Structure-based drug design of novel carborane-containing nicotinamide phosphoribosyltransferase inhibitors

Article abstract of DOI:10.1016/j.bmc.2019.05.013

A series of carborane-containing NAMPT inhibitors were designed and synthesized based on the structure of compounds 1 and the NAMPT inhibitory activity was evaluated using NAMPT Colorimetric Assay. Among the compounds synthesized, compounds 2b and 2c show

Full text of DOI:10.1016/j.bmc.2019.05.013

Accepted Manuscript
Structure-based drug design of novel carborane-containing nicotinamide phos-
phoribosyltransferase inhibitors
Yasunobu Asawa, Kiyotaka Katsuragi, Akira Sato, Atsushi Yoshimori, Sei-ichi
Tanuma, Hiroyuki Nakamura
PII:
S0968-0896(19)30652-2
https://doi.org/10.1016/j.bmc.2019.05.013
BMC 14900
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
19 April 2019
8 May 2019
8 May 2019
Please cite this article as: Asawa, Y., Katsuragi, K., Sato, A., Yoshimori, A., Tanuma, S-i., Nakamura, H., Structure-
based drug design of novel carborane-containing nicotinamide phosphoribosyltransferase inhibitors, Bioorganic &
Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.05.013
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Structure-based drug design of novel carborane-containing nicotinamide
phosphoribosyltransferase inhibitors
Yasunobu Asawa,a,b Kiyotaka Katsuragi,c Akira Sato,c Atsushi Yoshimori,d
Sei-ichi Tanumac,* Hiroyuki Nakamura,a,*
a
Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of
Technology, Nagatsuta-cho, Midori-ku, Yokohama 226-8503, Japan
b School of Life Science and Technology, Tokyo Institute of Technology, Nagatsuta-cho, Midori-ku,
Yokohama 226-8503, Japan
c Faculty of Pharmaceutical Science, Tokyo University of Science, Noda, Chiba 278-8510, Japan
d
Institute for Theoretical Medicine, Inc., 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa
251-8555, Japan
* Corresponding authors. Tel.: +81-45-9245244; +81-4-7124-1501
e-mail: hiro@res.titech.ac.jp; tanuma@rs.noda.tus.ac.jp
Abstract
A series of carborane-containing NAMPT inhibitors were designed and synthesized based on the
structure of compounds 1 and the NAMPT inhibitory activity was evaluated using NAMPT
Colorimetric Assay. Among the compounds synthesized, compounds 2b and 2c showed significant
NAMPT inhibitory activity with IC50 values of 0.098 ± 0.008 and 0.057 ± 0.001 µM, respectively.
Docking simulation of compound 2 toward NAMPT using the crystal structure of the FK866-
NAMPT complex (PDB code: 2GVJ) with replacing the boron atom type by the C3 atom type of
carboranes predicted that the NAMPT inhibitory activity of 2c was improved by the hydrogen bond
formation between the carborane amide and H191 of NAMPT. Although dicarborane compounds 38,
50, 51, and 55 were synthesize aiming to two hydrophobic pockets present in the binding pocket of
NAMPT, their inhibitory activity was moderate.
Keywords
Carborane, nicotinamide phosphoribosyltransferase, inhibitor, structure-based drug design, docking
simulation

1. Introduction
Dicarba-closo-dodecaborane (carborane) is an icosahedral cluster containing two carbon atoms, ten
boron atoms (one for each vertex), and ten hydrogen atoms. Depending on the position of the two
carbon atoms, the three structural isomers of carborane are labeled o-, m-, and p-. Because of its
characteristic size and hydrophobicity, being comparable to that of hydrocarbons, carboranes have
been used as hydrophobic pharmacophores for drug design over the past 20 year.1,2 Various bioactive
carborane derivatives have been reported including: an estrogen receptor agonist,3 a transthyretin
amyloidosis inhibitor,4 a HSP60 inhibitor,5 a LNCaP inhibitor,6 a carbonic anhydrase 2 inhibitor,7
and a cyclooxgenase-2 inhibitor.8 In addition to hydrophobic interactions, carboranes can bind
strongly with biomolecules through a unique hydrogen bonding mechanism.9 Interestingly, the
strength of this interaction is different among the carborane isomers. Lee and coworkers reported the
synthesis of carborane-containing nicotinamide phosphoribosyltransferase (NAMPT) inhibitors 1a-c
and compared the dihydrogen bonding interactions among the three carborane isomers (o-, m-, and p-,
Figure 1).10 They found that compound 1b, containing the m-carborane, was the most active NAMPT
inhibitor.
NAMPT is the first rate-limiting enzyme in the mammalian nicotinamide adenine dinucleotide
(NAD+) recycling pathway, which converts nicotinamide to nicotinamide mononucleotide (NMN). It
has recently been elucidated that NAMPT plays a central role in metabolism, cell proliferation, cell
survival, and inflammatory responses. Inhibitors of this enzyme are novel and useful for the
treatment of many diseases, including cancer, Alzheimer's disease, diabetes, and arthritis. Therefore,
NAMPT is an attractive target for new drug discovery.11,12
Various NAMPT inhibitor scaffolds have been (Figure 1).13 For example, compound X, having a
1H-pyrrolo[3,2-c]pyridine moiety, was found to be a highly potent, amide-containing, NAMPT
inhibitor.14 Compound Y, having a 1,2,3-triazole ring, showed activity in xenograft and allograft
models, strengthening the potential of NAMPT inhibitors to act as anti-tumor drugs.15 FK866 is the
first known specific and highly potent inhibitor of NAMPT: it is now in clinical trials.16 Although the
superimposition of compounds 1a-c in the hydrophobic site of NAMPT was demonstrated based on
the crystal structure of the FK866-NAMPT complex,17,18 their detailed binding modes to NAMPT
have not been elucidated. In this paper, we developed several carborane-containing NAMPT
inhibitors using a structure-based drug design model supported by the Autodock vina calculation
program.19

O
m
o
p
N
O
O
N
N
H
H
= C
= BH
N
N
FK866
1a : o-carborane
1b : m-carborane
1c : p-carborane
O
S
N
N
O
O
N
N
H
N
N
N
H
N
N
X
Y
Figure 1. Structures of reported NAMPT inhibitors
The crystal structure of NAMPT/FK866 (PDB code: 2GVJ) was used as the template for the
docking simulations. However, the docking studies of carborane derivatives are trivial due to the lack
of a carborane forcefield. Although vigorous studies have been carried out for the docking
simulations of carborane derivatives using QM/MM modeling20 or MD simulations21, these methods
require extensive computation time and few compounds can be subjected to these calculation
methods. Therefore, carboranes are often approximated by substituting the boron atom for a C3-type
atom and boron clusters can be artificially treated as clusters with only carbon atoms.22,23
2. Results and Discussion
We designed carborane-containing NAMPT inhibitors based on the structure of compounds 1. We
divided compounds 1 into three moieties: head group, linker, and carborane moiety, as shown in
Figure 2. Based on the structures of NAMPT inhibitors FK866, X, and Y in Figure 1, three skeletons
were selected as the head group: pyridinylacrylamide skeleton A, pyrrolopyridine skeleton B, and
pyridinyltriazole skeleton C, respectively (Figure 2).13 For the linker, four and five carbon chains
were introduced between the head group and the carborane because these linkers will likely be
located in the tubular hydrophobic site of NAMPT. Finally, o-carborane was selected as the first
carborane moiety to employ for the expected ease of preparation. Additionally, methylene (CH2),
piperidyl carbonyl (Pip-CO), and amide (NHCO) groups were selected for the connection point
between the carborane and the linker. Both methylene and piperidyl carbonyl connections were
observed in compounds 1 and FK866, respectively. The amide group was selected based on our
docking simulation. As shown in Figure 3, the docking study of compound 2 suggested the formation
of a hydrogen bond interaction between the amide carbonyl group of compound 2 and histidine 191
(H191) of NAMPT (calculated as ~2.2 Å). The H191 residue is known to be an important amino acid
residue for the enzymatic activity of wild-type NAMPT: In fact, the mutation of H191R yields a
resistant cell line to FK866.24

O
N
H
N
1
Head group
Linker
C4, C5
Carborane
O
O
O
H
N
CB
N
CB
N
N
H
H
H
CH2
NHCO
N
N
O
A
B
N
CB
N
N
N
N
Pip-CO
C
Figure 2. Design of carborane-containing NAMPT inhibitors based on compound 1
m
o
p
O
H
N
N
H
O
N
2
2.2 Å
Figure 3. Docking simulation of compound 2 with the enzymatic site of NAMPT.
Compounds 1a, 2, 6-8, 10, and 11 were synthesized from alkynes 3 via the decaborane coupling
reaction according to Scheme 1. 1a was synthesized according to literature procedures.10 Next,
alkynes 3a-b were treated with decaborane in chlorobenzene under microwave irradiation25 to form
o-carborane derivatives 4a-b. Reduction with NaBH4 followed by quenching with HCl yielded the
corresponding ammonium salts 5a-b. Ammonium salts 5a-b were treated with (E)-3-(pyridin-3-
yl)acrylic acid or 1H-pyrrolo[3,2-c]pyridine-2-carboxylic acid to affording 6-8. For alkynes 3c-d,
they were treated with decaborane in chlorobenzene under microwave irradiated conditions at 130 ℃
to form the o-carborane derivatives. Treatment thereafter with NaN3 yielded the corresponding azides
9a-b. Hüisgen-type click reaction of the azide 9a-b was carried out with 3-ethynyl pyridine in the
presence of CuI catalyst to afford the corresponding pyridinyltriazoles 10 and 11, respectively.
Scheme 1. Synthesis of 1a, 2, 6-8, 10, and 11

O
N
n
H
o
N
1a : n=2
6 : n=3
a
b
PhthN
H3N
Cl
c
n
n
o
o
O
4a : n=2
4b : n=3
5a : n=2
5b : n=3
H
N
N
H
n
X
o
n
N
7 : n=2
8 : n=3
3a: X = NPhth, n=2
3b: X = NPhth, n=3
3c: X = Cl, n=2
N3
N
N
n
d
e
n
3d: X = Br, n=3
N
o
N
o
9a : n=2
9b : n=3
10 : n=2
11 : n=3
(a) B10H14, N,N-dimethylaniline, chlorobenzene, MW, 130 ℃, 86%-quant.; (b) (i) NaBH4,
IPA/H2O, r.t.; (ii) HCl, 80 ℃; (c) RCOOH, HATU, DIPEA, DMF, r.t. 26-81% 2 steps; (d) (i)
B10H14, N,N-dimethylaniline, chlorobenzene, MW, 130 ℃; (ii) NaN3, DMF, 80 ℃, 61–79% 2
steps; (e) 3-ethynylpyridine, CuI, Na ascorbate, DMF/H2O, 34-40%.
Next, the synthesis of compounds 2a-c and 19 from carborane carboxylic acids 15a-c was
performed according to Scheme 2. Amine 12 was prepared according to literature procedures.26 12
was treated with (E)-3-(pyridin-3-yl)acrylic acid in the presence of 1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide (EDCI), 1-hydroxybenzotriazole (HOBt), and N,N-diisopropylethylamine
(DIPEA) to afford compound 13. After deprotection of the tert-butoxycarbonyl (Boc) group on 13,
the resulting 14 was treated with acid chlorides 16a-c (which were prepared from the carborane
carboxylic acids 15a-c).27 Although the corresponding m- and p-carborane derivatives 2b and 2c
were obtained in good yields (43% and 63%, 2 steps, respectively), o-carborane derivative 2a was
unstable and readily decomposed, probably due to deboronation of o-carborane. It is known that o-
carborane is highly polarized in comparison with the other isomers. Furthermore, the boron atom at
the 3-position was likely activated by the electron withdrawing amide group, thus enhancing the
electrophilicity of o-carborane in compound 2a. In fact, the deboronation reaction could be
monitored by 11B NMR over time.
Compound 17 was prepared according to literature procedures28 and was reacted with (E)-3-
(pyridin-3-yl)acrylic acid in a similar manner to the preparation of compound 13. The resulting 18
was treated with p-carborane carboxylic acid 15c, after Boc deprotection, to afford the piperidyl
carbonyl p-carborane 19.
Scheme 2. Synthesis of 2 and 19

