Structure-activity relationship study of small molecule inhibitors of the DEPTOR-mTOR interaction

Article abstract of DOI:10.1016/j.bmcl.2017.09.002

DEPTOR is a 48 kDa protein that binds to mTOR and inhibits this kinase within mTORC1 and mTORC2 complexes. Over-expression of DEPTOR specifically occurs in the multiple myeloma (MM) tumor model and DEPTOR knockdown is cytotoxic to MM cells, suggesting it is a potential therapeutic target. Since mTORC1 paralysis protects MM cells against DEPTOR knockdown, it indicates that the protein–protein interaction between DEPTOR and mTOR is key to MM viability vs death. In a previous study, we used a yeast two-hybrid screen of a small inhibitor library to identify a compound that inhibited DEPTOR/mTOR binding in yeast. This therapeutic (compound B) also prevented DEPTOR/mTOR binding in MM cells and was selectively cytotoxic to MM cells. We now present a structure–activity relationship (SAR) study around this compound as a follow-up report of this previous work. This study has led to the discovery of five new leads – namely compounds 3g, 3k, 4d, 4e and 4g – all of which have anti-myeloma cytotoxic properties superior to compound B. Due to their targeting of DEPTOR, these compounds activate mTORC1 and selectively induce MM cell apoptosis and cell cycle arrest.

Full text of DOI:10.1016/j.bmcl.2017.09.002

Accepted Manuscript
Structure-Activity Relationship Study of Small Molecule Inhibitors of the DEP-
TOR-mTOR Interaction
Jihye Lee, Yijiang Shi, Mario Vega, Yonghui Yang, Joseph Gera, Michael E.
Jung, Alan Lichtenstein
PII:
S0960-894X(17)30889-2
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.09.002
BMCL 25266
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
18 May 2017
1 September 2017
1 September 2017
Please cite this article as: Lee, J., Shi, Y., Vega, M., Yang, Y., Gera, J., Jung, M.E., Lichtenstein, A., Structure-
Activity Relationship Study of Small Molecule Inhibitors of the DEPTOR-mTOR Interaction, Bioorganic &
Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.09.002
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Structure-Activity Relationship Study of Small Molecule Inhibitors of the
DEPTOR-mTOR Interaction
Jihye Lee,a,d,f Yijiang Shi,b,c,d Mario Vega,b,c Yonghui Yang,b,c Joseph Gera,b,c,d,e Michael E.
Jung,*,a,c,d and Alan Lichtenstein*,b,c,d,e
aDepartment of Chemistry and Biochemistry, bDepartment of Medicine, David Geffen School of Medicine, cJonnson Comprehensive Cancer
Center, and dMolecular Biology Institute, University of California Los Angeles, Los Angeles, California 90095 and eDepartment of
Research and Development, Greater Los Angeles Veterans Affairs Healthcare System, Los Angeles, California, USA 91343. dThese authors
contributed equally. eThese authors contributed equally. fCurrent address: Neuroscience Research Institute, Gachon University, 24,
Namdong-daero 774 beon-gil, Namdong-gu, Incheon 20565, Korea.
ABSTRACT
DEPTOR is a 48 kDa protein that binds to mTOR and inhibits this kinase within mTORC1 and mTORC2 complexes. Over-
expression of DEPTOR specifically occurs in the multiple myeloma (MM) tumor model and DEPTOR knockdown is
cytotoxic to MM cells, suggesting it is a potential therapeutic target. Since mTORC1 paralysis protects MM cells against
DEPTOR knockdown, it indicates that the protein-protein interaction between DEPTOR and mTOR is key to MM viability
vs death. In a previous study, we used a yeast two-hybrid screen of a small inhibitor library to identify a compound that
inhibited DEPTOR/mTOR binding in yeast. This therapeutic (compound B) also prevented DEPTOR/mTOR binding in
MM cells and was selectively cytotoxic to MM cells. We now present a structure-activity relationship (SAR) study around
this compound as a follow-up report of this previous work. This study has led to the discovery of five new leads – namely
compounds 3g, 3k, 4d, 4e and 4g – all of which have anti-myeloma cytotoxic properties superior to compound B. Due to
their targeting of DEPTOR, these compounds activate mTORC1 and selectively induce MM cell apoptosis and cell cycle
arrest.
DEPTOR is a recently described protein that
binds to mTOR and inhibits this kinase within
mTORC1 and mTORC2 complexes.1 As an inhibitor
of mTOR, it is not surprising that DEPTOR’s
expression is quite low in most tumor types.1
However, over-expression of DEPTOR occurs in the
multiple myeloma (MM) tumor model with highest
levels found in the specific genetic categories of MM
that contain translocations between the IgH and MAF
genes or copy number gains at chromosome 8q242, a
region that contains the DEPTOR gene. DEPTOR
knockdown in high DEPTOR-expressing MM cell
lines induces growth arrest and apoptosis. The anti-
MM effects of DEPTOR silencing and singular over-
expression in MM suggest DEPTOR is a potential
therapeutic target in this malignancy.
Since DEPTOR is an mTOR inhibitor, the
proximal molecular effect of DEPTOR knockdown is
activation of mTORC1 and mTORC2 activity. The
finding that mTORC1 paralysis protects MM cells
against DEPTOR knock-down1,3 indicates that
DEPTOR binding to mTOR with resulting mTORC1
inhibition contributes to MM viability and
proliferation. To this end, in a previous study4, we
designed a yeast two-hybrid screen, interrogating a
small inhibitor library for compounds that could
inhibit DEPTOR/mTOR binding in yeast. We
identified a hit compound (compound B) which also
prevents mTOR/DEPTOR binding within MM cell
lines, acutely activates mTORC1 and selectively
induces MM cell apoptosis and cell cycle arrest.4
Anti-MM cell cytotoxicity was directly correlated
with the levels of DEPTOR expression in a panel of
MM cell lines. This compound is also effective in
vivo in a murine xenograft model of MM growth, is
cytotoxic to primary MM cells, and is remarkably
non-toxic to normal hematopoietic colony forming
cells and without observable side effects when used in
mice. In a surface plasmon resonance assay, our lead
compound B bound to recombinant DEPTOR but not
mTOR.
The IC50 for compound B’s cytotoxic effect
against MM cell lines with the highest DEPTOR
expression was 0.8-2.5 µM. Thus, in a follow-up
study to our previous work, we undertook a structure-
activity relationship (SAR) study with the expectation
of discovering chemically modified derivatives with
enhanced efficacy. This report details our findings of
this SAR study.
As previously described4 we conducted a pilot
screening of ~150,000 compounds from the NCI
small molecule inhibitor library utilizing the yeast-2-
hybrid screen, and identified four compounds (‘hits’)
that specifically inhibited the DEPTOR-mTOR
interaction (Figure 1). Of these four, the first two
compounds, NSC119055 and NSC119670, did not
offer many opportunities for structural variation since
1

they are quite simple structures. The third compound,
NSC118305, presented several positions for variation
but we were worried about the conjugated diene unit
since such polyolefinic units might give a rise to non-
selective toxicity. Indeed, this compound was toxic to
normal hematopoietic colony forming cells,
completely preventing colony formation when used as
low as 0.5 µM (not shown). The final compound,
NSC126405, was not toxic to colony formation (in
concentrations as high as 10 µM) and yet
demonstrated molecular efficacy (enhanced mTORC1
activity) and anti-MM cytotoxicity (MTT assays).
Therefore we chose the final compound shown,
NSC126405, which was called simply compound B,
as the first compound to modify to try to improve its
activity.
1 with the selected hydrazine unit 2 in THF generally
proceeded quite well. One could also use the HCl
salts of the hydrazines and added base.6 The best
procedure was often to use the hydrazine HCl salt in
pyridine as solvent. The desired compounds 3 were
normally purified by flash column chromatography on
silica gel and several could be recrystallized as well.
The parent compound B was prepared by this route in
62% yield. We prepared some N-alkyl derivatives 3a-
3c and also used this route to prepare some N-aryl
derivatives 3d-3l by using either the alkyl hydrazines
or the N-aminoanilines 2, where R1 and/or R2 was an
aryl group. The compounds 3a-3l were generally
quite deeply colored, e.g., dark orange or red.7
Scheme 1. Reagents and conditions: (a) 2 free form,
THF, 22 °C; (b) 2 HCl salt, TEA, THF, 22 °C; (c) 2
HCl salt, pyr, 22 °C.
Fig 1. Hit compounds from NCI inhibitor library.
We next prepared several N-mono and di-acyl
derivatives, 4a-4f (Scheme 2).8 The monoacyl com-
pounds 4a and 4c-4d were synthesized by selective
mono-acylation of the parent compound B with either
acid anhydrides or acyl chlorides in the presence of
base as shown. If B were treated with two equivalents
of the acyl chloride and base, one obtained the
diacylated derivatives 4b and 4e-4f. And we also
prepared a few N-mono-carbamoyl derivatives 4g-4i,
which were prepared from B using di-tert-butyl
dicarbonate or the corresponding alkyloxycarbonyl
chloride.
The possible modifications of all parts of compound
B are shown in Figure 2. Since it has been reported
that unsubstituted analogues of B, those with the
chlorines removed, are quite reactive nucleophiles,5
likely due to the strong resonance structure which
puts positive charge on the amino group and negative
charge on the cyclopentadiene system, we decided to
not pursue completely unsubstituted cyclopentadiene
systems, hoping that more substituted ones would be
more stable and less reactive. A series of compounds
were prepared and tested for their biological activity
in order to establish a comprehensive structure-
activity relationship (SAR) for this series.
Scheme 2. Reagents and conditions: (a) 1 or 2 eq
(RCO)2O or RCOCl, TEA, THF, 22 °C, 0.5-3 h; (b)
(ROCO)2O or ROCOCl, pyr, DMAP, THF, 0 to 22
°C, 16-24 h.
Fig. 2. Modification and SAR relationship of
NSC126405 (B).
Since there was some concern that compounds
with the dichloroalkene unit might show some non-
specific toxicity, we introduced a few cyclic and
acyclic moieties in place of the tetrachlorocyclo-
pentadiene ring system (Scheme 3).9,10 We also
wanted to expand our substrate scope by further
modifying the bottom part of the molecule. The
hydrazones 6a,9 6b,10 and 6d, were prepared by
reaction of the simple ketones, fluorenone and
First we decided to modify the hydrazone unit
and in particular to vary the substituents on the
hydrazone amine nitrogen, namely the top part of
compound B as shown in Figure 2. The synthesis of
these compounds was accomplished by several
relatively easy routes (Scheme 1).9 Thus condensation
of commercially available hexachlorocyclopentadiene
2

