Tetrazolylhydrazides as selective fragment-like inhibitors of the JumonjiC-domain-containing histone demethylase KDM4A

Article abstract of DOI:10.1002/cmdc.201500335

The JumonjiC-domain-containing histone demethylase 2A (JMJD2A, KDM4A) is a key player in the epigenetic regulation of gene expression. Previous publications have shown that both elevated and lowered enzyme levels are associated with certain types of cancer, and therefore the definite role of KDM4A in oncogenesis remains elusive. To identify a novel molecular starting point with favorable physicochemical properties for the investigation of the physiological role of KDM4A, we screened a number of molecules bearing an iron-chelating moiety by using two independent assays. In this way, we were able to identify 2-(1H-tetrazol-5-yl)acetohydrazide as a novel fragment-like lead structure with low relative molecular mass (M_r=142 Da), low complexity, and an IC₅₀ value of 46.6 μm in a formaldehyde dehydrogenase (FDH)-coupled assay and 2.4 μm in an antibody-based assay. Despite its small size, relative selectivity against two other demethylases could be demonstrated for this compound. This is the first example of a tetrazole group as a warhead in JMJD demethylases. Anchor fragment: To develop non-promiscuous metalloenzyme inhibitors, a metal-complexing acetohydrazide group was integrated in a tetrazolyl fragment, which can be matured into a scaffold to promote further selectivity at the ligand backbone binding site of these emerging drug targets.

Full text of DOI:10.1002/cmdc.201500335

DOI: 10.1002/cmdc.201500335
Full Papers
Tetrazolylhydrazides as Selective Fragment-Like Inhibitors
of the JumonjiC-Domain-Containing Histone Demethylase
KDM4A
Nicole Rüger,[a] Martin Roatsch,[b] Thomas Emmrich,[a] Henriette Franz,[c] Roland Schüle,[c]
Manfred Jung,[b] and Andreas Link*[a]
The JumonjiC-domain-containing histone demethylase 2A
(JMJD2A, KDM4A) is a key player in the epigenetic regulation
of gene expression. Previous publications have shown that
both elevated and lowered enzyme levels are associated with
certain types of cancer, and therefore the definite role of
KDM4A in oncogenesis remains elusive. To identify a novel mo-
lecular starting point with favorable physicochemical proper-
ties for the investigation of the physiological role of KDM4A,
we screened a number of molecules bearing an iron-chelating
moiety by using two independent assays. In this way, we were
able to identify 2-(1H-tetrazol-5-yl)acetohydrazide as a novel
fragment-like lead structure with low relative molecular mass
(Mr =142 Da), low complexity, and an IC50 value of 46.6 mm in
a
formaldehyde dehydrogenase (FDH)-coupled assay and
2.4 mm in an antibody-based assay. Despite its small size, rela-
tive selectivity against two other demethylases could be dem-
onstrated for this compound. This is the first example of a tet-
razole group as a warhead in JMJD demethylases.
Introduction
The human enzyme lysine-specific demethylase 4A is encoded
by the KDM4A gene,[1] a member of the Jumonji domain 2
(JMJD2/KDM4) subfamily (isoforms A–E).[2] Under non-patho-
logical conditions, the expression of this gene in many normal
tissues is low, but there is ample evidence that elevated
KDM4A levels are associated with certain types of malignant
neoplasms. The enzyme could be identified as integral to the
proliferation of bladder,[3] colon,[4] lung,[5] and prostate can-
cers,[6] isolated breast cancer cell lines,[7] and also in manifest
breast cancer.[8] Overexpression of KDM4A was demonstrated
at the RNA level by real-time PCR, as well as at the protein
level by immunohistochemical staining. Moreover, suppression
of KDM4A expression in lung and bladder cancer cells resulted
in significant suppression of cell growth in these experi-
ments.[3] As expression levels of KDM4A were low in normal tis-
sues in all aforementioned cases, specific inhibitors of this
enzyme may have a decreased risk of adverse reactions.[9] In
stark contrast, other investigations have shown that KDM4A
levels were significantly lower in malignant urothelium tissue
than in healthy tissue, which could also be associated with
a negative prognosis.[10] At this point, the reader is referred to
the extensive and in-depth reviews of KDM4A’s complex role
in cancer by Hoffmann et al.[9] and Guerra-Calderas et al.[11]
While there seems to be a general trend regarding the correla-
tion between elevated KDM4A expression levels and cancer,
the opposite was observed as well. This picture is even further
complicated by the influence of single-nucleotide polymorph-
isms within the KDM4A gene, as recently reported by Van Re-
chem et al.[12] Thus, the definite role of this enzyme in onco-
genesis remains to be determined.
Structures of the catalytic-core domain of KDM4A with and
without its natural substrate, 2-oxoglutarate (1), in the pres-
ence of Ni2+ have been determined by X-ray crystallography.[13]
The structure of the core domain, consisting of the JmjN
domain, the JmjC domain, the C-terminal domain, and a zinc
finger motif, revealed the unique elements that form a poten-
tial substrate binding pocket.[14] In addition, the enzyme fea-
tures a JD2H domain, two TUDOR domains, and another PHD-
type zinc finger,[15] which have not yet been resolved crystallo-
graphically. This nuclear protein functions as 2-oxoglutarate-
and FeII-dependent oxygenase, which catalyzes a diverse set of
reactions.[16] In vitro, KDM4A specifically demethylates di- and
[a] N. Rüger, Dr. T. Emmrich, Prof. Dr. A. Link
Institute of Pharmacy, Ernst-Moritz-Arndt-Universität Greifswald
Friedrich-Ludwig-Jahn-Str. 17, 17487 Greifswald (Germany)
E-mail: Link@uni-greifswald.de
trimethylated residues of lysine 9 and 36 of histone H3 (H3K9/
[17]
36me2/3
)
through initial hydroxylation of the N-(e)methyl
groups, which results in the formation of unstable hemiaminal
intermediates that spontaneously decompose to yield formal-
dehyde and the residual di- or monomethylated lysine residue;
in vivo, however, the enzyme only demethylates trimethylated
residues.[17a,18] Accordingly, the binding of 2-oxoglutarate (1) is
crucial for the molecular interaction. Compounds that mimic
this acid might interfere with the catalytic mechanism.[19] Re-
cently, it could be shown that the well-known and widely used
[b] M. Roatsch, Prof. Dr. M. Jung
Institute of Pharmaceutical Sciences, Albert-Ludwigs-Universität Freiburg
Albertstr. 25, 79104 Freiburg (Germany)
[c] Dr. H. Franz, Prof. Dr. R. Schüle
University of Freiburg Medical Center
Department of Urology, Women’s Hospital and Center for Clinical Research
Breisacher Str. 66, 79106 Freiburg (Germany)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201500335.
