(Bis)urea and (Bis)thiourea inhibitors of lysine-specific demethylase 1 as epigenetic modulators

Article abstract of DOI:10.1021/jm100217a

The recently discovered enzyme lysine-specific demethylase 1 (LSD1) plays an important role in the epigenetic control of gene expression, and aberrant gene silencing secondary to LSD1 overexpression is thought to contribute to the development of cancer. W

Full text of DOI:10.1021/jm100217a

J. Med. Chem. 2010, 53, 5197–5212 5197
DOI: 10.1021/jm100217a
(Bis)urea and (Bis)thiourea Inhibitors of Lysine-Specific Demethylase 1 as Epigenetic Modulators
Shiv K. Sharma,†,§ Yu Wu,‡,§ Nora Steinbergs,‡ Michael L. Crowley,† Allison S. Hanson,† Robert A. Casero, Jr.,‡ and
Patrick M. Woster*,†
†Department of Pharmaceutical Sciences, Wayne State University, 259 Mack Avenue, Detroit, Michigan 48202, and ‡Sidney Kimmel
Comprehensive Cancer Center, Johns Hopkins University, Baltimore, Maryland 21231. §Contributed equally to this work.
Received February 22, 2010
The recently discovered enzyme lysine-specific demethylase 1 (LSD1) plays an important role in the
epigenetic control of gene expression, and aberrant gene silencing secondary to LSD1 overexpression is
thought to contribute to the development of cancer. We recently reported a series of (bis)guanidines and
(bis)biguanides that are potent inhibitors of LSD1 and induce the re-expression of aberrantly silenced
tumor suppressor genes in tumor cells in vitro. We now report a series of isosteric ureas and thioureas
that are also potent inhibitors of LSD1. These compounds induce increases in methylation at the histone
3 lysine 4 (H3K4) chromatin mark, a specific target of LSD1, in Calu-6 lung carcinoma cells. In addition,
these analogues increase cellular levels of secreted frizzle-related protein (SFRP) 2 and transcription
factor GATA4. These compounds represent an important new series of epigenetic modulators with the
potential for use as antitumor agents.
Introduction
Chromatin architecture is a key determinant in the regulation
of gene expression, and this architecture is strongly influenced
by post-translational modifications of histones.1,2 Histone pro-
tein tails contain lysine residues that interact with the negative
charges on the DNA backbone. These lysine-containing tails,
consisting of up to 40 amino acid residues, protrude through the
DNA strand and act as a site for post-translational modifica-
tion of chromatin, allowing alteration of higher-order nucleo-
some structure.3 Multiple post-translational modifications of
histones can mediate epigenetic remodeling of chromatin, with
acetylation being the best characterized process.4 Transcrip-
tional repression is associated with specific CpG island DNA
methylation and recruitment of histone deacetylases (HDACsa)
to gene promoters that cooperate in the epigenetic silencing of
specific genes.5,6 Normal mammalian cells exhibit an exquisite
level of control of chromatin architecture by maintaining a
balance between histone acetyltransferase (HAT) and HDAC
activity.7
In cancer, CpG island DNA promoter hypermethylation in
combination with other chromatin modifications, including
decreased activating marks and increased repressive marks
on histone proteins 3 and 4, have been associated with the
silencing of tumor suppressor genes.8 The important role of
promoter CpG island methylation and its relationship to
covalent histone modifications has recently been reviewed.9
Figure 1. (Bis)guanidine and (bis)biguanides with potent antitry-
panosomal activity in vitro.
As was mentioned above, the N-terminal lysine tails of histones
can undergo numerous post-translational modifications, in-
cluding phosphorylation, ubiquitination, acetylation, and
methylation.4,10,11 To date, 17 lysine residues and 7 arginine
residues on histone proteins have been shown to undergo
methylation,12 and lysine methylation on histones can signal
transcriptional activation or repression depending on the
specific lysine residue involved.13-15 All known histone lysine
methyltransferases contain a conserved SET methyltransferase
*To whom correspondence should be addressed. Phone: 313-577-
1523. Fax: 313-577-2033. E-mail: pwoster@wayne.edu. Address: 3132
Applebaum Hall, 259 Mack Avenue, Wayne State University, Detroit,
Michigan 48202.
a Abbreviations: LSD1, lysine-specific demethylase 1; H3K4, histone
3 lysine 4; JmjC, Jumonji C domain-containing demethylase; HAT,
histone acetyltransferase; HDAC, histone deacetylase; SET, (Su(var.)3-9
enhancer of zeste; APAO, acetylpolyamine oxidase; SMO, spermine
oxidase; SFRP, secreted frizzle-related protein; PCNA, proliferating cell
nuclear antigen.
r
2010 American Chemical Society
Published on Web 06/22/2010
pubs.acs.org/jmc

