3-Deazaneplanocin A and neplanocin a analogues and their effects on apoptotic cell death

Article abstract of DOI:10.1002/cmdc.201402315

3-Deazaneplanocin A (DzNep) is a potential epigenetic drug for the treatment of various cancers. DzNep has been reported to deplete histone methylations, including oncogenic EZH2 complex, giving rise to epigenetic modifications that reactivate many silenced tumor suppressors in cancer cells. Despite its promise as an anticancer drug, little is known about the structure-activity relationships of DzNep in the context of epigenetic modifications and apoptosis induction. In this study, a number of analogues of DzNep were examined for DzNeplike ability to induce synergistic apoptosis in cancer cells in combination with trichostatin A, a known histone deacetylase (HDAC) inhibitor. The structure-activity relationship data thus obtained provide valuable information on the structural requirements for biological activity. The studies identified three compounds that show similar activities to DzNep. Two of these compounds show good pharmacokinetics and safety profiles. Attempts to correlate the observed synergistic apoptotic activities with measured S-adenosylhomocysteine hydrolase (SAHH) inhibitory activities suggest that the apoptotic activity of DzNep might not be directly due to its inhibition of SAHH.

Full text of DOI:10.1002/cmdc.201402315

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DOI: 10.1002/cmdc.201402315
3-Deazaneplanocin A and Neplanocin A Analogues and
Their Effects on Apoptotic Cell Death
Eric K. W. Tam,[a] Tuan Minh Nguyen,[a] Cheryl Z. H. Lim,[b] Puay Leng Lee,[b] Zhimei Li,[b]
Xia Jiang,[b] Sridhar Santhanakrishnan,[a] Tiong Wei Tan,[a] Yi Ling Goh,[a] Sze Yue Wong,[a]
Haiyan Yang,[a] Esther H. Q. Ong,[c] Jeffrey Hill,[c] Qiang Yu,*[b] and Christina L. L. Chai*[a, d]
3-Deazaneplanocin A (DzNep) is a potential epigenetic drug for
the treatment of various cancers. DzNep has been reported to
deplete histone methylations, including oncogenic EZH2 com-
plex, giving rise to epigenetic modifications that reactivate
many silenced tumor suppressors in cancer cells. Despite its
promise as an anticancer drug, little is known about the struc-
ture–activity relationships of DzNep in the context of epigenet-
ic modifications and apoptosis induction. In this study,
a number of analogues of DzNep were examined for DzNep-
like ability to induce synergistic apoptosis in cancer cells in
combination with trichostatin A, a known histone deacetylase
(HDAC) inhibitor. The structure–activity relationship data thus
obtained provide valuable information on the structural re-
quirements for biological activity. The studies identified three
compounds that show similar activities to DzNep. Two of these
compounds show good pharmacokinetics and safety profiles.
Attempts to correlate the observed synergistic apoptotic activi-
ties with measured S-adenosylhomocysteine hydrolase (SAHH)
inhibitory activities suggest that the apoptotic activity of
DzNep might not be directly due to its inhibition of SAHH.
1. Introduction
Epigenetic alterations play important roles in cancer develop-
ment. These alterations, including DNA hypermethylation and
chromatin modifications such as histone methylation and de-
acetylation,[1] have provided new targets for therapeutic inter-
ventions in cancer cells. While advancements have been made
in the last decade in the development of inhibitors targeting
histone deacetylation or DNA methylation for cancer therapy,[2]
there is little progress in the development of histone methyla-
tion inhibitors. Aberrant histone methylation, such as histone
H3 lysine trimethylation (H3K27me3) induced by oncogenic
polycomb protein histone methyltransferase EZH2, has been
frequently linked to tumorigenesis,[3] so there has been tre-
mendous effort directed towards the development of small
molecules that target EZH2.
We previously reported 3-deazaneplanocin A (DzNep)
(Figure 1), an S-adenosylhomocysteine (AdoHcy) hydrolase
(SAHH) inhibitor that can effectively deplete cellular levels of
[a] Dr. E. K. W. Tam, Dr. T. M. Nguyen, Dr. S. Santhanakrishnan, T. W. Tan,
Y. L. Goh, S. Y. Wong, H. Yang, Prof. C. L. L. Chai
Institute of Chemical & Engineering Sciences
Figure 1. Structures of 3-deazaneplanocin A (DzNep) and neplanocin (NPA).