O
H
H
N
a
N
N
R
H2N
Boc
H
m
N
13: R = Boc
14: R = H
12
o
p
O
b
H
d
N
N
H
O
N
m
m
2a : o-carborane
2b : m-carborane
2c : p-carborane
o
p
o
p
c
HO
Cl
O
O
15a : o-carborane
15b : m-carborane
15c : p-carborane
16a : o-carborane
16b : m-carborane
16c : p-carborane
O
e
f
O
NBoc
NBoc
O
N
N
H
PhthN
N
p
H
17
N
N
18
19
(a) (E)-3-(pyridin-3-yl)acrylic acid, EDCI, HOBt, DIPEA, THF, r.t., 82%; (b) HCl, dioxane, r.t.;
(c) (COCl)2, toluene, reflux; (d) TEA, DCM, r.t., 43-63% 2 steps; (e) (i) NH2NH2-H2O, EtOH,
reflux; (ii) (E)-3-(pyridin-3-yl)acrylic acid, EDCI, HOBt, THF, r.t. quant. 2 steps; (f) (i) HCl,
dioxane, r.t.; (ii) 15c, HATU, DIPEA, THF, r.t. 3.6%.
For further investigation, we designed and synthesized compound 2c derivatives according to
Scheme 3. Compound 22 is one carbon shorter and compound 25 is one carbon longer type than 2c,
respectively. Compounds 30 and 31 have benzoyl and trimethylsilyl (TMS) substituents, respectively,
at the p-position of p-carborane. Compounds 34 and 35 are m- and p-carborane derivatives with a
hexyl linker instead of a butylamide linker (2c). Furthermore, to compare the stereoelectronic
properties of carborane and adamantane, compound 37 was also synthesized from the corresponding
adamantylcarboxylic acid 36.
Scheme 3. Synthesis of 2c derivatives

p
p
p
p
H
N
H
a
b
e
H
H
N
N
HO
N
N
Boc
O
O
O
O
O
21
15c
16c
22
25
p
H
p
N
H
N
c
N
H
X
N
Cl
O
O
O
23: X = OH
d
24: X = NPhth
R
R
O
p
p
R
h
i
H
N
p
HO
N
H
H
N
O
O
p-Carborane : R = H
26: R = COPh
28: R = COPh
29: R = TMS
30: R = COPh
31: R = TMS
f
g
27: R = TMS
o
p
O
o
p
o
p
j
e
N
3
H
PhthN
3
N
o-Carborane
p-Carborane
32: o-Carborane
33: p-Carborane
34: o-Carborane
35: p-Carborane
O
O
k
H
OSO2
CH3
HO
N
N
H
N
3
N
O
O
O
36
37
PhthN(CH2)6OTs
(a) 20, HATU, DIPEA, THF, r.t.; (b) (i) HCl, dioxane, r.t.; (ii) (E)-3-(pyridin-3-yl)acrylic acid,
HATU, DIPEA, THF, r.t., 41%, 3 steps; (c) 5-aminopentan-1-ol , TEA, DCM, r.t.; (d) Phthalimide,
PPh3, DIAD, THF, r.t.; (e) (i) NH2NH2-H2O, EtOH, reflux; (ii) (E)-3-(pyridin-3-yl)acrylic acid,
HATU, DIPEA, DMF, r.t. (25: 11%, 4 steps, 34: 10% 3 steps, 35: 12%, 3 steps); (f) n-BuLi,
PhCOCl, THF, -78 ℃ to 0 ℃, quant.; (g) n-BuLi, TMSCl, Et2O, -78 ℃ to 0 ℃; (h)
diisopropylamine, n-BuLi, CO2, THF, -78 ℃ to 0 ℃; (i) 14, HATU, DIPEA, DMF, r.t. (30: 43% 2
steps and 31: 25%, 3 steps); (j) n-BuLi, PhthN(CH2)6OTs,29 THF, -78 ℃ to 0 ℃; (k) 14, HATU,
DIPEA, DMF, r.t., 30%.
NAMPT enzymatic activity was evaluated using the CycLex® NAMPT colorimetric assay kit
according to the manufacturer’s protocols. The results are summarized in Table 1. Surprisingly, 6-11
did not exhibit higher inhibitory activity toward NAMPT than 1a and 1b. When designing the
carborane-containing NAMPT inhibitor, to the docking calculations indicated selecting a pyridyl
acrylamide head group (A) was optimal. Notably, the IC50 value of 1b was 0.37 ± 0.03 μM, which
was a little higher than the reported value.10 Next, the IC50 values of 2b, 2c, and 19, having a
carborane amide, was significantly lower than 1a, 1b, or FK866. In particular, 2c had the best
NAMPT inhibitory activity: 10-fold higher than that of FK866. These results could be explained by
the hydrogen bonding interaction between His191 and the carborane amide, as suggested by the
docking simulations (Figure 3). Also, unlike the previous studies, compounds having a p-carborane

group were more effective than the m-carborane derivative by 2-fold. Therefore, the dihydrogen
bonding, caused by polarization of carborane, may contribute further to the effectiveness of the p-
carborane.
On the other hand, compound 19, in which the phenyl group of FK866 was replaced by p-
carborane, exhibited significant inhibition of NAMPT, in a level similar to that of 1b (IC50 = 0.32 ±
0.05 μM), which was about twice as high as FK866. These results suggested that the three-
dimensional volume, in addition to hydrophobicity, of the carborane skeleton was more suitable for
the hydrophobic pocket of NAMPT.
Next, the NAMPT inhibitory activity among the compounds with carborane amide linkages, like
2b or 2c, were compared. First, the NAMPT inhibitory activity was markedly affected by linker
length because the IC50 value of 22, which had a shorter linker, was significantly increased while that
of 25, which had a longer linker, did not increase. We believe the binding site in NAMPT has a
cylindrical shape, and when the linker was short (22), the head group could not reach the active site
so that little inhibitory activity was observed. For 30, the longer linker could be contained within the
binding site; however, the distance between the carborane amide of 30 and H191 was too long for
creating a hydrogen bond. Thus, the affinity of 30 with NAMPT was lower than that of 2c.
Second, the NAMPT inhibitory activity of 30 and 31 were lower than that of 2c. This decrease in
affinity with NAMPT may have resulted from the steric hindrance of the additional substituent on
the carborane.
Third, we elucidated that the hydrogen bond between amide bond of 2c and H191 of NAMPT could
increase the binding affinity. Comparing the IC50 value of 2c with 35, the NAMPT inhibitory activity
of 35 was significantly lower than that of 2c. Moreover, 34, in which the p-carborane in 35 was
changed to an o-carborane, showed the same result.
Finally, we elucidated that the carborane moiety increased the NAMPT inhibitory activity from
comparing 2c and 37. 2c had a 100-fold stronger inhibitory activity than adamantane-containing 37.
Thus, 2c was a potent NAMPT inhibitor because of the appropriate linker length, having a
carborane moiety, and the presence of a hydrogen bond between the carborane amide and H191.
Table 1. IC50 values of the various carborane-containing derivatives
Compound
NAMPT IC50 [µM]
0.61 ± 0.01
0.37 ± 0.03
4.9± 0.2
> 30
1a
1b
6
7
8
> 30
10
11
2b
> 30
> 30
0.098 ± 0.008

2c
19
0.057 ± 0.001
0.32 ± 0.05
> 30
22
25
0.17 ± 0.02
0.63 ± 0.04
0.18 ± 0.01
0.37 ± 0.02
0.25 ± 0.06
8.0 ± 1
30
31
34
35
37
FK866
0.62 ± 0.05
Each compound was mixed with NAMPT in advance and the 1-step assay buffer from the
CycLex® kit was added. The absorbance at 450 nm was measured and the inhibition rate of the
compound against NAMPT could be calculated (n = 3).
To investigate the anticancer properties of the synthesized compounds, the cell viability of
compounds 1a, 2b, and 2c, which showed significant NAMPT inhibition, toward A549 (lung),
MDA-MB 468 (breast), and HeLa (cervical) cells was evaluated by a MTT assay. The results are
summarized in Table 2. Unfortunately, the synthesized compounds did not show higher cytotoxicity
than FK866 for any of the cells tested. We believe that the water solubility of 2b or 2c was improved
due to the carborane amide, resulting in decreased cell membrane permeability. On the other hand, a
comparison between 2b and 2c indicated that the cytotoxicity difference between carborane isomers
was independent of the specific cell line and the cytotoxicity increased proportionally to the NAMPT
inhibitory activity.
Table 2. Cytotoxicity of the synthesized compounds against cancer cell lines.
A549 [nM]
17.8 ± 8
HeLa [nM]
32.3 ± 7
MDA-MB 468 [nM]
21.2 ± 3
1a
2b
14.4 ± 5
25.6 ± 7
11.8 ± 3
2c
3.77 ± 0.8
1.74 ± 0.5
14.4 ± 2
7.19 ± 0.6
FK866
3.33 ± 0.3
2.83 ± 0.4
Comparing the NAMPT inhibitory activity of 19 with FK866, it was found that the activity was
improved by replacing the phenyl group with a carborane. The activity was thought to be improved
by the three-dimensional hydrophobicity of carborane. Furthermore, as shown in Figure 5-A, the
docking simulation performed with 19 and NAMPT suggested that the carborane of compound 1
would be located at the position of the piperidine moiety of 19. In addition, the IC50 value of 1a, with
a C4-linker, was 0.61 ± 0.01 µM and that of 34, with a C6-linker, was 0.37 ± 0.02 µM, whereas that

of 6, with a C5-linker, was 4.9 ± 0.2 µM. This activity cliff was interesting and lead us to believe that
there were two potential carborane binding pockets in the active site of NAMPT.
Based on the hypothesis, dicarborane compound 38, containing two carboranes, was designed. The
docking simulation showed good overlap with 19 (Figure 5-C). Each carborane of 38 was located in
the carborane binding sites for 1a and 34 (Figure 5-D). Thus, we decided to synthesize the
dicarborane compounds for further study.
A:
C:
D:
B:
O
p
m
O
N
2
H
N
38
O
N
H
n
o
N
1a: n = 4
6: n = 5
34: n = 6
Figure 5. Docking experiments leading to the design of dicarborane compound 38: (A)
Comparisons of the docking models of 1a (yellow) and 19 (green). (B) Structure of dicarborane
compound 38. (C) Comparisons of the docking models of 1a (yellow), 19 (green), and 38 (black).
(D) Merged images of dicarborane compound 38 with 1a (green), 6 (blue), and 34 (yellow).
The synthesis of the dicarborane derivatives 38, 50, 51, and 55 are shown in Scheme 4. p-
Carborane was treated with n-BuLi in THF to generate the lithiated p-carborane. This was then
reacted in situ with tert-butyl(4-iodobutoxy)dimethylsilane (39) to yield compound 40. The lithiation
of 40 with n-BuLi in THF was followed by formylation with methyl formate to afford 41. This was
then treated with lithiated m- or p-carboranes and followed by a HCl in dioxane work-up to yield the
desilylated dicarboranederivatives 42 and 43, respectively. The primary alcohol of 42 was tosylated

and then substituted with azide, and the resulting azide 45 was oxidized with Dess-Martin
periodinane to yield dicarboranyl ketone 46. After Staudinger reduction of the azide in 46 to an
amino group, the resulting amine 47 was treated with (E)-4-(pyridin-4-yl)acrylic acid to afford
dicarboranyl ketone 38. On the other hand, p-carborane derivative 43 was converted to phthalimide
by a Mitsunobu reaction to afford compound 48, which was then converted to the corresponding
amine 49 using hydrazine. Amine 49 was treated with the (E)-4-(pyridin-4-yl)acrylic acid to yield
dicarborane carbinol 50. This was then oxidized to dicarboranyl ketone 51. In a similar manner,
dicarboranyl ketone 55, in which the m-carborane was bonded to the linker instead of p-carborane,
was also synthesized.
Scheme 4. Synthesis of the dicarborane compounds
O
OH
p
p
p
H
p
m
p
a
b
c
TBSO
TBSO
HO
2
2
2
p-Carborane
40
41
42: m-Carborane
43: p-Carborane
OH
O
d
f
p
p
m
m
42
X
N3
2
2
46
44: X = OTs
45: X = N3
e
O
O
p
g
h
m
O
p
m
N
H2N
2
H
2
N
47
38
OH
Z
p
p
O
i
h
43
p
p
Y
N
H
2
2
N
48: Y = NPhth
49: Y = NH2
50: Z = CHOH
51: Z = CO
j
f
p
a,b
k
H
m
m
m
TBSO
HO
2
2
O
O
m-Carborane
52
53
p
p
O
d,e
g,h
m
m
N3
N
2
2
H
O
O
N
54
55
(a) 39, n-BuLi, THF, 0 ℃; (b) n-BuLi, HCO2CH3, THF, -78 ℃; (c) (i) Carborane, n-BuLi, Et2O, -
78 ℃ to 0 ℃; (ii) HCl, dioxane, r.t.; (d) TsCl, TEA, DMAP, DCM, r.t.; (e) NaN3, DMF, 80 ℃,
11%, 6 steps; (f) Dess-Martin periodinane, DCM, r.t., 53%; (g) PPh3, THF/H2O, r.t.; (h) (E)-4-

