8H), 2.75 (bs, 5H),
2.6-1.6 (m, 50H), 1.4 (m, 9H), 1.2 (m, 9H), 1.0 (dd, 9H), 0.7 (m,
11H), 0.6 (d, 3H).
Examples. Conjugates of EC1392
##STR00177##
[0403] Example. Conjugates of EC59
[0404] A solution of EC59 (13.2 mg) in 20 mM pH7.1 phosphate buffer
(2.4 mL) was added to a solution of EC0312 (14.2 mg) in MeOH (2.4
mL). The resulting homogeneous solution was stirred at ambient
temperature under argon for 20 min. and then loaded directly onto a
preparatory HPLC (Mobile phase A=50 mM NH.sub.4HCO.sub.3 buffer,
pH=7.0. B=ACN. Method: 5-80% B in 20 min.) for purification.
Fractions containing the desired product were collected, combined,
and freeze-dried to give the product (15.3 mg) as a fluffy yellow
solid.
##STR00178##
Examples. Conjugates of EC1347
##STR00179##
[0405] EC1208: LCMS [ESI (M+H).sup.+: 1918]. .sup.1Selected .sup.1H
NMR data for EC145 (D.sub.2O, 300 MHz): .delta. 8.67 (s, 1H, FA
H-7), 7.50 (br s, 1H, VLB H-11'), 7.30-7.40 (br s, 1H, VLB H-14'),
7.35 (d, 2H, J=7.8 Hz, FA H-12 &16), 7.25 (m, 1H, VLB H-13'),
7.05 (br s, 1H, VLB H-12'), 6.51 (d, 2H, J=8.7 Hz, FA H-13
&15), 6.4 (s, 2H, VLB H-14 & 17), 5.65 (m, 1H, VLB H-7),
5.5 (m, 1H, VLB H-6), 4.15 (m, 1H, VLB H-8'), 3.82 (s, 3H, VLB
C.sub.18' --CO.sub.2CH.sub.3), 3.69 (s, 3H, VLB C.sub.16
--OCH.sub.3), 2.8 (s, 3H, VLB N--CH.sub.3), 1.35 (br s, 1H, VLB
H-3'), 1.15 (m, 1H, VLB H-2'), 0.9 (t, 3H, J=7 Hz, VLB H-21'), 0.55
(t, 3H, J=6.9 Hz, VLB H-21) ppm.
##STR00180##
EC1209: LCMS [ESI (M+H).sup.+: 1918]. .sup.1Selected .sup.1H NMR
data for EC145 (D.sub.2O, 300 MHz): .delta. 8.67 (s, 1H, FA H-7),
7.50 (br s, 1H, VLB H-11'), 7.30-7.40 (br s, 1H, VLB H-14'), 7.35
(d, 2H, J=7.8 Hz, FA H-12 &16), 7.25 (m, 1H, VLB H-13'), 7.05
(br s, 1H, VLB H-12'), 6.51 (d, 2H, J=8.7 Hz, FA H-13 &15), 6.4
(s, 2H, VLB H-14 & 17), 5.65 (m, 1H, VLB H-7), 5.5 (m, 1H, VLB
H-6), 4.15 (m, 1H, VLB H-8'), 3.82 (s, 3H, VLB C.sub.18'
--CO.sub.2CH.sub.3), 3.69 (s, 3H, VLB C.sub.16 --OCH.sub.3), 2.8
(s, 3H, VLB N--CH.sub.3), 1.35 (br s, 1H, VLB H-3'), 1.15 (m, 1H,
VLB H-2'), 0.9 (t, 3H, J=7 Hz, VLB H-21'), 0.55 (t, 3H, J=6.9 Hz,
VLB H-21) ppm.
##STR00181##
EC1575: A solution of EC1577 (9.5 mg) in 20 mM pH7 phosphate buffer
(2.0 mL) and a saturated NaHCO.sub.3 solution (0.50 mL) were added
to a solution of EC0312 (10.1 mg) in MeOH (2.0 mL) in tandem. The
resulting homogeneous solution was stirred at ambient temperature
under argon for 20 min and then loaded directly onto a preparatory
HPLC (Mobile phase A=50 mM NH.sub.4HCO.sub.3 buffer, pH=7.0. B=ACN.
Method: 5-80% B in 20 min.) for purification. Fractions containing
the desired product were collected, combined, and freeze-dried to
afford the product (9.5 mg) as a pale yellow solid. LCMS [ESI
(M+H).sup.+: 2627]. .sup.1H NMR (D.sub.2O, 300 MHz): 8.70 (s), 8.11
(s), 7.62 (d), 7.00 (d), 6.71 (dd), 6.11 (d), 5.80 (d), 5.33 (d),
4.60-4.50 (m), 4.40-4.15 (m), 3.88-3.51 (m), 3.50-3.20 (m),
3.19-2.80 (m), 2.76 (s), 2.60-1.43 (m), 1.40-1.27 (m), 1.18 (d),
1.02 (d), 0.97-0.82 (m), 0.76-0.63 (m).
##STR00182##
EC1548: A solution of EC1544 (55.1 mg) in 20 mM pH7 phosphate
buffer (1.95 mL) and a saturated NaHCO.sub.3 solution (0.30 mL)
were added to a solution of EC1248 (58.0 mg) in MeOH (2.30 mL) in
tandem. The resulting homogeneous solution was stirred at ambient
temperature under argon for 20 min and then loaded directly onto a
preparatory HPLC (Mobile phase A=50 mM NH.sub.4HCO.sub.3 buffer,
pH=7.0. B=ACN. Method: 5-80% B in 20 min.) for purification.
Fractions containing the desired product were collected, combined,
and freeze-dried to afford the product (61.5 mg) as a pale yellow
solid. MS (ESI, M+1) 1993
##STR00183##
EC1549: A solution of EC1547 (23.5 mg) in 20 mM pH7 phosphate
buffer (2.0 mL) and a saturated NaHCO.sub.3 solution (0.30 mL) were
added to a solution of EC1248 (24.7 mg) in MeOH (2.3 mL) in tandem.
The resulting homogeneous solution was stirred at ambient
temperature under argon for 20 min and then loaded directly onto a
preparatory HPLC (Mobile phase A=50 mM NH.sub.4HCO.sub.3 buffer,
pH=7.0. B=ACN. Method: 5-80% B in 20 min.) for purification.