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plant growth regulator daminozide (2, succinic acid 2,2-dime-
thylhydrazide) selectively inhibits KDM2A but neither KDM3A,
KDM4E, KDM5C, KDM6B nor PHD2 (hypoxia-inducible factor
prolyl hydroxylase), predominantly by competitive inhibition
with respect to 1 (Ki =1.97 mm).[20] In contrast, other known in-
hibitors such as 8-hydroxyquinoline-5-carboxylic acid (IOX1, 3)
are pan-KDM inhibitors that inhibit KDM2A, KDM3A, KDM4E,
KDM5C, and KDM6B to a similar extent (Figure 1).[21] One of the
amide group, this a-keto acid mimic complexes a divalent
cation (here Ni2+ instead of Fe2+ for stability reasons) that is
held in place by His188, His276, and Glu190 (Figure 2).
The conformationally unconstrained 5 inhibits KDM4A with
an IC50 value of 33 mm. Although the crystal structure of 5 with
Figure 2. Interactions of O-benzyl-N-(carboxycarbonyl)-d-tyrosine (5) and
KDM4A (image based on PDB 2WWJ).[26]
KDM4A indicates occupation of a large hydrophobic pocket
adjacent to the substrate binding cleft, the contribution of the
lipophilic interactions or p–p stacking of the phenyl and
benzyl moieties to Phe185 and Lys241 is only weakly pro-
nounced.[26] Considering this information, we designed a series
of acidic fragment-like molecules as potential inhibitors of 2-
oxoglutarate-dependent demethylases. Among the considera-
ble arsenal of iron chelating warheads such as hydroxamic
acids or diphenols, there is also the acylhydrazide function.
Thus, we decided to start with this motif as a replacement for
the a-keto acid moiety due to the success of this functional
group present in the proof-of-principle selective inhibitor 2.
The known binding mode of this inhibitor served as a starting
point for the selection of our iron binding warhead. In contrast
to the dimethyl substitution of 2, unsubstituted hydrazides
were prepared with a focus on ease of generation of ana-
logues by condensation with carbonyl compounds as, still
metal complexing, hydrazones.
Figure 1. Endogenous substrate 2-oxoglutarate (1) and a selection of pub-
lished inhibitors of JumonjiC-domain-containing histone demethylases 2–7.
earlier discovered KDM inhibitors with remarkable potency but
low selectivity is pyridine-2,4-dicarboxylic acid (4),[22] which fre-
quently serves as a standard against which other inhibitors are
compared in vitro. Due to their ionic character, none of the
aforementioned ligands is able to penetrate intact cell mem-
branes, which renders them unfeasible for any cell-based stud-
ies. This drawback was overcome for the first time by Hamada
et al. with their design of 2-oxoglutarate mimics that contain
a long alkyl chain (compound 6).[23] A structurally unique ap-
proach in this field was recently published by Wang et al.[24]
The E isomer of JIB-04 (7) is a strong, cell-active KDM4 inhibitor
with highest selectivity for KDM4D (JMJD2D); however, it is not
competitive against 1. An up-to-date review regarding known
demethylase inhibitors is provided by Thinnes et al.[25]
At the same time, we reasoned that it would be beneficial
to replace the second carboxylic acid of 2-oxoglutarate by
a tetrazole moiety which was unprecedented in this area. How-
ever, the bioisosteric replacement of carboxylic acids by tetra-
zoles has led to active compounds including many drugs in
the market, for example, diuretics and AT1 receptor antago-
nists. Size, pKa and electrostatic potential of carboxyl and tetra-
zolyl groups are similar, whereas tetrazoles are more lipophilic,
thus facilitating membrane penetration.[27] Based on these find-
ings, it should be possible to identify fragment-like inhibitors
that complex the divalent cation and form hydrogen bonds
and ionic interactions with protonated Lys206 and the phenol-
ic hydroxy group of Tyr132 in a favorable manner.
The pioneering work of the group of Schofield et al. has led
to a deeper understanding of the binding mode of experimen-
tal inhibitors of KDM4A.[2] Compounds with a core structure
closely resembling that of 1, such as O-benzyl-N-(carboxycar-
bonyl)-d-tyrosine (5),[26] which is essentially a derivative of the
known JmjC inhibitor N-oxalylglycine,[13] bind in a way that the
carboxylate group of the tyrosine moiety forms hydrogen
bonds and ionic interactions with protonated Lys206 and the
phenolic hydroxy group of Tyr132. The second carboxyl group
acts as a hydrogen bond donor toward Ser288 and a hydrogen
bond acceptor toward Ser196. Together with the adjacent
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moiety. Subsequently, intermediates 10a–c were subjected to
hydrazinolysis in quantitative yields.
Results and Discussion
The optimal distance between the two interaction motifs, that
is, metal chelating moiety and anionic center, was uncertain.
