Design and regioselective synthesis of a new generation of targeted therapeutics. Part 3: Folate conjugates of aminopterin hydrazide for the treatment of inflammation

Article abstract of DOI:10.1016/j.bmcl.2010.12.085

Efficient syntheses of folate receptor (FR) targeting conjugates of the anti-inflammatory, aminopterin hydrazide, are described. 2-{4-Benzoylamino}-5- oxo-5-{N′-[2-(pyridin-2-yldisulfanyl)-ethoxycarbonyl]-hydrazino} -pentanoic acid is synthesized from commercially available 4-[(2-amino-4-imino- 3,4-dihydro-pteridin-6-yl-methyl)-amino]-benzoic acid. Conjugation of this novel, activated aminopterin hydrazide to folic acid through cysteine-terminating (C-terminus), peptide/carbohydrate spacers results in highly water soluble conjugates which allow for the release of free aminopterin hydrazide within the endosomes of targeted cells.

Full text of DOI:10.1016/j.bmcl.2010.12.085

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1202–1205
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and regioselective synthesis of a new generation of targeted
therapeutics. Part 3: Folate conjugates of aminopterin hydrazide
for the treatment of inflammation
⇑
Iontcho R. Vlahov , Fei You, Hari Krishna R. Santhapuram, Yu Wang, Jeremy F. Vaughn, Spencer J. Hahn,
Paul J. Kleindl, Mingjin Fan, Christopher P. Leamon
Endocyte Inc., 3000 Kent Ave, Suite A1-100, West Lafayette, IN 47906, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Efficient syntheses of folate receptor (FR) targeting conjugates of the anti-inflammatory, aminopterin
hydrazide, are described. 2-{4-Benzoylamino}-5-oxo-5-{N0-[2-(pyridin-2-yldisulfanyl)-ethoxycarbonyl]-
hydrazino}-pentanoic acid is synthesized from commercially available 4-[(2-amino-4-imino-3,4-dihy-
dro-pteridin-6-yl-methyl)-amino]-benzoic acid. Conjugation of this novel, activated aminopterin
hydrazide to folic acid through cysteine-terminating (C-terminus), peptide/carbohydrate spacers results
in highly water soluble conjugates which allow for the release of free aminopterin hydrazide within the
endosomes of targeted cells.
Received 15 October 2010
Revised 14 December 2010
Accepted 16 December 2010
Available online 22 December 2010
Keywords:
Folate
Ó 2010 Elsevier Ltd. All rights reserved.
Folate receptor targeting
Folate-aminopterin hydrazide conjugate
Inflammation
Disulfide linkers
The vitamin folic acid (folate, FA) binds with high affinity
(KD <10ꢀ9 M)1 to a glycosylphosphatidylinositol anchored cell-sur-
face glycoprotein called the folate receptor (FR). After binding, FA is
transported into the cell via FR-mediated endocytosis.2 Conse-
quently, FA can be exploited as a molecular ‘Trojan horse’ for the
targeted delivery of covalently-attached, biologically active mole-
macrophages produce and release pro-inflammatory cytokines
such as TNF-a, IL-6, and IL-1, promoting and intensifying the dis-
ease.9 Consequently, developing therapies which target FR-b might
lead to an elimination of the activated macrophages and the result-
ing auto-immune/inflammatory disease while diminishing the
inherent collateral toxicity to normal cells.
cules.3 To date, three isoforms of the FR have been identified and
Previously, we reported the design and synthesis of a folate
targeted chemotherapeutic conjugate, EC145, which is in phase 2
clinical trials.10 In EC145, an anticancer drug, desacetyl vinblastine
hydrazide (DAVLBH) is attached to folic acid (FA) via a water-solu-
4
cloned: FR-
a, FR-b, and FR-
c
and its truncated cogener, FR-c0
.
In
normal tissues, the distribution of measurable levels of FR-
a
is lim-
ited only to the apical membrane surface of certain polarized epi-
thelial cells, in placental trophoblasts, and on the apical side of
kidney proximal tubule cells, the latter serving as a salvaging route
for folates prior to urinary excretion.5 In contrast, in many human
ble peptidic spacer and
a reducible disulfide linker system.
Recently, we published the synthesis of folate-DAVLBH conjugates
using carbohydrate-based peptide spacers designed to decrease
undesired hepatobiliary clearance of free DAVLBH without affect-
ing the conjugate’s targeted anti-tumor activity.11
malignant cells the FR-
a is highly overexpressed, especially in
aggressively growing cancers.6 FR-b is present on the cell surface
of human activated macrophages but not on resting/quiescent
macrophages.7 In response to pro-inflammatory stimuli, activated
macrophages concentrate at areas of inflammation. Excessive and
persistent build-up can cause tissue damage, resulting in such
inflammatory diseases as: rheumatoid arthritis, psoriasis, Crohn’s
disease, systemic lupus erythematosus, atherosclerosis, type II
diabetes, ulcerative colitis, osteoarthritis, organ transplant rejec-
tion, and ischemia/reperfusion injury.8 In addition, activated
In this Letter, we utilize a peptidic or a carbohydrate-based
spacer along with a disulfide linker system to synthesize targeted
folate-aminopterin hydrazide conjugates 1 and 2, the first conju-
gates in a novel class of compounds designed to suppress inflam-
matory disease ( Fig. 1). Aminopterin, a highly active antifolate
originally developed to treat cancer, was reported in 1951 to
produce rapid improvement in patients with rheumatoid arthritis
and psoriasis; however, due to manufacturing problems and a poor
therapeutic index, the clinical use of aminopterin was discontinued
in favor of the safer alternative, methotrexate.12 In our approach,
we use a novel derivative of aminopterin: aminopterin hydrazide.
⇑
Corresponding author.
E-mail address: ivlahov@endocyte.com (I.R. Vlahov).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.12.085