Fractions containing the desired product were collected, combined,
and freeze-dried to afford the product (29.2 mg) as a pale yellow
solid. MS (ESI, M+1) 1993
Example
##STR00184##
[0406] Example. EC1299
##STR00185##
[0407] A solution of EC0259 (35 mg) in 20 mM pH7 phosphate buffer
(3.0 mL) and a saturated NaHCO.sub.3 solution (1.5 mL) were added
to a solution of EC0312 (39 mg) in ACN (5.5 mL) in tandem. The
resulting homogeneous solution was stirred at ambient temperature
under argon for 20 min. and then loaded directly onto a preparatory
HPLC (Mobile phase A=50 mM NH.sub.4HCO.sub.3 buffer, pH=7.0. B=ACN.
Method: 5-80% B in 20 min.) for purification. Fractions containing
the desired product were collected, combined, and freeze-dried to
afford the product (25 mg) as a pale yellow solid. MS (ESI, M+1)
1993
Example
##STR00186##
[0408] Example
##STR00187##
[0409] Example
##STR00188##
[0410] EC153: MS(ESI, [M+H]) 1023; (ESI, [M-H]) 1021; .sup.1H NMR
(DMSO-d6, 300 MHz): 8.84 (s, 1H), 7.70 (d, 2H), 6.80 (d, 2H), 4.60
(m, 1H), 4.56 (s, 2H), 4.34 (m, 2H), 4.10 (m, 2H), 3.85 (m, 2H),
3.60-3.30 (m, 5H), 3.20 (s, 2H), 318-3.05 (m, 1H), 3.0(br, 2H),
2.90-2.70 (m, 2H), 2.40-2.00 (m, 4H), 1.95 (m, 3H).
[0411] EXAMPLES. The following compounds were prepared according to
the processes described herein starting from EC0059:
##STR00189##
Example
##STR00190##
[0413] COMPARATIVE EXAMPLES. The following comparative compounds
are disclosed:
Comparative Example
##STR00191##
[0414] Comparative Example
##STR00192##
[0415] Comparative Example. EC0923
##STR00193##
[0416] METHODS AND EXAMPLES
[0417] General. The following abbreviations are used herein:
partial response (PR); complete response (CR), three times per week
(M/W/F) (TIW).
[0418] METHOD. Relative Affinity Assay. The affinity for folate
receptors (FRs) relative to folate is determined according to a
previously described method (Westerhof, G. R., J. H. Schornagel, et
al. (1995) Mol. Pharm. 48: 459-471) with slight modification.
Briefly, FR-positive KB cells are heavily seeded into 24-well cell
culture plates and allowed to adhere to the plastic for 18 h. Spent
incubation media is replaced in designated wells with folate-free
RPMI (FFRPMI) supplemented with 100 nM .sup.3H-folic acid in the
absence and presence of increasing concentrations of test article
or folic acid. Cells are incubated for 60 min at 37.degree. C. and
then rinsed 3 times with PBS, pH 7.4. Five hundred microliters of
1% SDS in PBS, pH 7.4, is added per well. Cell lysates are then
collected and added to individual vials containing 5 mL of
scintillation cocktail, and then counted for radioactivity.
Negative control tubes contain only the .sup.3H-folic acid in
FFRPMI (no competitor). Positive control tubes contain a final
concentration of 1 mM folic acid, and CPMs measured in these
samples (representing non-specific binding of label) are subtracted
from all samples. Relative affinities are defined as the inverse
molar ratio of compound required to displace 50% of .sup.3H-folic
acid bound to the FR on KB cells, where the relative affinity of
folic acid for the FR is set to 1.
[0419] EXAMPLE. EC1669 shows high binding affinities towards folate
receptors as determined by an in vitro competitive binding assay
that measures the ability of the ligand to compete against
.sup.3H-folic acid for binding to cell surface folate receptors
(FR). EC1669 ( ). The relative affinity values of EC1669
(normalized against folic acid, which is set to (1) are determined
to be 0.53 and 0.13 on KB and CHO-FRO cells, respectively (see,
FIG. 1A and FIG. 1B). In comparison, methotrexate (MTX) showed poor
binding to the cell surface FRs. Without being bound by theory, it
is believed herein that the high binding affinity of EC1669 allows
for efficient cellular uptake via FR-mediated endocytosis.
[0420] METHOD. Inhibition of Cellular DNA Synthesis. The compounds
described herein are evaluated using an in vitro cytotoxicity assay
that predicts the ability of the drug to inhibit the growth of
folate receptor-positive cells, such as KB cells, RAW264.7
macrophages, and the like. It is to be understood that the choice
of cell type can made on the basis of the susceptibility of those
selected cells to the drug that forms the conjugate. The test
compounds are comprised of folate linked to a respective
chemotherapeutic drug, as prepared according to the processes
described herein. The test cells are exposed to varying
concentrations of folate-drug conjugate, and also in the absence or
presence of at least a 100-fold excess of folic acid to assess
activity as being specific to folate receptor mediation.
[0421] EXAMPLE. Conjugates of cytotoxic drugs described herein are
active against KB cells. The activity is mediated by the folate
receptor as indicated by competition experiments using
co-administered folic acid. KB cells are exposed for up to 7 h at
37.degree. C. to the indicated concentrations of folate-drug
conjugate in the absence or presence of at least a 100-fold excess
of folic acid. The cells are then rinsed once with fresh culture
medium and incubated in fresh culture medium for 72 hours at
37.degree. C. Cell viability was assessed using a .sup.3H-thymidine
incorporation assay. For compounds described herein, dose-dependent
cytotoxicity is generally measurable, and in most cases, the
IC.sub.50 values (concentration of drug conjugate required to
reduce .sup.3H-thymidine incorporation into newly synthesized DNA
by 50%) are in the low nanomolar range. Though without being bound
by theory, when the cytotoxicities of the conjugates are reduced in
the presence of excess free folic acid, it is believed herein that
such results indicate that the observed cell death is mediated by
binding to the folate receptor.
[0422] EXAMPLE. EC1669 shows a potent cytostatic effect against
murine RAW264.7 macrophages. The anti-proliferative activity of
EC1669 is measured in a XTT cell viability assay (FIG. 2) on
RAW264.7 cells after a 2-h exposure and a total of 72 h incubation.