xanthone, 5, R = H, R = (CH)4, with hydrazine or
with hydrazine HCl salt and KOH in refluxing
ethanol. The benzophenone hydrazine 6c was
commercially available. The N-Boc derivative 6e was
prepared from 6a by treating the hydrazine with di-
tert-butyl dicarbonate and pyridine and DMAP in
THF.11 The yields of the hydrazones 6a-6e were
generally quite good. We also prepared the
exhibit a significant increase of activity compared to
compound B, while the tert-butyl compound 3c
showed only modest activity. Again, the mono-
benzoylated compound 4a was toxic with
considerable cell death seen even at this low
concentration (0.5 µM). Follow-up experiments on
compound 4a at lower concentrations which did not
show toxicity (0.05-0.2 µM) demonstrated no
enhancement of p70 phosphorylation suggesting
compound 4a was non-specifically toxic. A summary
of the p70 phosphorylation data from 4 separate
experiments where derivatives were used at 0.5 µM, is
shown in Fig. 3B. These first 8 derivatives were also
screened for cytotoxicity against the same 8226 MM
cell line in 48 h MTT assays. The IC50s for these
assays are shown below the bar graphs in Fig. 3B and
an example of one experiment is shown in Fig. 3C. In
general, the analogs showed a correlation between the
molecular effects (i.e., the ability to increase p70
phosphorylation) and their anti-MM cytotoxic effects.
The four analogs (4b, 3d, 3e and 3f) with enhanced
molecular effects compared to the parent compound B
also demonstrated lower IC50s. In contrast,
compounds 3a-3c, which showed little or no
enhancement of p70 phosphorylation compared to the
parent compound B, also did not show enhanced anti-
MM cytotoxicity. As mentioned above, compound 4a
was cytotoxic without effects on p70 phosphorylation;
thus, its anti-MM effects were assumed to be non-
specific.
unsubstituted
hydrazones
of
indanone,
cyclopentanone and acetophenone, but these
compounds were unstable with respect to
rearrangement to the dimeric azines.12 Finally we
prepared the oxime derivative 7a and the dimethoxy
analogue 7b from hexachlorocyclopentadiene
(Scheme 4).
1
Scheme 3. Reagents and conditions: (a) N2H4, EtOH
or triethylene glycol (TEG), reflux; (b) H2N-NHR
HCl, KOH, EtOH, reflux; (c) Di-t-butyl-dicarbonate,
pyr, DMAP, THF, 0 to 22 °C, 3 h.
On the basis of the initial result from the first set
of analogues 3a-3f and 4a-4b, we designed and
synthesized further analogues in four categories, and
evaluated their molecular and anti-MM cytotoxic
activities in p70 phosphorylation and MTT assays.
P70 phosphorylation was measured in 8226 cells
treated for 6 hrs. Since the parent compound B was
Scheme 4. Reagents and conditions: (1)
Hydroxylamine HCl, KOH, MeOH, reflux, 8 h; (b)
KOH, MeOH, 22 °C, 18 h.
The first set of analogs, 3a-3f and 4a-4b, were
compared against the parent compound B in assays
for mTORC1 activity. We used the 8226 cell line for
these experiments as it demonstrates marked over-
expression of DEPTOR.1,3 As drugs that prevent
binding of the mTOR inhibitor, DEPTOR, to mTOR,
effective drugs should increase mTOR kinase activity.
In mTORC1, mTOR phosphorylates the p70S6
kinase. Thus, we used Western blot to test for
induction of p70 phosphorylation in this secondary
screen. Compounds were tested at 0.5, 1, and 2 µM
with 6 h in vitro exposure. Induction of p70
phosphorylation by all 8 derivatives was comparable
to that of parent compound B when used at 1 or 2 µM
except for the mono-benzoylated compound 4a which
was toxic and showed degradation of p70. However,
at 0.5 µM, the three N-aryl compounds 3d-3f and the
dibenzoylated compound 4b were more effective than
the parent compound B for induction of p70
phosphorylation (selected immunoblot shown in Fig.
3A). In contrast, the N-alkyl compounds 3a-3b did not
consistently
ineffective
in
inducing
p70
phosphorylation following exposure of 8226 cells to
0.5 µM, we used 0.5 µM concentration to screen these
additional derivatives for enhanced molecular activity.
MTT (48 h) assays exploited 8226 cells as well as an
additional DEPTOR-over-expressing MM cell line,
MM1.S. In general, MM1.S cells are less sensitive
than 8226 to the cytotoxic activity of the parent
compound B (IC50 of 1.3 µM and 3.0 µM for 8226
and MM1.S, respectively). The structures and
biological activity of all derivatives are shown in
Tables 1-4, categorized by structural modification.
First since the three N-arylated compounds 3d-3f
showed promising results, we prepared more aryl
derivatives having either electron-withdrawing or
electron-donating groups on the 3-position of phenyl
ring 3g-3i to examine the effect of phenyl substituents
on the hydrazone in more detail. In Table 1, we
present p70 phosphorylation and MTT assay results of
3

Table 1. p70 phosphorylation and cytotoxicity of
alkyl and aryl analogues 3a-l
p70
phophorylation
vs Ba
MTT (IC50, µM)
R1
H
R2
8226 MM1.S
B
H
1.0
NI
NI
NI
1.8
1.3
>3
>3
>3
0.6
3.0
>3
>3
>3
2.0
3a CH3
CH3
H
3b C6H11
3c C(CH3)3
3d C6H5
H
H
3e 3-FC6H4
3f 3,5-Cl2C6H3
3g 3-CF3C6H4
3h 3-MeC6H4
3i 3-MeOC6H4
3j 2-FC6H4
3k 4-FC6H4
3l C6H5
H
2.2
1.9
4.0
NI
0.5
0.8
0.17
2.0
>3
1.5
2.0
1.0
>3
>3
>3
1.3
>3
H
H
H
H
NI
H
NI
>3
H
5.0
NI
0.7
>3
C6H5
a) fold increase, measured at 0.5 µM. NI = no increase
Figure 3. A) 8226 cells exposed to drugs at 0.5 µM
for 6 h, followed by immunoblot for expression of
phosphorylated p70S6K, total p70 or actin. B-1 =
compound B from NCI; B-2 = compound B
synthesized at UCLA. B) Summary of p70
phosphorylation data from 4 separate experiments
(n=4) where derivatives were used at 0.5 µM, mean
SD; IC50 (mean) from MTT cytotoxicity assays (n=4)
shown below bars. C) MTT cytotoxicity data (mean
SD of triplicate samples) of four initially tested
derivatives (4b, 3d, 3e and 3f).
MM1.S, respectively. The mono-acetylated analogue
4c also showed no increase of p70 phosphorylation at
0.5 µM compared to compound B treatment, and
similar cytotoxicity (vs drug B) to 8226 cells. On the
other hand, the mono-pivaloylated analogue 4d
showed a substantial 7-fold increase in p70 phosphor-
ylation which correlated nicely with lowered IC50
values for both cell lines in the MTT assays, 0.12 µM
and 2.0 µM for 8226 and MM1.S, respectively.
Table 2. p70 Phosphorylation and cytotoxicity of acyl
and carbamate derivatives 4a-4i
p70
compound 3a-3l. The best activity was shown by the
3-trifluoromethylphenyl analogue 3g, namely a 4-fold
increase in p70 phosphorylation and an IC50 of 0.17
µM and 1.0 µM for the 8226 and MM1.S MM cell
lines, respectively. The analogues having electron-
donating group, 3h-3i, did not show good activity.
Generally the analogues with the more electron-
withdrawing substituents showed the best activity,
e.g., 3g and 3k. We also examined the effect of the
position of the substituent. Although the 3-fluoro and
4-fluoro analogues, 3e and 3k, showed similar IC50
values in the 8226 and MM1.S cell lines, curiously
the 4-fluoro analogue 3k showed much better activity
in the p70 phosphorylation assay, while the 2-fluoro
analogue 3j showed no activity. Finally the diphenyl
analogue 3l did not show any activity.
MTT (IC50, µM)
phospho
rylation
vs Ba
compd
R
8226
MM1.S
B
1.0
NI
1.3
0.6
0.8
1.0
0.12
0.25
0.5
0.1
0.8
>5
3.0
0.8
2.0
ND
2.0
0.3
0.5
0.6
ND
ND
4a
4b
4c
4d
4e
4f
C6H5
C6H5
2.0
NI
CH3
C(CH3)3
4-FC6H4
4-MeC6H4
C(CH3)3
CH2C≡CH
C(CH3)2C≡CH
7.0
5.0
2.5
6.0
1.5
1.2
4g
4h
4i
Next, we examined a series of analogues of the
mono- and di-acyl derivatives 4a-4f as well as some
carbamate derivatives 4g-4i (Table 2). The presumed
non-specific toxicity of the mono-benzoylated
analogue 4a was again shown by the MTT assays
with IC50 values of 0.6 and 0.8 µM for 8226 and
a) fold increase, measured at 0.5 µM; ND, not
determined; NI, no increase.
Since the N-dibenzoylated compound 4b was
quite promising in the initial testing, we then prepared
two additional dibenzoylated compounds having
different substituents on the 4-position of the phenyl
4