Agency for Science, Technology & Research (A*STAR)
8 Biomedical Grove, Neuros #07-01, Singapore 138665 (Singapore)
E-mail: christina_chai@ices.a-star.edu.sg
the EZH2 complex, inhibit H3K27me3 and other histone meth-
ylations, and induce apoptosis in cancer cells, but not in
normal cells, through the reactivation of many silenced tumor
suppressors in cancer cells.[4] Although this compound does
not appear to be specific for EZH2–H3K27me3, its anticancer
activity associated with the depletion of EZH2 has been dem-
onstrated in vitro and in vivo in many cancer types, including
breast, lung, liver, prostate, leukemia.[5] Recent efforts have led
to the discovery of a number of selective EZH2 inhibitors, for
example, EPZ005687,[6] GSK126,[7] and EI1.[8] However, these in-
hibitors seem to have anticancer activity only in B cell lympho-
mas that carry activating mutations of EZH2, but not in epithe-
lial tumors in which enhanced EZH2 activity is mainly caused
[b] C. Z. H. Lim, P. L. Lee, Z. Li, X. Jiang, Dr. Q. Yu
Cancer Therapeutics & Stratified Oncology, Genome Institute of Singapore
Agency for Science, Technology and Research (A*STAR)
60 Biopolis Street, #02-01, Singapore 138672 (Singapore)
E-mail: yuq@gis.a-star.edu.sg
[c] E. H. Q. Ong, Dr. J. Hill
Experimental Therapeutics Centre
Agency for Science, Technology and Research (A*STAR)
31 Biopolis Way, Nanos Level 3, Singapore 138669 (Singapore)
[d] Prof. C. L. L. Chai
Department of Pharmacy, National University of Singapore
18 Science Drive 4, Singapore 117543 (Singapore)
E-mail: phacllc@nus.edu.sg
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201402315.
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by increased gene expression.
Intriguingly,
a
recent study
showed that although inhibitors
of EZH2, such as GSK126, have
robust activity in the depletion
of H3K27me3 in epithelial
tumors cells, they have little
effect on cancer cell growth.[9]
Moreover, a peptide that induces
the depletion of EZH2 protein
complex is sufficient to inhibit
cell growth.[10] This raises a possi-
bility that targeting the EZH2
complex, rather than inhibiting
H3K27me3, is required for block-
ing the EZH2-mediated onco-
genic effect. Given the remark-
able activities of DzNep in the
depletion of EZH2 and in the in-
hibition of tumor growth, we
were intrigued with the possibili-
ties of examining DzNep deriva-
tives so as to obtain a better un-
derstanding of the structure–ac-
tivity relationships.
In this study, we report a com-
prehensive cell-based structure–
activity relationship study where
we screened for DzNep-like ac-
tivities for the induction of
apoptosis in cancer cells. We
also report the IC50 values of se-
lected DzNep analogues against
human SAHH enzymes with the
objective of determining if a cor-
relation exists between SAHH in-
hibition and the ability of the
compounds to induce apoptosis.
2. Results
2.1 Synthesis of the target
compounds
Our early studies have shown
that the natural product, nepla-
nocin A (NPA) (Figure 1) is able
to induce the same level of
apoptosis as DzNep in cellular
models (unpublished observa-
tions). We thus designed and
synthesized a total of 40 com-
Figure 2. Compounds used for structure–activity determination. Series A includes variations in substituents at-
tached to the carbocyclic ring system of NPA. Series B and D include variations in the carbocyclic ring system.
Series C includes variations in the heterocyclic ring attached. For D7, D8, D14, D18, and D19, the relative stereo-
chemistry is shown.
pounds based on variations in the structure of DzNep and NPA
(Figure 2). They include 1) variations in the substituents at-
tached to the carbocyclic ring system of NPA (series A, com-
pounds A1–A10), 2) variations in the carbocyclic ring system
(series B and D, compounds B1–B4, D1–D19), 3) variations in
the nature of the heterocyclic ring attached to the carbocyclic
ring (series C, compounds C1–C9). Only the synthesis of select-
ed compounds is discussed here. The full experimental details
on the synthesis of the target compounds can be found in the
Supporting Information.
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In general, the target compounds were synthesized by react-
ing the carbocyclic scaffold (“core”) with the appropriate heter-
ocyclic bases under Mitsunobu or SN2 conditions (Scheme 1).
For compounds in series A and C, the core is comprised of
functionalized cyclopentene 1 and 2, while the compounds in
series B and D were either commercially available or were syn-
thesized following literature procedures (see Supporting Infor-
mation).