Based on visual inspection of available crystal structures (e.g.,
PDB 4AI9[20]) we envisioned the synthesis of three homologous
hydrazides by established procedures in a three-step reaction
and subjecting them to biological evaluation (see Scheme 1,
Table 1).
Counterintuitively, the tetrazolyl acylhydrazide with the
smallest distance of the interaction motifs, 11 a, was the most
active compound with an IC50 value of 46.64Æ0.94 mm in
a
formaldehyde dehydrogenase (FDH)-coupled assay (see
Table 2; enzyme concentration cenz =2.4 mm). In the antibody-
based LANCE assay that employs far less enzyme (cenz =60 nm),
First, the chlorinated acid esters 8b–c were converted into
the corresponding nitriles 9b–c in good yields (79–83%),[28] fol-
lowed by ammonium chloride catalyzed cyclization with
sodium azide in DMF to obtain the corresponding 5-tetrazoles
10a–c.[29] Yields ranged between 15–56% and decreased with
increasing chain length between the tetrazole ring and the car-
bonyl group, because of the diminished activation of the nitrile
Table 2. In vitro activity of derivatives against isolated KDM4A in the FDH
and LANCE assays.
Compd[a]
IC50 [mm]
FDH
LANCE
4 (control)
11 a
11 b
11 c
12 f
12g
1.37Æ0.39
46.64Æ0.94
64.1Æ12.8
69.8Æ15.5
391Æ61
0.0337Æ0.007
2.38Æ0.37
8.32Æ1.08
7.68Æ1.54
11.80Æ0.38
6.2Æ1.5
154Æ42
[a] All other compounds of the series 11 and 12 were tested as well, but
were devoid of activity (see Supporting Information S5).
the potency is as high as 2.38Æ0.37 mm. Pyridine-2,4-dicarbox-
ylic acid (4) was used as positive control in both assays. Similar
differences have also been observed for the reference inhibitor
JIB-04 (7), which includes a hydrazone moiety, with 17.6 mm in
the FDH assay and 4.1 mm in the LANCE assay.[24] As 7 showed
cellular activity even at 1 mm, at least in this case the FDH
assay rather seems to underestimate potency which is encour-
aging for our results. By employing the LANCE assay, we were
able to exclude FDH inhibition as a confounder. Either way,
with respect to the relative molar mass (Mr =142 Da) of this
fragment-like inhibitor, this activity is remarkable. To evaluate
the importance of the acidic tetrazole ring for inhibitory activi-
ty, a series of related acetohydrazides with substituted and un-
substituted aromatic or heteroaromatic rings (Figure 3), as well
as the ring-methylated derivative 20 of lead 11 a were pre-
pared (see Scheme 3 below). Derivative 20 lacks the acidic
properties of an unsubstituted 5-tetrazole, while still exhibiting
chelating properties.
Scheme 1. Synthesis of tetrazolylhydrazides 11 a–c with varying spacer
length. Reagents and conditions: a) NaCN, DMSO, 3 h, 508C!15 h, RT;
b) NaN3, NH4Cl, DMF, 8–24 h, 95–1258C; c) H2N-NH2, microwave, 5 min,
115 8C; d) appropriate carbonyl component, MeOH, microwave, 2 min, 808C.
Table 1. Residues R1 and R2 in 12a–u.
Compd[a]
R1
R2
12a
12b
12c
12d
12e
12 f
12g
12h
12i
CH3
CH3
H
CH3
H
4-pyridyl
4-methoxyphenyl
phenyl
phenyl
4-methoxyphenyl
CH3
While the tetrazole ring could be built up via cycloaddition
reaction, hydrazides 11 d–l were prepared by different routes
(Scheme 2). Pyridylacetohydrazides 11 d–f were synthesized by
chlorinating the corresponding pyridylmethanols 13a–c, fol-
lowed by Kolbe nitrile synthesis of the chloromethylpyridyl hy-
drochlorides 14a–c[30] and saponification of the resulting ni-
triles 15a–c in the presence of a short-chained alcohol to
obtain esters 10d–f, directly.[31] These esters were subsequently
subjected to hydrazinolysis. Pyrazole and imidazole acetohy-
drazides 11 g and 11 h were obtained by alkylating the hetero-
cycles with ethyl bromoacetate (16), followed by hydrazinolysis
of intermediates 10g and 10h.
CH3
CH3CH2
CH3CH2
À(CH2)4À
H
H
H
H
H
CH3
H
CH3
H
H
H
4-(dimethylamino)phenyl
4-(trifluoromethyl)phenyl
4-chlorophenyl
4-cyanophenyl
4-bromophenyl
4-methylphenyl
4-nitrophenyl
12j
12k
12l
12m
12n
12o
12p
12q
12r
12s
12t
12u
4-nitrophenyl
4-methylphenyl
2-methylphenyl
cinnamyl
4-ethylphenyl
2-methoxyphenyl
H
H
Hydrazide 11 i was prepared from the commercially available
ethyl ester precursor, and compounds 11 j–l could be obtained
directly from the corresponding carboxylic acids, following
[a] See Supporting Information S5 for compound characterization.
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Scheme 3. Synthesis of tetrazolylhydrazide 20. Reagents and conditions:
a) CH3NHOH, Na2CO3, 16 h, RT; b) NaN3, H2O, 7 h, RT; c) H2N-NH2, EtOH, 8 h,
608C.
Figure 3. Inactive hydrazides 11 d–l devoid of an acidic heterocyclic ring.
active are summarized in the Supporting Information (see Sup-
porting Information S5).