(pyridin-4-yl)acrylic acid, EDCI, HOBt, DIPEA, THF, r.t., (38: 43%, 3 steps, 50: 29%, 3 steps, 55:
37%, 7 steps; (i) Phthalimide, PPh3, DIAD, THF, r.t.; (j) NH2NH2-H2O, EtOH, reflux; (k) (i) p-
Carborane, n-BuLi, Et2O, -78 ℃ to 0 ℃; (ii) Dess-Martin periodinane, DCM, r.t. (iii) HCl,
dioxane, r.t.
Next, we evaluated the NAMPT inhibitory activity of the synthesized compounds (Table 4). First,
it was clear that the two hydrophobic pockets were indeed present since the dicarborane compounds
showed a NAMPT inhibitory activity on the μM order. However, these compounds were less active
than FK866 or 1a and we believed that correct placement of the two carboranes was critical. In fact,
the flexible secondary alcohol linker for the two carboranes was superior to the carbonyl linker in 51
for yielding an activity lower than 10 μM. Compound 55 was the most active diborane compound, so
the placement of a terminal carborane was important for NAMPT inhibitory activity. Interestingly,
comparing between 38 and 51, the NAMPT inhibitory activity of 38, in which the terminal carborane
was a m-isomer, was higher than that of 51, in which the terminal carborane was a p-isomer. This
may suggest a difference in the manner that carborane isomers interact with proteins, specifically
through a dihydrogen bond .
Table 4. Inhibition of NAMPT by the dicarborane compounds
Compound
IC50 [µM]
0.61 ± 0.01
3.76 ± 0.5
4.51 ± 0.9
> 10
1a
38
50
51
55
2.92 ± 0.5
3. Conclusion
We designed and synthesized a series of carborane derivatives as NAMPT inhibitors. Among the
compounds synthesized, 2c was found to be the most potent NAMPT inhibitor with an inhibitory
activity more than 10 times higher than that of FK866. By performing docking simulations, the
NAMPT inhibitory activity of 2c was predicted to be improved because of the formation of a
hydrogen bond between the carborane amide in 2c and H191 in NAMPT. In fact, compound 35,
without a carborane amide, was approximately 4-fold less potent than 2c, indicating that the
carborane amide moiety is important for high NAMPT inhibitory activity. The activity was also
believed to have improved by the three-dimensional hydrophobicity of carborane (comparing 2c and
37). Using 2c to further our structure-activity relationship study, the following three issues were
critically important: (1) a 4 carbon-chain linker was optimal, (2) a little space around the carborane is
needed for flexibility and carborane positioning, and (3) the carborane amide skeleton was needed to
establish a hydrogen bond with H191. Although dicarborane compounds 38, 50, 51, and 55, designed

by docking simulations, showed moderate NAMPT inhibitory activity, they were less effective than
2c. The findings in this study are not only useful for the further design of NAMPT inhibitors, but
also for carborane-based drug development.
4. Experimental Section
General Methods
NMR spectra were recorded on a Bruker biospin AVANCE II (400 MHz for 1H, 100 MHz for 13C)
or a Bruker biospin AVANCE III (500 MHz for 1H, 125 MHz for 13C) instrument in the indicated
solvent. Chemical shifts are reported in units per million (ppm) relative to the signal (0.00 ppm) for
internal tetramethylsilane for solutions in CDCl3 (7.26 ppm for 1H, 77.16 ppm for 13C).
Multiplicities are reported using the following abbreviations: s; singlet, d; doublet, dd; doublet of
doublets, t; triplet, q; quartet, m; multiplet, br; broad, J; coupling constants in Hertz. Mass spectra
were measured using a JMS-700 Mstation. HRMS (EI, 70 eV) was calibrated as perfluorokerosene.
All reactions were monitored by thin-layer chromatography carried out on 0.2 mm E. Merck silica
gel plates (60F-254) with UV light (254 nm) and were visualized using an aqueous alkaline KMnO4
solution, ninhydrin AcOH solution and/or p-anisaldehyde EtOH solution. Column chromatography
was performed on Silica Gel 60 N, purchased from Fuji Silysia Chemical Ltd. Microwave-assisted
synthesis were performed on microwave synthesizer (Biotage, Initiator). Compound 1a was known
and synthesized from 3a according to the literature procedures.
Synthesis of 1-(Phthalimido-N-pentyl)-1,2-dicarba-closo-dodecaborane (4b)
To a solution of alkyne 3b (131 mg, 0.541 mmol) and N,N-dimethylaniline (103 µL, 0.812 mmol) in
chlorobenzene, was added decaborane (79 mg, 0.649 mmol). The resulting mixture was stirred at
130 ℃ under argon atmosphere for 10 min with microwave synthesizer. The resulting mixture was
added 1M HCl and Et2O after which the product was partitioned between the aqueous and organic
layers. The aqueous layer was washed with Et2O and combined organic layers were washed with
brine, dried over sodium sulfate and concentrated under vacuum. Then, the crude materials were
purified by column chromatography on silica gel (40% EtOAc in hexane) afforded 4b as a white
solid (184 mg, 0.531 mmol, quant.). 1H NMR (400 MHz; CDCl3): δ 7.85-7.83 (m, 2H), 7.73-7.71 (m,
2H), 3.67 (t, J = 5.8 Hz, 2H), 3.56 (s, 1H), 2.19 (t, J = 6.0 Hz, 2H), 1.67 (q, J = 6.0 Hz, 2H), 1.51 (q,
13
J = 6.0 Hz, 2H),1.31 (q, J = 6.0 Hz, 2H), 2.5-1.6 (m, 10H); C NMR (125 MHz; CDCl3): δ 168.5,
134.1, 132.2, 123.4, 75.2, 61.1, 38.0, 37.6, 28.8, 28.2, 26.2; 11B NMR (160 MHz; CDCl3): δ -2.36, -
5.824, -9.35, -11.5, -12.3, -13.2; HRMS (ESI, pos) for C15H25B10NO2 (m/z): calcd 382.2788
(M+Na)+, found 303.2796.
Synthesis of 1-((E)-4-(3-(pyridin-3-yl)acrylamido)pentyl)-1,2-dicarba-closo-dodecaborane (6)
To a solution of phthalimide 4b (84 mg, 0.234 mmol) in 2-propanol: water (6:1, v/v), was added

NaBH4 (44 mg, 1.17 mmol). The resulting mixture was reacted at room temperature until the full
conversion of a starting material was observed. After which, 12 M HCl aq. was slowly added and
stirred at 80 ℃ for 6 h under argon atmosphere. The reaction mixture was concentrated under
vacuum. The resulting ammonium salt 5b was dissolved in water and washed twice with CH2Cl2.
The aqueous phase was concentrated under vacuum, and the residue was dissolved in ethanol and
filtered through a small cotton plug. After removal of ethanol under vacuum, the crude product was
used for the next step without further purification. To a solution of the crude 5b, (E)-3-(pyridin-3-
yl)acrylic acid (35 mg, 0.234 mmol), and 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-
b]pyridinium 3-oxid hexafluorophosphate (HATU) (89 mg, 0.234 mmol) in THF was slowly added
N,N-diisopropylethylamine (DIEA) (102 µL, 0.585 mmol). The resulting mixture was stirred at room
temperature under argon atmosphere until the full conversion of a starting material was observed.
The resulting mixture was added aq. NaHCO3 and EtOAc after which the product was partitioned
between the aqueous and organic layers. The aqueous layer was washed with EtOAc and combined
organic layers were washed with brine, dried over sodium sulfate and concentrated under vacuum.
Then, the crude material was purified by column chromatography on silica gel (10% MeOH in
CH2Cl2) afforded 6 as a white solid (20 mg, 0.0554 mmol, 27% from 4b). m.p. 167-168 ℃; 1H NMR
(500 MHz; CDCl3): δ 8.75 (s, 1H), 8.58 (d, J = 2.2 Hz, 1H), 7.78 (d, J = 4.0 Hz, 1H), 7.62 (d, J = 8.0
Hz, 1H), 7.33-7.30 (m. 1H), 6.43 (d, J = 8.0 Hz, 1H), 5.67 (s. 1H), 3.57 (s. 1H), 3.38 (ddd, J = 10,
5.0 Hz, 2H), 2.21 (t, J = 7.5 Hz, 2H), 1.61-1.50 (m. 4H), 1.37-1.33 (m. 2H), 2.5-1.9 (br. 10H); 13C
NMR (125 MHz; CDCl3): δ 165.3, 150.6, 149.3, 137.8, 134.5, 130.7, 123.8, 122.6, 61.17, 39.6, 38.1,
29.8, 29.5, 29.0, 26.4; 11B NMR (160 MHz; CDCl3): δ -2.29, -5.77, -9.33, -11.5, -12.2, -13.2; HRMS
(ESI, pos) for C15H28B10N2O (m/z): calcd 383.3104 (M+Na)+, found 383.3108.
Synthesis
of
1-(5-(1H-Pyrrolo[3,2-c]pyridine-2-carboxamido)butyl)-1,2-dicarba-closo-
dodecaborane (7)
This compound was prepared from compound 4a (42 mg, 0.123 mmol) and 1H-pyrrolo[3,2-
c]pyridine-2-carboxylic acid (20 mg, 0.123 mmol) using the procedure described for 6. Purification
1
by column chromatography (10% MeOH in CH2Cl2) gave 7 in 62% yield as amorphous. H NMR
(500 MHz; CD3CN): δ 8.92 (s, 1H), 8.26 (d, J = 6.0 Hz, 1H), 7.40 (d, J = 5.0 Hz, 1H), 7.06 (s, 1H),
4.12 (s, 1H), 3.34 (t, J = 6.0 Hz, 2H), 2.25 (t, J = 8.0 Hz, 2H), 1.94-1.91 (m. 2H), 1.53-1.48 (m. 2H),
13
2.5-1.6 (br. 10H); C NMR (125 MHz; CD3CN): δ161.8, 146.2, 143.0, 140.5, 133.8, 125.9, 108.0,
102.1, 77.4, 63.3, 39.2, 37.7, 29.4, 27.3; 11B NMR (160 MHz; CD3CN): δ -3.03, -6.31, -9.88, -11.60,
-12.02, -13.26; ; HRMS (ESI, pos) for C15H26B10N2O1 (m/z): calcd 361.3072 (M+H)+, found
361.3070.
Synthesis
dodecaborane (8)
This compound was prepared from compound 4b (18 mg, 52.4 µmol) and 1H-pyrrolo[3,2-
of
1-(5-(1H-Pyrrolo[3,2-c]pyridine-2-carboxamido)pentyl)-1,2-dicarba-closo-

