Clickable prodrugs bearing potent and hydrolytically cleavable nicotinamide phosphoribosyltransferase inhibitors

Article abstract of DOI:10.2147/DDDT.S152685

Purpose: Our previous study indicated that carborane containing small-molecule 1-(hydroxymethyl)-7-(4′-(trans-3″-(3′″-pyridyl)acrylamido)butyl)-1,7-dicarbadodecaborane (hm-MC4-PPEA), was a potent inhibitor of nicotinamide phosphoribosyltransferase (Nampt)

Full text of DOI:10.2147/DDDT.S152685

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Open access FullText article
O r i g i n a l r e s e a r c h
clickable prodrugs bearing potent and
hydrolytically cleavable nicotinamide
phosphoribosyltransferase inhibitors
This article was published in the following Dove Press journal:
Drug Design, Development andTherapy
Keivan sadrerafi
emilia O Mason
Mark W lee Jr
Purpose: Our previous study indicated that carborane containing small-molecule
1-(hydroxymethyl)-7-(4′-(trans-3″-(3′″-pyridyl)acrylamido)butyl)-1,7-dicarbadodecaborane
(hm-MC4-PPEA), was a potent inhibitor of nicotinamide phosphoribosyltransferase (Nampt).
Nampt has been shown to be upregulated in most cancers and is a promising target for the
treatment of many different types of cancers, including breast cancers.
Department of chemistry, University
of Missouri, columbia, MO, Usa
Patients and methods: To increase the selectivity of hm-MC4-PPEA toward cancer cells,
three prodrugs were synthesized with different hydrolyzable linkers: ester, carbonate, and car-
bamate. Using click chemistry a fluorophore was attached to these prodrugs to act as a model
for our conjugation strategy and to serve as an aid for prodrug stability studies. The stabilities of
these drug conjugates were tested in phosphate-buffered saline (PBS) at normothermia (37°C)
using three different pH levels, 5.5, 7.5, and 9.5, as well as in horse serum at physiological pH.
The stability of each was monitored using reversed-phase HPLC equipped with both diode array
and fluorescence detection. The inhibitory activity of hm-MC4-PPEA was also measured using
a commercially available colorimetric assay. The biological activities of the drug conjugates as
well as those of the free drug (hm-MC4-PPEA), were evaluated using the MTT assay against
the human breast cancer cell lines T47D and MCF7, as well as the noncancerous, transformed,
Nampt-dependent human breast epithelium cell line 184A1.
Results: hm-MC4-PPEA showed to be a potent inhibitor of recombinant Nampt activity, exhibiting
anIC50concentrationof6.8nM.Theprodrugsshowedgreatstabilitytowardshydrolyticdegradation
under neutral, mildly acidic and mildly basic conditions. The carbamate prodrug also showed to be
stable in rat serum. However, the carbonate and the ester prodrug release at various rates in serum
presumably owing to the presence of several different classes of esterase. The biological activities
of the drug conjugates correlate with the stability of their cleavable linkers observed in serum.
Conclusion: The targeted and selective delivery of potent Nampt inhibitors to cancer cells is a
potentially new route for the treatment of many cancers. These prodrugs linked to small cancer-
associated peptides may be optimum for their use as targetable Nampt inhibitors.
Keywords: carboranes, Nampt, cancer, prodrugs, cleavable linker
Plain language summary
Why was the study done? To develop a platform for targeted delivery of 1-(hydroxymethyl)-
7-(4′-(trans-3″-(3‴-pyridyl)acrylamido)butyl)-1,7-dicarbadodecaborane (hm-MC4-PPEA),
a potent Nampt inhibitor.
correspondence: Mark W lee Jr
Department of chemistry, University
of Missouri, 601 south college avenue,
columbia, MO 65211, Usa
Tel +1 573 884 2424
email leemw@missouri.edu
What did the researchers do and find? Three prodrugs were developed, and their stability
was tested under various conditions.
What do these results mean? By incorporating a cleavable linker bearing an azide group,
these prodrugs may be attached to a variety of delivery vehicles to achieve targeted delivery.