5198 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Sharma et al.
Table 1. Structures of Compounds 1c, 2d, and 3-33, and Inhibition of LSD1 in Vitro Following Treatment with Each Analogue at 10 μM

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14 5199
Table 1. Continued

5200 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Table 1. Continued
Sharma et al.
domain, and it has been shown that aberrant methylation
of histones due to SET domain deregulation is linked to
carcinogenesis.16 Histone methylation, once thought to be an
irreversible process, has recently been shown to be a dynamic
process regulated by the addition of methyl groups by histone
methyltransferases and removal of methyl groups from mono-
and dimethyllysines by lysine specific demethylase 1 (LSD1),
and from mono-, di, and trimethyllysines by specific Jumonji C
(JmjC) domain-containing demethylases.10,11,17,18 Additional
demethylases in the JmjC demethylase class are continuing to
be identified.19,20 Recent evidence suggests that LSD1 is re-
quired for maintenance of global DNA methylation,21 indicat-
ing that the LSD1-mediated demethylation is a general me-
chanism for transcriptional control.
A key positive chromatin mark found associated with promo-
ters of active genes is histone 3 dimethyllysine 4 (H3K4me2).22,23
LSD1, also known as BHC110 and KDM1,10,24 catalyzes the
oxidative demethylation of histone 3 methyllysine 4 (H3K4me1)
andH3K4me2andisassociatedwithtranscriptionalrepression.10
H3K4me2 is a transcription-activating chromatin mark at
gene promoters, and demethylation of this mark by LSD1
may prevent expression of tumor suppressor genes important
in human cancer.25 Thus, LSD1 is emerging as an important
new target for the development of specific inhibitors as a new
class of antitumor drugs.26
To date, only a few existing compounds have been shown to
act as inhibitors of LSD1. The active site structure of LSD1
has considerable sequence homology to monoamine oxidases
A and B (MAO A and B) and to N1-acetylpolyamine oxidase
(APAO) and spermine oxidase (SMO).10,24,27 It has been
shown that classical MAO inhibitors phenelzine and tranyl-
cypromine inactivate nucleosomal demethylation by the re-
combinant LSD1/CoRest complex and increase global levels
of H3K4me2 in the P19 cell line.24,27 The synthetic substrate
analogue aziridinyl-K4H31-21 reversibly inhibited LSD1 with
an IC50 of 15.6 μM, while propargyl-K4H31-21 produced
time-dependent inactivation with a Ki of 16.6 μM.28 Propargyl-
K4H31-21 was later shown to inactivate LSD1 through for-
mation of a covalent adduct with the enzyme-bound flavin
cofactor.27,29 McCafferty et al. recently described the synth-
esis of a series of trans-2-arylcyclopropylamine analogues that
inhibit LSD1 with Ki values between 188 and 566 μM.30