ring. As expected, both compounds 4e and 4f showed
good results for p70 phosphorylation and MTT
assays. Moreover, in line with the results of the aryl
derivatives, compound 4e having an electron-
withdrawing substituent on the 4-position showed
even better results than the other two diacyl
derivatives. We also prepared and examined the
carbamate derivatives 4g-4i. The tert-butyloxy-
carbonyl analogue 4g showed a substantial 6-fold
increase in p70 phosphorylation and also enhanced
cytotoxicity, 0.1 µM and 0.6 µM IC50 for 8226 and
MM1.S, respectively. The other carbamate derivatives
4h and 4i having a terminal acetylene were prepared
for the possible further investigation of biotin protein
labeling by click chemistry, but these compounds
showed a minimal increase in mTORC1 activation.
Only the simple propargyl compound 4h showed
some cytotoxicity in the MTT assay.
It is perhaps interesting to compare the three
analogues having a tert-butyl unit, namely the alkyl
analogue, 3c, the acyl analogue 4d, and the carba-
mate 4g. The alkyl analogue 3c showed the least
activity in p70 phosphorylation, while the pivaloyl
(tert-butylcarbonyl) analogue 4d and the t-Boc (tert-
butyloxycarbonyl) analogue 4g showed very good
activities in both p70 phosphorylation and MTT
assay.
analogues showed enhanced biological activities
reasons that are still unclear. Finally, we examined the
oxime and dimethoxy derivatives, 7a and 7b, and they
both showed poor activity in the p70 phosphorylation
and the MTT cytotoxicity assay (Table 4).
Table 4. p70 Phosphorylation, cytotoxicity and
apoptosis of derivatives 7a-7b
p70
phosphorylation
MTT (IC50, µM)
structure
8226
MM1.S
vs Ba
B
hydrazone
oxime
1.0
NI
NI
1.5
>5
>5
3.0
ND
ND
7a
7b
dimethoxy
a) fold increase, measured at 0.5 µM; ND, not
determined; NI, no increase.
All the derivatives in each category showed a
rough correlation between the derivatives that were
successful molecularly, namely able to increase p70
phosphorylation at 0.5 µM, and were effective
cytotoxic compounds. Of the nine molecules (3d, 3e,
3f, 3g, 3k, 4b, 4d, 4e and 4g) active in the p70
phosphorylation assay (≥ 1.8-fold increase compared
to compound B at 0.5 µM), all had enhanced
cytotoxic effects (i.e., lower IC50) compared to the
parent B. Of the many compounds without molecular
Next we examined the derivatives 6a-6e having
cyclic and acyclic moieties other than the
tetrachlorocyclopentadiene ring system (Table 3). We
left unchanged the unsubstituted hydrazine as the top
unit and modified the bottom part to 9-fluorenyl 6a,
9-xanthyl 6b and benzophenone 6c. However these
new analogues did not show any improvement in both
the p70 phosphorylation and the MTT assay. Since
the 3-fluorophenyl (3e) and t-Boc carbamate (4g)
efficacy (i.e.,
phosphorylation), only two
demonstrated enhanced cytotoxic effects, which were
presumably non-specific.
<
1.8-fold increase in p70
4a and 4h
–
–
From these screening experiments, we identified
analogues 3g, 3k, 4d, 4e and 4g as being the most
active compounds in the p70 phosphorylation assay.
These were then studied in more detail. As shown in
Fig. 4A and 4B, although inducing comparable
amounts of p70 phosphorylation at 1 µM, when
compared to parent compound B at lower concen-
trations,, these biochemically modified compounds
were significantly more effective as low as 0.25 µM.
An additional molecular effect of either DEPTOR
knockdown or parent compound B is an upregulation
of p21 expression,3,4 believed to result from decreased
mTORC1-dependent expression of p21-targeting
miRNAs.3 Upregulated expression of p21 contributes
to the anti-MM cytotoxicity of DEPTOR targeting.3
As shown in Fig. 4C, some of these derivatives with
enhanced mTORC1 activation compared to parent
Table 3. p70 Phosphorylation, cytotoxicity and
apoptosis of derivatives 6a-6e
p70
phosphor
MTT (IC50, µM)
R1
R2
R
ylation vs
Ba
8226
1.5
MM1.S
3.0
2,3,4,5-
Cl4C5
B
H
1.0
6a
6b
6c
9-Fluorenyl
H
H
H
NI
NI
NI
>5
>5
>5
ND
>5
9-Xanthyl
Ph
Ph
>5
3-
FC6H4
6d
6e
9-Fluorenyl
9-Fluorenyl
NI
NI
>5
>5
>5
>5
compound
B also demonstrated enhanced p21
t-Boc
expression, further strengthening the notion that their
biochemical modifications allow more efficacious
DEPTOR targeting. This was clearly shown for 4d,
a) fold increase, measured at 0.5 µM; ND, not determined;
NI, no increase.
5

Fig. 4. A) Representative experiment of p70 phosphorylation w/ increasing concentrations of derivatives vs
compound B (exposure 6 hrs); B) Summary of p70 phosphorylation data (mean SD, n=4) shown as fold
increase vs compound B (compound B kept at ‘1’) of densitometric ratio of phospho-p70/total p70 after
exposure to derivatives for 6 hrs; C) Upregulated p21 expression due to derivatives. D) MTT assays of all
derivatives vs compound B (48 h assay), mean SD, n=4; E) % apoptosis (mean SD, n=4) at 48 h after
exposure to different derivatives. In all immunoblot experiments, untreated control groups (uM=0) contain an
equal amount of DMSO.
4e, 4g, and 3g. Fig. 4D also demonstrates the
enhanced anti-MM cytotoxicity of these agents in
8226 MTT assays. We also tested the ability of these
drugs to enhance apoptosis in 8226 cells and, as
shown in Fig. 4E, their apoptosis activity was
enhanced compared to parent compound B.
To compare anti-myeloma efficacy to non-
specific toxicity, we compared each of these 5 active
derivatives to compound B in their ability to inhibit
survival of 8226 MM cells versus normal peripheral
blood lymphocytes (PBLs). In head-to-head
experiments, IC50 values for each target were
calculated and compared. As shown in Fig. 5A,
although each of the derivatives demonstrated
significantly reduced IC50 values for the MM cells
compared to compound B, they also showed variably
enhanced toxicity to PBLs. However, three of the
derivatives, 3g, 3k and 4g, showed significantly
compound B. To further support the fact that the
ability of these three active derivatives (3g, 3k and
4g) to induce MM cell death was specifically related
to their successful interference of DEPTOR/mTOR
binding and mTORC1 activation, we performed co-
immunoprecipitation experiments. Compound
prevents DEPTOR/mTOR binding in MM cells when
B
6

Fig. 5. A) IC50s of drug B and derivatives against 8226 MM cells or PBLs (48 h assays, results are means of 5
separate experiments). Therapeutic indices (TIs) calculated as IC50 PBLs/IC50 for 8226 cells; B) 8226 cells treated
with DMSO or drug B (6 h) followed by immunoprecipitation of DEPTOR and precipitate then immunoblotted for
DEPTOR or bound mTOR. C) 8226 cells treated with DMSO or 0.5 µM of derivatives (6 h) followed by similar
co-immunoprecipitation assay. D) 8226 cells infected with lentivirus expressing either shRAPTOR or control
shSCRAMBLE followed by immunoblot assay for RAPTOR, phosphorylated p70, total p70, DEPTOR or tubulin;
E) MM cells expressing either shSCRAMBLE or shRAPTOR incubated with increasing concentrations of
derivatives, followed by MTT assay (48 h). Cytotoxicity (i.e., decreased cell survival) induced in RAPTOR-silenced
cells was significantly reduced (p<0.05) compared to control shSCRAMBLE cells.
used at 1 µM but at 0.5 µM it is ineffective (Fig. 5B).
However, all three derivatives with enhanced TIs
prevented DEPTOR/mTOR binding when used at 0.5
µM (Fig. 5C). To test if drug cytotoxicity was due to
mTORC1 activation, we assessed derivatives for their
anti-MM cytotoxicity in isogenic lines with RAPTOR
knockdown. Fig. 5D demonstrates the inhibitory
effects of RAPTOR knockdown on mTORC1
activation. As shown in Fig. 5E, MTT cytotoxicity
Although RAPTOR knockdown, by itself, had no
discernable cytotoxic effects (i.e., normal viability,
(not shown)), it was always possible that RAPTOR
silencing altered the growth characteristics of the
cells. Thus, we independently tested the effects of
pp242, an mTOR kinase inhibitor, which would also
theoretically prevent mTORC1 activation, on the
cytotoxic effects of drugs 3g and 4g. As shown in
suppl figure 1, MTT cytotoxicity and apoptosis
induction was significantly decreased when pp242
was added to our compounds in a concentration that
prevented mTORC1 activation. Thus, in two
independent strategies where mTORC1 activation was
prevented (RAPTOR knockdown and pp242
assays demonstrate
a
significantly decreased
cytotoxicity induced by all three derivatives when
tested against the RAPTOR-silenced MM cells. This
indicates mTORC1 activation contributes to
cytotoxicity.
7