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Sadrerafi et al
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Introduction
not depend heavily on the NAD+ from the Nampt-catalyzed
Globally, there exists a tremendous unmet need for more recycle pathway.23,24 Given sufficient exposure time, Nampt
efficacious and affordable treatments for nearly every type inhibitors are exceptionally potent when compared with the
of cancer. The need for new treatments is even greater small-molecule anticancer agents used clinically today.25,26
for advanced and/or recurrent cancers. A limitation of all It must be noted that high levels of Nampt inhibition lead to
small-molecule anticancer agents is toxicity toward healthy thrombocytopenia, retinal, and hematopoietic toxicities.27,28
cells. The development of such agents is necessarily a com- However, the toxic effects may be minimized by lead opti-
promise between maximizing a drug’s biological activity mization of the inhibitor and its targeted delivery system.
with its absorption, distribution, metabolism, and excre-
A small molecule discovered through compound screen-
tion. To achieve a clinically meaningful therapeutic effect, ing designated FK866/APO866 was the first known potent
most anticancer agents must be administered at, or near, inhibitor of Nampt and has been investigated in a number of
their maximum tolerated dose (MTD). Unfortunately, the Phase I/II clinical trials against several cancers.25 For this class
drugs clinically used to treat cancer patients today lack the of inhibitors, cell death is preceded by a marked decrease in
efficacy required to achieve complete disease remission in the concentration of cellular adenine triphosphate following
many patients. Improvements to the therapeutic indices of 1–2 days of treatment; cells then remain viable for an addi-
anticancer agents will come from either 1) increased agent’s tional 2–3 days before sharply declining in numbers.25 While
potency, which will tend to decrease the minimum effective treatment of patients with the drug was determined to be well
dose required or 2) an increase in agent selectivity for cancer, tolerated and safe at the doses used (Phase II recommended
which should improve MTD. Ideally, both the potency and dose: continuous intravenous infusion 0.126 mg/m2/h for
selectivity of an agent could be improved.
One possible route toward such improvements is achieved topenia) prevented the delivery of an efficacious dose.29
through the conjugation of an anticancer or cytotoxic agent to
Our research group has recently reported a new family of
4 consecutive days), the dose-limiting toxicity (thrombocy-
a tumor-targeting vector, such as a peptide or antibody, pro- carborane-containing Nampt inhibitors, which exhibit up to
ducing peptide–drug conjugates (PDCs), and antibody–drug 10-fold higher activities than FK866 in several cancer cell
conjugates (ADCs).1–5 The number of molecules of a cytotoxic lines in vitro and a ~100-fold greater inhibition of Nampt.26
agent that is necessary to kill a cancer cell must be ideally The most potent of these molecules exhibit sub-nanomolar
much less than the maximum number able to be delivered by half-maximal inhibitory concentrations (IC50) in cancer
the targeting vector. This limitation has led to the focus of cur- cells, in vitro. Recently, these molecules were tested through
rent research efforts on the use of exceptionally potent small the National Cancer Institute’s Developmental Therapy
molecules, such as the microtubule agents auristatin and may- Program (DTP) NCI-60 and our molecule 1-(4′-(trans-3″-
tansine for the syntheses of new bioconjugates.6–8 However, the (3‴-pyridyl)acrylamido)butyl)-1,7-dicarbadodecaborane
cytotoxicity of these molecules is not limited to cancer cells (MC4-PPEA) was the most potent Nampt inhibitor tested, to
and their delivery to healthy cells must be minimized.
date, exhibiting low growth inhibition of 50% of cells (GI50)
A new and promising target for the treatment of cancer is concentrations in all 60 of the standardized cancer cell lines,
nicotinamide phosphoribosyltransferase (Nampt; aka visfatin, representing many different types of cancer. In addition,
pre-B-cell colony enhancing factor 1 [PBEF1]). Nampt is the the sub-nanomolar potency of our agent MC4-PPEA was
first and rate-limiting enzyme in the mammalian nicotinamide observed in vivo using small animal experiments.30
adenine dinucleotide (NAD)+ recycling pathway, catalyzing
We recently reported 1-(hydroxymethyl)-7-(4′-(trans-
the conversion of nicotinamide-to-nicotinamide mononucle- 3″-(3‴-pyridyl)acrylamido)butyl)-1,7-dicarbadodecaborane
otide. Over the past several years, it has been revealed that (hm-MC4-PPEA), a new derivative of MC4-PPEA.31 The
Nampt activity is highly implicated with many diseases, structure of these inhibitors is depicted in Figure 1. In vitro
including cancer.9–11 Overexpression of Nampt increases assays against three breast cancer cell lines revealed that
SIRT1 activity.12 SIRT1 and PARP, two NAD+-dependent the presence of the hydroxymethyl group does not greatly
enzymes, obtain NAD+ primarily from the Nampt-controlled diminish the activity of this new molecule and the group pro-
recycling pathway.12 Nampt has been shown to be upregulated vides a convenient linkage for covalent attachment through
in many/most cancers,13–20 and this overexpression is highest carbonates, carbamates, esters, etc.