Cyclopentenol 1 (Z=H) was synthesized in eight steps start-
ing from d-ribose,[11] and subsequent transformations gave
protected NPA 3 in good yields. With the protected NPA in
hand, protected precursors of A2–A10 were accessed from this
advanced intermediate as summarized in Scheme 2. Com-
pound 4 was directly accessed from 3 through the reaction of
the alcohol with 1,1’-thiocarbonyl diimidazole and tributyltin
hydride, while the synthesis of 5 was achieved using dimethy-
laminosulfur trifluoride (DAST). Methylation, ethylation, and
benzylation of 3 gave the O-analogues 6, 7, and 9, respectively.
Phenoxy derivative 8 was obtained under Mitsunobu condi-
tions, while methyl ester 10 was accessed via a three-step pro-
Scheme 1. Key coupling step to target compounds.
Scheme 2. Reagents and conditions: a) 1. 1,1’-thiocarbonyl diimidazole, RT, 18 h, CH2Cl2, 56%, 2. tributyltinhydride, 1108C, 8h, 56%; b) DAST, RT, 3 h, 72%;
c) MeI, NaH, DMF, RT, 16 h, 54%; d) EtI, NaH, DMF, RT, 16 h, 25%; e) PhOH, PPh3, DIAD, THF, RT, 18 h, 78%; f) BnBr, NaH, DMF, RT, 16 h, 39%; g) 1. Dess–Martin
periodinane, CH2Cl2, RT, 17 h, 95%, 2. oxone, DMF, RT, 48 h, 74%, 3. (trimethylsilyl)diazomethane, MeOH/toluene (2:3), RT, 48 h, 47%; h) 11. Dess–Martin peri-
odinane, CH2Cl2, RT, 17 h, 95%; 2) MeMgBr, THF, 08C!RT, 16 h, 86%.
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cedure (oxidation to the aldehyde with Dess–Martin periodi-
nane, followed by oxidation to the acid and esterification)
from alcohol 3. Compounds 11 and 12 were derived from the
same aldehyde by treatment with methyl magnesium iodide.
In all cases, global deprotection of the tert-butyloxycarbonyl
(Boc) and acetonide protecting groups under acidic conditions
afforded the target compounds.
For the synthesis of cyclopentenol 15, d-ribose was convert-
ed, through a sequence of reactions, to a diastereomeric mix-
ture of dienes 13 following modified procedures (Scheme 3).[12]
Scheme 3. Reagents and conditions: a) 1. 2nd-gen. Hoveyda–Grubbs catalyst,
CH2Cl2, RT, 4 h, 2. pyridinium chlorochromate, Celite, CH2Cl2, RT, 4 h (Overall
yield of 55% from the aldehyde; see Supporting Information for details);
b) NaBH4, CeCl3, 08C!RT 85–95%.
Ring-closing metathesis followed by pyridinium chlorochro-
mate (PCC) oxidation gave enone 14 in reasonable yields. The
ketone functionality of the enone was then reduced under
Luche’s conditions to afford a single stereoisomer (15) in high
isolated yields. With this alcohol 15 in hand, coupling with
N,N-diprotected adenine followed by deprotection gave com-
pound A1 in good yields (Scheme 4). Subsequent hydrogena-
tion of A1 gave compound D9 in quantitative yields.
Scheme 5. Reagents and conditions: a) I2, pyridine, CH2Cl2/THF (10:1), RT,
18 h, 75%; b) NaBH4, CeCl3, MeOH, 08C!RT, 1 h, 98%; c) TBDPSCl, imidazole,
CH2Cl2/THF (10:1), RT, 18 h, 93%; d) nBuLi, NFSI, THF, ꢀ608C, 4 h; e) 1. TBAF,
THF, RT, 18 h, 2. TsCl, Et3N, DMAP, CH2Cl2, RT, 18 h; f) adenine, NaH, DMF,
808C, 18 h, 28% over four steps for 23; g) 2n HCl, RT, 48 h, 79%; h) 10%
Pd/C, H2 (50 psi), MeOH, RT, 16 h, 73%; i) 2n HCl, RT, 48 h, 95%.
Scheme 4. Reagents and conditions: a) 1. PPh3, DIAD, diBoc adenine, THF,
80%, 2. 10% HCl in H2O/MeOH (1:1), 90%; b) H2, 10% Pd/C, H2O, RT, 24 h,
95%.
using tetrabutylammonium fluoride (TBAF) provided an insepa-
rable mixture of alcohols. Tosylation of the mixture to give 21
and 22, followed by alkylation with adenine in N,N-dimethyl-
formamide (DMF), yielded a mixture of 23 and 24, which was
separable by column chromatography. The reduction of the
double bond of 23 in the presence of Pd/C under high pres-
sure (50 psi) provided 25 in 73% yield. The stereochemistry of
the fluorine substituent and the basic skeleton of 25 was un-
ambiguously characterized by X-ray crystallography (see Sup-
porting Information). Hydrolysis of 25 in acidic medium gave
D16 in good yields. Similarly, hydrolysis of 23 furnished D4 in
good yields.