I. R. Vlahov et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1202–1205
1203
Figure 1. Aminopterin hydrazide-folate conjugates 1 (peptidic spacer region) and 2 (carbohydrate based peptidic spacer region).
Aminopterin and aminopterin hydrazide both displayed very sim-
ilar in vitro activities in a series of cells (unpublished results). We
selected the hydrazide because base drugs which contain this moi-
ety are readily releasable from their derived conjugates using our
linker technology. In contrast, a conjugate derived from aminop-
terin and linked through an ester moiety would be expected to
be readily releasable, but have poor serum stability.
pyrrolidinophosphonium hexafluorophosphate (PyBop) and DIPEA,
yielded glutamic acid derivative 6. 4-Dimethylaminopyridine
(DMAP) mediated Fmoc deprotection of 6 followed by in situ cou-
pling with commercially available sodium 4-[(2-amino-4-imino-
3,4-dihydro-pteridin-6-yl-methyl)-amino]-benzoate 5 using PyBop
and hydroxybenzotriazol (HOBt) resulted in fully protected aminop-
terin hydrazide 11. Treatment of 11 with TFA expediently removed
the t-butyl moiety to yield pyridinedisulfanyl-activated aminop-
terin hydrazide 4.14 Finally, treatment of a suspension of FA-spacer
3 in H2O under Argon with 0.1 N NaHCO3 resulted in a clear yellow
solution at pH >6.9. To this mixture was added at once under exten-
sive stirring a solution of 4 in THF.15 According to the HPLC profile,
the reaction was completed in less then 15 min. HPLC purification
gave pure conjugate 1.16
Second generation folate-aminopterin conjugate 2 was de-
signed using carbohydrate-based folate-spacer 12 (Scheme 3).
FA-spacer unit 12 was synthesized using a standard Fmoc-SPPS
protocol as described in our previous publication.11 Treatment of
a suspension of 12 in phosphate buffer and under argon with NaH-
CO3 resulted in a clear yellow solution. To this mixture was added
at once under vigorous stirring a dimethylsulfoxide (DMSO) solu-
tion of 4 to yield the final conjugate 2.17
As outlined in the retrosynthetic analysis (Scheme 1), disulfide-
linked conjugate 1 can be assembled from the corresponding
thiol-containing FA-spacer
3 and thiol-reactive derivative of
aminopterin hydrazide 4. The disulfide bond is readily reduced in
the internalized endosome of the targeted cell, of crucial impor-
tance for drug delivery applications.13 Aminopterin derivative 4
can be readily derived from commercially available 4-aminoptero-
ic acid sodium salt 5 and activated glutamic acid 6. The peptide-
based FA-spacer 3 is designed to introduce a discrete number of
charged amino acids for better water solubility and was synthe-
sized using standard fluorenylmethyloxycarbonyl-based solid
phase peptide synthesis (Fmoc-SPPS).10
The nucleophile- and thiol-activated heterobifunctional carbon-
ate 7 (Scheme 2) served as an important starting material for the
releasable drug conjugate synthesis.10 Reaction of this mixed
carbonate with t-butyl-carbazate in the presence of diisopropyleth-
ylamine (DIPEA) gave the corresponding t-butyl-carbazate 8. Triflu-
oroacetic acid (TFA) mediated Boc deprotection of 8 in the presence
of triisopropylsilane (TIPS) resulted in 9 as a TFA salt. Coupling of 9
with protected glutamic acid 10, using benzotriazol-1-yl-oxytri-
Treatmentof conjugate 2 with a reducing agent demonstrates the
release of free aminopterin hydrazide (Scheme 4). In brief, a 1 mM
solution of 2 in phosphate buffer (pH 7.4) was treated with 40 equiv
of dithiothreitol (DTT) at room temperature.18 The HPLC profile
(Fig. 2) showed cleavage of the disulfide bond (t1/2 ꢁ22 min.) with
Scheme 1.