in aggressive and refractory cancers.9,21 Nampt overexpres-
In the present study, we focus on tailoring a hydrolyzable
sion is predictive of poor response to the treatment of various linker, covalently attached to our therapeutic molecule for
cancers,9,21 including breast cancer.22 Most healthy cells do possible conjugation with various targeting vectors. Herein,
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clickable prodrugs bearing potent and cleavable nampt inhibitors
1
1
+
1
+
1
+
+2
2
2
0&ꢁꢀ33($
KPꢀ0&ꢁꢀ33($
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Figure 1 Molecular structure of Mc4-PPea and hm-Mc4-PPea.
Abbreviations: hm-Mc4-PPea, 1-(hydroxymethyl)-7-(4′-(trans-3″-(3′″-pyridyl)acrylamido)butyl)-1,7-dicarbadodecaborane; Mc4-PPea, 1-(4′-(trans-3″-(3′″-pyridyl)
acrylamido) butyl)-1,7-dicarbadodecaborane.
the synthesis of three prodrugs of hm-MC4-PPEA – 5, 7, and TopSpin. Chemical shifts (δ, ppm) for 1H and 13C were refer-
11 each bearing a terminal azide moiety for the attachment to enced to residual solvent peaks. Mass spectra were obtained
vectors through the facile click-chemistry cycloaddition reac- on an ABI Q-Star under both positive and negative modes,
tion – is described.32 This scheme promises drug conjugation reported in m/z, and analyzed using the Mariner Biospectrom-
using mild conditions, while protecting the integrity of both etry Workstation by PerSeptive Biosystems. HPCORE Chem-
the targeting vector and the cleavable linker. Stability of these Station was used for data analysis. Triply distilled water was
prodrugs was tested under various conditions, and the data used as the eluent with 5%–95% gradient of high-performance
obtained will be used to prepare future PDCs and ADCs for liquid chromatography (HPLC) grade acetonitrile.
targeted delivery. A simple fluorophore (14) bearing a terminal
alkyne is utilized to click onto our target compounds and moni- Prodrug stability studies
tor the stability of these prodrugs under various conditions. To Samples for stability tests were prepared by dissolving each
the best of our knowledge, these new molecules are the first analyte in a 50:50 solution of acetonitrile and the appropriate
reported conjugatable prodrugs of a potent Nampt inhibitor.
buffer. For serum stability experiments, a 5 μL solution of each
prodrug dissolved in acetonitrile was added to 200 μL of horse
serum and sonicated briefly in a water bath. At time intervals,
which were dependent on the apparent stability of each prodrug
Materials and methods
Materials
All manipulations were performed under nitrogen atmo- under the test conditions, samples were injected into an Agilent
sphere unless stated otherwise. All anhydrous solvents, 1100 Series HPLC System equipped with both a UV–visible
n-butyllithium (n-BuLi; 2.5 M solution in hexane), para- detector and a fluorescence detector. Agilent ChemStation
formaldehyde (PFA), 1-chloro-4-iodobutane (Cl(CH2)4I), was used for both data collection and analysis. Triply distilled
HCl (37%), sodium azide (NaN3), sodium iodide (NaI), water was used as the eluent with a 5%–95% gradient of HPLC
potassium iodide (KI), triphenylphosphine (PPh3), sodium grade acetonitrile. The retention time and the percent area of
sulfate (Na2SO4), magnesium sulfate (MgSO4), sodium each peak were recorded. The retention time of the free drug
thiosulfate (Na2S2O3), trans-3-(3′-pyridyl)acrylic acid was measured using a sample containing pure compound (hm-
(C5NH4(CH)2COOH), thionyl chloride (SOCl2), Hünig’s base MC4-PPEA). This sample produced a single peak on the UV
or N,N-diisopropylethylamine (DIPEA), pyridine (C5H5N), chromatogram, whileeachprodrugwasobservedassinglepeak
chloromethyl chloroformate (ClCH2CO2Cl), diethylene onboththeUVandfluorescencechromatograms. Thecleavage
glycol, tosyl chloride (TsCl), silver oxide (Ag2O), prop- of each prodrug resulted in the appearance of two peaks, one of
argyl bromide (CHCCH2Br), chromium trioxide (CrO3), these corresponding with free drug (hm-MC4-PPEA) and the
and dansyl chloride were purchased from Aldrich and used other associated with the liberated fluorescent linker.