The synthesis of D4 and D16 are summarized in Scheme 5.
Using enone 14 as described in Scheme 4, iodination followed
by reduction of ketone 16 provided alcohol 17 in good yields.
The free hydroxy group of 17 was protected as its tert-butyldi-
phenylsilyl (TBDPS) ether 18. Fluorination of iodo-alkene 18
with excess N-fluorobenzenesulfonimide (NFSI) and BuLi fur-
nished an inseparable mixture of compounds 19 and 20 in
a ratio of 3:1, which was used as is in the subsequent steps.
Deprotection of the TBDPS groups of the mixture of 19 and 20
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Compounds D5, D6, D15, and D17 were synthesized from
versatile iodocyclopentenes 17 and 18 through palladium-cata-
lyzed reactions followed by a sequence of transformations sim-
ilar to that described previously (Scheme 6; see Supporting In-
formation for details). The stereochemistry of the vicinal hy-
droxy groups of the cyclopentane ring in compounds D15 and
D17 is presumed to be syn, based on observation made with
D16.
placement of the carbocyclic ring of NPA with other carbocy-
clic core structures (series B) indicated that the free hydroxy
substituents are critical for DzNep-like apoptotic activity as is
the stereochemistry of the hydroxy groups, as B1 and B2 do
not show any synergistic activity. Surprisingly, saturation of the
double bond (cyclopentane vs cyclopentene; B3) retained the
synergistic apoptotic activity.
In view of the simplicity of the structure of A1, further struc-
tural modifications were centered on variations of this deriva-
tive. Compounds in series C examined the effect of varying the
heterocyclic bases, and none of the compounds tested in this
series show any significant DzNep-like apoptotic activity. Series
D further examined the effect of varying the carbocyclic core.
Consistent with the observations made previously, saturation
of the double bond of A1 (D9) retained the apoptotic activity.
The vinyl fluoride derivative D4 and the saturated compound
D16 showed synergistic apoptotic cell death, while all other
variations of the carbocyclic core resulted in loss of apoptotic
activity. This indicates the sensitivity of the carbocyclic core in
inducing apoptosis. As observed with compounds in series B,
the location, stereochemistry, and presence of both the secon-
dary hydroxy groups are critical for apoptosis. In addition, the
ring size is also important. From these studies, three com-
pounds D4, D9, and D16 have been identified as showing syn-
ergistic apoptotic activity comparable with DzNep.
A secondary screen with selected positive hits was carried
out with HCT116 cells in which E2F1 is fused to the 4-hydroxy-
tamoxifen (4-OHT)-responsive ligand-binding domain of the es-
trogen receptor (ER) to form an ER–E2F1 fusion protein, which
becomes activated after addition of 4-OHT. We have previously
shown that DzNep, upon addition of 4-OHT (to activate E2F1)
in cell culture, potently induced apoptosis in this system, while
only a modest level of apoptosis was induced in the absence
of 4-OHT.[14]
Scheme 6. Synthetic routes to D5, D6, D15, and D17. Reagents and condi-
tions: a) PdCl2(PPh3)2, AlMe3, THF, 508C, 18 h; b) Phenylboronic acid, PdCl2-
(PPh3)2, Na2CO3, toluene/MeOH, 508C, 18 h. (See Supporting Information for
details).
2.2 Cell-based structure–activity relationship studies
We have previously shown that DzNep is able to synergize
with histone deacetylase (HDAC) inhibitor, trichostatin A (TSA),
to potently induce apoptosis in colon cancer DLD1 cells.[13] We
also showed that DzNep potently induces apoptosis upon acti-
vation of transcription factor E2F1 in colon cancer cell
HCT116.[14] We thus used these two independent in vitro cellu-
lar systems to determine the cellular activities of the DzNep-
like compounds described here. Initially, DLD1 cells were used
to screen for DzNep analogues that can increase apoptosis in
combination with TSA. DLD1 cells were treated with DzNep an-
alogues (5 mm) for 48 h followed by the HDAC inhibitor TSA
(150 nm) for an additional 24 h before harvesting for apoptosis
assessment using fluorescent-activated cell sorting (FACS) anal-
ysis of DNA content. The positive hits were compounds that
show increased apoptotic cell death on drug combination,
that is to say, where the apoptotic cell death is greater than
the sum of that caused by treatment with the DzNep analogue
or TSA alone. The results are summarized in the Supporting In-
formation.