c]pyridine-2-carboxylic acid (10 mg, 52.4 µmol) using the procedure described for 6. Purification by
column chromatography (10% MeOH in CH2Cl2) gave 8 in 26 % yield as a white solid. m.p. 148-
149 ℃; 1H NMR (500 MHz; CDCl3): δ 9.83 (bs, 1H), 9.00 (s, 1H), 8.35 (d, J = 5.5 Hz, 1H), 7.36 (d,
J = 6.0 Hz, 1H), 6.91 (s, 2H), 6.36 (s, 1H), 3.57 (s, 1H), 3.52-3.48 (m, 2H), 2.21 (t, J = 5.6 Hz, 2H)
1.66 (q, J = 7.6 Hz, 2H), 1.53 (q, J = 8.0 Hz, 2H), 1.38 (q, J = 8.5 Hz, 2H), 2.5-1.9 (br. 10H); 13C
NMR (125 MHz; CDCl3): δ 165.4, 150.6, 149.3, 138.0, 134.6, 130.6, 123.9, 122.5, 75.1, 61.4, 39.0,
37.6, 29.2, 26.5; 11B NMR (160 MHz; CDCl3): δ -2.17, -5.60, -9.25, -11.5, -12.2, -13.1; HRMS (ESI,
pos) for C15H27B10N3O1 (m/z): calcd 374.3237 (M+H)+, found 374.3238.
Synthesis of 1-(4-Azidebutyl)-1,2-dicarba-closo-dodecaborane (9a)
To a solution of alkyne 3c (48 µL, 0.400 mmol) and N,N-dimethylaniline (76 µL, 0.600 mmol) in
chlorobenzene, was added decaborane (57 mg, 0.480 mmol). The resulting mixture was stirred at
130 ℃ under argon atmosphere for 10 min with microwave synthesizer. The resulting mixture was
added 1M HCl and Et2O after which the product was partitioned between the aqueous and organic
layers. The aqueous layer was washed with Et2O and combined organic layers were washed with
brine, dried over sodium sulfate and concentrated under vacuum. The resulting crude material was
used for the next step without further purification. To a solution of the crude material in
dimethylformamide (DMF), was added sodium azide (78 mg, 1.20 mmol) at room temperature and
then the mixture was stirred at 80 ℃ under argon atmosphere for 12 h. The reaction was quenched
with water and the mixture was extracted with Et2O, washed with brine, dried over sodium sulfate
and concentrated under vacuum. The residue was purified by column chromatography on silica gel
1
(30% EtOAc in hexane) to afford azide 9a as color less oil (55 mg, 0.228 mmol, 61%). H NMR
(500 MHz; CDCl3): δ 3.57 (s, 1H), 3.30 (s, 2H), 2.25-2.21 (m, 2H), 1.56 (m, 4H), 2.80-1.50 (m,
13
11
10H); C NMR (125 MHz; CDCl3): δ 75.3, 61.7, 51.4, 38.2, 28.8, 27.0; B NMR (160 MHz;
CDCl3): δ -2.17, -5.60, -9.28, -11.5, -12.2, -13.1; HRMS (ESI, nega) for C6H19B10N3 (m/z): calcd
278.2241 (M+Cl)-, found 278.2242.
Synthesis of 1-(5-Azidepentyl)-1,2-dicarba-closo-dodecaborane (9b)
This compound was prepared from compound 3d (100 mg, 0.571 mmol) and decaborane (84 mg,
0.685 mmol) using the procedure described for 9a. Purification by column chromatography (30%
EtOAc in hexane) gave 9b in 79% yield as a colorless liquid. 1H NMR (500 MHz; CDCl3): δ 3.56 (s,
1H), 3.27 (t, J = 6.6 Hz, 2H), 2.21 (dd, J = 8.5 Hz, 17 Hz, 2H), 1.61-1.56 (m, 2H), 1.53-1.47 (m, 2H),
13
11
1.38-1.34 (m, 2H) ; C NMR (125 MHz; CDCl3): δ 75.1, 61.2, 51.2, 38.1, 28.9, 28.6, 26.2; B
NMR (160 MHz; CDCl3): δ -2.17, -5.60, -9.28, -11.5, -12.2, -13.1; HRMS (ESI, nega) for
C7H21B10N3 (m/z): calcd 254.2658 (M-H)-, found 254.2651.
Synthesis of 1-(4-(4-(Pyridin-3-yl)-1H-1,2,3-triazol-1-yl)butyl)-1,2-dicarba-closo-dodecaborane
(10)

To a solution of 3-ethynylpyridine (17 mg, 0.164 mmol) in DMF:H2O (4:1, v/v), were added CuI
(3.5 mg, 6.85 µmol), sodium ascorbate (7.2 mg, 0.0137 mmol) and azide 9a (33mg, 0.137 mmol).
The mixture was stirred at room temperature under argon atmosphere for 8 h. The reaction was
quenched with water and the mixture was extracted with CH2Cl2, washed with brine, dried over
sodium sulfate and concentrated under vacuum. The residue was purified by column chromatography
on silica gel (100% EtOAc) to afford 10 as a white solid (16.1 mg, 0.0467 mmol, 34%). m.p. 150-
151 ℃; 1H NMR (500 MHz; CDCl3): δ 8.99 (d, J = 1.8 Hz, 1H), 8.59 (dd, J = 1.8 Hz, 6.0 Hz, 1H),
8.20 (td, J = 2.4 Hz, 6.0 Hz, 1H), 7.83 (s, 1H), 7.39 (dd, J = 6.0 Hz, 9.6 Hz, 1H), 4.43 (t, J = 8.7 Hz,
2H), 3.59 (s, 1H), 2.27 (dd, J = 11 Hz, 21 Hz, 2H), 2.00-1.93 (m, 2H), 1.61-1.53 (m, 2H), 3.00-1.50
(m, 10H).13C NMR (125 MHz;CDCl3): δ 149.5, 147.2, 145.1, 133.1, 126.7, 123.9, 119.9, 74.5, 61.3,
49.8, 37.4, 29.6, 26.2; 11B NMR (160 MHz; CDCl3): δ -2.24, -5.65, -9.24, -11.6, -12.2, -13.0; HRMS
(ESI, pos) for C13H24B10N4 (m/z): calcd 345.3083 (M+H)+, found 345.3091.
Synthesis
of
1-(4-(4-(Pyridin-3-yl)-1H-1,2,3-triazol-1-yl)pentyl)-1,2-dicarba-closo-
dodecaborane (11)
This compound was prepared from azide 9b (58 mg, 0.198 mmol) and 3-ethynylpyridine (25 mg,
0.238 mmol) using the procedure described for 10. Purification by column chromatography (100%
EtOAc) gave 10 in 40% yield as a white solid. m.p. 85-86 ℃; 1H NMR (500 MHz; CDCl3): δ 9.00 (d,
J = 1.6 Hz, 1H), 8.60 (dd, J = 1.6 Hz, 4.8 Hz, 1H), 8.22 (td, J = 1.9 Hz, 6.0 Hz, 1H), 7.85 (s, 1H),
7.40 (dd, J = 4.8 Hz, 8.0 Hz, 1H), 4.44 (t, J = 7.0 Hz, 2H), 3.58 (s, 1H), 2.20 (dd, J = 8.7 Hz, 17 Hz,
2H), 2.00 (tt, J = 7.7 Hz, 7.7 Hz, 2H), 1.57-1.50 (m, 2H), 1.38-1.33 (m, 2H), 3.00-1.60 (m, 10H); 13C
NMR (125 MHz;CDCl3): δ 149.4, 147.1, 145.0, 133.1, 126.8, 124.0, 119.9, 74.9, 61.3, 50.3, 37.9,
30.0, 28.8, 26.0; 11B NMR (160 MHz; CDCl3): δ -2.24, -5.65, -9.24, -11.6, -12.2, -13.0; HRMS (ESI,
pos) for C14H26B10N4 (m/z): calcd 381.3060 (M+Na)+, found 381.3056.
Synthesis of tert-Butyl (E)-(4-(3-(pyridin-3-yl)acrylamido)butyl)carbamate (13)
To a solution of 12 (226 mg, 1.20 mmol), carboxylic acid (149 mg, 1.00 mmol), and HATU (380 mg,
1.00 mmol) in THF (3 mL) was slowly added DIEA (261 µL, 1.50 mmol). The resulting mixture was
stirred at room temperature under argon atmosphere until the full conversion of a starting material
was observed. The resulting mixture was added aq. NaHCO3 and EtOAc after which the product was
partitioned between the aqueous and organic layers. The aqueous layer was washed with EtOAc and
combined organic layers were washed with brine, dried over sodium sulfate and concentrated under
vacuum. Then, the crude material was purified by column chromatography on silica gel (10% MeOH
1
in CH2Cl2) afforded 13 (390 mg, quant.) as a white solid. H NMR (500 MHz; CDCl3): δ 8.72 (s,
1H), 8.53 (d, J = 2.2 Hz, 1H), 8.07 (d, J = 4.0 Hz, 1H), 7.57-7.49 (m. 2H), 6.74 (d, J = 8.0 Hz, 1H),
3.76-3.70 (m, 2H), 3.42-3.37 (m, 2H), 1.63-1.55 (m, 4H), 1.44 (s, 9H); 13C NMR (125 MHz; CDCl3):
δ 165.4, 156.4, 150.4, 149.2, 137.1, 134.4, 130.9, 123.8, 123.2, 79.5, 40.1, 39.6, 28.5, 28.0, 28.0,
26.2; HRMS (ESI, pos) for C17H25N3O3 (m/z): calcd 342.1788 (M+Na)+, found 342.1787.

Synthesis
of
(E)-N-(4-(1,7-Dicarba-closo-dodecaboranyl)amidebutyl)-3-(pyridin-3-
yl)acrylamide (2b)
To a solution of the dicarba-closo-dodecaborane-1-acetic acid 15b (43 mg, 0.221 mmol) in toluene,
was slowly added excess oxalyl chloride. After the resulting mixture was stirred at 60 ℃ under argon
atmosphere for 12 h, the reaction mixture was concentrated under pressure. The crude materials 16b
were used to the next reaction without further purification.
In another flask, to a solution of 13 (47 mg, 0.147 mmol) in CH2Cl2, was slowly added TFA in
dioxane. The resulting mixture was stirred at room temperature under argon atmosphere until the full
conversion of a starting material was observed. After evaporation of the solvent, 14 was used to the
next reaction without further purification.
To a solution of the unpurified amine 14 and triethylamine (60 µL, 0.441 mmol) in CH2Cl2, was
slowly added dicarba-closo-dodecaborane-1-acetyl chloride (16b) in CH2Cl2 at 0 ℃. After the
resulting mixture was stirred 0 ℃ under argon atmosphere for 1 h, the resulting mixture was added
NH4Cl aq. and CH2Cl2 after which the product was partitioned between the aqueous and organic
layers. The aqueous layer was washed with CH2Cl2 and combined organic layers were dried over
sodium sulfate and concentrated under vacuum. Then, the crude materials were purified by column
chromatography on silica gel (10% MeOH in CH2Cl2) afforded 2b as a white solid (24.4 mg, 0.063
1
mmol, 43%). m.p. 131-132 ℃; H NMR (500 MHz; CD3OD): δ 8.71 (s, 1H), 8.51 (dd, J = 5.0 Hz,
1H), 8.03 (d, J = 8.0 Hz, 1H), 7.53 (d, J = 16 Hz, 1H), 7.47 (dd, J = 5.0 Hz, 8.0 Hz, 1H), 6.72 (d, J =
16 Hz, 1H), 3.65 (s, 1H), 3.27 (t, J = 5.5 Hz, 2H), 3.13 (t, J = 7.8 Hz, 2H), 1.48-1.45 (m, 4H), 1.53-
13
1.52 (m, 4H), 2.87-1.59 (m, 10H); C NMR (125 MHz; CD3OD): δ 167.7, 162.5, 150.7, 149.7,
137.4, 136.2, 132.9, 125.5, 124.8, 77.4, 56.8, 41.3, 40.1, 27.6, 27.5; 11B NMR (160 MHz; CD3OD): δ
-5.61, -7.45, -11.1, -11.6, -13.4, -15.6; HRMS (ESI, pos) for C15H27B10N3O2 (m/z): calcd 390.3187
(M+H)+, found 390.3195.
Synthesis
of
(E)-N-(4-(1,12-Dicarba-closo-dodecaboranyl)amidebutyl)-3-(pyridin-3-
yl)acrylamide (2c)
Preparation and purification of compound 2c was carried out according to the procedure of
1
compound 2b (Yield: 63%). m.p. 173-174 ℃; H NMR (500 MHz; CD3OD): δ 8.70 (d, J = 2.0 Hz,
1H), 8.51 (dd, J = 1.3 Hz, 4.9 Hz, 1H), 8.03 (d, J = 8.0 Hz, 1H), 7.53 (d, J = 16 Hz, 1H), 7.47 (dd, J
= 1.3 Hz, 8.0 Hz, 1H), 6.70 (d, J = 16 Hz, 1H), 3.35 (s, 1H), 3.27-3.25 (m, 2H), 3.27 (t, J = 13 Hz,
2H), 3.07 (t, J = 13 Hz, 2H), 1.47-1.46 (m, 2H), 2.87-1.59 (m, 10H); 13C NMR (125 MHz; CD3OD):
δ 166.4, 163.5, 149.3, 148.3, 136.0, 134.9, 131.5, 124.2, 123.5, 54.5, 38.9, 38.7, 30.3, 26.2, 26.1; 11B
NMR (160 MHz; CD3OD): δ -13.6, -15.2; HRMS (ESI, pos) for C15H27B10N3O2 (m/z): calcd
413.2975 (M+Na)+, found 413.2966.
Synthesis of tert-Butyl (E)-4-(4-(3-(pyridin-3-yl)acrylamido)butyl)piperidine-1-carboxylate (18)