Because both tetrazole derivatives and acyl hydrazides are
widely recognized as versatile metal chelators, we considered
it necessary to determine the inhibition mechanism of 11 a, in
order to exclude ejection of enzyme-bound metal ions as the
modus operandi. In fact, zinc-ejecting KDM4A inhibitors have
been reported, for example, ebselen and disulfiram,[34] and
such a behavior was not unexpected for 11 a. However, inhibi-
tion experiments in the presence of various concentrations of
1 clearly revealed 11 a to be a competitive inhibitor with
regard to the endogenous substrate (see Figure 4). The Ki
value was 1.974 mm, which again demonstrates that values ob-
tained with the FDH assay seem to underestimate compound
potency. The iron chelators EDTA and deferoxamine were used
as controls, and gave markedly different results (see Support-
ing Information S4).
Scheme 2. Synthesis of hydrazides 11 d–h. Reagents and conditions: a) SOCl2,
3 h, 608C; b) NaCN, DMSO, 2.5 h, 30–408C; c) HCl, ROH, 21 h, reflux; d) H2N-
NH2, 8 h, 808C; e) Cs2CO3, DMF, 1–24 h, RT; f) H2N-NH2, 1–16 h, 808C.
a procedure described by Rabini and Vita.[32] All these varia-
tions proved to be inactive (Figure 3), underlining the impor-
tance of the tetrazole moiety for efficient binding.
For the synthesis of ring-methylated tetrazolylhydrazide 20,
diethyl 2-(ethoxymethylene)malonate (17) was transformed
into isoxazolone 18, which could be easily converted in the
course of a ring-opening-recyclization tandem reaction into
ethyl tetrazolylacetate 19.[33] As before, this ester was subjected
to hydrazinolysis, thus yielding the desired nonacidic tetrazole
20 (Scheme 3), which expectedly did not show any notable ac-
tivity against KDM4A at concentrations up to 200 and 400 mm
in the LANCE and FDH assays, respectively.
Figure 4. Determination of the inhibition kinetics of 11 a against KDM4A in
the LANCE assay. Various concentrations were assayed against the endoge-
nous substrate, 2-oxoglutarate (1), in a competition assay. The results show
that ejection of protein-bound metal can be excluded as the inhibiting
mechanism; 11 a rather inhibits KDM4A competitively.
As further proof of principle, we tested the ester 10a lacking
the hydrazide function, and could show that this interaction
motif is essential for enzyme inhibition by the expected nega-
tive outcome. The in vitro data for all compounds tested as in-
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We were furthermore surprised to find that 11 a displayed
a relative selectivity of ~4- and 41-fold for KDM4A relative to
KDM5A (JARID1A) and KDM6B (JMJD3), respectively, in the
LANCE assay (Table 3). This is quite remarkable, considering the
simple structure and the fragment-like character of this com-
pound. As control, we employed again 4, which has been de-
scribed as a rather unselective, broad-spectrum KDM inhibi-
Conclusions
To identify a novel starting point for inhibitors of histone lysine
demethylase 4A, we developed a fragment-like compound, 5-
tetrazolyl acetohydrazide (11 a). The half-maximal inhibitory
concentrations were found to be 46.64Æ0.94 and 2.38Æ
0.37 mm in enzyme-coupled and antibody-based assays, respec-
tively. It is competitive with regard to the co-substrate 2-oxo-
glutarate (1) with a Ki value of ~2 mm. Based on the mecha-
nism of inhibition and its structural features, we propose
a binding mode similar to those of well-known KDM4A inhibi-
tors, that is, via complexation of the active center FeII ion with
the acyl hydrazide moiety, and formation of a salt bridge with
Lys206 and a hydrogen bond with Tyr132 through the acidic
tetrazole ring (Figure 5). The latter is thus far unprecedented.
Table 3. Selectivity screening of 11 a and two selected reference inhibi-
tors: activity against isolated KDM4A, KDM5A, and KDM6B in the FDH
and LANCE assays.
Compd
IC50 [mm]
KDM5A
KDM4A (JMJD2A)
KDM6B
(JMJD3)
LANCE[e]
(JARID1A)
LANCE[d]
FDH[b]
LANCE[c]
11 a
4
46.6Æ0.9
2.38Æ0.37
0.034Æ0.007
54.4Æ1.6
10.41Æ1.80
98.0Æ7.2
0.70Æ0.03
0.1000Æ0.028 36.0Æ4.7
GSK-J1[a] NI[f]
0.418Æ0.153
0.128Æ0.018
[a] 3-{[2-(Pyridin-2-yl)-6-(1,2,4,5-tetrahydro-3H-benzo[d]azepin-3-yl)pyrimi-
din-4-yl]amino}propanoic acid.[32] [b] Enzyme 1.75 mm, peptide 35 mm,
60 min, 378C. [c] Enzyme 60 nm, peptide 400 nm, 45 min, RT. [d] Enzyme
25 nm, peptide 100 nm, 45 min, RT. [e] Enzyme 50 nm, peptide 400 nm,
120 min, RT. [f] No inhibition at 400 mm.
tor,[35] a finding that we were able to confirm. To also include
a control with relative selectivity, we used the bipyridyl deriva-
tive GSK-J1, which was recently discovered in a high-through-
put screening by researchers at GlaxoSmithKline.[36] The com-
pound was initially described as being highly selective for de-
methylases KDM6A and KDM6B, although Heinemann et al.
shortly after demonstrated that this statement did not repre-
sent the whole picture. In fact, several KDM5 isoenzymes, in
particular KDM5B, were only five- to tenfold less affected.[37]
Nonetheless is GSK-J1 still one of the most potent and (rela-
tively) selective KDM inhibitors known to date and was there-
fore considered a suitable control. In comparison with GSK-J1,
our acylhydrazide 11 a showed a unique selectivity profile,
which cannot be found with any other known KDM inhibitor.
We also tested 11 a against histone deacetylase 1 (HDAC1) in
a standard trypsin-based assay,[38] where it did not show any
notable activity (data not shown).
