Improved synthesis of MC4-PPEA and the biological evaluation of its hydroxymethyl derivative

Article abstract of DOI:10.1016/j.bmcl.2015.11.068

Nicotinamide phosphoribosyltransferase (Nampt) is an intriguing target for the treatment of many diseases, including cancer. Previously, our group demonstrated that carborane clusters may be used to increase the potency of small molecule inhibitors of Nam

Full text of DOI:10.1016/j.bmcl.2015.11.068

Bioorganic & Medicinal Chemistry Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Improved synthesis of MC4-PPEA and the biological evaluation of its
hydroxymethyl derivative
⇑
Keivan Sadrerafi, Emilia O. Zargham, Mark W. Lee Jr.
Department of Chemistry, University of Missouri, 601 South College Avenue, Columbia, MO 65211, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Nicotinamide phosphoribosyltransferase (Nampt) is an intriguing target for the treatment of many dis-
eases, including cancer. Previously, our group demonstrated that carborane clusters may be used to
increase the potency of small molecule inhibitors of Nampt over other, similarly sized organic moieties.
Herein, we report a greatly improved, gram-scale synthesis of our most potent agent: 1-(40-(trans-300-
(3000-pyridyl) acrylamide)butyl)-1,7-dicarba-closo-dodecaborane (MC4-PPEA). Additionally, the carborane
moiety of the molecule has been modified with a hydroxymethyl functional group to allow for its cova-
lent attachment to targeted prodrugs, the synthesis of which are underway. Using cell viability assays, we
demonstrate that this new derivative exhibits low, to mid-nanomolar potencies against human breast
cancer cell lines in vitro.
Received 18 August 2015
Revised 13 November 2015
Accepted 19 November 2015
Available online xxxx
Keywords:
Carborane
Nampt
Anticancer
Cancer
Ó 2015 Elsevier Ltd. All rights reserved.
There is a need for new antitumoral drugs which are more effi-
cacious against a wide range of cancers. This need is even greater
for advanced or recurrent cancers which too often respond poorly
to current treatments. A promising new target for the treatment of
cancer is nicotinamide phosphoribosyltransferase (Nampt), the
first and rate limiting enzyme in the mammalian NAD+ recycling
pathway. This enzyme catalyzes the conversion of nicotinamide
to nicotinamide mononucleotide. NAD+ is a vital cofactor used
throughout cellular respiration and thus, Nampt plays a key role
in regulating cellular metabolism. Nampt has been shown to be
upregulated in many/most cancers1–8 and this overexpression is
highest in aggressive and refractory cancers.9,10 FK866 was the first
known small molecule inhibitor of Nampt and has been investi-
gated in Phase I/II clinical trials against several cancers.11,12
Our research group recently reported a small family of carbo-
rane-containing Nampt inhibitors which exhibit low, to sub-
nanomolar potencies against four human cancer cell lines in vitro.13
Owing to the high boron content of carboranes and the extremely
high thermal neutron cross sectional area of the isotope boron-10,
these clusters have been extensively studied as building blocks for
the development of agents for boron neutron capture therapy
(BNCT).14,15 Apart from this area, there is an increasing interest
in the use of carboranes as ridged, hydrophobic moieties for drug
discovery. Several excellent reviews and books have been pub-
lished in this area.16–20 Our most potent derivative, MC4-PPEA
(4), exhibited a 10-fold higher activity and an approximately
100-fold greater inhibition of Nampt in vitro when compared with
the lead molecule FK866.13 We demonstrated that the carborane
clusters incorporated into the drug structure increased the potency
of the new molecules over other purely organic groups, such as
phenyl, or adamantly moieties. We also compared the potencies
for molecules incorporating ortho-, meta-, or para-carborane and
found that the molecule containing the m-isomer exhibited the
highest potency, suggesting that strong carborane–enzyme inter-
actions are formed, possibly through the formation of dihydrogen
bonds.21 Recent results obtained through the National Cancer
Institute’s Developmental Therapeutics Program confirm that 4 is
significantly more potent than FK866 in the 60 human cancer cell
lines employed by the NCI-60 screen.22
In the present study, we have optimized the synthesis of 4 using
a new route towards the intermediate amine and a new coupling
strategy, significantly improving the scalability, yield and purity
of the final product. We have also synthesized a new derivative
11 bearing a hydroxymethyl group appended to the meta-carbo-
rane moiety. This group will serve as a reactive moiety for the
future covalent attachment of the molecule, through various
Abbreviations: Nampt, nicotinamide phosphoribosyltransferase; MC4-PPEA,
1-(40-(trans-300-(3000-pyridyl)
acrylamide)butyl)-1,7-dicarba-closo-dodecaborane;
BNCT, boron neutron capture therapy; BOP, benzotriazole-1-yl-oxy-tris-(dimethy-
lamino)-phosphonium hexafluorophosphate; HMPA, hexamethylphosphoramide;
PAC, trans-3-(30-pyridyl)acryloyl chloride; IC50, half maximal inhibitory concentra-
tions; MTT assay, microculture tetrazolium assay.