To a solution of 17 (204 mg, 0.528 mmol) in EtOH (6.3 mL) was added hydrazine monohydrate (70
µL, 1.32 mmol). The resulting mixture was stirred at reflux temperature under argon atmosphere
until the full conversion of a starting material was observed. After evaporation of the solvent, to the
reaction mixture were added water and CH2Cl2. The product was partitioned between the aqueous
and organic layers. The aqueous layer was washed with CH2Cl2 and combined organic layers were
dried over sodium sulfate and concentrated under vacuum. The crude materials were used to the next
reaction without further purification.
To a solution of adamantane-1-carboxylic acid (79 mg, 0.528 mmol), HATU (95 mg, 0.634 mmol) in
THF (1.5 mL) was slowly added DIEA (138 µL, 0.792 mmol). The resulting mixture was stirred at
room temperature under argon atmosphere until the full conversion of a starting material was
observed. The resulting mixture was added aq. NaHCO3 and EtOAc after which the product was
partitioned between the aqueous and organic layers. The aqueous layer was washed with EtOAc and
combined organic layers were washed with brine, dried over sodium sulfate and concentrated under
vacuum. Then, the crude material was purified by column chromatography on silica gel (5% MeOH
in CH2Cl2) afforded 18 as a white solid (231 mg, 0.596 mmol, quant.). 1H NMR (400 MHz; CDCl3):
δ 8.72 (s, 1H), 8.53 (d, J = 2.2 Hz, 1H), 8.07 (d, J = 4.0 Hz, 1H), 7.57-7.49 (m. 2H), 6.74 (d, J = 8.0
Hz, 1H), 3.76-3.70 (m, 2H), 3.42-3.37 (m, 2H), 1.63-1.55 (m, 4H), 1.44 (s, 9H); HRMS (ESI, pos)
for C22H33N3O3 (m/z): calcd 410.2414 (M+Na)+, found 410.2420.
Synthesis of (E)-N-(4-(1-(1,12-Dicarba-closo-dodecaboranyl)piperidin-4-yl)butyl)-3-(pyridin-3-
yl)acrylamide (19)
To a solution of 4 N HCl in dioxane was added 18 (54.0 mg, 0.139 mmol). The resulting mixture
was stirred at room temperature for 12 h under argon atmosphere. The resulting mixture was
concentrated under vacuum. It is used to the next step without further purification.
To a solution of the crude material, 15c (32 mg, 0.167 mmol), and HATU (63 mg, 0.167 mmol) in
THF was slowly added DIEA (50 µL, 0.278 mmol). The resulting mixture was stirred at room
temperature under argon atmosphere until the full conversion of a starting material was observed.
The resulting mixture was added aq. NaHCO3 and EtOAc after which the product was partitioned
between the aqueous and organic layers. The aqueous layer was washed with EtOAc and combined
organic layers were washed with brine, dried over sodium sulfate and concentrated under vacuum.
Then, the crude material was purified by column chromatography on silica gel (10% MeOH in
1
CH2Cl2) afforded products as a solid (2.3 mg, 0.0050 mmol, 3.6% 2 steps). m.p. 138-139 ℃; H
NMR (500 MHz; CD3OD): δ 8.71 (d, J = 2.0 Hz, 1H), 8.51 (dd, J = 2.0 Hz, 4.9 Hz, 1H), 8.04 (dt, J =
2.0 Hz, 8.0 Hz 1H), 7.53 (d, J = 20 Hz, 1H), 7.47 (dd, J = 4.9 Hz, 8.0 Hz, 1H), 6.72 (d, J = 20 Hz,
1H), 4.35 (d, J = 10 Hz, 2H) 3.45 (s, 1H), 3.30-3.29 (m, 3H), 2.77 (t, J = 12.5 Hz, 2H), 1.72 (d, J =
12.5 Hz, 1H), 1.59-1.51 (m, 3H), 1.42-1.35 (m, 2H), 1.30-1.25 (m, 2H), 1.01-0.93 (m, 2H), 3.10-1.70
(m, 10H).13C NMR (125 MHz; CDCl3): δ 165.3, 159.2, 150.3, 149.1, 137.2, 134.4, 130.9, 123.8,
11
123.1, 83.8, 64.0, 47.3, 39.8, 35.9, 35.8, 32.6, 29.9, 24.0; B NMR (160 MHz; CD3OD): δ -12.6, -

15.3; HRMS (ESI, pos) for C20H35B10N3O2 (m/z): calcd 493.3401 (M+Cl)-, found 493.3391.
Synthesis of tert-Butyl (1,12-dicarba-closo-dodecaboranylamido)propyl)carbamate (22)
To a solution of 15c (77 mg, 0.408 mmol), 20 (71 mg, 0.408 mmol) HATU (155 mg, 0.408 mmol) in
THF (2 mL) was slowly added DIEA (100 µL, 0.612 mmol). The resulting mixture was stirred at
room temperature under argon atmosphere until the full conversion of a starting material was
observed. The resulting mixture was added aq. NaHCO3 and EtOAc after which the product was
partitioned between the aqueous and organic layers. The aqueous layer was washed with EtOAc and
combined organic layers were washed with brine, dried over sodium sulfate and concentrated under
vacuum. It was used to the next step without further purification.
To a solution of 4 N HCl (100 µL, 0.408 mmol) in dioxane was added the crude material. The
resulting mixture was stirred at room temperature for 12 h under argon atmosphere. The resulting
mixture was concentrated under vacuum. It is used to the next step without further purification.
To a solution of the crude material, (E)-3-(pyridin-3-yl)acrylic acid (61 mg, 0.408 mmol), and
HATU (155 mg, 0.408 mmol) in THF was slowly added DIEA (142 µL, 0.816 mmol). The resulting
mixture was stirred at room temperature under argon atmosphere until the full conversion of a
starting material was observed. The resulting mixture was added aq. NaHCO3 and EtOAc after which
the product was partitioned between the aqueous and organic layers. The aqueous layer was washed
with EtOAc and combined organic layers were washed with brine, dried over sodium sulfate and
concentrated under vacuum. Then, the crude material was purified by column chromatography on
silica gel (10% MeOH in CH2Cl2) afforded products as amorphous (48.6 mg, 0.129 mmol, 41%, 3
1
steps). H NMR (500 MHz; CD3OD): δ 8.72 (s, 1H), 8.52 (d, J = 4.8 Hz, 1H), 8.04 (d, J = 8.0 Hz,
1H), 7.50 (d, J = 20 Hz, 1H), 7.43 (dd, J = 6.1 Hz, 8.6Hz, 1H), 6.67 (d, J = 20 Hz, 1H), 3.36 (bs, 1H),
3.23 (t, J = 10 Hz, 2H), 3.10 (t, J = 10 Hz, 2H), 1.62 (td, J = 5 Hz, 10 Hz, 2H); 11B NMR (160 MHz;
13
CD3OD): δ -12.6, -15.3; C NMR (125 MHz; CD3OD): δ 168.0, 163.1, 150.7, 149.7, 137.6, 136.2,
132.8, 125.5, 124.6, 63.3, 55.8, 38.9, 37.7, 30.0; HRMS (ESI, pos) for C14H25B10N3O2 (m/z): calcd
398.2855 (M+Na)+, found 398.2849.
Synthesis
of
(E)-N-(4-(1,12-Dicarba-closo-dodecaboranyl)amidepentyl)-3-(pyridin-3-
yl)acrylamide (25)
To a solution of 5-aminopentan-1-ol (103.2 mg, 1.00 mmo) and triethylamine (340 µL, 2.00 mmol)
in CH2Cl2, was slowly added carbonic acid chloride in CH2Cl2 at 0 ℃. After the resulting mixture
was stirred 0 ℃ under argon atmosphere for 1 h, the resulting mixture was added NH4Cl aq. and
CH2Cl2 after which the product was partitioned between the aqueous and organic layers. The
aqueous layer was washed with CH2Cl2 and combined organic layers were dried over sodium sulfate
and concentrated under vacuum. It was used to the next step without further purification.
To a solution of PPh3 (262 mg, 1.00 mmol), phthalimide (220 mg, 1.00 mmol) in THF was slowly
added 1.9 M diisopropyl azodicarboylate in Toluene (526 µL, 1.00 mmol) at 0 ℃. The resulting