Figure 5. Proposed binding mode of lead fragment 11 a. The depicted inter-
actions were anticipated in analogy to published resolved crystal structures
with co-crystallized inhibitors.[13,15,16,17b,20,21,26,40] Further support comes from
the results of the in vitro testing of our compounds that either lack the acyl-
hydrazide motif (10a), the tetrazole ring (11 d–l), or where the tetrazole ring
was methylated, thus abolishing its acidic nature (20).
As Schofield et al. previously demonstrated by using sub-
strate analogous peptide fragments, linking of ligands of the
2-oxoglutarate and substrate binding sites enables potent and
highly selective inhibition of JmjC histone demethylases.[16]
Thus, we suggest use of the novel lead presented herein as an
anchor point to target the 2-oxoglutarate binding site and to
grow this fragment into a selective and tight-binding inhibitor
of Jumonji-domain-containing histone demethylases with
drug-like properties.
The combined results encouraged us to select tetrazolyl ace-
tohydrazide 11 a as a lead structure for the synthesis of deriva-
tives, because its small size results in high ligand efficiencies of
0.59 (FDH assay) and 0.78 (LANCE assay),[39] which is a very
good starting point for further studies. To generate arrays of
derivatives of 11 a, we prepared hydrazones 12a–u with vari-
ous carbonyl compounds aiming to increase the binding affini-
ty. Disappointingly, none of these derivatives showed im-
proved activity, and the majority of the synthesized com-
pounds were actually devoid of any activity (Table 2). Seeming-
ly, all prepared hydrazones were sterically too demanding. Fur-
ther work regarding the derivatization of our newly found lead
is under way and will be reported soon.
The central question, whether metal complexing drugs are
more promiscuous than other drugs due to nonspecific metal
chelation, is still under debate. Experimental results from Day
and Cohen should help to spark renewed interest in such com-
pounds.[41] They screened marketed and experimental metal-
loenzyme-inhibiting drugs against a panel of metalloproteins
such as HDAC and ACE. Even at high inhibitor concentrations
(10 mm), off-target inhibition was limited. As expected, off-
target inhibition was most pronounced among metalloen-
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NaOH (1.50 g), compound 9b was obtained as a colorless oil
(15.1 g, 119 mmol, 79%); purity: 92% (at 220 nm); 1H NMR
([D6]DMSO): d=1.20 (t, J=7.1 Hz, 3H), 2.68 (s, 4H), 4.11 ppm (q,
J=7.1 Hz, 2H); 13C NMR ([D6]DMSO): d=12.4, 14.0, 29.2, 60.5, 119.8,
170.6 ppm; IR (ATR): n˜ =2250 (w), 1729 cmÀ1 (s); HRMS (ESI) m/z
[M+H+] calcd for C6H10NO2+: 128.0712, found: 128.0701.
zymes incorporating the same catalytic metal, active site struc-
ture, and functionality, and was more common among early-
stage lead compounds than in fully developed inhibitors.
Almost all approved drugs showed no off-target inhibition, re-
gardless of the complexing warhead, suggesting that inhibitor
selectivity is a combination of metal binding and the suppor-
tive backbone interactions. Therefore, metal chelating inhibi-
tors of metalloproteins should no longer be automatically as-
sociated with high risk for indiscriminate off-target inhibition
of metalloenzymes, and their development poses no more risk
for nonspecific activity than small-molecule inhibitors of metal-
independent enzymes. After all, the relative selectivity of our
fragment-like lead 11 a for KDM4A, and the lack of activity
against HDAC1 seem to support these findings.
Ethyl 4-cyanobutanoate (9c): With halogenated ester 8c (50.0 g,
332 mmol), NaCN (23.9 g, 498 mmol), DMSO (113 mL) and NaOH
(3.00 g), compound 9c was obtained as a colorless oil (38.9 g,
276 mmol, 83%); purity: 100% (at 220 nm); 1H NMR ([D6]DMSO):
d=1.19 (t, J=7.1 Hz, 3H), 1.81 (qu, J=7.3 Hz, 2H), 2.40 (t, J=
7.3 Hz, 2H), 2.53 (t, J=7.3 Hz, 2H), 4.07 ppm (q, J=7.1 Hz, 2H);
13C NMR ([D6]DMSO): d=14.0, 15.6, 20.5, 32.2, 60.0, 120.2,
171.8 ppm; IR (ATR): n˜ =2245 (w), 1727 cmÀ1 (s); HRMS (ESI) m/z
[M+H+] calcd for C7H11NO2+: 142.0868, found: 142.0869.
General procedure for the synthesis of compounds 10a–c: The
appropriate cyano acid ester (1.0 equiv), NaN3 (1.1 equiv) and
NH4Cl (0.2 equiv) were suspended in DMF. The reaction mixture
was heated at 95–1258C for 8–24 h. Subsequently the solvent was
removed under reduced pressure. The resulting residue was dilut-
ed with H2O and adjusted to pH 1 with concentrated hydrochloric
acid (CAUTION: Release of HN3!). The desired product was ob-
tained by precipitation or extraction.
Experimental Section
General. All starting materials (aldehydes and ketones, halogenat-
ed acid esters, aromatic heterocycles) and solvents, unless other-
wise noted, were obtained from Sigma Aldrich or ABCR GmbH &
Co. KG and used without further purification. Medium pressure
liquid chromatography (MPLC) was done on silica gel from Macher-
ey–Nagel (particle size 50–100 mm, 140–270 mesh ASTM) with
Büchi devices C-630, C-601 and C-660 (column length 40 cm,
column diameter 3.5 cm). Melting points were determined on
a hot stage microscope by Kofler PHMK 81/3035 “Botius” (VEB
Wägetechnik Rapido) with 16-fold amplification or Büchi melting
point apparatus M-565 and are uncorrected. Microwave-assisted
synthesis was performed using a Discover LabMate (‘closed vessel’
mode, 10 mL total capacity vessel, temperature control via IR
sensor) from CEM. The parameter ‘PowerMax’ indicates a perma-
nent radiation of microwaves simultaneously to intense cooling.