RAW264.7 macrophages are susceptible to the drug forming the EC1669
conjugate, aminopterin. The cell viability is assessed by adding
XTT
(2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide-
) following the manufacturer's instructions. EC1669 showed a
dose-dependent inhibition of cell proliferation with a relative
IC.sub.50 value of .about.1.2 nM. The observed anti-proliferative
effect was 100% competable in the presence of 100-fold excess folic
acid (FA), indicating a FR-specific mode of action for EC1669.
[0423] METHOD. In vitro activity against various cancer cell lines.
IC.sub.50 values are generated for various cell lines. Cells are
heavily seeded in 24-well Falcon plates and allowed to form nearly
confluent monolayers overnight. Thirty minutes prior to the
addition of the test compound, spent medium is aspirated from all
wells and replaced with fresh folate-deficient RPMI medium
(FFRPMI). A subset of wells are designated to receive media
containing 100 .mu.M folic acid. The cells in the designated wells
are used to determine the targeting specificity. Without being
bound by theory it is believed herein that the cytotoxic activity
produced by test compounds in the presence of excess folic acid,
i.e. where there is competition for FR binding, corresponds to the
portion of the total activity that is unrelated to FR-specific
delivery. Following one rinse with 1 mL of fresh FFRPMI containing
10% heat-inactivated fetal calf serum, each well receives 1 mL of
medium containing increasing concentrations of test compound (4
wells per sample) in the presence or absence of 100 .mu.M free
folic acid as indicated. Treated cells are pulsed for 2 h at
37.degree. C., rinsed 4 times with 0.5 mL of media, and then chased
in 1 mL of fresh medium up to 70 h. Spent medium is aspirated from
all wells and replaced with fresh medium containing 5 .mu.Ci/mL
.sup.3H-thymidine. Following a further 2 h 37.degree. C.
incubation, cells are washed 3 times with 0.5 mL of PBS and then
treated with 0.5 mL of ice-cold 5% trichloroacetic acid per well.
After 15 min, the trichloroacetic acid is aspirated and the cell
material solubilized by the addition of 0.5 mL of 0.25 N sodium
hydroxide for 15 min. A 450 .mu.L aliquot of each solubilized
sample is transferred to a scintillation vial containing 3 mL of
Ecolume scintillation cocktail and then counted in a liquid
scintillation counter. Final results are expressed as the
percentage of .sup.3H-thymidine incorporation relative to untreated
controls.
[0424] EXAMPLE. Compounds described herein exhibit potent in vitro
activity against pathogenic cells, such as KB cells. Compounds
described herein exhibit greater specificity for the folate
receptor compared to compounds that do not include at least one
unnatural amino acid. For Example, EC1456 exhibits ca. 1000-fold
specificity for the folate receptor as determined by folic acid
competition (specificity=difference in IC.sub.50 between competed
group and non-competed group), and a 4-fold improvement in
specificity compared to comparator compound EC0531, which does not
include a linker L having an unnatural amino acid.
[0425] EXAMPLE. Selectivity for folate receptor expressing cells.
Compounds described herein show high activity for folate receptor
expressing cells. Compounds described herein do not show
significant binding to folate receptor negative cells. EC1456 show
high competable binding to low and high FR expressing cells (FR+),
and does not show binding to cells that do not express FR
(FR-).
Activity of EC1456 in (FR+) and (FR-) Cell Lines
TABLE-US-00007 [0426] Competable FR Activity up to Cell Line
Expression (lC.sub.50) 100 nM KB Human Cervical Carcinoma +++ 2.3
nM Yes NCl/ADR-RES-Cl.sub.2 Human ovarian Carcinoma ++ 1.4 nM Yes
IGROV1 Human ovarian + 0.72 nM Yes adenocarcinoma MDA-MB-231 Human
breast + 0.47 nM Yes adenocarcinoma (triple negative) A549 Human
Lung Carcinoma - Inactive (a) NA H23 Human Lung - Inactive NA
adenocarcinoma HepG2 Human hepatocellular - Inactive NA Carcinoma
AN3CA Human endometrial - Inactive NA adenocarcinoma LNCaP Human
prostate - ~850 nM NA adenocarcinoma (a) activity was evaluated
from 0.1-100 nM against these specifically selected (FR-) cell
lines (A549, H23, HepG2, AN3CA, LNCaP); NA = not applicable.
[0427] METHOD. Inhibition of Tumor Growth in Mice. Four to seven
week-old mice (Balb/c or nu/nu strains) are purchased from Harlan
Sprague Dawley, Inc. (Indianapolis, Ind.). Normal rodent chow
contains a high concentration of folic acid (6 mg/kg chow);
accordingly, test animals are maintained on a folate-free diet
(Harlan diet #TD00434) for about 1 week before tumor implantation
to achieve serum folate concentrations close to the range of normal
human serum, and during the Method. For tumor cell inoculation,
1.times.10.sup.6 M109 cells (a syngeneic lung carcinoma) in Balb/c
strain, or 1.times.10.sup.6 KB cells in nu/nu strain, in 100 .mu.L
are injected in the subcutis of the dorsal medial area (right
axilla). Tumors are measured in two perpendicular directions every
2-3 days using a caliper, and their volumes are calculated as
0.5.times.L.times.W.sup.2, where L=measurement of longest axis in
mm and W=measurement of axis perpendicular to L in mm Log cell kill
(LCK) and treated over control (T/C) values are then calculated
according to published procedures (see, e.g., Lee et al.,
"BMS-247550: a novel epothilone analog with a mode of action
similar to paclitaxel but possessing superior antitumor efficacy"
Clin Cancer Res 7:1429-1437 (2001); Rose, "Taxol-based combination
chemotherapy and other in vivo preclinical antitumor studies" J
Natl Cancer Inst Monogr 47-53 (1993)). Dosing is initiated when the
s.c. tumors have an average volume between 50-100 mm.sup.3
(t.sub.0), typically 8 days post tumor inoculation (PTI) for KB
tumors, and 11 days PTI for M109 tumors. Test animals (5/group) are
injected i.v., generally three times a week (TIW), for 3 weeks with
varying doses, such as with 1 mol/kg to 5 .mu.mol/kg, of the drug
delivery conjugate or with an equivalent dose volume of PBS
(control), unless otherwise indicated. Dosing solutions are
prepared fresh each day in PBS and administered through the lateral
tail vein of the mice.