⇑
Corresponding author. Tel.: +1 (573)884 2424; fax: +1 (573)882 2754.
E-mail address: leemw@missouri.edu (M.W. Lee).
http://dx.doi.org/10.1016/j.bmcl.2015.11.068
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Sadrerafi, K.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.11.068

2
K. Sadrerafi et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
C
C
C
C
C
hydrolysable linkers, to cancer-associated targeting vectors; efforts
which are now underway in our group.
H
H
H
Li
Li
Li
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
-BuLi
n
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
Our initial attempts to synthesize a conjugate of 4 focused on
the substitution of the nitrogen atom of the pyridyl moiety.
However, preparation of an oxymethylenecarbonyl derivative, sim-
ilar to that found in the Nampt inhibitor GMX1777, suffered from
very poor yields.23 Furthermore, the conjugate produced was
hydrolytically unstable. Attempts were also made to prepare an
ester derivative from a carboxyl group bound to the unsubstituted
carbon atom on the carborane cluster of 4 without success. New
efforts to introduce other reactive groups for producing conjugates
with enhanced potency are currently underway.
= BH
= C
Scheme 2. Metalation of m-carborane using n-butyllithium.
by converting trans-3-(30-pyridyl)acrylic acid to the acid chloride,
making it more susceptible to react with the amine without the
The potency of the new hydroxymethyl derivative was tested
using cell viability assays in two human breast cancer cell lines,
as well as a non-cancerous, chemically transformed breast epithe-
lial cell line which expresses Nampt.
In our initial report, we prepared 40 mg of 4 using a total of four
synthetic steps in seven percent yield overall.13 Here, we have
improved the overall synthetic yield to twenty one percent, while
increasing the scale of the reactions by a factor of 20. The synthesis
of 4, as previously described,13 and the sequence of reactions lead-
ing to optimized and improved synthesis of 4 are depicted in
Scheme 1.
To synthesize 4, m-carborane was first activated using n-butyl-
lithium, allowing for a metalation type reaction on the weakly
acidic C–H proton to create nucleophilic sites.16 These nucleophiles
can be used to prepare a wide range of carborane based derivatives
(Scheme 2).16
The lithiated m-carborane was then reacted with 1-chloro-4-
iodobutane, producing 1-(4-chlorobutyl)-m-carborane (1a)13 as a
colorless oil with a yield of 51%. The di-substituted side product
(1b) (yield of 32%) and some starting material were also recovered.
Both mono- and di-substituted products were isolated using col-
umn chromatography on silica gel. The reactions following this
step involved manipulation of the terminal end of the substituted
alkyl group placed on the carborane cage and did not involve the
cage itself.
need for a coupling agent, such as the benzotriazole-1-yl-
oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate (BOP)
used previously. In a one-pot, two-step reaction, trans-3-(30-pyri-
dyl)acrylic acid was treated with thionyl chloride; DMF was used
as a catalyst to achieve trans-3-(30-pyridyl)acryloyl chloride. Sulfur
dioxide and hydrochloric acid gas were produced as byproducts
and removed in vacuo. The product was redissolved in THF and
the remaining acid was captured using Hünig’s base. A THF solu-
tion of 3 was then added drop-wise and the reaction mixture
was allowed to stir overnight. This reaction afforded 1-(40-(trans-
300-(3000-pyridyl)acrylamido)butyl)-1,7-dicarbadodecaborane (4) as
an orange foam with 69% yield, a significant improvement over
the 36% yield previously observed.13
One reason that the use of BOP as a coupling reagent was
avoided was owing to the challenge of isolating the product from
the many byproducts that result from that reaction. One such
byproduct was hexamethylphosphoramide (HMPA) which is a
polar aprotic solvent having a high boiling point (232.5 °C). HMPA
is known to be carcinogenic and its complete removal was not pos-
sible using flash chromatography. Due to the presence of HMPA,
the product stayed in solution as a thick yellow oil. Previously, it
was necessary to utilize high pressure liquid chromatography
(HPLC) to recover 4 as a pure solid product. The purification of pro-
duct using HPLC was time consuming and was not practical at the
gram scale. Under the present conditions, the pure product can be
obtained using flash chromatography on silica gel.