The specified purities were determined by the 100% method of
the DAD chromatogram at wavelengths as indicated. Acylhydra-
zones are subject to isomerism at the amide moiety; when both
isomers could be resolved chromatographically, purity values apply
for both isomers together. NMR spectra were recorded using an
Avance III instrument with Ultrashield 400 (1H: 400.2 MHz, 13C:
100.6 MHz) from Bruker at 258C with tetramethylsilane (TMS) as in-
ternal standard, using the ppm scale. High-resolution mass spectra
(HRMS) were obtained after high-performance liquid chromatogra-
phy (HPLC) with a mass spectrometer (LC-IT-TOF) from Shimadzu
based on a deviance tolerance limit ꢀ5 ppm. Mid-infrared spectra
were recorded on a Nicolet IR200 FT-IR from Thermo Electron Cor-
poration with diamond ATR accessory. Figures 2 and 5 were pre-
pared using LigPlot.[42]
Ethyl 2-(1H-tetrazol-5-yl)acetate (10a): With cyano acid ester 9a
(23.0 g, 200 mmol), NaN3 (14.3 g, 220 mmol), NH4Cl (2.30 g,
43 mmol), DMF (220 mL) and H2O (220 mL) for 8 h at 958C. The re-
action was worked up by filtering off the precipitate, followed by
recrystallization from propan-2-ol, to obtain compound 10a as
a colorless solid (17.6 g, 113 mmol, 56%); purity: 70% (at 220 nm);
mp: 1258C; 1H NMR ([D6]DMSO): d=1.21 (t, J=7.0 Hz, 3H), 4.12–
4.17 (q, J=7.0 Hz, 2H), 4.17 (s, 2H), 16.34 ppm (s, 1H); 13C NMR
([D6]DMSO): d=14.0, 29.5, 61.3, 150.4, 167.7 ppm; IR (ATR): n˜ =
+
1741 cmÀ1 (s); HRMS (ESI) m/z [M+H+] calcd for C5H9N4O2
:
157.0726, found: 157.0727.
Ethyl 3-(1H-tetrazol-5-yl)propanoate (10b): With cyano acid ester
9b (17.8 g, 140 mmol), NaN3 (10.0 g, 154 mmol), NH4Cl (1.80 g,
34 mmol), DMF (70 mL) and H2O (70 mL) for 8 h at 1058C. The
crude solution was worked up by diluting with brine and extract-
ing exhaustively with EtOAc, drying the combined organic phases
over Na2SO4 and concentrating under reduced pressure. The result-
ing colorless oil was cooled to À188C, filtering off the resulting
precipitate and washing with Et2O gave compound 10b as a color-
less solid (9.47 g, 55.7 mmol, 42%); mp: 84–858C; 1H NMR
([D6]DMSO): d=1.16 (t, J=7.0 Hz, 3H), 2.83 (t, J=7.2 Hz, 2H), 3.12
(t, J=7.2 Hz, 2H), 4.06 (q, J=7.0 Hz, 2H), 16.04 ppm (s, 1H);
13C NMR ([D6]DMSO): d=14.0, 18.5, 30.7, 60.2, 155.2, 171.4 ppm; IR
(ATR): n˜ =1719 cmÀ1 (s); HRMS (ESI) m/z [M+H+] calcd for
C6H11N4O2+: 171.0882, found: 171.0878.
General procedure for the synthesis of compounds 9a–c: NaCN
(1.5 equiv) was suspended in DMSO at room temperature. Subse-
quently, the appropriate chlorinated acid ester (1.0 equiv) was
added dropwise while the temperature of the resulting mixture
was kept at 408C. The suspension was then heated at 508C for 3 h
and stirred for another 15 h at room temperature. NaOH was
added and the reaction mixture was diluted with H2O until all
solids had dissolved. The solution was exhaustively extracted with
Et2O, dried over Na2SO4 and concentrated under reduced pressure
to obtain a colorless oil.
Ethyl 4-(1H-tetrazol-5-yl)butanoate (10c): With cyano acid ester
9c (38.0 g, 270 mmol), NaN3 (19.3 g, 297 mmol), NH4Cl (3.80 g,
72 mmol), DMF (135 mL) and H2O (135 mL) for 24 h at 1258C. To
work up the solution, it was exhaustively extracted with EtOAc, the
collected organic phases dried over Na2SO4 and concentrated
under reduced pressure. The orange oil was cooled to À188C, fil-
tering off the resulting precipitate and recrystallization from Et2O
gave compound 10c as a colorless solid (7.50 g, 40.8 mmol, 15%);
Ethyl 2-cyanoacetate (9a): This compound was commercially
available.
1
mp: 49–528C; H NMR ([D6]DMSO): d=1.18 (t, J=7.1 Hz, 3H), 1.96
(qu, J=7.4 Hz, 2H), 2.39 (t, J=7.4 Hz, 2H), 2.92 (t, J=7.4 Hz, 2H),
4.06 (q, J=7.1 Hz, 2H), 16.04 ppm (s, 1H); 13C NMR ([D6]DMSO): d=
14.0, 21.9, 22.2, 32.4, 59.8, 155.4, 172.2 ppm; IR (ATR): n˜ =
Ethyl 3-cyanopropanoate (9b): With halogenated ester 8b
(20.0 g, 150 mmol), NaCN (10.5 g, 220 mmol), DMSO (45 mL) and
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+
1723 cmÀ1 (s); HRMS (ESI) m/z [M+H+] calcd for C7H13N4O2
185.1039, found: 185.1030.