[0428] METHOD. General 4T-1 Tumor Assay. Six to seven week-old mice
(female Balb/c strain) are obtained from Harlan, Inc.,
Indianapolis, Ind. The mice are maintained on Harlan's folate-free
chow for a total of three weeks prior to the onset of and during
the method. Folate receptor-negative 4T-1 tumor cells
(1.times.10.sup.6 cells per animal) are inoculated in the subcutis
of the right axilla. Approximately 5 days post tumor inoculation
when the 4T-1 tumor average volume is .about.100 mm.sup.3
(t.sub.0), mice (5/group) are injected i.v. three times a week
(TIW), for 3 weeks with varying doses, such as 3 .mu.mol/kg, of
drug delivery conjugate or with an equivalent dose volume of PBS
(control), unless otherwise indicated herein. Tumor growth is
measured using calipers at 2-day or 3-day intervals in each
treatment group. Tumor volumes are calculated using the equation
V=a.times.b.sup.2/2, where "a" is the length of the tumor and "b"
is the width expressed in millimeters.
[0429] METHOD. Drug Toxicity. Persistent drug toxicity is assessed
by collecting blood via cardiac puncture and submitting the serum
for independent analysis of blood urea nitrogen (BUN), creatinine,
total protein, AST-SGOT, ALT-SGPT plus a standard hematological
cell panel at Ani-Lytics, Inc. (Gaithersburg, Md.). In addition,
histopathologic evaluation of formalin-fixed heart, lungs, liver,
spleen, kidney, intestine, skeletal muscle and bone (tibia/fibula)
is conducted by board-certified pathologists at Animal Reference
Pathology Laboratories (ARUP; Salt Lake City, Utah).
[0430] METHOD. Toxicity as Measured by Weight Loss. The percentage
weight change of the test animals is determined on selected days
post-tumor inoculation (PTI), and during dosing. The results are
graphed.
[0431] EXAMPLE. In vivo activity against tumors. Compounds
described herein show high potency and efficacy against KB tumors
in nu/nu mice. Compounds described herein show specific activity
against folate receptor expressing tumors, with low host animal
toxicity. For example, EC1456 shows a complete response in 4/4 test
animals when administered intravenously at 1 mol/kg TIW, 2 wk.
EC1456 also shows specific activity mediated by the folate receptor
as evidenced by being competable with excess comparator compound
EC0923 (50 or 100 mol/kg), as shown in FIG. 3A. EC1456 does not
show any evidence of whole animal toxicity, as shown in FIG.
3B.
[0432] EXAMPLE. The therapeutic performance of EC1663 was evaluated
against the human KB tumors. The data in FIG. 4A show 4/4 partial
responses where the tumor volume was significantly decreased
compared to control, but did not go to zero, and the tumor began to
regrow after dosing ended. It is believed herein that a higher dose
may result in a complete response and/or cure. The data in FIG. 4B
show that at the administered efficacious dose, whole animal
toxicity was not observed.
[0433] METHOD. TNBC Tumor Assay. Triple negative breast cancer
(TNBC) is a subtype characterized by lack of gene expression for
estrogen, progesterone and Her2/neu. TNBC is difficult to treat,
and the resulting death rate in patients is reportedly
disproportionately higher than for any other subtype of breast
cancer. A TNBC xenograft model was generated in an analogous way to
the KB and M109 models described herein by implanting MDA-MB-231
breast cancer cells in nu/nu mice. Dosing is initiated when the
s.c. tumors have an average volume between 110-150 (generally 130)
mm.sup.3 (t.sub.0), typically 17 days post tumor inoculation (PTI).
Test animals (5/group) are injected i.v., generally three times a
week (TIW), for 2-3 weeks with varying doses, such as with 1
.mu.mol/kg to 5 .mu.mol/kg, of the drug delivery conjugate or with
an equivalent dose volume of PBS (control), unless otherwise
indicated. Dosing solutions are prepared fresh each day in PBS and
administered through the lateral tail vein of the mice.
[0434] EXAMPLE. When tested against an established triple negative
FR-positive subcutaneous MDA-MB-231 breast cancer xenografts,
EC1456 is found to be highly active at 2 .mu.mol/kg intravenous
dose administered on a three times per week, 2 consecutive week
schedule. The treatment produced 4 of 5 complete responses, where
tumor volume was reduced to zero, and regrowth did not occur during
the observation window over nearly 135 days. Without being bound by
theory, it is believed herein that the test animals were cured of
the triple negative breast cancer. The results for EC1456 are shown
in FIG. 5A. The anti-tumor activity was not accompanied by
significant weight loss in the test animals, as shown in FIG.
5B.
[0435] METHOD. Human cisplatin-resistant cell line. A human
cisplatin-resistant cell line is created by culturing FR-positive
KB cells in the presence of increasing cisplatin concentrations
(100.fwdarw.2000 nM; over a >12 month period). The
cisplatin-resistant cells, labeled as KB-CR2000 cells, are found to
be tumorigenic, and are found to retain their FR expression status
in vivo. KB-CR2000 tumors are confirmed to be resistant to
cisplatin therapy. Treatment with a high, toxic dose of cisplatin
(average weight loss of 10.3%, as shown in FIG. 6B), did not
produce even a single partial response (PR), as shown in FIG. 6A.
In contrast, EC1456 is found to be very active against KB-CR
tumors, where 5/5 CRs are observed. In addition, regrowth of the
tumor was only observed in 1/5 test animals. Without being bound by
theory, it is believed herein that 4/5 test animals were cured of
the cisplatin-resistant cancer, where regrowth did not occur during
the nearly 70 day observation period. Furthermore, unlike
cisplatin, EC1456 did not cause any weight loss in this cohort of
mice, and therefore did not display any evidence of gross animal
toxicity during the dosing period.
[0436] EXAMPLE. Comparison of conjugated and unconjugated drugs.
The therapeutic performance of unconjugated tubulysin B and
unconjugated TubB-H (EC0347) drugs is evaluated in vivo against
human KB tumors in mice. The anti-tumor efficacy and gross
toxicity, as determined by body weight changes, of each
unconjugated drug are compared to the EC1456 conjugate. EC1456
produced dose responsive anti-tumor activity in this model.