As shown in Figure 3, in HCT116 ER-E2F1 cells, 4-OHT and
DzNep alone induced approximately 13% and 20% of cells in
apoptosis, respectively, as determined by counting cells in sub-
G1 phase using FACS analysis, while the combination of 4-OHT
and DZNep resulted in 54% of cells in apoptosis. Compounds
A4, D4, D9, and D16 exhibited comparable activities to DzNep,
resulting in synergistic apoptosis of 45%, 47%, 49%, and 47%,
From the screening studies, the effect of substitution (R) at
the C-4 position of the carbocyclic ring of NPA was investigat-
ed. Positive hits are compounds A1–A4, A9, and A10 (i.e.,
compounds possessing H, Me, CH2F, CH2OMe, CH(OH)CH3 sub-
stituents instead of the CH2OH substituent present in NPA). Re-
Figure 3. In vitro assessment of DzNep analogues. p53-null HCT116-ER–
E2F1-expressing cells were treated with DzNep (5 mm) or DzNep analogues
A4, D4, D9 or D16 in the presence or absence of 4-OHT (300 nm). After
72 h, cells were harvested, and cell death was assessed by propidium iodide
staining using fluorescent-activated cell sorting (FACS).
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respectively, when combined with 4-OHT. We also showed that
the DzNep analogues have the capacity to deplete histone
methylations including H3K27me3, H3K4me3, and H3K20me3,
though D9 seems to be more selective on H3K27me3 as
shown in Figure 4.
can be concluded that neither D9 and D16 show any signifi-
cant inhibition of the hERG channel.
2.5. Antitumor effects of D9 in a xenograft tumor mouse
model
To evaluate the anticancer effect of D9 in vivo, an HCT116 sub-
cutaneous xenograft tumor mouse model was used. In this
model, 5ꢁ106 HCT116 cells were injected into Balb/c nude
mice subcutaneously (s.c.), and when the tumors reached an
average of 200 mm3 in size, D9 was administered through in-
traperitoneal (i.p.) injection. As shown in Figure 5a D9 adminis-
tration at 30 or 80 mgkgꢀ1 five days a week for two weeks re-
sulted in marked decrease in tumor growth in a dose-depen-
dent manner. During the course of drug treatments, the body
weight of mice was monitored daily. D9 did not give rise to
obvious signs of toxicity, and only a minimum loss of body
weight was observed (Figure 5b). These data suggest that D9
has significant antitumor effects while at the effective doses it
Figure 4. Western blot showing the effect of DzNep analogues on histone
methylation. HCT116 cells were treated with increasing concentrations of in-
dicated compounds (0.5, 1, 2.5 and 5 mm) for 72 h before being harvested
for Western blot analysis.
2.3 SAHH inhibition by selected compounds
A total of 16 compounds (DzNep, NPA, A1, A3–A6, A9–10, D4–
6, D9, D15–17) were tested for inhibition of human SAHH
using an in vitro SAHH activity assay. Although the IC50 or Ki
values of some of these compounds have already been report-
ed in the literature, comparison of the literature values proved
to be difficult due to the different assay platforms and the spe-
cies and form of SAHH used (recombinant or purified native).
A summary of the results is shown in Table 1. From these stud-
ies, it is noted that NPA and DzNep are potent inhibitors of
SAHH, while D9 is approximately sixfold less potent than
DzNep. Interestingly D16 was 18 fold less potent than D9,
while the activity of D4 and D16 are similar. The ranking of the
compounds in terms of the IC50 values with SAHH inhibition is
NPA (most potent)>A3>DzNep>D9>A9–A4>A10>A6>
A5>D17>D16ꢁD4ꢁD15>A1>D5ꢁD6 (least potent).
2.4 In vitro ADME and hERG studies
Compounds D9 and D16 were selected for in vitro ADME eval-
uation and hERG studies. Measurement of the logD showed
that both compounds have comparable values (ꢀ1.6 at pH 3.0
and ꢀ0.5 at pH 7.4). In addition, the solubility of the com-
pounds at pH 2 and 6.5 did not change and is approximately
300 mm for both D9 and D16. The half-lives of D9 and D16
with human liver microsomes were measured to be 2636 min
and 1029 min, respectively, while that with male rat liver micro-
somes and male mouse liver microsomes were 183 min and
307 min for D9 and 836 and 982 min for D16, respectively.
hERG safety studies were performed, and the IC50 values of
both D9 and D16 were found to be greater than 100 mm using
a FluxOR hERG fluorescence assay, and greater than 10 mm
using the electrophysiological hERG test. From these data, it
Figure 5. Effects of D9 in vivo on a) HCT116 xenograft tumor growth and
^
b) body weight change of mice. Group I: 10% DMSO ( ); Group II: D9
30 mgkgꢀ1 (&); Group III: D9 80 mgkgꢀ1 (~).