1204
I. R. Vlahov et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1202–1205
Scheme 2.
Scheme 3.
Scheme 4.
concomitant release of the FA-Spacer 12 and 2-thioethyl carbazate
13. Subsequently, 13 fragments into free aminopterin hydrazide
14, thus demonstrating the self-immolative nature of the linker
system.19
Following positive in vitro and in vivo results and toxicological
evaluation, 2 (known also as EC0746) was selected as a lead candi-
date for further (pre)clinical development. Results of the complex
biological investigations will be reported soon in the appropriate
scientific journals.
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Figure 2. HPLC profile (280 nm) of the treatment of 2 with DTT.

I. R. Vlahov et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1202–1205
1205
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14. Analytical data for activated aminopterin hydrazide 4: 1H NMR (DMSO-d6 & D2O)
d 8.82 (s, 1H), 8.44 (d, J = 4.7 Hz, 1H), 7.80 (m, 2H), 7.71 (d, J = 8.8 Hz, 2H), 7.24
(t, J = 4.6 Hz, 1H), 6.74 (d, J = 8.8 Hz, 2H), 4.60 (s, 2H), 4.32 (dd, J = 5.0 Hz, 1H),
4.20 (t, J = 6.0 Hz, 2H), 3.07 (t, J = 6.0 Hz, 2H), 2.22 (t, J = 7.8 Hz, 2H), 2.15 - 1.94
(m, 2H). ESI-MS: (M+H)+ = Calculated 668.2; found 668.2.
15. General procedure for conjugation: To H2O (bubbled with argon for 10 min before
use) was added folate linker in a centrifuge tube. To this suspension, while
bubbling with argon, was added drop-wise saturated NaHCO3 solution (bubbled
with argon for 10 min before use) until the pH of the resulting solution reached
6.9. 1 equiv of 4 in THF was added quickly and the resulting homogenous
solution was stirred under argon for 15 min. The reaction progress was checked
by analytical HPLC. The mixture was diluted with of phosphate buffer and the
THF was removed under vacuum. The cloudy solution was centrifuged and
filtered. The yellow filtrate was purified by preparative HPLC.
16. Analytical data for aminopterin folate 1: 1H NMR (DMSO-d6 & D2O) d 8.68 (s, 1H)
8.62 (s, 1H), 7.64 (d, J = 8.8 Hz, 2H), 7.58 (d, J = 8.8 Hz, 2H), 6.72 (d, J = 8.8 Hz,
2H), 6.60 (d, J = 8.8 Hz, 2H), 4.55 (t, J = 6.5 Hz, 2H), 4.46 (m, 7H), 4.36(t,
J = 6.5 Hz, 2H), 4.18 (m, 4H), 3.29 (m, 3H), 3.08 (m, 3H), 2.87 (m, 4H), 2.15 (m,
4H), 2.02 (m, 2H), 1.88 (m, 2H). ESI-MS: [(M+2H)2+]/2 = Calculated 767.2;
found 767.4.
17. Analytical data for aminopterin carbohydrate-based folate 2: 1H NMR (DMSO-d6
& D2O) d 8.67 (s, 1H) 8.60 (s, 1H), 7.62 (d, J = 9 Hz, 2H), 7.59 (d, J = 9 Hz, 2H),
6.71 (d, J = 8.7 Hz, 2H), 6.62 (d, J = 8.1 Hz, 2H), 4.47 (m, 4H), 4.26–4.04 (m, 10H),
3.70–3.30 (m, 22H), 3.30–3.10 (m, 6H), 3.10–2.76 (m, 9H), 2.40–2.04 (m, 15H),
2.04–1.60 (m, 4H). ESI-MS: [(M+2H)2+]/2 = Calculated 1119.09; found 1119.10.
18. The species which reduces the disulfide bond to free thiol in the endosome is
not known, but the reduction is unlikely to result from disulfide exchange with
small thiol-containing molecules due to the relatively low pH. The use of DTT
or another reductant such as glutathione does not accurately mimic the
biological system. See Ref. 13.
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