exposure), anti-myeloma cytotoxicity was abated,
confirming that mTORC1 activation participates in
the effectiveness of our compounds.
Blocking the interaction between mTOR and
DEPTOR (see fig 5C) should enhance kinase activity
We also considered an outside possibility that the
most effective agent, compound 3g, was inducing
mTOR kinase activity independent of DEPTOR.
Enhanced kinase activity with phosphorylation of
DEPTOR, by itself, could theoretically prevent
mTOR-DEPTOR binding (explaining the co-IP
results) and induce p70 phosphorylation. To test this,
we compared the effect of drug 3g on DEPTOR-
silenced cells. As expected (Supplemental fig 3),
DEPTOR knockout by itself (achieved by shRNA)
resulted in enhanced P70 phosphorylation (6x fold
increase). However, drug 3g was incapable of further
mTORC1 activation in these cells while inducing
significant p70 phosphorylation (4x fold increase) in
control cells (transfected with shScramble lentivirus).
Thus, the activation of mTORC1 in drug 3g-treated
cells is dependent on the presence of DEPTOR.
We have initiated in vivo toxicity studies with the
most promising derivative, 3g. Immunodeficient
mice were challenged subcutaneously with 8226 cells
(as described in ref 4) and, when tumors reached 500
mm3, daily treatment with 20 mg/kg of compound B
or drug 3g was initiated. The aim of the experiment
was to test short-term toxicity of drug 3g on
hematopoiesis with a dose of drug that demonstrated
anti-MM efficacy. Although a more thorough dose-
dependent toxicity vs efficacy study is planned in the
future, this initial experiment with analogue 3g
indicates an enhanced anti-myeloma effect in vivo vs
drug B. As shown in supplemental fig 4, compound
3g had no toxic effects on RBC, WBC or platelet
levels but it induced a significantly more rapid tumor
cytoreductive response compared to drug B which
was evident by day +3 of treatment.
of both mTORC1 and mTORC2.
To further
investigate these downstream molecular effects for
two of the most promising anti-myeloma derivatives
(compounds 4g and 3g), we performed additional
Western blot experiments (supplemental fig 2). The
previous experiments demonstrated an increase in p70
phosphorylation indicating the expected activation of
mTORC1.
Both
compounds
also
induced
phosphorylation of 4E-BP1 (supplemental fig 2), an
additional substrate of mTORC1. Phosphorylation of
S6 was also increased indicating not only increased
phosphorylation of p70S6kinase but increased kinase
activity as well. These data confirm the activation of
mTORC1 in drug-treated myeloma cells. We also
assayed mTORC2 activity by analysis of AKT serine
473 phosphorylation, as serine 473 is a substrate of
mTORC2. As shown in supplemental fig 2, exposure
to both compounds resulted in enhanced S473
phosphorylation. In addition, SGK, a direct substrate
of AKT kinase activity, was increased in its
phosphorylation.
These data strongly support
activation of mTORC2 as well as mTORC1 following
a block of the DEPTOR-mTOR interaction. It is
remarkable that, even in the presence of enhanced
AKTS473 phosphorylation and AKT activity (i.e.,
SGK phosphorylation), these compounds are so
significantly cytotoxic to myeloma cells.
When DEPTOR is silenced by shRNA and
mTORC1 is upregulated, activated TORC1 results in
feedback downregulation of the IGF/IRS-1/PI3-
K/PDK1 pathway1,3.
This results in decreased
Although activating mTORC1 and mTORC2, the
compounds described in this study have therapeutic
potential in multiple myeloma. However, in other
tumor models where DEPTOR expression is low and
mTOR activity is high, these therapeutics may be less
promising and even, possibly, harmful, due to
possible stimulation of tumor cell proliferation.
Nevertheless, they have promise in myeloma. Thus,
we have prepared a series of analogues of the initial
hit, NSC126405, compound B, from our high-
throughput screen. Several derivatives of the parent
compound B showed promising activity with N-aryl,
N-mono and diacyl and N-carbamoyl analogues being
the most promising. From these studies, we have
identified five new leads, 3g, 3k, 4d, 4e and 4g
(Figure 6). The ability of these more efficacious
derivatives to inhibit mTOR-DEPTOR binding in
MM cells when used at lower concentrations (vs drug
B) was demonstrated by co-immunoprecipitation
studies. Further support for their specificity is the
finding that their enhanced cytotoxicity to MM cells
phosphorylation of AKT on serine 308 (a PDK1
substrate). A comparable effect is seen in myeloma
cells treated with 3g or 4g (supplemental fig 2),
supporting the notion that, similar to effects of
DEPTOR gene silencing, this negative feedback
pathway is impacted. The difference between our
anti-myeloma drugs and gene silencing is the effects
on AKT S473 phosphorylation where our compounds
increase activation and DEPTOR knockdown
decreases activation. It is likely that the resulting
increase in mTORC2 activity in drug-treated cells
(due to the prevention of DEPTOR-mTOR binding)
overcomes any inhibitory effect on the PI3-
K/PDK1/AKT pathway. Nevertheless, It is evident
that our therapeutic compounds do not kill myeloma
cells by down-regulating the prosurvival effects of
AKT as suggested by Peterson1 in DEPTOR-silenced
cells. This is consistent with our previous work4 on
drug B, the initial 'hit' in the yeast screen.
8

is significantly prevented by mTORC1 inactivation.
This indicates that, similar to drug B, their acute
activation of mTORC1 subsequent to DEPTOR
targeting participates in the injury to MM cells. Our
previous work demonstrates direct binding of drug B
to recombinant DEPTOR.4 One possible explanation
for enhanced activity of the derivatives is that their
binding to DEPTOR may be quantitatively or
qualitatively different than that of drug B. Further
research into the mechanism of action and selectivity
of these compounds, as well as additional analogues,
is currently underway.
integration. Splitting patterns are designated as
follows: s, singlet; d, doublet; t, triplet; q, quartet; dd,
doublet of doublets; dt, doublet of triplets; td, triplet
of doublets; tt, triplet of triplets; qd, quartet of
doublets; qt, quartet of triplets; m, multiplet; and br,
broad. 13C NMR spectra were recorded on Bruker
Spectrometers at 125 MHz and are reported relative to
deuterated solvent signals (CHCl3 δ 77.0; DMSO δ
40.0 ppm). 19F NMR spectra were recorded on Bruker
Spectrometers at 376.3 MHz and are reported relative
to external Freon-113 in benzene (δ -73.75 ppm).
Data for 13C and 19F NMR spectra are reported in
terms of chemical shift. The chemical shifts are
reported in parts per million (ppm, δ). Melting points
were obtained using Buchi B-545 melting point
apparatus and are uncorrected. The reactions were
monitored with a silica gel TLC plate under UV light
(254 and 365 nm) followed by visualization with a
ninhydrin or phosphomolybdic acid staining solution.
Column chromatography was performed on silica gel
60, 230-400 mesh. DART-HRMS spectra were
collected on a Thermo Exactive Plus MSD (Thermo
Scientific) equipped with an ID-CUBE ion source and
a Vapur Interface (IonSense). Both the source and
MSD were controlled by Excalibur, version 3.0. The
purity of the compounds was assayed by high field
proton and carbon NMR and was ≥95%.
Synthetic procedures and Characterization data.
(Perchlorocyclopenta-2,4-dien-1-ylidene)hydrazine
(B). To a solution of hexachlorocyclopentadiene (1.6
mL, 10.0 mmol, 1.0 eq) in tetrahydrofuran (50 mL)
was added hydrazine monohydrate (1.45 mL, 30.0
mmol, 3.0 eq) dropwise at 0 °C. The reaction mixture
was stirred for 10 min at room temperature and then
concentrated in vacuo. The residue was purified by
flash column chromatography over silica gel
(hexane/ethyl acetate, 10:1, v/v) to afford the desired
product B (1.44 g, 62%) as red brown solid: Rf = 0.4
Fig. 6. Most active analogues.
3. Experimental
Chemistry. General Methods
1
All reactions were carried out under open-air
condition unless otherwise specified. Tetrahydrofuran
(THF) was distilled from benzophenone ketyl radical
(hexane/ethyl acetate, 5:1, v/v); mp 187-189 °C; H
NMR (DMSO-d6, 500 MHz) δ 10.67 (d, J = 3.2 Hz,
1
1H), 9.93 (d, J = 3.6 Hz, 1H); H NMR (CDCl3, 500
MHz) δ 8.09 (br, 2H); 13C NMR (DMSO-d6, 125
MHz) δ 129.2, 125.6, 119.8, 118.2, 104.2; 13C NMR
(CDCl3, 125 MHz) δ 132.7, 131.2, 124.6, 119.5,
105.8 ppm; DART-HRMS found 230.88672 [M+H]+,
calcd for C5H3Cl4N2 230.90448.
under
an
argon
atmosphere.
Methanol,
dichloromethane (DCM) and triethylamine (TEA)
were distilled from calcium hydride under an argon
atmosphere.
Hexachlorocyclopentadiene
was
purchased from Chemieliva Pharmaceutical Co. in
China and various hydrazines were purchased from
Sigma-Aldrich, Alfa Aesar and TCI in ≥95% purity,
all other solvents or reagents were purified according
Representative procedure for synthesis of alkyl and
aryl hydrazones.
1
to literature procedures if necessary. H-NMR spectra
Method A. 1,1-Dimethyl-2-(perchlorocyclopenta-
2,4-dien-1-ylidene)hydrazine (3a). To a solution of
hexachlorocyclopentadiene (0.16 mL, 1.0 mmol, 1.0
eq) in tetrahydrofuran (10 mL) was added unsym-
dimethyl-hydrazine (0.23 mL, 3.0 mmol, 3.0 eq)
dropwise at 0 °C. The reaction mixture was stirred for
were recorded on Bruker spectrometers at 500 MHz
and are reported relative to deuterated solvent signals
(CHCl3 δ 7.26; DMSO δ 2.48 ppm). Data for 1H
NMR spectra are reported as follows: chemical shift
(δ ppm), multiplicity, coupling constant (Hz) and
9