Complete responses were observed under treatment conditions that
produced little to no weight loss. In contrast, both unconjugated
tubulysin-based drugs failed to yield any anti-tumor response, even
when very toxic doses were administered to the mice. The results
are shown in the following table.
TABLE-US-00008 Toxicity Dose Dosing PR CR Cures Deaths Avg. Weight
Example (.mu.mol/kg) Schedule (%) (%) (%) (%) Loss EC1456 0.5 TIW,
3 weeks 60 0 0 0 <5%* 0.67 TIW, 2 weeks 60 20 0 0 <2% 1.0
TIW, 2 weeks 40 60 60 0 <1.5% 2.0 TIW, 2 weeks 0 100 100 0
<3% Tubulysin B 0.1 (4 doses) TIW, 2 weeks 0 0 0 100 >20% 0.2
(3 doses) TIW, 2 weeks 0 0 0 100 >18% 0.5 (1 dose) TIW, 2 weeks
0 0 0 100 >15% TubB-H 0.5 TIW, 2 weeks 0 0 0 0 <5.5% 0.75
TIW, 2 weeks 0 0 0 20 >10% 1.0 (2 doses).sup.1 TIW, 2 weeks 0 0
0 20 >15% *Untreated control group had an average weight loss of
2.4% .sup.1Group received only 2 doses due to toxicity.
[0437] These results confirm that despite tubulysin B and TubBH
being highly cytotoxic to cells in culture (typical
IC.sub.50.about.1 nM), both agents yielded dose-limiting toxicities
in mice at levels that did not produce measurable anti-tumor
effect. Thus, the unconjugated compounds do not exhibit a
therapeutic window. In contrast, the conjugated forms of the drugs,
such as conjugated TubBH (EC1456) produce anti-tumor responses
without significant toxicity to mice bearing well-established human
tumor xenografts. Conjugation as described herein provides a
therapeutic window to highly toxic drugs.
[0438] EXAMPLE. Compounds described herein exhibit high folate
receptor affinity compared to folic acid (relative affinity=1) in
10% serum/FDRPMI, potent in vitro activity, potent in vivo
activity, specificity for the folate receptor, and a sufficiently
high therapeutic index compared to unconjugated drug.
TABLE-US-00009 In vitro Therapeutic Relative IC.sub.50 50% In vitro
index over Affinity (nM) competition specificity In vivo
unconjugated Example (a) (b) (nM) (c) (fold) (d) activity (e) drug
(f) EC1299 0.29 0.9 700 778 CR Yes EC1393 0.25 2.2 600 300 NT NT
EC1456 0.27 1.5 1416 944 CR Yes EC1548 0.23 4.4 350 78 CR Yes
EC1549 0.90 4.5 350 78 CR Yes EC1586 0.56 NT NT NT NT NT EC0531 NT
1.5 355 237 CR Yes (comparator example) (a) compared to folic acid;
(b) as determined by thymidine incorporation; (c) IC.sub.50 for
test compound when competed with excess folic acid; the higher the
IC.sub.50 the more specific is the folate mediation.; (d) in vitro
specificity = difference in IC.sub.50 between competed group and
non-competed group; (e) as determined in subcutaneous KB tumor in
nu/nu mice; CR = complete response, where tumor volume, as defined
herein, during the observation period was zero for all test animals
in the group; (f) parent tubulysin; NT = not tested.
[0439] METHOD. Adjuvant-Induced Arthritis (AIA) Model. Female Lewis
rats are fed a folate-deficient diet (Harlan Teklad, Indianapolis,
Ind.) for 9-10 days prior to arthritis induction. The
adjuvant-induced arthritis (AIA) is induced by intradermal
inoculation (at the base of tail) of 0.4-0.5 mg of heat-killed
Mycobacteria butyricum (BD Diagnostic Systems, Sparks, Md.) in 100
.mu.L light mineral oil (Sigma). Ten days after arthritis
induction, paw edema (degree of arthritis) in rats is assessed
using a modified arthritis scoring system: 0=no arthritis;
1=swelling in one type of joint; 2=swelling in two types of joint;
3=swelling in three types of joint; 4=swelling of the entire paw. A
total score for each rat is calculated by summarizing the scores
for each of the four paws, giving a maximum score of 16 for each
rat. On Day 10 post arthritis induction, rats with a total
arthritis score of .gtoreq.2 were removed from the study and the
remaining rats are distributed evenly across the control and
treatment groups (n=5 for all groups except that n=2-3 for healthy
controls). All treatments are started on Day 10 unless indicated
otherwise. Rat paws are also evaluated radiographically to assess
and determine bone damage.
[0440] EXAMPLE. Compounds described herein are potent in treating
inflammatory diseases, such as inflammation and bone damage
accompanying arthritis. EC1496 is potent and efficacious in the
reducing paw inflammation in a rat model of adjuvant-induced
arthritis, as shown in FIG. 7. FIG. 7 shows that EC1496 is
efficacious in preventing the development of arthritis based on the
evaluation of paw edema. EC1496 (trace (d)) is significantly
different from untreated control (trace (b)). In addition, the data
indicate that the effect is folate receptor mediated because EC1496
(trace (d)) is also significantly different from the competition
control group where EC1496 is co-administered with excess folic
acid (trace (d)).
[0441] EXAMPLE. Compounds described herein are potent in treating
inflammatory diseases, such as inflammation and bone damage
accompanying arthritis. Illustratively, EC1496 is potent and
efficacious in reducing and/or preventing bone damage in a rat
model of adjuvant-induced arthritis, as determined by radiographic
analysis. The radiography shows that the treated animals do not
exhibit the bone damage seen in the untreated control animals,
based on visual scoring. Instead, the treated animals and the
healthy animals show similar bone structure.
[0442] EXAMPLE. EC1669 displays folate receptor-specific activity
against adjuvant arthritis. Starting 9 days after induction, rats
with developing AIA are distributed according to arthritis scores
into three groups (n=5): (1) the untreated AIA control, (2) the
EC1669 treated group, and (3) the EC1669 plus EC0923 competition
group. All treatments last for 2 consecutive weeks. The animals in
the AIA control group are left untreated. The animals in the EC1669
treatment group are given twice-a-week subcutaneous doses of EC1669
at a dosage of 375 nmol/kg. The animals in the EC1669 plus EC0923
group are given twice-a-week subcutaneous doses of EC1669 at a
dosage of 375 nmol/kg in conjunction to EC0923 at a dosage of 187.5
.mu.mol/kg. The study endpoints are shown in FIG. 8A, FIG. 8B, FIG.