mixture was stirred at room temperature for 1 h under argon atmosphere. To the resulting mixture
was added the crude material at 0 ℃. The resulting mixture was stirred at room temperature for 16
h. After evaporation of solvent, it was used to the next step without further purification.
To a solution of the crude material in EtOH was added Hydrazine Monohydrate (125 µL, 2.50
mmol). The resulting mixture was stirred at reflux temperature for 12 h. After evaporation of the
solvent, the crude product was resolved in CH2Cl2 and filtered. The filtrate was concentrated under
vacuum. The crude materials were used as amine to the next reaction without further purification.
To a solution of the crude material, (E)-3-(pyridin-3-yl)acrylic acid (149 mg, 1.00 mmol), and
HATU (380 mg, 1.00 mmol) in THF (1 mL) was slowly added DIEA (348 µL, 2.00 mmol). The
resulting mixture was stirred at room temperature under argon atmosphere until the full conversion
of a starting material was observed. The resulting mixture was added aq. NaHCO3 and EtOAc after
which the product was partitioned between the aqueous and organic layers. The aqueous layer was
washed with EtOAc and combined organic layers were washed with brine, dried over sodium sulfate
and concentrated under vacuum. Then, the crude material was purified by column chromatography
on silica gel (10% MeOH in CH2Cl2) afforded products as a white solid (45.5 mg, 0.113 mmol, 11%,
4 steps). m.p. 85-86 ℃; 1H NMR (500 MHz; CD3OD): δ 8.71 (d, J = 2.0 Hz, 1H), 8.51 (dd, J = 6.1
Hz, 2.0 Hz, 1H), 8.04 (d, J = 8.6 Hz, 1H), 7.54 (d, J = 20 Hz, 1H), 7.46 (dd, J = 6.1 Hz, 8.6 Hz, 1H),
6.73 (d, J = 20 Hz, 1H), 3.32 (bs, 1H), 3.28 (t, J = 7.0 Hz, 2H), 1.54 (quin, J = 7.0 Hz, 2H), 1.44
11
(quin, J = 7.0 Hz, 2H), 1.29-1.23 (m, 2H), 2.97-1.85 (m, 10H); B NMR (160 MHz; CD3OD): δ -
12.6, -15.3; 13C NMR (125 MHz; CD3OD): δ 167.6, 163.0, 150.7, 149.7, 137.3, 136.2, 132.9, 125.5,
124.8, 84.4, 63.2, 41.3, 40.4, 29.9, 29.6, 24.9; HRMS (ESI, pos) for C15H29B10N3O2 (m/z): calcd
426.3163 (M+Na)+, found 426.3169.
Synthesis of 1-Benzoyl-1,12-dicarba-closo-dodecaborane (26)
To a solution of p-carborane (200 mg, 1.39 mmol) in Et2O, was slowly added n-BuLi 1.6 M solution
in hexane (984 μL, 1.53 mmol) at -78 ℃. After the resulting mixture was stirred -78 ℃ under argon
atmosphere for 1 h, benzoyl chloride (176 µL, 1.53 mmol) was added. Then, the resulting mixture
was stirred at room temperature for 2 h. After that, the reaction mixture was concentrated under
pressure. The resulting mixture was added water and hexane after which the product was partitioned
between the aqueous and organic layers. The aqueous layer was washed with hexane and combined
organic layers were dried over sodium sulfate and concentrated under vacuum. The crude product
was purified by column chromatography on silica gel (10 % Et2O in hexane) afforded products as
white solid (356.4 mg, 1.435 mmol, quant.). 1H NMR (500 MHz; CDCl3): δ 7.48-7.44 (m, 3H), 7.36-
11
13
7.32 (m, 2H), 2.88 (s, 1H), 3.01-1.57 (m, 10H); B NMR (160 MHz; CDCl3): δ -12.6, -15.3; C
NMR (125 MHz; CDCl3): δ 189.7, 136.4, 131.9, 128.3, 87.3, 63.6; HRMS (ESI, pos) for C9H16B10O
(m/z): calcd 271.2101 (M+Na)+, found 271.2093.
Synthesis
of
(E)-N-(4-(1-Benzoyl-1,12-dicarba-closo-dodecaboran-12-yl)amidebutyl)-3-

(pyridin-3-yl)acrylamide (30)
To a solution of isopropyl amine (18 µL, 0.127 mmol) in Et2O, was n-BuLi 1.6 M solution in hexane
(71 μL, 0.110 mmol) slowly added at 0 ℃. After the resulting mixture was stirred 0 ℃ under argon
atmosphere for 1 h, was added a solution of 26 (21 mg, 0.0846 mmol). Then, the resulting mixture
was stirred at room temperature CO2 atmosphere for 3 h. After that, the reaction mixture was
concentrated under pressure. The resulting mixture was added and hexane after which the product
was partitioned between the aqueous and organic layers. The aqueous layer was washed with hexane.
Then, the aqueous layer was added 6 M HCl and hexane after which the product was partitioned
between the aqueous and organic layers. The aqueous layer was washed with hexane and combined
organic layers were dried over sodium sulfate and concentrated under vacuum. The crude materials
were used to the next reaction without further purification.
To a solution of the crude material, excess amount of 14 and HATU (48 mg, 0.127 mmol) in THF
was slowly added DIEA (44 µL, 0.254 mmol). The resulting mixture was stirred at room temperature
under argon atmosphere until the full conversion of a starting material was observed. The resulting
mixture was added aq. NaHCO3 and EtOAc after which the product was partitioned between the
aqueous and organic layers. The aqueous layer was washed with EtOAc and combined organic layers
were washed with brine, dried over sodium sulfate and concentrated under vacuum. Then, the crude
material was purified by column chromatography on silica gel (10% MeOH in CH2Cl2) afforded
1
products as a solid (18.0 mg, 0.0365 mmol, 43%, 2 steps). m.p. 64-65 ℃; H NMR (500 MHz;
CD3OD): δ 8.71 (d, J = 2.0 Hz, 1H), 8.51 (dd, J = 6.1 Hz, 2.0 Hz, 1H), 8.04 (d, J = 8.6 Hz, 1H),
7.56-7.40 (m, 7H), 6.70 (d, J = 20 Hz, 1H), 3.29-3.26 (m, 2H), 3.09-3.06 (m, 2H), 1.47-1.45 (m, 4H),
3.20-1.88 (m, 10H); 11B NMR (160 MHz; CD3OD): δ -12.6, -15.3; 13C NMR (125 MHz; CD3OD): δ
189.3, 166.3, 161.2, 149.3, 148.3, 136.1, 136.0, 134.9, 131.8, 131.5, 127.8, 127.6, 124.1, 123.4, 85.2,
83.4, 39.8, 38.7, 26.2, 26.1; HRMS (ESI, pos) for C22H31B10N3O3 (m/z): calcd 517.3242 (M+Na)+,
found 517.3233.
Synthesis of (E)-N-(4-(1-Trimethylsilyl-1,12-dicarba-closo-dodecaboran-12-yl)amidebutyl)-3-
(pyridin-3-yl)acrylamide (31)
To a solution of p-carborane (27 mg, 0.185 mmol) in Et2O, was slowly added n-BuLi 1.6 M solution
in hexane (120 µL, 0.185 mmol) at -78 ℃. After the resulting mixture was stirred -78 ℃ under argon
atmosphere for 1 h, TMSCl (23 µL, 0.185 mmol) was added. Then, the resulting mixture was stirred
at room temperature for 2 h. After that, the reaction mixture was concentrated under pressure. The
resulting mixture was added water and hexane after which the product was partitioned between the
aqueous and organic layers. The aqueous layer was washed with hexane and combined organic
layers were dried over sodium sulfate and concentrated under vacuum. It is used to the next step
without further purification.
To a solution of the crude material in Et2O, was slowly added n-BuLi 1.6 M solution in hexane (120
µL, 0.185 mmol) at -78 ℃. The resulting mixture was stirred at room temperature under argon

atmosphere for 1.5 h. The resulting mixture was stirred at room temperature CO2 atmosphere for 3 h.
After that, the reaction mixture was concentrated under pressure. The resulting mixture was added
water and hexane after which the product was partitioned between the aqueous and organic layers.
The aqueous layer was washed with hexane. Then, the aqueous layer was added 6 M HCl and hexane
after which the product was partitioned between the aqueous and organic layers. The aqueous layer
was washed with hexane and combined organic layers were dried over sodium sulfate and
concentrated under vacuum. The crude material was used to the next reaction without further
purification.
To a solution of the crude material, excess amount of 14 and HATU (70 mg, 0.185 mmol) in THF (1
mL) was slowly added DIEA (64 µL, 0.370 mmol). The resulting mixture was stirred at room
temperature under argon atmosphere until the full conversion of a starting material was observed.
The resulting mixture was added aq. NaHCO3 and EtOAc after which the product was partitioned
between the aqueous and organic layers. The aqueous layer was washed with EtOAc and combined
organic layers were washed with brine, dried over sodium sulfate and concentrated under vacuum.
Then, the crude material was purified by column chromatography on silica gel (10% MeOH in
CH2Cl2) afforded products as a solid (21.0 mg, 0.0455 mmol, 25%, 3 steps). m.p. 65-66 ℃; 1H NMR
(500 MHz; CD3OD): δ 8.72 (d, J = 2.0 Hz, 1H), 8.53 (dd, J = 6.1 Hz, 2.0 Hz, 1H), 8.06 (d, J = 8.6
Hz, 1H), 7.52 (d, J = 20 Hz, 1H), 7.49 (dd, J = 6.1 Hz, 8.6 Hz, 1H), 6.72 (d, J = 20 Hz, 1H), 3.33-
11
3.22 (m, 2H), 3.09-3.08 (m, 2H), 1.49-1.47 (m, 4H), 2.95-1.71 (m, 10H), 0.01 (s, 9H) ; B NMR
13
(160 MHz; CD3OD): δ -12.6, -15.3; C NMR (125 MHz; CD3OD): δ 166.3, 161.7, 149.3, 148.3,
148.3, 136.0, 134.9, 131.5, 124.2, 123.4, 85.8, 71.6, 39.7, 38.7, 26.2, 26.1, -2.69; HRMS (ESI, pos)
for C18H35B10N3O2Si (m/z): calcd 485.3374 (M+Na)+, found 485.3376.
Synthesis of 1-((E)-4-(3-(Pyridin-3-yl)acrylamido)hexyl)-1,2-dicarba-closo-dodecaborane (34)
To a solution of o-carborane (40 mg, 0.277 mmol) in THF (2 mL) was slowly added n-BuLi 1.6 M
solution in hexane (179 µL, 0.277 mmol) at 0 ℃. The resulting mixture was stirred at 0 ℃ for over 1
h under argon atmosphere. Then PhthN(CH2)6OTs29 (111 mg, 0.277 mmol) was added at 0 ℃. After
the resulting mixture was stirred for 12 h at room temperature, the resulting mixture was added water
and hexane after which the product was partitioned between the aqueous and organic layers. The
aqueous layer was washed with hexane and combined organic layers were washed with brine, dried
over sodium sulfate and concentrated under vacuum. Then, the crude material was purified by
column chromatography on silica gel afforded products. The crude material was used to the next
reaction without further purification.
To a solution of the crude material in 2-propanol:water (2.0 mL, 6:1, v/v), was added NaBH4 (52 mg,
1.39 mmol). The resulting mixture was stirred at room temperature. After 2 h, conc.HCl (200 µL)
was added after which was reacted at 80 ℃ for 6 h under argon atmosphere. The reaction mixture
was dried by repeated co-concentration with methanol. The product was dissolved in water and
washed twice with CH2Cl2. The aqueous phase was concentrated in vacuo, dissolved in ethanol and