3 h at room temperature and then concentrated in
vacuo. The residue was diluted with ethyl acetate (80
mL) and washed with water (2 X 20 mL) and brine
(20 mL). The organic layer was dried with MgSO4,
filtered and concentrated in vacuo. The residue was
purified by flash column chromatography over silica
gel (hexane/ethyl acetate, 10:1, v/v) to afford the
desired product 3a (254 mg, 98%) as dark brown
solid: Rf = 0.45 (hexane/ethyl acetate, 3:1, v/v); mp
69-71 °C; 1H NMR (CDCl3, 500 MHz) δ 3.59 (s, 6H);
13C NMR (CDCl3, 125 MHz) δ 129.5, 128.4, 121.8,
119.4, 103.0, 50.5 ppm; DART-HRMS found
258.93448 [M+H]+, calcd for C7H7Cl4N2 258.93634.
eq) in tetrahydrofuran (5 mL) was added the
previously generated free form of cyclohexyl-
hydrazine through filtration at room temperature. The
reaction mixture was stirred for 12 h at room
temperature and then concentrated in vacuo. The
residue was diluted with ethyl acetate (80 mL) and
washed with water (2 X 20 mL) and brine (20 mL).
The organic layer was dried with MgSO4, filtered and
concentrated in vacuo. The residue was purified by
flash column chromatography over silica gel (hexane
only) to afford the desired product 3b (60 mg, 19%)
as red brown solid: Rf = 0.5 (hexane only); mp 79-81
1
°C; H NMR (DMSO-d6, 500 MHz) δ 10.36 (d, J =
4.0 Hz, 1H), 3.61-3.56 (m, 1H), 1.93-1.89 (m, 2H),
1.76-1.72 (m, 2H), 1.60-1.56 (m, 1H), 1.53 (qd, J =
12.5, 3.5 Hz, 2H), 1.31 (qt, J = 12.5, 3.5 Hz, 2H), 1.13
(qt, J = 12.5, 3.5 Hz, 1H); 13C NMR (DMSO-d6, 125
MHz) δ 127.3, 124.6, 118.7, 117.2, 103.5, 61.4, 31.6,
25.3, 24.7 ppm; DART-HRMS found 312.96460
[M+H]+, calcd for C11H13Cl4N2 312.98329.
1-(Perchlorocyclopenta-2,4-dien-1-ylidene)-2-
phenylhydrazine (3d). Dark brown solid (94%
yield): Rf = 0.65 (hexane/ethyl acetate, 3:1, v/v); mp
1
130-131 °C; H NMR (CDCl3, 500 MHz) δ 10.7 (s,
1H), 7.40 (td, J = 7.5, 1.5 Hz, 2H), 7.34 (dd, J = 9.0,
1.0 Hz, 2H), 7.15 (tt, J = 7.5, 1.0 Hz, 1H); 13C NMR
(CDCl3, 125 MHz) δ 141.4, 130.94, 130.91, 129.7,
124.9, 123.9, 119.4, 115.1, 104.9 ppm; DART-HRMS
found 306.91754 [M+H]+, calcd for C11H7Cl4N2
306.93634.
1-(tert-Butyl)-2-(perchlorocyclopenta-2,4-dien-1-
ylidene)hydrazine (3c). Red solid (5% yield): Rf =
0.4 (hexane only); mp 80-82 °C; 1H NMR (DMSO-d6,
500 MHz) δ 9.98 (s, 1H), 1.34 (s, 9H); 13C NMR
(CDCl3, 125 MHz) δ 127.3, 125.1, 119.1, 117.3,
103.9, 59.2, 28.2 ppm; DART-HRMS found
286.96594 [M+H]+, calcd for C9H11Cl4N2 286.96764.
1-(Perchlorocyclopenta-2,4-dien-1-ylidene)-2-(3-
(trifluoromethyl)phenyl)hydrazine (3g). Red brown
solid (46% yield): Rf = 0.45 (hexane/ethyl acetate,
1
10:1, v/v); mp 146-148 °C; H NMR (CDCl3, 500
MHz) δ 10.69 (s, 1H), 7.53-7.52 (m, 3H), 7.38 (d, J =
5.0 Hz, 1H); 13C NMR (CDCl3, 125 MHz) δ 142.0,
132.2 (q, JCF = 32.0 Hz, 1C), 130.3, 126.9, 125.2,
124.8, 122.6, 121.1 (q, JCF = 3.5 Hz, 1C), 119.8,
117.9, 111.7 (q, JCF = 3.8 Hz, 1C), 105.4; 19F NMR
(CDCl3, 376 MHz, 1H-dc) δ -62.90 ppm; DART-
HRMS found 374.90492 [M+H]+, calcd for
C12H6Cl4F3N2 374.92372.
1-(3,5-dichlorophenyl)-2-(perchlorocyclopenta-2,4-
dien-1-ylidene)hydrazine (3f). Brown solid (16%
yield): Rf = 0.6 (hexane/ethyl acetate, 5:1, v/v); mp
1
188-190 °C; H NMR (CDCl3, 500 MHz) δ 10.52 (s,
1H), 7.22 (d, J = 2.0 Hz, 2H), 7.10 (t, J = 2.0 Hz, 1H;
13C NMR (CDCl3, 125 MHz) δ 143.3, 136.2, 133.0,
132.7, 125.7, 124.3, 119.9, 113.4, 105.6 ppm; DART-
HRMS found 374.83957 [M+H]+, calcd for
C11H5Cl4N2 374.85839.
1-(Perchlorocyclopenta-2,4-dien-1-ylidene)-2-(m-
tolyl)hydrazine (3h). Red brown solid (32% yield):
Rf = 0.65 (hexane/ethyl acetate, 5:1, v/v); mp 139-141
Method
(perchlorocyclopen-ta-2,4-dien-1-
ylidene)hydrazine (3e). To
C.
1-(3-Fluorophenyl)-2-
1
°C; H NMR (DMSO-d6, 500 MHz) δ 11.55 (s, 1H),
a
solution of
7.36 (s, 1H), 7.34 (d, J = 9.0 Hz, 1H), 7.30 (t, J = 7.5
Hz, 1H), 7.00 (d, J = 7.5 Hz, 1H); 13C NMR (CDCl3,
125 MHz) δ 141.4, 139.8, 130.8, 130.7, 129.5, 125.9,
123.7, 119.3, 115.6, 112.3, 104.8, 21.5 ppm; DART-
HRMS found 320.93277 [M+H]+, calcd for
C12H8Cl4N2 320.95199.
hexachlorocyclopentadiene (0.16 mL, 1.0 mmol, 1.0
eq) in pyridine (5 mL) was added 3-fluorophenyl
hydrazine HCl (244 mg, 1.5 mmol, 1.5 eq) at room
temperature. The reaction mixture was stirred for 12 h
at room temperature and then concentrated in vacuo.
The residue was diluted with ethyl acetate (80 mL)
and washed with water (2 X 20 mL) and brine (20
mL). The organic layer was dried with MgSO4,
filtered and concentrated in vacuo. The residue was
purified by flash column chromatography over silica
gel (hexane only) to afford the desired product 3e
(212 mg, 65%) as brown solid: Rf = 0.6 (hexane/ethyl
Method B. 1-Cyclohexyl-2-(perchlorocyclopenta-
2,4-dien-1-ylidene)hydrazine (3b). To a suspension
of cyclohexylhydrazine HCl (527 mg, 3.5 mmol, 3.5
eq) in tetrahydrofuran (5 mL) was added
triethylamine (0.49 mL, 3.5 mmol, 3.5 eq) and the
mixture was stirred for 0.5 h. To a solution of
hexachlorocyclopentadiene (0.16 mL, 1.0 mmol, 1.0
1
acetate, 5:1, v/v); mp 134-136 °C; H NMR (CDCl3,
500 MHz) δ 10.63 (s, 1H), 7.33 (dt, J = 6.5, 8.5 Hz,
10