9A, and FIG. 9B are: (a) arthritis score; (b) change in body
weight; and (c) paw swelling, assessed by percent increase in paw
weight (collected 4 days after the last dose), and (d) bone
radiography. EC1669 is found to be highly effective in alleviating
paw swelling (by .about.80% compared to control) and bone damage
(by .about.80% compared to control). The anti-arthritic activity of
EC1669 is competable (blocked) with the folate competitor
(EC0923).
[0443] EXAMPLE. In a subsequent dosing study, various EC1669 dosing
regiments were evaluated including once-a-week at 1000 nmol/kg,
twice-a-week at 250 nmol/kg, and twice-a-week at 500 nmol/kg.
Surprisingly, twice-a-week dosing at 250 nmol/kg was superior to
once-a-week dosing at 1000 nmol/kg, a two-fold decrease in total
dose. EC1669 was found more efficacious when dosed biweekly than
once weekly in reducing paw swelling at 81% reduction at 500
nmol/kg, biw, and 64% reduction at 250 nmol/kg, biw, compared to a
44% reduction at 1000 nmol/kg, siw.
[0444] EXAMPLE. EC1669 plus CellCept is more effective than either
agent alone against adjuvant-induced arthritis. CellCept is a
prodrug of mycophenolic acid, an immunosuppressant drug used to
prevent organ rejection in transplantation. CellCept is activated
in vivo and releases its active product that can inhibit T cell
proliferation and interfere with leukocyte adhesion to endothelial
cells. To test the combination effect of EC1669 and CellCept, rats
with developing AIA are distributed according to arthritis scores
into four groups (n=5): (1) the untreated AIA control, (2) the
EC1669 treated group, (3) the CellCept treated group, and (4) the
EC1669 and CellCept combination group. All treatments start on day
9 after AIA induction and last 2 consecutive weeks. The animals in
the AIA control group are untreated. The animals in the EC1669
treatment group are given weekly subcutaneous doses of EC1669 at a
dosage of 1000 nmol/kg. The animals in the CellCept treatment group
are given daily oral doses of CellCept at a dosage of 30 mg/kg, 5
days per week. The animals in the EC1669 and CellCept combination
treatment group are given weekly subcutaneous doses of EC1669 at a
dosage of 1000 nmol/kg and daily oral doses of CellCept at a dosage
of 30 mg/kg, 5 days per week. As shown in FIG. 10A and FIG. 11, the
EC1669 and CellCept combination therapy is more effective than
either agent alone in reducing arthritis scores, paw swelling, and
weight loss due to disease progression. FIG. 10B shows that the
EC1669 and CellCept combination therapy causes lower toxicity than
either drug given alone.
[0445] METHOD. Collagen-Induced Arthritis (CIA) Model. The
collagen-induced arthritis (CIA) is induced in female Lewis rats on
folate-deficient diet (Harlan Teklad, Indianapolis, Ind.). On Day
0, rats are immunized with 500 .mu.g of bovine collagen Type II
(Chondrex, Redmond, Wash.) formulated with Freund's complete
adjuvant. A booster immunization is given on Day 7 with 250 .mu.g
of the bovine collagen formulated with Freund's incomplete
adjuvant. Arthritis disease is assessed by a qualitative clinical
score system described by the manufacturer (Chondrex, Redmond,
Wash.): 0=normal, 1=Mild, but definite redness and swelling of the
ankle or wrist, or apparent redness and swelling limited to
individual digits, regardless of the number of affected digits,
2=Moderate redness and swelling of ankle of wrist, 3=Severe redness
and swelling of the entire paw including digits, and 4=Maximally
inflamed limb with involvement of multiple joints. On Day 10 post
first immunization, rats are distributed evenly (according to the
arthritis score) across the control and treatment groups. The CIA
rats are given ten consecutive subcutaneous doses of test compound
on days 10-19. For each drug, an induction dose (for example, 500
nmol/kg) is given on days 10 and 15 and a maintenance dose (for
example, 100 nmol/kg) is given on days 11-14 and 16-19. The animals
in the arthritis control group are left untreated. The arthritis
score and animal body weight are recorded five times a week.
[0446] METHOD. Animal Experimental Autoimmune Uveitis Model.
Experimental autoimmune uveitis (EAU) is induced in female Lewis
rats maintained on a folate-deficient diet (Harlan Teklad,
Indianapolis, Ind.). On Day 0, the animals are immunized
subcutaneously with 25 .mu.g of bovine S--Ag PDSAg peptide
formulated with Freund's incomplete adjuvant containing 0.5 mg of
grounded M. Tuberculosis H37Ra. Purified pertussis toxin (PT) is
given at a dosage of 1 .mu.g per animal on the same day via
intraperitoneal injection. The severity of uveitis in each eye is
assessed by a qualitative visual score system: 0=No disease, eye is
translucent and reflects light (red reflex); 0.5 (trace)=Dilated
blood vessels in the iris, 1=Engorged blood vessels in iris,
abnormal pupil contraction; 2=Hazy anterior chamber, decreased red
reflex; 3=Moderately opaque anterior chamber, but pupil still
visible, dull red reflex; and 4=Opaque anterior chamber and
obscured pupil, red reflex absent, proptosis. This assessment
yields a maximum uveitis score of 8 per animal.