:
2% in all wells. Fluorescence intensity of the forming product
NADH was measured at lex =330 nm and lem =460 nm on a PO-
LARstar Optima microplate reader (BMG Labtech, Ortenberg, Ger-
many) immediately after addition (t=0) and after one hour of incu-
bation on a horizontal shaker at 378C. Values were blank-corrected
and the difference in intensity at t=1 h and t=0 was taken as
a measurement of enzyme activity. Activity in percent is in compar-
ison with compound-free DMSO control and no-substrate negative
control. Inhibition curves were analyzed by sigmoidal curve fitting
using GraphPad Prism 4.00 and IC50 values calculated from the fit
parameters as mean ÆSD from two independent experiments.
General procedure for the synthesis of compounds 11 a–c: The
appropriate tetrazole acid ester (1.0 equiv) and hydrazine hydrate
(80% =^ 50% hydrazine, 3.0–12.3 equiv) were heated by microwave
irradiation (pmax =17 bar, Tmax =115 8C, Pmax =200 W, ramp time=
1 min, hold time=5 min, continuous irradiation and stirring).
2-(1H-Tetrazol-5-yl)acetohydrazide (11a): With tetrazole acid ester
10a (16.6 g, 106 mmol) and hydrazine hydrate (20.6 g, 318 mmol).
EtOH (50 mL) was added and the solution cooled to 08C. Filtering
off the resulting precipitate and washing with EtOH gave com-
pound 11 a as a colorless solid (16.3 g, 110 mmol,>95%); purity:
100% (at 220 nm); mp: 158–1598C; 1H NMR ([D6]DMSO): d=
3.52 ppm (s, 2H); 13C NMR ([D6]DMSO): d=31.6, 155.5, 168.5 ppm;
IR (ATR): n˜ =1675 (m), 1624 (w), 1568 (w), 1525 cmÀ1 (w); HRMS
(ESI) m/z [M+H+] calcd for C3H7N6O+: 143.0681, found: 143.0669.
KDM4A LANCE assay. The commercial antibody-based LANCEUltra
demethylase activity assay (PerkinElmer, Waltham, MA, USA) was
performed in a total volume of 10 mL on white OptiPlate-384 mi-
crotiter plates (PerkinElmer) using a 50 mm HEPES buffer at
pH 7.50 containing 0.01% Tween-20 and 0.01% BSA. A solution of
60 nm KDM4A 1–359 was pre-incubated with compound solutions
of varying concentration (0–1000 mm) in DMSO at room tempera-
ture for 10 min. A substrate solution containing 100 mm ascorbic
acid, 5 mm FeSO4, 1 mm 2-oxoglutarate, and 400 nm of biotinylated
H3K9me3 substrate peptide ARTKQTARK(me3)-STGGKAPRKQLA-
GGK(biotin) (BPS Bioscience, San Diego, CA, USA) was added (final
concentrations). Final DMSO concentration was 5% in all wells.
Plates were incubated on a horizontal shaker at room temperature
for 45 min. Reactions were stopped by addition of 10 mL of detec-
tion mix containing 2 nm europium-labeled anti-H3K9me2 LANCE
antibody (PerkinElmer), 50 nm ULight-streptavidin dye (PerkinElm-
er), and 1 mm EDTA in 1” LANCE detection buffer (PerkinElmer)
(final concentrations). Plates were again incubated on a horizontal
shaker at room temperature for 60 min. FRET intensity was mea-
sured on an EnVision 2102 multilabel plate reader (PerkinElmer) at
lex =340 nm and lem =665 nm with a delay of 100 ms. Values were
blank-corrected and activity in percent is in comparison with com-
pound-free DMSO control and no-enzyme negative control. Inhibi-
tion curves were analyzed by sigmoidal curve fitting using Graph-
Pad Prism 4.00 and IC50 values calculated from the fit parameters
as mean ÆSD from two independent experiments.
3-(1H-Tetrazol-5-yl)propanehydrazide (11b): With tetrazole acid
ester 10b (7.91 g, 46 mmol) and hydrazine hydrate (30.9 g,
469 mmol). EtOH (20 mL) was added and the solution cooled to
À188C. Filtering off the resulting precipitate and washing with
EtOH gave compound 11 b as a colorless solid (7.38 g, 47 mmol,
>95%); purity: 100% (at 220 nm); mp: 136–1398C; 1H NMR
([D6]DMSO): d=2.38–2.42 (m, 2H), 2.85–2.90 ppm (m, 2H); 13C NMR
([D6]DMSO): d=21.4, 33.1, 159.6, 171.3 ppm; IR (ATR): n˜ =1596 (m),
1567 cmÀ1 (m); HRMS (ESI) m/z [M+H+] calcd for C4H9N6O+:
157.0838, found 157.0835.
4-(1H-Tetrazol-5-yl)butanehydrazide (11c): With tetrazole acid
ester 10c (7.06 g, 38 mmol) and hydrazine hydrate (30.9 g,
469 mmol). All volatiles were removed under reduced pressure and
residual hydrazine removed by azeotropic distillation with EtOH.
Recrystallization from MeOH/Et2O 3:1 gave compound 11 c as a col-
orless solid (6.04 g, 36 mmol, 93%); purity: 100% (at 220 nm); mp:
1
113–1158C; H NMR ([D6]DMSO): d=1.82 (qu, J=7.6 Hz, 2H), 2.06
(t, J=7.6 Hz, 2H), 2.66 ppm (t, J=7.6 Hz, 2H); 13C NMR ([D6]DMSO):
d=24.4, 24.8, 33.1, 159.6, 171.5 ppm; IR (ATR): n˜ =3288 (m), 1648
(s), 1521 cmÀ1 (m); HRMS (ESI) m/z [M+H+] calcd for C5H11N6O+:
171.0994, found: 171.0990.