1H), 7.15 (dt, J = 10.0, 2.0 Hz, 1H), 7.01 (dd, J = 8.0,
1.5 Hz, 1H), 6.83 (td, J = 8.0, 1.5 Hz, 1H); 13C NMR
(CDCl3, 125 MHz) δ 163.8 (d, JCF = 245.3 Hz, 1C),
143.2 (d, JCF = 10.4 Hz, 1C), 131.9, 131.7, 131.0 (d,
JCF = 9.4 Hz, 1C), 124.8, 119.7, 1115. (d, JCF = 21.5
Hz, 1C), 110.7 (d, JCF = 2.9 Hz, 1C), 105.3, 102.3 (d,
JCF = 26.8 Hz, 1C); 19F NMR (CDCl3, 376 MHz, 1H-
dc) δ -110.42 ppm; DART-HRMS found 324.90814
[M+H]+, calcd for C11H6Cl4FN2 324.92691.
DART-HRMS found 381.95616 [M]+, calcd for
C17H10Cl4N2 381.95981.
Representative procedure for synthesis of mono and
diacyl hydrazones.
Method A. N'-(Perchlorocyclopenta-2,4-dien-1-yli-
dene)benzohydrazide (4a). To
a
solution of
(perchloro-cyclopenta-2,4-dien-1-ylidene)hydrazine
(B, 116 mg, 0.5 mmol, 1.0 eq) in tetrahydrofuran (10
mL) was added benzoic anhydride (113 mg, 0.5
mmol, 1.0 eq) and triethylamine (0.07 mL, 0.5 mmol,
1.0 eq) dropwise in ice-bath. The reaction mixture
was stirred for 3 h at room temperature and then
concentrated in vacuo. The residue was diluted with
ethyl acetate (150 mL) and washed with water (2 X
50 mL) and brine (50 mL). The organic layer was
dried with MgSO4, filtered and concentrated in
vacuo. The residue was purified by flash column
chromatography over silica gel (hexane/ethyl acetate,
10:1, v/v) to afford the desired product 4a (239 mg,
71%) as brown solid: Rf = 0.45 (hexane/ethyl acetate,
1-(3-Methoxyphenyl)-2-(perchlorocyclopenta-2,4-
dien-1-ylidene)hydrazine (3i). Red brown solid
(50% yield): Rf = 0.4 (hexane/ethyl acetate, 10:1,
1
v/v); mp 123-125 °C; H NMR (CDCl3, 500 MHz) δ
10.66 (s, 1H), 7.27 (t, J = 8.0 Hz, 1H), 6.96 (s, 1H),
6.84 (dd, J = 8.0, 1.0 Hz, 1H), 6.69 (dd, J = 8.0, 1.5
Hz, 1H), 3.85 (s, 3H); 13C NMR (CDCl3, 125 MHz) δ
161.0, 142.7, 131.0, 130.9, 130.5, 124.0, 119.4, 110.7,
107.7, 105.0, 100.6, 55.4 ppm; DART-HRMS found
336.92801 [M+H]+, calcd for C12H8Cl4N2O
336.94690.
1
5:1, v/v); mp 170-172 °C; H NMR (DMSO-d6, 500
1-(2-Fluorophenyl)-2-(perchlorocyclopenta-2,4-
dien-1-ylidene)hydrazine (3j). Red brown solid
(52% yield): Rf = 0.6 (hexane/ethyl acetate, 20:1,
v/v); mp 120-122 °C; 1H NMR (DMSO-d6, 500 MHz)
δ 11.16 (s, 1H), 7.65 (t, J = 8.0 Hz, 1H), 7.36 (dd, J =
10.0 Hz, 1H), 7.29 (t, J = 7.5 Hz, 1H), 7.20 (dd, J =
12.5, 6.0 Hz, 1H), 3.32 (s, 3H); 13C NMR (CDCl3,
125 MHz) δ 151.1 (d, JCF = 242.4 Hz, 1C), 132.7,
131.8, 130.1, (d, JCF = 8.4 Hz, 1C), 125.3 (d, JCF = 3.5
Hz, 1C), 124.8, 124.6 (d, JCF = 7.3 Hz, 1C), 119.5,
115.6, 115.5 (d, JCF = 17.4 Hz, 1C), 105.6; 19F NMR
MHz) δ 11.98 (s, 1H), 7.93 (d, J = 7.5 Hz, 2H), 7.69
(t, J = 7.5 Hz, 1H), 7.59 (t, J = 7.5 Hz, 2H); 13C NMR
(DMSO- d6, 125 MHz) δ 164.8, 140.8, 135.0, 133.7,
132.1, 129.5, 129.3, 128.7, 120.8, 109.7 ppm; DART-
HRMS found 334.91239 [M+H]+, calcd for
C12H7Cl4N2O 334.93125.
N-Benzoyl-N'-(perchlorocyclopenta-2,4-dien-1-
ylidene)benzohydrazide (4b). Dark brown solid
(52% yield): Rf = 0.65 (hexane/ethyl acetate, 5:1,
1
1
(CDCl3, 376 MHz, H-dc) δ -135.16 ppm; DART-
v/v); mp 123-124 °C; H NMR (CDCl3, 500 MHz) δ
HRMS found 324.90775 [M+H]+, calcd for
C11H6Cl4FN2 324.92691.
7.15 (dd, J = 8.5, 1.0 Hz, 2H), 8.02 (dd, J = 8.5, 1.0
Hz, 2H), 7.68 (tt, J = 7.5, 1.0 Hz, 1H), 7.56 (t, J = 7.5
Hz, 1H), 7.53 (t, J = 7.5 Hz, 2H), 7.48 (t, J = 7.5 Hz,
2H); 13C NMR (CDCl3, 125 MHz) δ 162.1, 151.0,
149.4, 137.7, 134.4, 132.6, 132.5, 130.6, 130.0, 128.9,
128.8, 128.1, 127.6, 120.2, 112.4 ppm; DART-HRMS
found 438.93702 [M+H]+, calcd for C19H11Cl4N2O2
438.95747.
1-(4-Fluorophenyl)-2-(perchlorocyclopenta-2,4-
dien-1-ylidene)hydrazine (3k). Brown solid (48%
yield): Rf = 0.6 (hexane/ethyl acetate, 5:1, v/v); mp
1
147-149 °C; H NMR (DMSO-d6, 500 MHz) δ 11.64
(s, 1H), 7.59-7.56 (m, 2H), 7.30-7.26 (m, 2H); 13C
NMR (CDCl3, 125 MHz) δ 160.0 (d, JCF = 243.3 Hz,
1C), 137.8, 131.0, 130.9, 124.1, 119.4, 116.5 (d, JCF
=
N'-(Perchlorocyclopenta-2,4-dien-1-ylidene)aceto-
hydrazide (4c). Brown solid (89% yield): Rf = 0.7
23.1 Hz, 1C), 116.4 (d, JCF = 7.9 Hz, 1C), 104.9; 19F
NMR (CDCl3, 376 MHz, 1H-dc) δ -117.44 ppm;
DART-HRMS found 324.90593 [M+H]+, calcd for
C11H6Cl4FN2 324.92691.
1
(hexane/ethyl acetate, 5:1, v/v); mp 145-147 °C; H
NMR (CDCl3, 500 MHz) δ 10.62 (s, 1H), 2.42 (s,
3H); 13C NMR (CDCl3, 125 MHz) δ 173.6, 136.3,
135.1, 129.1, 120.9, 107.5, 19.6 ppm; DART-HRMS
found 272.89722 [M+H]+, calcd for C7H4Cl4N2O
272.91560.
2-(Perchlorocyclopenta-2,4-dien-1-ylidene)-1,1-
diphe-nylhydrazine (3l). Dark red solid (78% yield):
Rf = 0.6 (hexane/ethyl acetate, 10:1, v/v); mp 128-130
°C; 1H NMR (DMSO-d6, 500 MHz) δ 7.48 (t, J = 7.5
Hz, 4H), 7.37 (t, J = 7.0 Hz, 2H), 7.34 (d, J = 7.5 Hz,
4H); 13C NMR (DMSO-d6, 125 MHz) δ 146.5, 132.2,
131.7, 130.4, 128.3, 123.6, 123.0, 121.7, 106.4 ppm;
N'-(Perchlorocyclopenta-2,4-dien-1-ylidene)pivalo-
hydrazide (4d). Red brown solid (71% yield): Rf =
0.5 (hexane/ethyl acetate, 5:1, v/v); mp 156-158 °C;
1H NMR (DMSO-d6, 500 MHz) δ 11.17 (s, 1H), 1.24
11