[0447] EXAMPLE. Compounds described herein are potent in treating
autoimmune uveitis. EC1669 displays folate receptor-specific
activity against autoimmune uveitis. Animals presenting EAU are
randomized and distributed into three groups: (1) the untreated EAU
control (n=11), (2) the test compound treated group (n=7), such as
EC1669, (3) the test compound and competitor compound treated group
(n=7), such as EC1669 plus EC0923, and (4) the positive control
treated group (n=7), such as methotrexate (MTX). All treatments
start on day 8 after EAU induction. The animals in the EAU control
group are untreated. The animals in the EC1669 treatment group are
given five subcutaneous doses of EC1669 at a dosage of 250 nmol/kg
every other day (q2d). The animals in the EC1669 plus EC0923
treatment group are given five subcutaneous doses of EC1669 at a
dosage of 250 nmol/kg every other day plus a 500-fold excess of
EC0923 at a dosage of 125 .mu.mol/kg as the folate competitor. The
animals in the MTX treatment group are given five subcutaneous
doses of MTX at a dosage of 250 nmol/kg every other day. The
uveitis score and animal body weight are recorded for each animal
at predetermined frequencies. The clinical severity of EAU is
monitored on a daily basis using an ophthalmoscope and graded on a
scale of 0 to 4 per eye with a maximum possible score of 8 per
animal. On day 16, the animals are euthanized and rat eye balls are
fixed in formalin for histology. As shown in FIG. 12A, EC1669
treatment at disease on-set effectively reduces the symptoms of EAU
in a FR-dependent manner and its activity is competitive with
subcutaneous MTX. Treatment-related weight loss was not observed
with the conjugate compounds described herein that include a linker
comprising at least one unnatural amino acid, as shown in FIG.
12B.
[0448] EXAMPLE. EC1496 is potent and efficacious against folate
receptor specific autoimmune uveitis, as shown in FIG. 13A. Tissues
are evaluated by histology as shown in FIG. 13B.
[0449] METHOD. Autoimmune Encephalomyelitis (EAE) Model. EAE is
induced in rats by immunization against 25 .mu.g of guinea pig
myelin basic protein (MBP) formulated with CFA containing 1 mg of
grounded Mycobacterium tuberculosis H37Ra. Pertussis toxin is given
intraperitoneally (1 .mu.g/rat) to enhance the organ-specific
autoimmunity. Starting 8 days after induction, rats are divided
into 4 groups: (1) untreated control (n=8), (2) test compound
(n=7), and (3) test compound plus competitor compound (n=7), such
as EC0923 competition. All treatments start on day 8 after EAE
induction. The animals in the EAE control group are left untreated.
The animals in the test compound treatment group are given four
subcutaneous doses of test compound at a dosage of 250 nmol/kg
every other day (q2d). The animals in the test compound plus
competitor compound treatment group are given four subcutaneous
doses of test compound at a dosage of 250 nmol/kg every other day
plus a 500-fold excess of competitor compound, such as EC0923 at a
dosage of 125 .mu.mol/kg as an illustrative folate receptor
competitor. The clinical severity of EAE is monitored on a daily
basis and graded on a scale of 0 to 5 per animal. The clinical
signs of ascending paralysis of EAE rats are divided into a 0-5
scale: 0=No disease, 0.5=distal limp tail, 1=limp tail, 2=mild
paraparesis; ataxia-weakened hind limbs, 3=moderate paraparesis;
hind limbs paresis, 4=complete hind limb paralysis, 5=complete hind
limb paralysis and incontinence (euthanasia). On day 16, the
animals are euthanized and brain and spinal cords are fixed in
formalin for histology.
[0450] EXAMPLE. Compounds described herein are potent in treating
experimental autoimmune encephalomyelitis (EAE). EC1669 displays
folate receptor-specific activity against EAE. As shown in FIG.
14A, EC1669 treatment at disease on-set effectively suppresses the
neurological symptoms during the acute phase of EAE.
Treatment-related weight loss was not observed with EC1669 when
dose alone, as shown in FIG. 14B. The therapeutic effect of EC1669
is blocked by the folate receptor competitor EC0923.
[0451] EXAMPLE. EC1496 is potent and efficacious against EAE, as
shown in FIG. 15. Treatment-related weight loss was not observed
with EC1496 when dosed alone.
[0452] METHOD. Human serum stability. Compounds described herein
are tested in human serum for stability using conventional
protocols and methods. Briefly, test compound is administered to
the test animal, such as by subcutaneous injection. The plasma
concentration of the conjugate, and optionally one or more
metabolites, is monitored over time. The results are graphed to
determine Cmax, Tmax, half-life, and AUC for the test compound and
metabolites.
[0453] EXAMPLE. Conjugate compounds described herein that include a
linker comprising at least one unnatural amino acid are more stable
in plasma than comparator conjugate compounds that do not have a
linker comprising at least one unnatural amino acid. EC1495 and
EC0746 (comparator compound) are each administered at 500 nmol/kg
by subcutaneous injection. The plasma concentration of the
conjugate and the metabolites (aminopterin and aminopterin
hydrazide) are monitored over time. EC1496 shows a higher Cmax than
EC0746, as shown in FIG. 16A and FIG. 16B, respectively. In
addition, FIG. 16A and FIG. 16B show that EC1496 releases
substantially less drug in plasma than does EC0746. As also shown
in the following table, free drug is released as the parent
aminopterin and the hydrazide derivative (EC0470).
TABLE-US-00010 From From Free drug released (%) EC0746 EC1496 AMT
11.0 5.13 AMT-hydrazide 7.4 3.15 (EC0470) Total 18.4 8.28
[0454] Without being bound by theory, it is believed herein that
the data indicate that the compounds described herein that include
a linker comprising at least one unnatural amino acid, such as
EC1496, exhibit greater plasma stability. In addition, the
comparative example EC0746, which does not include a linker
comprising at least one unnatural amino acid, releases more than
2-fold more drug than the EC1496 after a subcutaneous dose in rats.
EC1496 also shows a higher Cmax than EC0746 leading to a higher
effective therapeutic dose. Finally, EC1496 shows a shorter
half-life. Without being bound by theory, it is believed herein
that rapid clearance may further lead to lower toxicity because the
duration of exposure to prematurely released drug from the
conjugates described herein, compared to compounds that do not
include a linker comprising at least one unnatural amino acid, will
also be decreased.
[0455] METHOD. Plasma clearance. In vivo studies include a minimum
of 3 test animals, such as rats, per time point. Illustratively,
female Lewis rats with jugular vein catheters (Harlan, regular
rodent diet) are given a single subcutaneous injection of test
compound, such as EC1669 at 500 nmol/kg. Whole blood samples (300
.mu.L) are collected at the following time points: 1 min, 10 min,
30 min, 1 h, 2 h, 3 h, 4 h, 8 h, and 12 h after injection. The
blood samples are placed into anti-coagulant tubes containing 1.7
mg/mL of K.sub.3-EDTA and 0.35 mg/mL of N-maleoyl-beta-alanine
(0.35 mg/mL) in a 0.15% acetic acid solution. Plasma samples are
obtained by centrifugation for 3 min at -2,000 g and stored at
-80.degree. C. The amounts of test compound in the plasma and any
metabolites, such as EC1669 and its two active metabolites
aminopterin (AMT) and AMT hydrazide (EC0470), respectively, are
quantified by LC-MS/MS.