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Table 1. Inhibition (IC50) of human S-adenosylhomocysteine hydrolase (SAHH) by DzNep derivatives.[a]
Structure
IC50 [mm]
Structure
IC50 [mm]
0.23ꢂ0.07
[Lit: Ki=23 nm,
human SAHH][25]
0.04ꢂ0.01
DzNep
NPA
[Lit: IC50 =0.82 mm,
human SAHH][26]
2.22ꢂ0.24
A4
A6
A9
A1
D9
D5
D6
[Lit: IC50 >10 mgmLꢀ1
,
A5
A3
13.1[b]
rabbit erythrocyte][19]
0.13ꢂ0.04
7.47ꢂ4.30
[Lit: IC50 >10 mgmLꢀ1
,
rabbit erythrocyte][19]
1.71ꢂ0.24
A9/A10
(15:85)[c]
[Lit: IC50 =0.2 mgmLꢀ1
rabbit erythrocyte][19]
,
3.46ꢂ0.87
44.59ꢂ13.34
[Lit: Ki=35 nm,
bovine liver][27]
D4
24.39ꢂ0.40
25.18ꢂ2.17
25.7[b]
1.35ꢂ0.35
[Lit: IC50 =9 mm,
human SAHH][28]
D16
D15
D17
>200
>200
20.9[b]
[a] Inhibition was determination using an enzyme assay with recombinant expressed human SAHH; data represent the meanꢂSD of n=2 independent ex-
periments, unless specified otherwise. [b] Data from a single independent assay. [c] Tested as a mixture of compounds.
is well tolerated in the recipient mice (Figure 5). The pharma-
cokinetic profile of D9 in Sprague–Dawley rats through oral
(p.o.) administration (5 mgkgꢀ1) also suggested that D9 is
readily absorbed and has a half-life of 5 h.
3. Discussion
Our studies above have delineated some of the critical struc-
tural features required for DzNep-like apoptotic activity
(Figure 6). In brief, the structure–activity relationship study
identified that the 2,3-dihydroxycarbocyclic core of DzNep,
whether saturated or unsaturated, is needed for synergistic ac-
tivity (Figure 6). The heterocyclic base at the C-1 position of
Figure 6. Critical structural features for DzNep-like apoptotic activity.
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the carbocycle can either be adenine or deazaadenine, but all
the other heterocyclic combinations led to loss in activity. Thus
compound A1 and D9 contain the minimum pharmacophore
required for synergistic apoptotic activity. When considering
the structure–activity relationships of DzNep and NPA, it is ap-
parent that variation in the substituents at the C-4 position
cannot be easily rationalized. The presence of a substituent is
not necessary for synergistic apoptotic activity, as seen with
compound A1. There is also some tolerance for small substitu-
ents at the C-4 position of the carbocyclic ring. Interestingly,
a secondary alcohol at the C-4 position does not lead to large
losses in biological activity. The presence of substituents at the
C-5 position of the carbocyclic ring causes large losses in syn-
ergistic apoptotic activity—the exception is when a fluorine
atom replaces the hydrogen atom at C-5 of A1 and D9.
4. Conclusions
The toxicities and the short half-lives of DzNep and NPA have
long been known, and this is in part due to the rapid phos-
phorylation by adenosine kinase.[20] Through these studies, we
have identified two compounds (D9 and D16) that have
longer half-lives than NPA and DzNep. In addition, animal stud-
ies with D9 show that it has lower toxicity than DzNep and is
effective at shrinking tumors. Thus, we have successfully identi-
fied at least two compounds, which are potentially superior to
DzNep and NPA, for further development as chemotherapeu-
tics for epigenetic therapy. Future mechanistic studies will un-
doubtedly shed more information on the mode of action of
these compounds.
SAHH is an important enzyme that catalyzes the hydrolysis
of S-adenosylhomocysteine (AdoHcy) to adenosine and l-ho-
mocysteine.[15] Accumulation of AdoHcy through inhibition of
SAHH can, in turn, lead to inhibition of methyltransferases and
thus indirectly affect the epigenetic mechanisms of a cell.