as received. All solvents were dried using 3 Å molecular
sieves (20%, m/v).33 Silica gel was purchased from Sorbent cell cultures and treatment
Technologies (60 Å, 63–200 μm).
All cell lines were obtained from American Type Culture
Collection (ATCC) (Manassas, VA, USA). An MTT assay
kit was purchased from Promega Corporation (Fitchburg,
instrumentation
Nuclear magnetic resonance spectra were recorded on Bruker WI, USA). FK866 was purchased from Enzo Life Sciences
DRX300, DRX500, Bruker Ultrashield 400, and Ultrashield (Farmingdale, NY, USA). T47D and MCF-7 cells were
500 Plus Nuclear Magnetic Spectrometers and analyzed using maintained in DMEM containing 350 μL/L gentamicin and
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Sadrerafi et al
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25 mL/L fetal bovine serum. 184A1 cells were maintained
To prepare the carbamate prodrug 5, 4-nitrophenyl
in HuMEC media containing 175 μL/L gentamicin, HuMEC chloroformate was first reacted with the hydroxyl group of
supplement, and bovine pituitary extract. The cells were hm-MC4-PPEA, forming the amine reactive intermediate 4
grown to confluency in either T-12.5 or T-25 cm2 tissue (Scheme 1).
culture flasks using an incubator maintained at 5% CO2
The carbonate prodrug 7 was prepared in two steps by first
humid atmosphere at 37°C. The MTT assay34,35 was car- reacting hm-MC4-PPEA with 2-chloroethyl chloroformate to
ried out as follows: T47D, MCF-7, and 184A1 cells were form the intermediate 6, followed by the displacement of the
plated in 96-well plates at a density of 10,000 cells/well for chloro group with an azide in a SN2 type reaction (Scheme 2).
overnight at 37°C. The cells were then treated with different In a similar fashion, chloroformate derivatized polyethylene
concentrations of the test agent and incubated for 72 h. The glycol linkers of varying lengths may be synthesized using
MTT reagent was added to the cells for 4 h for developing phosgene and PEGs bearing a single terminal halide.
formazan crystals. Following this, solubilization buffer was
The ester prodrug 11 was prepared by coupling an acid
added to the wells and the absorbance was measured at chloride derivatized linker with 10. Azido-diethylene glycol 8
570 nm. Each experiment was repeated four times, and the was first oxidized using Jones reagent. Postoxidation, the
standard deviations are reported.
newly formed carboxylic acid 9 was treated with thionyl
chloride to make the acid chloride 10. This intermediate, in
turn, was reacted with the hydroxyl group present on hm-
MC4-PPEA to form 11 in high yield (Scheme 3). To increase
Results and discussion
hydrolyzable linkers
Three different hydrolyzable groups were investigated to the solubility of the prodrug, different length of PEG linkers
attach hm-MC4-PPEA with an azido-linker, such as an ester, may be utilized in place of diethylene glycol.
carbonate, and carbamate. These moieties have been previ-
ously utilized for the preparation of drug conjugates.36 They Clickable fluorophore
range in stability and are known to hydrolyze under varying To act as a model for our conjugation strategy using click
conditions, including those found in the acidic environments chemistry and to serve as an aid for prodrug stability studies,
of many solid tumors.36
a fluorophore bearing an alkyne was synthesized. Diethylene
(a)
O
O
O
OTs
OTs
OH
+
HO
TsO
HO
77%, 8%
1a
1b
(b)
90%
(c)
66%
O
O
1b
N3
N3
NH2
N3
2
3
O2N
N
O
(d)
88%
H
N
hm-MC4-PPEA
O
O
O
4
N
O
H
(e)
3+4
N
O
N3
N
O
98%
H
O
5
Scheme 1 synthesis of the carbamate prodrug 5.