The chlorine on compound 1a was exchanged with an azide
using sodium azide in an SN2 type reaction to afford 1-(4-azi-
dobutyl)-m-carborane (2) as a colorless oil with a yield of 100%.
Compound 2 was reduced using palladium on activated carbon
under a hydrogen atmosphere to afford aminobutyl-m-carborane
(3) as a yellow oil with 60% yield. The product 4 was synthesized
The synthetic route for hydroxymethyl functionalized 11 is
depicted in Scheme 3. An acidic proton on one of the carbon atoms
of m-Carborane was removed using n-butyllithium and the lithi-
ated m-carborane was then reacted with paraformaldehyde.24,25
After quenching the reaction with 2 N HCl(aq)
,
1-(hydrox-
O
O
C
C
C
C
C
C
C
C
N
H
H
(CH2)4
H
(CH2)4Cl
N
H
H
H
(CH2)4NH2
H
(CH2)4
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
b
C
C
C
C
a
C
c
C
d
C
C
C
C
(I)
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
N
C
O
C
C
C
C
C
63%
77%
36%
41%
O
C
C
C
C
C
C
C
C
N
H
(CH2)4NH2
H
(CH2)4
H
H
H
(CH2)4Cl
H
(CH2)4N3
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
g
C
C
C
C
C
C
C
C
C
C
C
f
e
H
h
(II)
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
N
C
C
C
C
C
51%
100%
60%
69%
1a
4
2
3
= BH
= C
Scheme 1. Previously reported synthetic pathway (I)13 and improved procedure (II) for compound 4. Reagents and conditions: (a) n-BuLi, THF, 4 h, rt, 1-chloro-4-iodobutane,
overnight, rt. (b) Potassium phthalimide, DMF, reflux, overnight. (c) Hydrazine, EtOH, 4.5 h reflux. (d) DMF, BOP, Et3N, trans-3-(30-pyridyl)acrylic acid, overnight rt. (e) n-BuLi,
THF, 0 °C–rt, 1-chloro-4-iodobutane, ꢀ78 °C to rt, overnight. (f) NaN3, NaI (cat.), DMF, 70 °C, overnight. (g) Pd/C, H2, MeOH, overnight, rt. (h) trans-3-(3-Pyridinyl)-2-propenyl
chloride, DIPEA, THF, ꢀ78 °C to rt, overnight.
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K. Sadrerafi et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
3
OTr
OH
H
C
C
C
C
C
c
a
C
C
C
b
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
92%
100%
C
59%
H
H
H
6
5a
OH
OTr
OH
C
C
C
C
C
e
C
C
C
C
C
C
d
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
N3
Cl
C
C
Cl
87%
99%
C
9
7
8
OH
N
H
N
C
C
C
f
C
C
C
C
C
C
C
g
C
C
C
HO
C
C
C
C
C
C
C
C
C
C
O
NH2
C
79%
82%
10
11
Scheme 3. Synthesis of hydroxymethyl functionalized MC4-PPEA (11). Reagents and conditions: (a) n-BuLi, benzene/Et2O, 0 °C–rt, paraformaldehyde, 0 °C–rt, 2 N HCl. (b)
Trityl chloride, DIPEA, DCM, rt. (c) n-BuLi, THF, 0 °C–rt, 1-chloro-4-iodobutane, ꢀ78 °C to rt, 2 h. (d) HCl (concd), MeOH:CHCl3, rt. (e) NaN3, NaI (cat.), DMF, 70 °C. (f) PPh3, H2O,
THF, rt. (g) trans-3-(3-Pyridinyl)-2-propenyl chloride, DIPEA, THF, ꢀ78 °C to rt.
Figure 1. Percent cell viability of human breast cancer cell lines: MCF7, T47D, and 184A1 at various concentrations of 11.
ymethyl)-m-carborane (5a) was afforded as a white solid with a
yield of 59.3%. The di-substituted product (5b) (yield of 31.1%)
and some starting material were also recovered and isolated using
column chromatography on silica gel. The hydroxyl group was
then protected using a trityl group so that the second carbon on
the m-carborane cage could be manipulated. This was achieved
using trityl chloride and Hünig’s base to afford 1-(methyl triph-
enylmethyl ether)-1,7-dicarbadodecaborane (6) as a white solid
with a yield of 92.3%.