filtered through a small cotton plug. The crude material was used without further purification.
To a solution of the crude material, (E)-3-(pyridin-3-yl)acrylic acid (41 mg, 0.277 mmol) and HATU
(105 mg, 0.277 mmol) in THF (500 µL) was slowly added DIEA (97 µL, 0.554 mmol). The resulting
mixture was stirred at room temperature under argon atmosphere until the full conversion of a
starting material was observed. The resulting mixture was added aq. NaHCO3 and EtOAc after which
the product was partitioned between the aqueous and organic layers. The aqueous layer was washed
with EtOAc and combined organic layers were washed with brine, dried over sodium sulfate and
concentrated under vacuum. Then, the crude material was purified by column chromatography on
silica gel (5% MeOH in CH2Cl2) afforded products as a solid (10.5 mg, 0.028 mmol, 10%, 3 steps).
m.p. 125-126 ℃; 1H NMR (500 MHz; CDCl3): δ 8.71 (d, J = 2.0 Hz, 1H), 8.51 (dd, J = 6.1 Hz, 2.0
Hz, 1H), 8.04 (d, J = 8.6 Hz, 1H), 7.54 (d, J = 20 Hz, 1H), 7.46 (dd, J = 6.1 Hz, 8.6 Hz, 1H), 6.73 (d,
J = 20 Hz, 1H), 4.52 (bs, 1H), 2.27 (m, 2H), 1.58-1.46 (m, 4H), 1.40-1.29 (m, 6H), 2.97-1.85 (m,
11
13
10H); B NMR (160 MHz; CDCl3): δ -2.24, -5.65, -9.24, -11.6, -12.2, -13.0; C NMR (125 MHz;
CDCl3): δ 167.6, 150.7, 149.6, 137.3, 136.2, 132.9, 125.5, 124.8, 86.0, 59.7, 40.4, 40.0, 30.4, 30.2,
29.7, 27.5; HRMS (ESI, pos) for C16H30B10N2O (m/z): calcd 374.3476 (M+H)+, found 374.3463.
Synthesis of 1-((E)-4-(3-(Pyridin-3-yl)acrylamido)hexyl)-1,12-dicarba-closo-dodecaborane (35)
To a solution of p-carborane (40 mg, 0.277 mmol) in THF (2 mL) was slowly added n-BuLi 1.6 M
solution in hexane (179 µL, 0.277 mmol) at 0 ℃. The resulting mixture was stirred at 0 ℃ for over 1
h under argon atmosphere. Then PhthN(CH2)6OTs (111 mg, 0.277 mmol) was added at 0 ℃. After
the resulting mixture was stirred for 12 h at room temperature. The resulting mixture was added
water and hexane after which the product was partitioned between the aqueous and organic layers.
The aqueous layer was washed with hexane and combined organic layers were washed with brine,
dried over sodium sulfate and concentrated under vacuum. Then, the crude material was purified by
column chromatography on silica gel afforded products. The crude material was used to the next
reaction without further purification.
To a solution of the crude material in EtOH (1 mL) was added hydrazine monohydrate (19 µL, 0.277
mmol). The resulting mixture was stirred at reflux temperature for 6.5 h under argon atmosphere.
After evaporation of solvent, to the reaction mixture were added water and CH2Cl2. The product was
partitioned between the aqueous and organic layers. The aqueous layer was washed with CH2Cl2 and
combined organic layers were washed with brine, dried over sodium sulfate and concentrated under
vacuum. The crude material was used without further purification.
To a solution of the crude material, (E)-3-(pyridin-3-yl)acrylic acid (41 mg, 0.277 mmol) and HATU
(105 mg, 0.277 mmol) in THF (500 µL) was slowly added DIEA (97 µL, 0.554 mmol). The resulting
mixture was stirred at room temperature under argon atmosphere until the full conversion of a
starting material was observed. The resulting mixture was added aq. NaHCO3 and EtOAc after which
the product was partitioned between the aqueous and organic layers. The aqueous layer was washed
with EtOAc and combined organic layers were washed with brine, dried over sodium sulfate and

concentrated under vacuum. Then, the crude material was purified by column chromatography on
silica gel (5% MeOH in CH2Cl2) afforded products as a white solid (12 mg, 0.033 mmol, 12%, 3
steps). m.p.147-148 ℃; 1H NMR (500 MHz; CDCl3): δ 8.71 (s, 1H), 8.51 (s, 1H), 8.04 (s, 1H), 7.57-
7.47 (m, 2H), 6.71 (m, 1H), 3.27 (m, 2H), 3.10 (bs. 1H), 1.64 (s. 2H), 1.51 (m, 2H), 1.30 (bs, 2H),
1.18 (m, 4H), 2.5-1.9 (br. 10H); 11B NMR (160 MHz; CD3OD): δ -13.0, -15.9; 13C NMR (125 MHz;
CDCl3): δ 167.6, 150.7, 149.6, 137.3, 136.2, 132.9, 125.5, 124.8, 86.0, 59.7, 40.4, 40.0, 30.4, 30.2,
29.7, 27.5; HRMS (ESI, pos) for C16H30B10N2O (m/z): calcd 397.3261 (M+Na)+, found 397.3268.
Synthesis of (3r,5r,7r)-N-(4-((E)-3-(pyridin-3-yl)acrylamido)butyl)adamantane-1-carboxamide
(37)
To a solution of adamantane-1-carboxylic acid (30 mg, 0.166 mmol), excess amount of 14, 1-(3-
Dimethylaminopropyl)-3-ethylcarbodiimide (EDCI) (34 mg, 0.177 mmol), 1-hydroxybenzotriazole
(HOBt) (34 mg, 0.249 mmol) in THF was slowly added DIEA (87 µL, 0.498 mmol). The resulting
mixture was stirred at room temperature under argon atmosphere until the full conversion of a
starting material was observed. The resulting mixture was added aq. NaHCO3 and EtOAc after which
the product was partitioned between the aqueous and organic layers. The aqueous layer was washed
with EtOAc and combined organic layers were washed with brine, dried over sodium sulfate and
concentrated under vacuum. Then, the crude material was purified by column chromatography on
silica gel (10% MeOH in CH2Cl2) afforded 37 as a white solid (19.2 mg, 0.0503 mmol, 30%). m.p.
194-195 ℃; 1H NMR (400 MHz; CD3OD): δ 8.71 (d, J = 1.6 Hz, 1H), 8.51 (dd, J = 6.0 Hz, 1.6 Hz,
1H), 8.04 (d, J = 8.0 Hz, 1H), 7.54 (d, J = 20 Hz, 1H), 7.47 (dd, J = 6.0 Hz, 8.0 Hz, 1H), 6.72 (d, J =
20 Hz, 1H), 3.34-3.22 (m, 2H), 3.09-3.08 (m, 2H), 2.00 (s, 3H), 1.86-1.71 (m, 12H), 1.56 (t, J = 3.2
13
Hz, 4H); C NMR (125 MHz; CD3OD): δ 181.0, 167.7, 150.7, 149.7, 137.4, 136.2, 132.9, 125.5,
124.9, 41.8, 20.3, 40.3, 40.2, 40.0, 37.6, 29.7, 28.0, 27.7; HRMS (ESI, pos) for C23H31N3O2 (m/z):
calcd 404.2308 (M+Na)+, found 404.2328.
Synthesis
of
(4-(4-Azidobutyl)-1,12-dicarba-closo-dodecaboranyl)
(1,7-dicarba-closo-
dodecaboranyl)methanol (45)
To a solution of p-carborane (72 mg, 0.50 mmol) in THF (4 mL) was slowly added n-BuLi 1.6 M
solution in hexane (318 µL, 0.50 mmol) at -78 ℃. The resulting mixture was stirred at 0 ℃ for over 1
h under argon atmosphere. Then 39 (157 mg, 0.50 mmol) was added at -78 ℃. After the resulting
mixture was stirred for 12 h at room temperature, the resulting mixture was added water and hexane
after which the product was partitioned between the aqueous and organic layers. The aqueous layer
was washed with hexane and combined organic layers were washed with brine, dried over sodium
sulfate and concentrated under vacuum. Then, the crude material was purified by column
chromatography on silica gel afforded products. Then, the crude material 40 was used to the next
reaction without further purification.
To a solution of the crude material 40 in THF (2 mL), was slowly added n-BuLi 1.6 M solution in

hexane (318 µL, 0.50 mmol) at 0 ℃. After the resulting mixture was stirred at 0 ℃ under argon
atmosphere for 1 h, the resulting mixture was cooled to -78 ℃. Methyl formate (62 µL, 1.00 mmol)
was slowly added the resulting mixture was stirred at the same temperature for 2 h. After that, the
resulting mixture was quenched by NH4Cl aq. and concentrated under pressure. The resulting
mixture was added water and hexane, after which the product was partitioned between the aqueous
and organic layers. The aqueous layer was washed with hexane and combined organic layers were
dried over sodium sulfate and concentrated under vacuum. Then, the crude material 41 was used to
the next reaction without further purification.
To a solution of m-carborane (72 mg, 0.50 mmol) in Et2O (2 mL), was slowly added n-BuLi 1.6 M
solution in hexane (318 µL, 0.50 mmol) at 0 ℃. After the resulting mixture was stirred at -78 ℃
under argon atmosphere for 1 h, the resulting mixture was cooled to -78 ℃. The crude material 41 in
Et2O was slowly added the resulting mixture was stirred at the same temperature for 2 h. After that,
the resulting mixture was quenched by NH4Cl aq., the resulting mixture was partitioned between the
aqueous and organic layers. The aqueous layer was washed with hexane and combined organic
layers were dried over sodium sulfate and concentrated under vacuum. Next, to a solution of 4M HCl
in Dioxane (500 µL) was added the crude material at room temperature. After the resulting mixture
was stirred for 12 h, and concentrated under vacuum. Then, the crude material 42 was used to the
next reaction without further purification.
To a solution of the crude material 42, triethylamine (205 µL, 1.50 mmol), and DMAP (6.1 mg,
0.050 mmol) in dichloromethane, was slowly added p-toluenesulfonylchloride (191 mg, 1.00 mmol)
at room temperature. After the resulting mixture was stirred at room temperature under argon
atmosphere until full conversion of a starting material was observed. The resulting mixture was
added water and CH2Cl2 after which the product was partitioned between the aqueous and organic
layers. The aqueous layer was washed with CH2Cl2 and combined organic layers were washed with
brine, dried over sodium sulfate and concentrated under vacuum. Then, the crude material 44 was
used to the next reaction without further purification.
To a solution of the crude material 44 in dimethylformamide, was added sodium azide (162 mg, 2.50
mmol). After the resulting mixture was stirred at 80 ℃ under argon atmosphere until full conversion
of a starting material was observed. The resulting mixture was added water and Et2O after which the
product was partitioned between the aqueous and organic layers. The aqueous layer was washed with
Et2O and combined organic layers were washed with brine, dried over sodium sulfate and
concentrated under vacuum. Then, the crude material was purified by column chromatography on
silica gel afforded 45 as a colorless oil (23.8 mg, 0.0570 mmol, 11%, 6 steps).
1H NMR (400 MHz; CDCl3): δ3.84 (d, J = 8.0 Hz, 1H), 3.20 (t, J = 10.8 Hz, 2H), 3.10-1.60 (m,
20H), 2.85 (s, 1H), 2.23 (d, J = 8.0 Hz, 1H), 1.67-1.62 (m, 2H), 1.44-1.37 (m, 2H), 1.26-1.23 (m,
2H); 11B NMR (160 MHz; CDCl3): δ –3.99, -6.94, -11.2, -13.2, -15.6; 13C NMR (125 MHz; CDCl3):
δ 84.4, 82.6, 74.5, 66.0, 51.1, 37.5, 28.5, 26.2; HRMS (ESI, pos) for C9H31B20N3O (m/z): calcd
436.4367 (M+Na)+, found 436.4367.

Synthesis of (E)-N-(4-(4-(1,7-Dicarba-closo-dodecaboranecarbonyl) -1,12-dicarba-closo-
dodecaboranyl)butyl)-3-(pyridin-3-yl)acrylamide (38)
To a solution of 45 (8.3 mg, 0.020 mmol) in CH2Cl2 was added Dess–Martin periodinane (20 mg,
0.040 mmol) at room temperature. The resulting mixture was stirred under argon atmosphere until
full conversion of a starting material was observed. The resulting mixture was quenched by NaHCO3
aq., after which the product was partitioned between the aqueous and organic layers. The aqueous
layer was washed with CH2Cl2 and combined organic layers were washed with brine, dried over
magnesium sulfate and concentrated under vacuum. Then, the crude material is used to the next step
without further purification.
To a solution of the crude material in THF : H2O (500 µL, 10:1, v/v), was added PPh3 (10 mg, 0.040
mmol). The resulting mixture was stirred at room temperature for 12 h under argon atmosphere.
After evaporation of solvent, the crude product 47 was used to the next reaction without further
purification.
To a solution of the crude material 47, (E)-3-(pyridin-3-yl)acrylic acid (3.0 mg, 0.020 mmol), EDCI
(4.6 mg, 0.024 mmol), HOBt (4.6 mg, 0.030 mmol) in THF was slowly added DIEA (10 µL, 0.060
mmol, 3.0). The resulting mixture was stirred at room temperature under argon atmosphere until full
conversion of a starting material was observed. The resulting mixture was added aq. NaHCO3 and
EtOAc after which the product was partitioned between the aqueous and organic layers. The aqueous
layer was washed with EtOAc and combined organic layers were dried over sodium sulfate and
concentrated under vacuum. Then, the crude material was purified by column chromatography on
silica gel (5% MeOH in CH2Cl2) afforded 38 as a white solid (4.4 mg, 8.5 µmol, 43%, 3 steps). m.p.
127-128 ℃; 1H NMR (500 MHz; CD3OD): δ 8.71 (s, 1H), 8.51 (d, J = 5.0 Hz, 1H), 8.04 (d, J = 8.0
Hz, 1H), 7.54 (d, J = 15 Hz, 1H), 7.47 (dd, J = 5.0 Hz, 8.0 Hz, 1H), 6.70 (d, J = 15 Hz, 1H), 3.72 (s,
1H), 3.22 (t, J = 7.0 Hz, 2H), 3.10-1.60 (m, 20H), 1.72 (t, J = 8.5 Hz, 1H), 1.41-1.37 (m, 2H), 1.24-
11
13
1.21 (m, 2H); B NMR (160 MHz; CDCl3): δ -4.56, -10.9, -13.1, -14.9; C NMR (125 MHz;
CD3OD): δ 181.7, 167.7, 150.7, 149.7, 137.5, 136.3, 132.9, 125.6, 124.7, 79.5, 57.5, 57.1, 54.8, 39.9,
39.0, 29.7, 27.8; HRMS (ESI, pos) for C17H36B20N2O2 (m/z): calcd 540.4652 (M+Na)+, found
540.4637.
Synthesis of (E)-N-(4-(4-(1,12-Dicarba-closo-dodecaboranyl(hydroxy)methyl) -1,12-dicarba-
closo-dodecaboranyl)butyl)-3-(pyridin-3-yl)acrylamide (50)
To a solution of p-carborane (34 mg, 0.240 mmol) in Et2O (2 mL), was slowly added n-BuLi 1.6 M
solution in hexane (151 µL, 0.240 mmol) at 0 ℃. After the resulting mixture was stirred at -78 ℃
under argon atmosphere for 1 h, the resulting mixture was cooled to -78 ℃. The crude material 41
(described above) was slowly added the resulting mixture was stirred at the same temperature for 2 h.
After that, the resulting mixture was quenched by NH4Cl aq., the resulting mixture was partitioned
between the aqueous and organic layers. The aqueous layer was washed with hexane and combined