Notes: reagents and conditions: (a) Tscl, ag2O, Ki, DcM, 0°c 4 h, rT 2 h; (b) nan3, DMF, 60°c 4 h; (c) PPh3, et2O:etOac, 5% hcl(aq), 0°c, rT, overnight; (d) 4-nitrophenyl
chloroformate, pyridine, ThF, 0°c, rT, overnight; and (e) DiPea, DcM, rT, overnight.
Abbreviations: ag2O, silver oxide; DcM, dichloromethane; DiPea, N,N-diisopropylethylamine; DMF, dimethylformamide; et2O, diethyl ether; etOac, ethyl acetate;
hcl(aq), hydrochloric acid; hm-Mc4-PPea, 1-(hydroxymethyl)-7-(4′-(trans-3″-(3‴-pyridyl)acrylamido)butyl)-1,7-dicarbadodecaborane; Ki, potassium iodide; nan3, sodium
azide; PPh3, triphenylphosphine; rT, room temperature; ThF, tetrahydrofuran; Tscl, tosyl chloride.
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clickable prodrugs bearing potent and cleavable nampt inhibitors
1
2
+
1
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Scheme 2 synthesis of the carbonate prodrug 7.
Note: reagents and conditions: (a) 2-chloroethyl chloroformate, DiPea, ThF, 0°c, rT, and (b) nan3, nai (cat.), DMF, 60°c.
Abbreviations: DiPea, N,N-diisopropylethylamine; DMF, dimethylformamide; hm-Mc4-PPea, 1-(hydroxymethyl)-7-(4′-(trans-3″-(3‴-pyridyl)acrylamido)butyl)-1,7-
dicarbadodecaborane; nai, sodium iodide; nan3, sodium azide; rT, room temperature; ThF, tetrahydrofuran.
glycol was modified with an amine at one terminal end and The drug conjugates 15–17 (Scheme 5) were synthesized
an alkyne at the other end. The amine was then reacted with using the same conditions in high yield (75%, 99%, and 60%,
dansyl chloride to afford the stable sulfonamide bond of respectively). Proton and carbon NMR spectra of compounds
compound 14 (Scheme 4).
15–17 can be found in the Supplementary materials. It is
The azide containing prodrugs 5, 7, and 11 were noteworthy that these reactions were carefully optimized to
covalently bound to the alkyne containing fluorophore 14 achieve high yields in just under an hour.
through click type chemistry in high yields. Anhydrous
dimethyl sulfoxide was degassed by bubbling nitrogen gas linker stability test
through the solvent to minimize the presence of molecular The stabilities of drug conjugates (15–17) were tested in
oxygen and subsequent oxidation of the metal catalyst. phosphate-buffered saline (PBS) at normothermia (37°C)
Copper(I) iodide was used as the catalyst, and N,N,N′,N′,N″- using three different pH levels, 5.5, 7.5, and 9.5, as well as
pentamethyldiethylenetriamine was used as a tridentate in horse serum at physiological pH. The stability of each was
chelating ligand to stabilize copper(I) and prevent its oxida- monitored using reversed-phase HPLC equipped with both
tion.32 DIPEA was used as a base, and the reaction mixture diode array and fluorescence detection, allowing a quantita-
was sonicated in a water bath for 1 h at room temperature. tive measurement of the disappearance of each conjugate, as
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Scheme 3 synthesis of the ester prodrug 11.
Notes: reagents and conditions: (a) nan3, h2O, 90°c, overnight; (b) crO3, h2sO4, acetone, 0°c, rT, 18 h; (c) sOcl2, pyridine, DcM; (d) pyridine, DcM, 0°c, rT.
Abbreviations: crO3, chromium trioxide; DcM, dichloromethane; h2sO4, sulfuric acid; hm-Mc4-PPea, 1-(hydroxymethyl)-7-(4′-(trans-3″-(3‴-pyridyl)acrylamido)butyl)-
1,7-dicarbadodecaborane; nan3, sodium azide; rT, room temperature; sOcl2, thionyl chloride.
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ꢀDꢁ
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2
2
2
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6
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2
1+
2
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Scheme 4 Clickable fluorophore.
Notes: reagents and conditions: (a) nah, DMF, rT, overnight; (b) 1) PPh3 and 2) h2O, ThF, rT, overnight; and (c) DiPea, ThF, 0°c, rT, overnight.