(s, 9H); 13C NMR (DMSO-d6, 125 MHz) δ 175.4,
139.5, 134.5, 128.7, 120.7, 109.3, 39.3, 27.0 ppm;
DART-HRMS found 314.94314 [M+H]+, calcd for
C10H11Cl4N2O 314.96255.
NMR (CDCl3, 500 MHz) δ 10.22 (s, 1H), 1.56 (s,
9H); 13C NMR (CDCl3, 125 MHz) δ 150.5, 135.9,
135.7, 128.2, 121.4, 107.2, 84.2, 28.0 ppm; DART-
HRMS found 330.95319 [M+H]+, calcd for
C10H11Cl4N2O2 330.95747.
4-Fluoro-N-(4-fluorobenzoyl)-N'-(perchlorocyclo-
penta-2,4-dien-1-ylidene)benzohydrazide (4e).
Brown solid (67% yield): Rf = 0.75 (hexane/ethyl
Prop-2-yn-1-yl 2-(perchlorocyclopenta-2,4-dien-1-
ylidene)hydrazine-1-carboxylate (4h). Red solid
(19% and 30% RSM): Rf = 0.5 (hexane/ethyl acetate,
1
acetate, 5:1, v/v); mp 126-128 °C; H NMR (CDCl3,
1
500 MHz) δ 8.17 (dd, J = 8.5, 5.0 Hz, 2H), 8.03 (dd, J
= 9.0, 5.0 Hz, 2H), 7.20 (t, J = 9.0 Hz, 2H), 7.17 (t, J
= 8.5 Hz, 2H); 13C NMR (CDCl3, 125 MHz) δ 166.6
(d, JCF = 255.4 Hz, 1C), 165.5 (d, JCF = 253.5 Hz, 1C),
161.1, 150.9, 149.9, 138.0, 133.4 (d, JCF = 9.6 Hz,
1C), 132.8, 130.6 (d, JCF = 9.0 Hz, 1C), 126.2 (d, JCF
= 3.0 Hz, 1C), 123.7 (d, JCF = 2.9 Hz, 1C), 120.2,
116.3 (d, JCF = 9.3 Hz, 1C), 116.2 (d, JCF = 9.3 Hz,
5:1, v/v); mp 170-172 °C; H NMR (CDCl3, 500
MHz) δ 10.37 (s, 1H), 4.91 (d, J = 2.5 Hz, 2H), 2.58
(t, J = 2.5 Hz, 1H); 13C NMR (CDCl3, 125 MHz) δ
151.3, 137.4, 136.9, 129.2, 121.5, 107.4, 76.5, 76.3,
54.6 ppm; DART-HRMS found 312.90698 [M+H]+,
calcd for C9H5Cl4N2O2 312.91052
2-Methylbut-3-yn-2-yl 2-(perchlorocyclopenta-2,4-
dien-1-ylidene)hydrazine-1-carboxylate (4i). Red
solid (50% yield): Rf = 0.5 (hexane/ethyl acetate, 5:1,
1
1C), 112.3; 19F NMR (CDCl3, 376 MHz, H-dc) δ -
102.42, -105.37 ppm; DART-HRMS found
474.91973 [M+H]+, calcd for C19H9Cl4F2N2O2
474.93862.
1
v/v); mp 143-145 °C; H NMR (CDCl3, 500 MHz) δ
10.27 (s, 1H), 2.62 (s, 1H), 1.80 (s, 6H); 13C NMR
(CDCl3, 125 MHz) δ 149.9, 136.6, 136.2, 128.6,
121.5, 107.3, 83.5, 75.2, 73.6, 28.9 ppm; DART-
HRMS found 340.98938 [M+H]+, calcd for
C11H9Cl4N2O2 340.94182.
4-Methyl-N-(4-methylbenzoyl)-N'-(perchlorocyclo-
penta-2,4-dien-1-ylidene)benzohydrazide (4f).
Brown solid (48% yield): Rf = 0.75 (hexane/ethyl
1
acetate, 5:1, v/v); mp 147-149 °C; H NMR (CDCl3,
500 MHz) δ 8.03 (d, J = 8.0 Hz, 2H), 7.91 (d, J = 8.0
Hz, 2H), 7.31 (d, J = 8.0 Hz, 2H), 7.27 (d, J = 10.5
Hz, 2H), 2.46 (s, 3H), 2.42 (s, 3H); 13C NMR (CDCl3,
125 MHz) δ 162.2, 151.8, 149.4, 145.3, 143.3, 137.5,
132.3, 130.7, 129.7, 129.5, 128.2, 127.4, 127.9, 120.2,
112.3 ppm; DART-HRMS found 466.97013 [M+H]+,
calcd for C21H15Cl4N2O2 466.98877.
(9H-Fluoren-9-ylidene)hydrazine (6a). To an
ethanol (10 mL) solution of fluoren-9-one (360 mg,
2.0 mmol, 1.0 eq) was added hydrazine monohydrate
(0.29 mL, 6.0 mmol, 3.0 eq) dropwise at room
temperature. The reaction mixture was refluxed for 6
h and then concentrated in vacuo. The residue was
diluted with ethyl acetate (100 mL) and washed with
water (2 X 30 mL) and brine (40 mL). The organic
layer was dried with MgSO4, filtered and
concentrated in vacuo. The residue was purified by
flash column chromatography over silica gel (hexane/
ethyl acetate, 10:1, v/v) to afford the desired product
6a (315 mg, 81%) as yellow solid: Rf = 0.15
(hexane/ethyl acetate, 10:1, v/v); mp 152-154 °C; 1H
NMR (CDCl3, 500 MHz) δ 7.91 (d, J = 7.5 Hz, 1H),
7.77 (d, J = 7.5 Hz, 1H), 7.73 (d, J = 7.0 Hz, 1H),
7.65 (d, J = 7.5 Hz, 1H), 7.44 (t, J = 7.5 Hz, 1H),
7.37-7.29 (d, 3H), 6.41 (s, 2H); 13C NMR (CDCl3,
125 MHz) δ 145.6, 141.3, 138.6, 137.7, 130.2, 129.7,
128.5, 127.9, 127.7, 125.5, 120.8, 120.5, 119.5 ppm;
DART-HRMS found 195.09088 [M+H]+, calcd for
C13H11N2 195.09222.
Method B. tert-Butyl 2-(perchlorocyclopenta-2,4-
dien-1-ylidene)hydrazine-1-carboxylate (4g). To a
solution
of
(perchlorocyclopenta-2,4-dien-1-
ylidene)hydrazine (B, 116 mg, 0.5 mmol, 1.0 eq) in
tetrahydrofuran (10 mL) was added pyridine (0.04
mL, 0.5 mmol, 1.0 eq) and 4-dimethylaminopyridine
(12 mg, 0.1 mmol, 0.2 eq) at room temperature and
the mixture was cooled down in ice bath. To the
mixture, di-tert-butyl dicarbonate (164 mg, 0.75
mmol, 1.5 eq) in tetrahydrofuran (2 mL) was added
dropwise at 0 °C. The ice bath was removed and the
reaction mixture was allowed to warm to room
temperature and stirred for 16 h at room temperature
and then concentrated in vacuo. The residue was
diluted with ethyl acetate (100 mL) and washed with
water (2 X 30 mL) and brine (30 mL). The organic
layer was dried with MgSO4, filtered and
concentrated in vacuo. The residue was purified by
flash column chromatography over silica gel
(hexane/ethyl acetate, 20:1, v/v) to afford the desired
product 4h (90 mg, 54%) as orange solid: Rf = 0.5
(9H-Xanthen-9-ylidene)hydrazine (6b). Yellow
solid (21% yield): Rf = 0.3 (hexane/ethyl acetate, 5:1,
1
v/v); mp 126-128 °C; H NMR (CDCl3, 500 MHz) δ
8.32 (d, J = 8.0 Hz, 1H), 7.91 (d, J = 7.5 Hz, 1H),
7.43 (t, J = 7.5 Hz, 1H), 7.34-7.30 (m, 2H), 7.21 (t, J
= 7.5 Hz, 1H), 7.18-7.15 (m, 2H), 5.80 (br, 2H); 13C
NMR (CDCl3, 125 MHz) δ 154.0, 151.8, 135.9,
1
(hexane/ethyl acetate, 5:1, v/v); mp 108-110 °C; H
12