organic layers were dried over sodium sulfate and concentrated under vacuum. Next, to a solution of
4M HCl in Dioxane (500 µL) was added the crude material at room temperature. After the resulting
mixture was stirred for 12 h, and concentrated under vacuum. Then, the crude product was used to
the next reaction without further purification.
To a solution of PPh3 (63 mg, 0.240 mmol), phthalimide (35 mg, 0.240 mmol) in THF (1.5 mL) was
slowly added 1.9 M diisopropyl azodicarboylate in toluene (126 μL, 0.240 mmol) at 0 ℃. The
resulting mixture was stirred at room temperature for 1 h under argon atmosphere. To the resulting
mixture was added the crude material at 0 ℃. The resulting mixture was stirred at room temperature
for 16 h. After evaporation of solvent, the crude product was used to the next reaction without further
purification.
To a solution of the crude material in EtOH, was added hydrazine monohydrate (30 µL, 0.600 mmo).
The resulting mixture was stirred at reflux temperature under argon atmosphere until full conversion
of a starting material was observed. After evaporation of solvent, to the reaction mixture were added
water and CH2Cl2. The product was partitioned between the aqueous and organic layers. The
aqueous layer was washed with CH2Cl2 and combined organic layers were dried over sodium sulfate
and concentrated under vacuum. The crude materials were used to the next reaction without further
purification.
To a solution of the crude material, (E)-3-(pyridin-3-yl)acrylic acid (36 mg, 0.240 mmol), EDCI (55
mg, 0.029 mmol), HOBt (55 mg, 0.360 mmol) in THF was slowly added DIEA (126 µL, 0.720
mmol). The resulting mixture was stirred at room temperature under argon atmosphere until full
conversion of a starting material was observed. The resulting mixture was added aq. NaHCO3 and
EtOAc after which the product was partitioned between the aqueous and organic layers. The aqueous
layer was washed with EtOAc and combined organic layers were dried over sodium sulfate and
concentrated under vacuum. Then, the crude material was purified by column chromatography on
silica gel (5% MeOH in CH2Cl2) afforded 50 as a white solid (36 mg, 0.0686 mmol, 29%, from 41).
m.p. 204-205 ℃; 1H NMR (500 MHz; CD3OD): δ 8.71 (s, 1H), 8.51 (d, J = 5.0 Hz, 1H), 8.04 (d, J =
8.0 Hz, 1H), 7.53 (d, J = 15 Hz, 1H), 7.47 (dd, J = 5.0 Hz, 8.0 Hz, 1H), 6.70 (d, J = 15 Hz, 1H), 3.44
(s, 1H), 3.25 (s, 1H), 3.21 (t, J = 8.0 Hz, 2H), 2.90-1.50 (m, 20H), 1.67 (t, J = 8.5 Hz, 1H), 1.40-1.37
11
13
(m, 2H), 1.24-1.19 (m, 2H); B NMR (160 MHz; CDCl3): δ -12.9, -15.7; C NMR (125 MHz;
CD3OD): δ 167.7, 150.7, 149.7, 137.5, 136.3, 132.9, 125.5, 124.7, 90.6, 85.3, 75.6, 64.4, 54.8, 40.0,
38.5, 29.8, 28.0; HRMS (ESI, pos) for C17H38B20N2O2 (m/z): calcd 542.4809 (M+Na)+, found
542.4813.
Synthesis of (E)-N-(4-(4-(1,12-Dicarba-closo-dodecaboranecarbonyl) -1,12-dicarba-closo-
dodecaboranyl)butyl)-3-(pyridin-3-yl)acrylamide (51)
To a solution of 50 (16.3 mg, 0.029 mmol) in CH2Cl2 (500 µL) was added Dess–Martin periodinane
(24.6 mg, 0.058 mmol) at room temperature. The resulting mixture was stirred under argon
atmosphere until full conversion of a starting material was observed. The resulting mixture was

quenched by NaHCO3 aq., after which the product was partitioned between the aqueous and organic
layers. The aqueous layer was washed with CH2Cl2 and combined organic layers were washed with
brine, dried over magnesium sulfate and concentrated under vacuum. The crude product was purified
by column chromatography on silica gel (5% MeOH in CH2Cl2) afforded products as a white solid
(8.1 mg, 0.0155 mmol, 53%). m.p. 176-177 ℃; 1H NMR (500 MHz; CDCl3): δ 8.74 (s, 1H), 8.57 (d,
J = 4.0 Hz, 1H), 7.77 (d, J = 8.0 Hz, 1H), 7.60 (d, J = 16 Hz, 1H), 7.30 (dd, J = 4.0 Hz, 8.0 Hz, 1H),
6.43 (d, J = 16 Hz, 1H), 5.72 (s, 1H), 3.28 (dd, J = 7.0 Hz, 14 Hz, 2H), 2.87 (s, 1H), 2.90-1.60 (m,
20H), 1.62 (t, J = 8.5 Hz, 1H), 1.41-1.35 (m, 2H), 1.25-1.16 (m, 2H); 11B NMR (160 MHz; CDCl3):
δ -13.0, -15.4; 13C NMR (125 MHz; CDCl3): δ 180.5, 165.2, 150.5, 149.2, 137.7, 134.5, 130.7, 123.8,
122.7, 86.6, 85.9, 70.7, 65.2, 39.4, 37.9, 29.2, 26.8; HRMS (ESI, pos) for C17H36B20N2O2 (m/z):
calcd 540.4652 (M+Na)+, found 540.4653.
Synthesis of (E)-N-(4-(4-(1,12-Dicarba-closo-dodecaboranecarbonyl) -1,7-dicarba-closo-
dodecaboranyl)butyl)-3-(pyridin-3-yl)acrylamide (55)
Preparation and purification of compound 55 was carried out according to the procedure of
compound 38 as a white solid (11.8 mg, 0.023 mmol, 37%, 7 steps). m.p. 209-211 ℃; 1H NMR (500
MHz; CDCl3): δ 8.74 (d, J = 1.5 Hz, 1H), 8.57 (dd, J = 1.5 Hz, J = 6.0 Hz, 1H), 7.78 (d, J = 8.0 Hz,
1H), 7.62 (d, J = 15 Hz, 1H), 7.31 (dd, J = 5.0 Hz, 8.0 Hz, 1H), 6.45 (d, J = 15 Hz, 1H), 5.80 (s, 1H),
3.35 (q, J = 6.7 Hz, 2H), , 2.91 (s, 1H) 3.10-1.50 (m, 20H), 1.95 (t, J = 8.5 Hz, 1H), 1.51-1.42 (m,
13
2H), 1.41-1.38 (m, 2H); 11B NMR (160 MHz; CDCl3): δ -2.24, -7.24, -11.0, -12.8, -15.3; C NMR
(125 MHz; CDCl3): δ 180.2, 165.3, 150.5, 149.3, 137.8, 134.5, 130.7, 123.8, 122.7, 85.8, 77.7, 75.6,
65.4, 39.4, 36.7, 29.3, 27.3; HRMS (ESI, pos) for C17H36B20N2O2 (m/z): calcd 539.4686 (M+Na)+,
found 539.4682.
Recombinant NAMPT Inhibition Assay
The assay was performed according to the manufacturers protocol (CycLex NAMPT Colorimetric
Assay Kit, MBL International Corp., Woburn, MA). The NAMPT assay was performed according
manufacturers protocol. Briefly we used the "1-Step Assay Method" for which following reagents
were mixed to make assay buffer and kept at ice before starting the assay: 10 µL each of 10X
NAMPT assay buffer, nicotinamide, PRPP, ATP and EtOH; 2 µL each of recombinant NMNAT1,
WST-1, ADH, diaphorase and dH2O. The NAMPT inhibition assay was performed by mixing 2 µL
of various concentrations (DMSO as vehicle control) with the following: 2 µL recombinant NAMPT
and 36 µL dH2O. The reaction was initiated by adding 60 μL of 1- Step Assay Buffer to each well
and mixed thoroughly followed by incubation at 30 ℃ for 20 mins. After this period, the
absorbance at 485 nm was measured and compared with the positive control.
MTT assay
Human epithelioid cervical carcinoma HeLa cells were used for the cell viability assay. The cells

(5×104 cells/mL of 96-well plate) were incubated at 37 °C for 72 h in 100 µL of RPMI-1640 medium
containing various concentrations of compounds. After the incubation, 10 µL of 3’-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, TCI) in PBS (5 mg/ml) was added
into the each well, and the cells were further incubated at 37 ºC for 2 h. After the removal of the
medium, 100 µL of DMSO was added and the absorbance at 595 nm was determined by a microplate
S6 reader. The drug concentration required to reduce cell viability by 50% (IC50) was determined
from semilogarithmic dose-response plots.
Docking study
Ligand structures and geometries were optimized with Discovery Studio 4.1 using CHARMm force
field. The crystal structures for NAMPT complexed with the NAMPT- selective inhibitor FK866
(PDB ID: 2GVJ) were downloaded from the Protein Data Bank (PDB). All ligands and water
molecules were removed. One monomer of NAMPT was prepared for docking with AutoDock Vina.
The optimized geometries of the ligands were prepared for docking. Ligand nonpolar hydrogens
(including carborane C−H protons) were merged to conform to the AutoDock atom types, and all of
the torsion angles within the carborane clusters were set to nonrotatable. A new atom type (B) was
defined for the boron atoms, utilizing the force field parameters reported by Tiwari et al. for docking
of carborane-containing ligands. The docking area was defined using AutoGrid, Three-dimensional
affinity grid with 1.0 Å grid point spacing was placed around the NAMPT active site. Docking was
performed with AutoDock Vina.
5. Reference
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Scholz, M.; Hey-Hawkins, E. Carbaboranes as Pharmacophores: Properties, Synthesis, and
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Endo, Y.; Iijima, T.; Yamakoshi, Y.; Yamaguchi, M.; Fukasawa, H.; Shudo, K. Potent
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Julius, R. L.; Farha, O. K.; Chiang, J.; Perry, L. J.; Hawthorne, M. F. Synthesis and Evaluation
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