Abbreviations: DiPea, N,N-diisopropylethylamine; DMF, dimethylformamide; nah, sodium hydride; PPh3, triphenylphosphine; rT, room temperature; ThF, tetrahydrofuran.
well as the emergence of the hydrolytic degradation products,
A marked difference was observed between the stability
including the appearance of a peak corresponding with that of the prodrug linkers incubated in PBS of varying acidity
of the free drug (hm-MC4-PPEA). Graphs depicting the and those measured in serum. While the carbamate linkage
stability of 15–17 over several days for each test condition was stable under all conditions tested, including in serum,
are shown in Figures S1–S3.
the ester and carbonates were far more labile in serum
1
2
1
+
1
2
1
1
2
2
2
6
2
2
+
1
1
2
1
+
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ꢀ
ꢀꢁꢂ
1
+
2
6
+
1
1
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2
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1
2
2
2
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2
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1
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2
1
2
6
2
2
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1
1
1
+
2
2
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Scheme 5 Prodrugs linked to a fluorophore through click chemistry.
Note: reagents and conditions (all three reactions follow the same procedure): DiPea, PMDeTa, cui, DMsO, sonicated in water bath for 1 h at room temperature.
Abbreviations: cui, copper(i) iodide; DiPea, N,N-diisopropylethylamine; DMsO, dimethyl sulfoxide; PMDeTa, N,N,N′,N′,N″-pentamethyldiethylenetriamine.
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clickable prodrugs bearing potent and cleavable nampt inhibitors
Table 1 Measured stability of prodrugs (15–17) in serum and in PBs of varying ph at 37°c
Compound
Structure
Half-life (t1/2)
PBS, pH 5.5 (days)
PBS, pH 7.4 (days)
PBS, pH 9.5 (days)
Horse serum (h)
2
15
stable
stable
stable
stable
2
1
+
2
2
16
17
.600
.200
18.9
5.4
1.7
1.5
4.6
1.0
2
2
2
2
2
Abbreviation: PBs, phosphate-buffered saline.
than in PBS. Human blood serum is known to contain
several classes of esterase enzymes, including albumin,
butyrylcholinesterase, and paraoxonase.37 The horse serum
that was utilized contained cholinesterase that resembles
those found in human blood serum and is responsible for the
hydrolysis of many aliphatic esters.38 The liability of the ester
and carbamate moieties is presumably owing to the presence
of these classes of esterases.
control vs inhibitor concentration (Figure S4) is found in the
Supplementary materials.
The biological activities of 15–17, as well as those of the
free drug (hm-MC4-PPEA), were evaluated using the MTT
assay against the human breast cancer cell lines T47D and
MCF7, as well as the noncancerous, transformed, Nampt-
dependent human breast epithelium cell line 184A1 (Figures
S5–S7).34 The concentration-dependent cell viability exhibited
by 15–17 against these cell lines is summarized in Table 2.
Compound 15 exhibited a cell-line-dependent IC50 of
32.0±0.4, .200, and 9.1±0.9 nM for the T47D, MCF7, and
184A1 cell lines, respectively. Compound 16 exhibited an IC50
of 2.9±0.3, 81.0±2.5, and 4.7±1.0 nM for T47D, MCF7, and
184A1 cell lines, respectively, while compound 17 exhibitedan
IC50 of 5.5±0.5, 34.0±1.3, and 1.8±0.5 nM for these same cell
lines. For each prodrug, the biological activities correlate with
the stabilities observed in serum. The carbamate prodrug (15),
which was observed to be stable under all conditions tested,
exhibited the lowest biological activity of the three, with an
~5-fold decrease in activity below the parent compound (hm-
MC4-PPEA). The carbonate prodrug (16), which exhibited a
half-life of 4.6 h in serum, possessed higher activity than the
carbamate prodrug (15) in each of the cell lines, presumably
The quantity of prodrug remaining in each test sample
was measured over a period of several hours by integrating
the area under the curve for the analyte peak in the fluores-
cence chromatogram. The half-lives (t1/2) were calculated
by fitting the data to an equation and extrapolating the time
for which 50% of the prodrug remained. The results of the
stability test for prodrug–fluorophore conjugates (15–17) are
summarized in Table 1. As stated, the carbamate prodrug
(15) was observed to be stable under all test conditions. The
half-lives of 16 in pH buffers 5.5, 7.4, and 9.5 were .600
(extrapolated), 18.9, and 1.7 days, respectively, while the
half-life of 16 in serum was 4.6 hours. The ester prodrug (17)
was observed to be the least stable, exhibiting half-lives of
200 (extrapolated), 5.4, and 1.5 days, respectively, at pH 5.5,
7.4, and 9.5. In serum, the half-life of 17 was 1.0 hours.