DzNep and NPA have long been recognized as potent inhibi-
tors of SAHH. Indeed, early studies have focused on the devel-
opment of these and related compounds as antivirals through
the inhibition of SAHH.[16] The mechanisms of SAHH inhibition
are suggested to occur either via a reversible Type I mecha-
nism through the oxidation of the cofactor NAD+ to NADH, or
via an irreversible Type II mechanism of covalent binding to
the active site by an inhibitor with a nucleophilic residue.[17] Of
the selection of the target compounds measured for SAHH ac-
tivity, the most potent SAHH inhibitor was NPA (IC50 =40 nm),
followed by the fluorinated compound A3 (IC50 =130 nm),
then DzNep (IC50 =230 nm). We note that these values for NPA
and DzNep differ from those reported in the literature for
human SAHH.[16b,18] The compounds that were tested from ser-
ies A showed that SAHH inhibition is sensitive to modifications
of the substituents at the C-4 position. The stereoisomers of
the secondary alcohol A9 showed a 1.5-fold difference in
SAHH activities as compared with the enriched mixture of A10
(containing 15% A9). From the literature, a large difference in
SAHH activities between A9 and A10 was reported, in favor of
A9, as measured with rabbit SAHH.[19] For the selected com-
pounds studied in series D, the most potent compound is D9
which showed very different activity in comparison with the
unsaturated compound A1. The vinyl fluoride D4 was the best
compound amongst the unsaturated compounds in series D,
and D4 has been reported to inhibit SAHH via the Type II
mechanism.[20] The saturated compounds D15–D17 have com-
parable SAHH activity.
Experimental Section
Chemistry
Unless otherwise specified, materials were purchased from com-
mercial suppliers and used without further purification. Solvents
for reactions were taken from a Glass Contour solvent purification
system under nitrogen. 1H NMR and 13C NMR spectra were ac-
quired on a Bruker400. UltraShield spectrometer operating at
400 MHz and 100 MHz, respectively, unless specified otherwise.
Chemical shifts (d) are given in ppm relative to respective residual
solvent peaks (CD3OD: d=3.31 ppm; [D6]DMSO: d=2.50 ppm;
D2O: d=4.79 ppm) except for those recorded in CDCl3, which were
referenced to tetramethylsilane (TMS) or residual solvent peak at
d=7.26 ppm. For 13C NMR spectra, chemical shifts are given rela-
tive to the respective residual solvent peak (CD3OD: d=49.0 ppm;
[D6]DMSO: d=39.5 ppm; CDCl3: d=77.2 ppm). Where necessary,
the signals of novel compounds were assigned by 1DNOE differen-
ces and/or 2DNMR techniques: 1H–1H COSY, 13C–1H HMQC, and
13C–1H HMBC. These experiments were performed using standard
Bruker microprograms. Low-resolution and high-resolution electron
impact mass spectra (EIMS) were measured using a Finnigan
MAT95XP double-focusing mass spectrometer. Low-resolution elec-
trospray ionization mass spectra (ESIMS) were recorded using
Waters Quattro MicroTM API, and high-resolution mass spectra
(HRMS) were obtained using an Agilent6210 Time-of-Flight LC–MS
system. Flash column chromatography was conducted manually
using Merck silica gel 60 (35–70 mm) with an overpressure of
200 mbars. Amination reactions using 33% aqueous ammonia
were carried out using a high-pressure reactor. Elemental analysis
was performed using a EuroEA3000 series CHNS analyzer.
Compounds B1,[22] B2,[23] and D12[24] were synthesized following lit-
erature procedures, while B4 is commercially available. Detailed
procedures and characterization data for all other compounds are
provided in the Supporting Information.
Our SAHH studies identified NPA, DzNep, methyl ether A4,
fluorinated derivative A3, alcohol A9, and D9 as the best inhib-
itors of SAHH. Broadly speaking, these compounds are also
potent inducers of apoptosis. However there is no discernible
direct relationship between SAHH inhibition and synergistic
apoptotic activities. Specifically compounds that have reasona-
ble SAHH inhibitory activities (e.g., D15) do not display syner-
gistic apoptotic activity.