130.8, 129.4, 127.4, 124.1, 123.9, 123.3, 122.6, 118.2,
117.5, 116.5 ppm; DART-HRMS found 211.08521
[M+H]+, calcd for C13H11N2O 211.08714.
7.5 Hz, 1H), 7.61 (d, J = 7.5 Hz, 1H), 7.47 (t, J = 7.5
Hz, 1H), 7.38-7.35 (m, 2H), 7.31 (t, J = 7.5 Hz, 1H),
1.61 (s, 9H); 13C NMR (CDCl3, 125 MHz) δ 152.8,
146.1, 142.4, 139.1, 137.1, 130.9, 129.9, 129.8, 128.3,
127.9, 125.3, 122.3, 120.9, 119.5, 82.4, 28.3 ppm;
DART-HRMS found 295.14353 [M+H]+, calcd for
C18H19N2O2 295.14353.
1-(9H-Fluoren-9-ylidene)-2-(3-fluorophenyl)hydra-
zine (6d). To an ethanol (10 mL) suspension of 3-
fluorophenylhydrazine HCl (325 mg, 2.0 mmol, 2.0
eq) was added triethylamine (0.29 mL. 2.1 mmol, 2.1
eq) and the mixture was stirred for 0.5 h. To the
reaction mixture was added fluoren-9-one (180 mg,
1.0 mmol, 1.0 eq) and refluxed for 24 h. After the
completion, the mixture was concentrated in vacuo.
The residue was diluted with ethyl acetate (100 mL)
and washed with water (2 X 30 mL) and brine (40
mL). The organic layer was dried with MgSO4,
filtered and concentrated in vacuo. The residue was
purified by flash column chromatography over silica
gel (hexane/ethyl acetate, 10:1, v/v) to afford the
desired product 6d (242 mg, 84%) as brown solid: Rf
= 0.4 (hexane/ethyl acetate, 5:1, v/v); mp 159-161 °C;
1H NMR (CDCl3, 500 MHz) δ 8.80 (s, 1H), 7.90-7.88
(m, 1H), 7.83 (d, J = 8.0 Hz, 1H), 7.78 (d, J = 7.5 Hz,
1H), 7.66-7.65 (m, 1H), 7.45 (td, J = 7.5, 0.5 Hz, 1H),
7.39-7.27 (m, 4H), 7.18 (dt, J = 11.0, 2.5, 1H), 6.98
(dd, J = 8.0, 2.0 Hz, 1H), 6.68 (td, J = 8.5, 2.5 Hz,
2,3,4,5-Tetrachlorocyclopenta-2,4-dien-1-one
oxime (7a). To hydroxylamine HCl (417 mg, 6.0
mmol, 6.0 eq) in 50 mL-round bottomed flask was
added methanol (5 mL) and potassium hydroxide (337
mL, 6.0 mmol, 6.0 eq) in methanol (5 mL) at room
temperature and the mixture was stirred for 1 h. Then
the resultant KCl was filtered and the hydroxylamine
solution was added to hexachlorocyclopentadiene
(0.16 mL, 1.0 mmol, 1.0 eq) in methanol (5 mL)
dropwise, and the mixture was refluxed for 6 h. After
all hexachlorocyclopentadiene was consumed, the
mixture was concentrated in vacuo. The residue was
purified by flash column chromatography over silica
gel (hexane/ethyl acetate, 10:1, v/v) to afford the
desired product 7a (80 mg, 34%) as red brown solid:
Rf = 0.5 (hexane/ethyl acetate, 5:1, v/v); mp 178-180
1
°C; H NMR (DMSO-d6, 500 MHz) δ 14.52 (s, 1H);
1H); 13C NMR (CDCl3, 125 MHz) δ 164.0 (d, JCF
=
13C NMR (DMSO-d6, 125 MHz) δ 145.8, 134.2,
128.3, 119.0, 109.3 ppm; DART-HRMS found
231.87045 [M+H]+, calcd for C5HCl4NO 231.88905.
242.8 Hz, 1C), 146.2 (d, JCF = 10.6 Hz, 1C), 141.5,
141.0, 138.1, 137.7, 130.5 (d, JCF = 9.8 Hz, 1C),
130.1, 129.8, 128.5, 128.0, 127.6, 124.4, 121.1, 120.9,
119.6, 109.3 (d, JCF = 2.5 Hz, 1C), 108.2 (d, JCF
=
1,2,3,4-Tetrachloro-5,5-dimethoxycyclopenta-1,3-
diene (7b). To a methanol (5 mL) solution of
hexachlorocyclopentadiene (0.48 mL, 3.0 mmol, 1.0
eq) was added potassium hydroxide (370 mL, 6.6
mmol, 2.2 eq) in methanol (5 mL) dropwise for 30
min at room temperature. The mixture was stirred for
18 h and the mixture was poured to chopped ice (70
mL). After the ice had melted, the mixture was
extracted with dichloromethane (3 X 100 mL). The
organic layer was dried with brine (100 mL) and
MgSO4, filtered and concentrated in vacuo. The
residue was purified by flash column chromatography
over silica gel (hexane/ethyl acetate, 10:1, v/v) to
afford the desired product 7b (230 mg, 29%) as light
brown oil: Rf = 0.15 (hexane/ethyl acetate, 10:1, v/v);
1H NMR (CDCl3, 500 MHz) δ 3.34 (s, 6H); 13C NMR
(CDCl3, 125 MHz) δ 129.4, 128.5, 104.8, 51.9 ppm.
21.6 Hz, 1C), 101.0 (d, JCF = 26.5 Hz, 1C) ppm ;
DART-HRMS found 289.11254 [M+H]+, calcd for
C19H14FN2 289.11355.
tert-Butyl
2-(9H-fluoren-9-ylidene)hydrazine-1-
carb-oxylate (6e). To a solution of (9H-Fluoren-9-
ylidene)hydrazine (6a, 97 mg, 0.5 mmol, 1.0 eq) in
tetrahydrofuran (8 mL) was added pyridine (0.04 mL,
0.5 mmol, 1.0 eq) and DMAP (12 mg, 0.1 mmol, 0.2
eq) at room temperature, and the mixture was cooled
with an ice-bath. To the mixture, di-tert-butyl
dicarbonate (164 mg, 0.75 mmol, 1.5 eq) in
tetrahydrofuran (2 mL) was added dropwise at 0 °C.
The ice bath was removed and the reaction mixture
was allowed to warm to room temperature and stirred
for 3 h at room temperature and then concentrated in
vacuo. The residue was diluted with ethyl acetate (100
mL) and washed with water (2 X 30 mL) and brine
(30 mL). The organic layer was dried with MgSO4,
filtered and concentrated in vacuo. The residue was
purified by flash column chromatography over silica
gel (hexane/ethyl acetate, 10:1, v/v) to afford the
desired product 6e (96 mg, 65%) as yellow solid: Rf =
Biological methods
Cell lines. The 8226 and MM1.S myeloma cell lines
were purchased from ATCC. The cell lines were
characterized by FISH analysis and shown to contain
MAF/Ig translocations. Western blot confirmed a
significant over-expression of DEPTOR protein. Both
lines were tested for mycoplasma within the last 6
months and were negative.
o
0.5 (hexane/ethyl acetate, 5:1, v/v); mp 106-107 C;
1H NMR (CDCl3, 500 MHz) δ 8.85 (s, 1H), 7.93 (d, J
= 7.5 Hz, 1H), 7.79 (d, J = 8.0 Hz, 1H), 7.74 (d, J =
13

Western blot assay. Protein was extracted and
separated by 12.5% SDS-PAGE as previously
* M.E.J.: e-mail, jung@chem.ucla.edu; phone, (310)
825-7954.
* A.L.: e-mail, alan.lichtenstein@med.va.gov; phone,
(310) 268-3622
described.14
Proteins
were
transferred
to
polyvinylidene difluoride membranes and their
expression was detected utilizing specific antibodies
purchased from Cell Signaling (Beverly, MA).
Author Contributions
All authors contributed to the writing of this
manuscript. All authors have given approval to the
final version of the manuscript.
MTT assay. The MTT assay was performed by
seeding 1-2 x 104 target cells in 0.1 ml of complete
media into wells of a 96 well microtiter plate. After
incubation with compounds, the reduction of MTT to
formazan by live cells was determined with a
microplate ELISA reader equipped with a 570 nm
filter. Quadrupliate wells were run for each group and
the SD of each group was always <5% of the mean.
Results are presented as % of control or % survival
where OD of exp group was compared to the OD of a
control group (cells incubated with DMSO alone)
where the latter was arbitrarily made to be 100%.
Notes
The authors declare no competing financial interest.
M.E.J., J.L., A.L., Y.S. and J.G. are coauthors on a
patent application that includes the molecules
described in this manuscript.
Acknowledgments
This study was supported by NIH grants
2RO1CA111448, RO1CA168700, R21CA168491 as
well as research funds of the Veteran’s
Administration and the Multiple Myeloma Research
Foundation. The NMR spectrometers were supported
by the National Science Foundation under equipment
grant no. CHE-1048804.
Apoptosis assay. To identify apoptotic cells, we used
a phycoerythrin (PE)-conjugated antibody specific for
activated caspase 3 (BD Biosciences). For staining,
106 cells were washed with PBS and fixed and
permeabilized with 0.5 ml cytofix/cytoperm solution.
The cells were then incubated with a 1:5 dilution of
PE-conjugated monoclonal anti-caspase 3 antibody
for 30 mins and analyzed by flow cytometry.
References and Notes
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Kang SA, Kuehl WM, Gray NS, Sabatini DM.
DEPTOR is an mTOR inhibitor frequently
overexpressed in multiple myeloma cells and required
for their survival. Cell 2009:137:873-886.
(2) Carrasco DR, Tonon G, Huang Y, Zhang Y, Sinha
R, Feng B. High resolution genomic profiles define
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RAPTOR knockdown. The short hairpin RNAs
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Lentivirus was produced by the UCLA Vector Core
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cells with lentivirus and selecting in geneticin.
Statistical analysis. The induction of p70
phosphorylation by derivative compounds was
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signals of phosphorylated p70 vs total p70. This ratio
was then compared to that resulting from parental
compound B, with the latter ratio arbitrarily placed at
‘1’. The IC50 for MTT cytotoxicity was determined
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activated caspase 3) in the DMSO control cultures
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Lockwood M. Chromogens based on Non-benzenoid
Aromatic Systems. Part III. Synthesis, Spectra, and
Supporting Information
Supplementary Figures 1-4, Schemes S1-S3, Tables
S1-S3, 1H NMR, 13C NMR and 19F NMR spectra.
Corresponding authors
14

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15