KDM5A LANCE assay. The antibody-based LANCEUltra activity assay
for KDM5A (JARID1A) was performed essentially as described for
JMJD2A (KDM4A) with the following modifications: A solution of
25 nm full-length JARID1A 1–1090 (BPS BioScience, San Diego, CA,
USA) was used with 100 nm biotinylated H3K4me3 1–21 substrate
peptide ARTK(me3)-QTARKSTGGKAPRKQLA-GGK(biotin) (AnaSpec,
Fremont, CA, USA). The detection mix contained the appropriate
europium-labeled anti-H3K4me2/me1 LANCE antibody (PerkinElm-
er).
Purification of KDM4A. The plasmid pNIC28-Bsa4 JMJD2A encoding
human KDM4A residues 1–359 was used for expression and purifi-
cation as described by Ng et al. with minor modifications.[13] Short-
ly, the expression construct was transformed in BL21-CodonPlus-Ril
competent cells. Six liters of TB media containing kanamycin
(50 mgmLÀ1) and chloramphenicol (34 mgmLÀ1) were inoculated
with a 15 mLLÀ1 overnight culture and grown at 378C. Expression
was induced by addition of 0.2 mm IPTG at A260 =0.6. Then the cul-
ture was incubated at 188C for another 18 h. After harvesting and
lysis of the bacteria, the protein was purified by Talon bead
column. The purity of KDM4A estimated by SDS-PAGE was >90%.
KDM6B LANCE assay. The antibody-based LANCEUltra activity assay
for KDM6B (JMJD3) was performed essentially as described for
KDM4A (JMJD2A) with the following modifications: A solution of
50 nm catalytic domain JMJD3 1043-end (BPS BioScience, San
Diego, CA, USA) was used with 400 nm of biotinylated H3K27me3
21–44 substrate peptide ATKAARK(me3)-SAPATGGVKKPHRYRPG-
GK(biotin) (PSL Peptide Specialty Laboratories, Heidelberg, Germa-
ny). Incubation time for the enzymatic reaction was 120 min and
the detection mix contained the appropriate europium-labeled
anti-H3K27me2 LANCE antibody (PerkinElmer).
KDM4A FDH assay. The formaldehyde dehydrogenase (FDH)
enzyme-coupled demethylase activity assay was performed in
a total volume of 20 mL on white OptiPlate-384 microtiter plates
(PerkinElmer, Waltham, MA, USA) using a 50 mm HEPES buffer at
pH 7.50 containing 0.01% Tween-20. A solution of 0.10 mgmLÀ1
(2.4 mm) KDM4A 1–359 was pre-incubated with compound solu-
tions of varying concentration (0–400 mm) in DMSO at room tem-
perature for 10 min. A substrate solution containing 100 mm ascor-
bic acid, 10 mm FeSO4, 0.001 U·mLÀ1 FDH, 500 mm NAD+, 50 mm 2-
oxoglutarate, and 35 mm of H3K9me3 substrate peptide ARK(me3)-
STGGK-NH2 (Peptide Specialty Laboratories, Heidelberg, Germany)
was added (final concentrations). Final DMSO concentration was
KDM4A 2-oxoglutarate competition assay. To assess the competitive
degree of enzyme inhibition by test compounds to 2-oxoglutarate,
the LANCEUltra assay was performed as described above with vary-
ing concentrations of 2-oxoglutarate (0–5.0 mm) and inhibitor (0–
2.5 mm). Each combination was tested in duplicate. The blank-cor-
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den, U. Oppermann, M. A. McDonough, C. J. Schofield, Angew. Chem.
Int. Ed. 2012, 51, 1631–1634; Angew. Chem. 2012, 124, 1663–1666.
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Tempst, J. M. Wong, Y. Zhang, Nature 2006, 442, 312–316; b) J. F. Cou-
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rected LANCE signal was compared with a pre-established calibra-
tion curve to determine the amount of demethylated product
formed and, thus, the reaction velocity over the incubation time.
Curve fitting was performed using the Michaelis–Menten equation:
vmax Á ½2-oxoglutarateŠ
ð1Þ
v ¼
KM þ ½2-oxoglutarateŠ
The apparent KM values were plotted against the concentration of
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[19] M. Mantri, T. Krojer, E. A. Bagg, C. J. Webby, D. S. Butler, G. Kochan, K. L.
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O. N. F. King, C. Lejeune, S. S. Ng, T. Krojer, M. C. Chan, A. M. Rydzik, R. J.
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ꢀ
ꢁ
½compoundŠ
KaMpp ¼ K0M 1 þ
ð2Þ
Ki
Acknowledgements
[21] O. N. F. King, X. S. Li, M. Sakurai, A. Kawamura, N. R. Rose, S. S. Ng, A. M.
Quinn, G. Rai, B. T. Mott, P. Beswick, R. J. Klose, U. Oppermann, A.
Jadhav, T. D. Heightman, D. J. Maloney, C. J. Schofield, A. Simeonov,
PLoS One 2010, 5, e15535.
M.J. and R.S. thank the Deutsche Forschungsgemeinschaft for
funding (SFB992 MEDEP—Medical Epigenetics). M.R. thanks the
Studienstiftung des deutschen Volkes for support. Furthermore,
we thank Johanna Senger (University of Freiburg) for performing
the histone deacetylase assay.
´
[22] N. R. Rose, S. S. Ng, J. Mecinovic, B. t. M. R. LiØnard, S. H. Bello, Z. Sun,
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[23] S. Hamada, T. Suzuki, K. Mino, K. Kosek, F. Oehme, I. Flamme, H. Ozasa,
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Keywords: acyl hydrazides · acyl hydrazones · epigenetics ·
histone demethylase · tetrazoles
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