Biology
In vitro evaluation of DzNep analogues
Induction of apoptosis in HCT116 cells: p53 Knock out (KO) HCT116
cells that express inducible E2F1 (ER–E2F1) were used to assess the
ability of DzNep analogues to induce E2F1-dependent apoptosis.[14]
To activate E2F1, 4-hydroxytamoxifen (4-OHT) (300 nm) was added
to the tissue culture medium. Cell apoptosis induced by DzNep an-
alogues was measured in the presence or absence of 4-OHT for
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72 h by analysis of DNA content via FACS. To measure the ability of
DzNep analogues to synergize with a histone deacetylase inhibitor
(HDACi) for apoptosis induction, DLD1 cells were treated with
DzNep analogues (5 mm) for 48 h followed by HDACi trichostatin A
(TSA) (150 nm) for an additional 24 h before being harvested for
FACS analysis. To assess the apoptosis induction, cells were har-
vested and fixed in 70% ethanol. Fixed cells were stained with pro-
treated with vehicle or DzNep analogue D9 at 30 and 80 mgkgꢀ1
by intraperitoneal (IP) injection for 14 days. Tumor growth and the
whole body weight changes of mice were monitored every other
day.
Student’s t-test is used to determine the statistical significance of
tumor volumes and body weight changes between groups. Statis-
tical analyses are conducted at a p level of 0.05. SPSS was used for
all statistical analyses and graphic presentations.
pidium iodide (50 mgmLꢀ1
)
after treatment with RNase
(100 mgmLꢀ1). The FACS analysis of stained cells was done in FACS-
calibur flow cytometer (Becton Dickinson Instrument, San Jose, CA,
USA). Cell cycle fractions were quantified using the CellQuest soft-
ware (Becton Dickinson). Apoptosis was determined by measuring
the DNA content of cells in sub-G1.
Acknowledgements
The authors thank the Agency for Science, Technology and Re-
search (A*STAR), Singapore (CCOG01-001-2008 and ETPL/10-
FS0002) for financial support of this project.
Production and purification of recombinant SAHH: The glutatione S-
transferase (GST)-tagged SAHH in the pDEST 565 vector was ex-
pressed in Escherichia coli BL21 (DE3) pLysS and inoculated over-
night in LB medium with chloramphenicol (34 mgmLꢀ1 ) and ampi-
cillin (100 mgmLꢀ1) at 378C with orbital shaking at 200 rpm. The
culture was then diluted 1:250 in LB medium with chloramphenicol
and ampicillin, and maintained at 308C until the OD600 value
reached 0.5–0.6 arbitrary units. After the addition of isopropyl-b-d-
1-thiogalactopyranoside (400 mm), the culture was incubated for
an additional 16 h and centrifuged at 6000 rpm for 40 min. at 48C.
The bacterial pellet was then collected by centrifugation and lysed
by sonication in buffer containing Na3(PO4) (10 mm), NaCl
(150 mm), DNAseI (5 mgmLꢀ1), lysozyme (0.625 mgmLꢀ1), 1X pro-
tease inhibitor (pH 7.4), and 2-mercaptoethanol (5 mm). The over-
expressed protein was purified using the Bio-Scale Mini Profinity
GST 5 mL cartridges (BioRad) followed by concentration and desalt-
ing in Tris buffer (2 mm, pH 8.0) with NaCl (100 mm). The concen-
trated SAHH–GST was cleaved with Tobacco Etch Virus (TEV) pro-
tease at 1 mg TEV per 50 mg GST-tagged protein in Tris buffer
(100 mm, pH 8.0) with NaCl (100 mm), and dithiothreitol (DTT)
buffer (2 mm). The cleaved GST was removed using glutathione Se-
pharose 4B beads (GE Healthcare) after 1 h incubation in Tris buffer
(10 mm, pH 7.5) with NaCl (50 mm).
Keywords: deazaneplanocin A · epigenetics · neplanocin A · S-
adenosylhomocysteine hydrolase (SAHH) · structure–activity
relationships
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Received: July 26, 2014
Published online on && &&, 0000
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Epigenetic agents against cancer: 3-
Deazaneplanocin A and neplanocin A
are known to reactivate tumor suppres-
sors leading to apoptosis in cancer cells;
however, their exact mode of action is
not fully understood. Here, 42 ana-
logues were evaluated in cell- and
enzyme-based assays. No direct correla-
tion between cytotoxicity and SAHH in-
hibitory activity was found. The SAR
studies identified two compounds with
good PK and safety profiles that warrant
closer investigation.
E. K. W. Tam, T. M. Nguyen, C. Z. H. Lim,
P. L. Lee, Z. Li, X. Jiang,
S. Santhanakrishnan, T. W. Tan, Y. L. Goh,
S. Y. Wong, H. Yang, E. H. Q. Ong, J. Hill,
Q. Yu,* C. L. L. Chai*
&& – &&
3-Deazaneplanocin A and Neplanocin
A Analogues and Their Effects on
Apoptotic Cell Death
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