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14 5201
However, in all but one instance, these analogues were 1-2orders
of magnitude more potent against MAO A and MOA B. Most
recently, Ueda and co-workers identified small molecule tranyl-
cypromine derivatives that are selective for LSD1 over MAO-A
and MAO-B,31 and Binda et al. described similar tranylcypro-
mine analogues that exhibited partial selectivity between LSD1
and the newly identified histone demethylase LSD2.32
acetylpolyamine oxidase (APAO) and spermine oxidase
(SMO).10,33 Several groups have identified amines, guani-
dines, or similar analogues that act as selective modulators
of these 2 amine oxidases.33-39 We previously reported the syn-
thesis of a novel series of (bis)guanidines and (bis)biguanides40
that are potent antitrypanosomal agents in vitro, with IC50
values against Trypanosoma brucei as low as 90 nM. Because
of their structural similarity to guanidine-based inhibitors
of APAO and SMO, we sought to determine whether (bis)-
guanidines 1a-g and (bis)biguanides 2a-f (Figure 1) were
inhibitors of LSD1 and whether this inhibition had any
influence on selected chromatin marks in tumor cells. Nine of
the 13 compounds tested were found to inhibit LSD1 activity
by >50% at 1 μM.25 The two most potent LSD1 inhibitors
exhibited noncompetitive kinetics at concentrations up to
2.5 μM. A 48 h exposure of HCT116 human colon carcinoma
cells to increasing concentrations of analogues 1c and 2d
(Figure 1) produced significant global increases in both
H3K4me1 and H3K4me2 while not affecting global H3K9-
me2 levels. These analogues also induced the re-expression of
multiple, aberrantly silenced genes important in the develop-
ment of colon cancer, including members of the secreted
frizzle-related proteins (SFRPs) and the GATA family of
transcription factors.
LSD1 was identified in part because its C-terminal domain
shares significant sequence homology with the amine oxidases
Scheme 1
Because of the promising cellular effects of 1c and
2d, the synthesis and evaluation of additional analogues
was proposed. To access a library of more diverse analo-
gues related to 1c and 2d, we adapted our previously
published syntheses40 to produce a series of 30 isosteric
Scheme 2

5202 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Sharma et al.
Figure 2. Effect of compounds 2d and 3-33 on LSD1 activity in vitro. Percent of LSD1 activity remaining was determined following
treatment with 10 μM of each test compound as determined by the luminol-dependent chemiluminescence method.
Scheme 3
(bis)alkylureas or (bis)alkylthioureas (compounds 3-33,
Table 1) and these analogues were evaluated for the ability
to inhibit LSD1 and induce increases in global H3K4me2
in vitro.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14 5203
Figure 3. Effect of compounds 25-27 on the expression of global H3K4me1 and H3K4me2. Calu-6 human anaplastic nonsmall cell lung
carcinoma cells were treated with a 10 μM concentration of 25, 26, or 27 for 24 h (A,B) or 48 h (C,D) as described in the Experimental Section.
(A,C) Global H3K4me1 expression and (B,D) global H3K4me2 expression. Proliferating cell nuclear antigen (PCNA) was used as a loading
control. Shown are Western blot images from a single representative experiment performed in triplicate. Relative protein expression levels were
determined by quantitative Western analysis using the Odyssey infrared detection system shown as bar graphs. The results represent the mean
of three treatments ( SD. The protein expression level for control samples was set to a value of 1.
Chemistry
groups (HCl in EtOAc)44 to afford the desired urea or thiourea
products 3-30.
Preparation of compounds 3-33 depended on the avail-
ability of the appropriate isocyanates and isothiocyanates. All
of these intermediates were commercially available, with the
exception of isocyanate 35c and isothiocyanates 37a-c, which
were synthesized as shown in Scheme 1. Isocyanate 35c could
be made in a single step by reacting the requisite diphenylalkyl-
amine 34c (m = 2) with trichloroacetic anhydride (toluene, N2,
reflux) for 5 h.41,42 To produce the corresponding isothiocya-
nates 37a-c (m = 0, 1 or 2, respectively), amines 34a-c were
allowed to react with carbon disulfide in the presence of
triethylamine in THF at 5 °C.43 The reaction was allowed to
warm to room temperature, and after 3 h, the intermediate
dithiocarbamates 36a-c could be isolated. Reaction of the
intermediate dithiocarbamates 36a-c with tosyl chloride in
THF then afforded the desired isothiocyanates 37a-c.43
To access a library of isosteric urea and thiourea analogues
related to 1c and 2d, we employed our previously published
synthesis40 of precursor molecules 41a-c, as shown in Scheme 2.
The appropriate diamine 38a,38b,or38c was (bis)cyanoethylated
(acrylonitrile, EtOH, reflux) toafford the corresponding(bis)-
cyano intermediates 39a-c. The central nitrogens in 39a-c
were then N-Boc protected ((Boc)2O, CH2Cl2/Aq. NaHCO3)44
to form 40a-c, and the cyano groups were reduced (Raney Ni)
to yield the desired diamines 41a-c.40,45 Compounds 41a-c
were then reacted with the appropriate isocyanates or isothio-
cyanates 42d-z, 42aa-ee, 35c, and 37a-c42 to produce the
correspondingprotected(bis)ureasor(bis)thioureas43d-zand
43aa-ee, followed by acid removal of the N-Boc protection
To access isosteric derivatives of the (bis)biguanide lead
compound 2d, the synthetic route outlined in Scheme 3 was
devised. Initially, primary amines of general structure 44 were
reacted with N-chlorocarbonyl isocyanate 45,41,46 with the
intention of forming the alkyl N-chlorocarbonylurea 46.
However, this reaction could not be controlled, even at low
temperature, to produce the monoalkylated derivative 46, but
immediately formed the bis-alkylated (bis)biguanide 47. To
form the desired monoalkylated product, it was necessary to
use a less reactive secondary amine in the initial step. Thus
diphenylamine 48 was added to N-chlorocarbonyl isocyanate
45,41,46 and the mixture was stirred for 20 min to form a
mixture of 49 and the bis-alkylated product 50. Addition of
compounds 41a,b, or c40 to this reaction mixture in the
presence of triethylamine produced the bis-N-Boc-protected
precursors 51-53, which were separated from 50 by silica gel
chromatography. Acid-catalyzed removal of the N-Boc pro-
tecting groups44 in 51-53 then afforded the desired target
compounds 31-33. Importantly, the syntheses described in
Schemes 1-3 can be adapted to produce a wide variety of
analogues with chemical diversity in the length of the alkyl
chains, and in the terminal alkyl- or aralkyl substituents.
Biological Evaluation
The ability of the target (bis)ureas and (bis)thioureas to
inhibit LSD1 was determined in an assay procedure utilizing
the recombinant human enzyme. Expression and purification