recombinant nampt inhibition assay,
MTT assay, and ic50 measurements
Table 2 Measure of ic50 for agents hm-Mc4-PPea and 15–17
against the human breast cancer cell lines T47D, McF7, and
184a1 (noncancerous)
Previously, we reported that the unsubstituted parent
molecule MC4-PPEA inhibited Nampt, (IC50 =4.2 nM)
demonstrating a ~100-fold higher inhibitory activity than
FK866.26 We measured the inhibitory activity of hm-MC4-
PPEA using a commercially available colorimetric assay
and found that this molecule is also a potent inhibitor of
recombinant Nampt activity, exhibiting an IC50 concentration
of 6.8 nM. Protocol for the recombinant Nampt inhibition
assay as well as a graph illustrating percentage inhibition of
Compound
IC50 (nM) ± SD (n)
T47D
MCF7
184A1
hm-Mc4-PPea
4.9±0.3 (4)
32.0±0.4 (4)
2.9±0.3 (4)
5.5±0.5 (4)
34.0±1.5 (4)
.200
1.9±0.4 (4)
9.1±0.9 (4)
4.7±1.0 (4)
1.8±0.5 (4)
15
16
17
81.0±2.5 (4)
34.0±1.3 (4)
Abbreviations: hm-Mc4-PPea, 1-(hydroxymethyl)-7-(4′-(trans-3″-(3‴-pyridyl)
acrylamido)butyl)-1,7-dicarbadodecaborane;
ic50,
half-maximal
inhibitory
concentration.
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Sadrerafi et al
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2. Sievers EL, Senter PD. Antibody-drug conjugates in cancer therapy.
Annu Rev Med. 2013;64(1):15–29.
due to its cleavage and the resultant release of (hm-MC4-
PPEA)byesterases, whicharepresentinthefetalbovineserum
containing cell culture assay medium. The ester prodrug (17)
exhibited the highest cell inhibitory activity and was nearly
equal to that of the free drug (hm-MC4-PPEA) in each cell line
(IC50 4.9±0.3, 34.0±1.5, and 1.9±0.4 nM for T47D, MCF7, and
184A1 cell lines, respectively). These results are consistent
with the observed 1.0 h half-life of 17 in serum; during the cell
viability assay, essentially all the prodrug would be cleaved,
releasing an equal molar amount of hm-MC4-PPEA.
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Conclusion
The targeted and selective delivery of potent Nampt inhibitors
to cancer cells is a potentially new route for the treatment of
many different cancers, including breast cancer. The three
new prodrugs of our potent Nampt inhibitor (hm-MC4-PPEA)
described herein, each bearing an azide moiety, may be read-
ily conjugated with alkyne-bearing linkers for the preparation
of future ADCs and/or PDCs using facile click chemistry and
in excellent yields. In principle, different cancer types may
be targeted through conjugation using antibodies or peptides
associated with a specific cancer. To our knowledge, these
are the first conjugatable Nampt inhibitor prodrugs.
The ester, carbamate, and carbonate covalent linkers
investigated herein are quite stable toward hydrolytic degrada-
tion under neutral, mildly acidic, and mildly basic conditions.
However, the ester and carbonate containing derivatives are
readily cleaved in serum, presumably owing to the presence
of several different classes of esterase. Experiments are now
underway to prepare PDCs using these three prodrugs linked
to small cancer-associated peptides and to test their stability
using different classes of serum esterases.
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Acknowledgments
The authors are grateful to Dr Raghuraman Kannan and
Dr Ajit Prakash Zambre for their assistance with cell studies.
This article was presented at the 49th Midwest Regional
Meeting of the American Chemical Society as a poster
presentation with interim findings. The poster is available
online at researchgate.net: https://www.researchgate.net/
publication/283462753_Development_of_a_Nampt_Inhibi-
tor_Prodrug_and_the_Stability_of_its_Cleavable_Linkers.
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Disclosure
The authors report no conflicts of interest in this work.
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