Compound 6 was further modified by activating the unsubsti-
tuted carbon on the m-carborane moiety using n-butyllithium
and then reacted with 1-chloro-4-iodobutane, producing 1-
(methyl triphenylmethyl ether)-7-(4-chlorobutyl)-m-carborane
(7) as a yellow oil with a yield of 100%. Following this, the trityl
protecting group on compound 7 was removed using concentrated
HCl to afford 1-(hydroxymethyl)-7-(4-chlorobutyl)-m-carborane
(8) as a yellow oil with a yield of 87.2%. In a manner similar to that
employed on 2, the chlorine on compound 8 was exchanged with
an azide using sodium azide to afford, 1-(hydroxymethyl)-7-(4-
azidobutyl)-1,7-dicarbadodecaborane (9) as a yellow oil with a
yield of 98.6%. Compound 9 was reduced using triphenylphosphine
(PPh3) instead of palladium on activated carbon. We observed the
presence of secondary and tertiary amines in small quantities
when reduction was done using palladium. Post reduction,
1-(hydroxymethyl)-7-(4-aminobutyl)-m-carborane (10) was
afforded as an orange oil with 87.5% yield.
The hydroxymethyl derivative of MC4-PPEA (11) was synthe-
sized using a method similar to that employed for 4 by converting
trans-3-(30-pyridyl)acrylic acid to trans-3-(30-pyridyl)acryloyl chlo-
ride (PAC) in a one-pot, two-step reaction. To minimize a reaction
between the hydroxyl group and PAC, both solutions of 10 and PAC
were prepared separately and chilled to ꢀ78 °C using an acetone/
dry-ice bath and the PAC solution was added drop-wise to the
solution containing 10. The reaction mixture was allowed to stir
overnight, while gradually being allowed to warm to ambient
temperature. This reaction afforded 1-(hydroxymethyl)-7-(40-
(trans-300-(3000-pyridyl)acrylamido)butyl)-m-carborane (11) as an
orange foam with 75.9% yield.
The activity of 11 was evaluated using MTT assay,26 against
three human cell lines: MCF7, T47D, (cancerous) and 184A1 (a
chemically transformed noncancerous breast epithelium). Figure 1
depicts the concentration dependent cell viability exhibited by 11
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4
K. Sadrerafi et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
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against these cell lines in vitro. The compound exhibited half max-
imal inhibitory concentrations (IC50 of 58.0 5.2 nM,
)
12.0 2.1 nM, and 3.9 1.3 nM for MCF7, T47D, and 184A1 cell
lines, respectively. Past studies, including our own, have demon-
strated that the biological activity of Nampt inhibitors is cell line
dependent and may vary over a decade, or more.27 The present cell
viability assays reveal that the addition of a hydroxymethyl moiety
to 4, producing compound 11, results in an approximate 10-fold
decrease in potency for the new molecule in these breast cancer
cell lines. This new molecule exhibits low, to mid- nanomolar
potencies in vitro and the presence of the hydroxymethyl group
should allow for the covalent attachment of this molecule, through
hydrolyzable linkers, to cancer-associated targeting vectors.
In conclusion, a significantly improved synthesis of MC4-PPEA
has been described. A new hydroxymethyl functionalized deriva-
tive of 4 was also synthesized in high yield. Low, to mid-nanomolar
potencies of the new derivative were observed in three human
breast cell lines in vitro. The hydroxymethyl moiety of 11 should
allow for the covalent attachment of this molecule, through
hydrolyzable linkages, to cancer-associated targeting vectors, such
as nanoparticles, proteins, or antibodies. The selective delivery of
highly potent Nampt inhibitors to tumor tissue might overcome
some of the limitations observed from early clinical trials of
FK866/APO866.
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This work was financially supported by a grant from MIZZOU
Advantage at the University of Missouri, United States. The authors
would like to thank Mrs. Ellen E. Moore for proof reading the Letter.
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Supplementary data
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Supplementary data (experimental procedures, characteriza-
tion data for compounds, copies of NMR spectra, and information
regarding cell culture and treatment) associated with this article
can be found, in the online version, at http://dx.doi.org/10.1016/j.
bmcl.2015.11.068.
References and notes
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Please cite this article in press as: Sadrerafi, K.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.11.068