[0456] EXAMPLE. EC1669 shows fast plasma clearance after
subcutaneous administration in rats. EC1669 is detectable in the
blood stream within minutes, with a Cmax of .about.472 nM occurring
at .about.30 min post dose, and it maintained a plateau until 60
min after the injection. The EC1669-derived AMT and EC0470 are
detected at similar Cmax values of 27 nM and 21 nM, respectively,
but there is a 30-min delay in comparison to the EC1669 Cmax. While
EC1669 itself is cleared rapidly from the blood with an elimination
half-life of .about.37 min, the elimination half-lives of the two
metabolites are approximately 2-3 times longer at 66 min (AMT) and
112 min (EC0470), respectively. The corresponding
area-under-the-curve (AUC) values for EC1669, AMT and EC0470 are
52, 5.9, and 3.9 nmol*min/mL, respectively. Based on their AUC
responses, .about.15.8% of active drug exposure/release (AMT plus
EC0470) is estimated in the plasma over the 12 h collection period,
a shown in the following table.
TABLE-US-00011 Metabolites % Released AMT/EC0470 Ratio Total 15.8
1.51 AMT 9.5 EC0470 6.3
[0457] Without being bound by theory, it is believed herein that
the fast plasma clearance observed for the compounds described
herein, such as EC1669, may result in lower host animal toxicity
because the duration of exposure to prematurely released drug from
the conjugates described herein, compared to compounds that do not
include a linker comprising at least one unnatural amino acid, will
also be decreased.
[0458] METHOD. Pharmacokinetic biodistribution. Studies in this
section included a minimum of 3 test animals (mice) per time point.
The pharmacokinetic biodistribution of test compound, such as
.sup.3H-EC1669 (label on the drug), compared to positive control,
such as .sup.3H-methotrexate (.sup.3H-MTX), is observed in female
Balb/c mice on folate-deficient diet. Compounds are administered as
a single subcutaneous (SC) injection at 500 nmol/kg. Whole blood
(>300 .mu.L) along with .about.100 mg each of various tissues of
interests are collected at various time points (such as 10 min, 30
min, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, and 72 h). The blood samples
are placed in BD microtainer tubes (heparin) and centrifuged (4,000
g.times.3 min, 4.degree. C.) to separate plasma (>100 .mu.L).
The remaining red blood cell (RBC) mass is washed 2.times. with
phosphate buffered saline (PBS, pH 7.4) to obtain RBCs. The
collected tissues are weighed and processed to determine
.sup.3H-EC1669 and .sup.3H-MTX distribution: plasma, RBC, heart,
lung, liver (the smallest lobe), spleen, kidney (1), intestine
(above cecum), fecal material (from colon), muscle, and brain.
[0459] EXAMPLE. Comparison of pharmacokinetic biodistribution of
.sup.3H-EC1669 and .sup.3H-methotrexate after subcutaneous
administration. The comparative pharmacokinetic biodistribution
results are shown in FIG. 17 (as percent injected dose per gram (%
ID/g)). At 10 min post-dose, 31% ID/g of .sup.3H-MTX is captured by
the liver. Twice as much .sup.3H-EC1669 is found in the plasma than
.sup.3H-MTX (12% versus 5.2% ID/g). .sup.3H-MTX retention in RBCs,
spleen, liver, intestine, and feces are also consistently higher
than that of .sup.3H-EC1669 during the entire sampling period. The
RBC data is also plotted in FIG. 18 showing that .sup.3H-MTX
retention is higher than EC1669. Without being bound by theory, it
is believed herein these data suggest that EC1669 differs
significantly from MTX in hepatic clearance, where MTX is
preferentially cleared by the liver. Without being bound by theory,
it is also believed herein these data suggest that EC1669 differs
significantly from MTX in RBC uptake, suggesting different methods
of cellular entry. MTX reportedly enters cells non-specifically,
typically via the ubiquitously expressed reduced folate carrier
(RFC). The compounds described herein are shown to enter cells
specifically through the functional folate receptor. Without being
bound by theory, it is believed herein that the RBC data further
support the folate receptor mediated activity of the conjugates
described herein.
[0460] In a subsequent renal/hepatic secretion study, mice are
housed in metabolic cages with a 6-h fast before subcutaneous
administration of .sup.3H-EC1669 or .sup.3H-MTX. At 24 h post-dose,
.about.14% more radioactivity is found in the pooled urine of
.sup.3H-EC1669 dosed animals than in .sup.3H-MTX dosed animals. In
contrast, twice as much radioactivity was found in the pooled feces
of .sup.3H-MTX dosed animals than in EC1669 dosed animals. Without
being bound by theory, it is believed herein these data suggest
that EC1669 differs significantly from MTX in renal to hepatic
clearance ratio, where EC1669 is preferentially cleared by the
kidneys, rather than the liver. MTX reportedly causes
hepatotoxicity as a major side effect, especially after long-term
use. Without being bound by theory, it is believed herein that the
preferential renal clearance of the compounds described herein will
lead to fewer side effects such as hepatotoxicity.
[0461] EXAMPLE. Compounds described herein are less toxic than
compounds that do not have a linker comprising at least one
unnatural amino acid. Test compounds are administered i.v. at
equivalent doses to folate deficient rats. As shown in FIG. 19,
conjugates described herein that include a linker comprising at
least one unnatural amino acid, such as EC1496, are less toxic than
the corresponding conjugate that does not, such as comparative
example EC0746.
[0462] EXAMPLE. Maximum tolerated dose (MTD). Conjugate compounds
described herein that include a linker comprising at least one
unnatural amino acid show high MTDs, which are improved over
compounds that do not have linkers comprising one or more unnatural
amino acids. Test compounds are administered by i.v., BIW, 2 wks in
female Sprague-Dawley rats. Comparator compound EC0531 has a MTD of
0.33 .mu.mol/kg, while EC1456 has a MTD of at least 0.51
.mu.mol/kg, a 65% improvement, as shown in FIG. 20. Histopathologic
changes were not observed with doses of EC1456 at or below the
MTD.
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