5204 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Sharma et al.
Figure 5. Effect of compounds 25-27 on Calu-6 human anaplastic
nonsmall cell lung carcinoma cell viability as measured by standard
MTS assay. Cells were treated with increasing concentrations of
each test compound for 96 h prior to measurement of cell viability.
%NT refers to the percent of viable cells remaining at time T (96 h)
as compared to the number of cells seeded, N0. Each data point is the
average of three determinations that differed in all cases by 5% or
less.
was observed at 48 h (Figure 3C,D). Compound 25 produced
the most dramatic increases in H3K4me1 and H3K4me2 at
both24and 48h. The reduction inH3K4me2 and correspond-
ing increase in H3K4me1 by 26 at both 24 and 48 h cannot be
readily explained and is the subject of continuing investiga-
tion. However, this anomalous finding seem to correlate with
the observed cytotoxicity of 26 (see below). These data strongly
suggest that intracellular inhibition of LSD1 by 25-27 leads to
significant increases in methylation at the H3K4 chromatin
mark. It is noteworthy that in HCT116 human colon tumor
cells, compounds 25-27 all produced at least a 2-fold increase
in global H3K4me2 (data not shown), with the most effective
analogue being compound 25 (17.4-fold increase).
The ability of compounds 25-27 to induce the re-expression
of aberrantly silenced tumor suppressor genes in vitro was
next measured using the Calu-6 human lung carcinoma cell
line. The tumor suppressor genes SFRP2 and GATA4 were
chosen because they are known to be under expressed in
human lung cancer and because they are thought to play a
role in tumorigenesis when silenced. Thus, the genes coding
for these proteins are well-documented LSD1 targets. Cells
were treated for 24 h with either a 5 or 10 μM concentration of
25, 26, or 27, after which the levels of secreted frizzle-related
protein (SFRP) 2, a soluble modulator of Wnt signaling, and
the zinc-finger transcription factor GATA4, were determined
by quantitative PCR (qPCR). The results of these studies are
shown in Figure 4. All three compounds produced increases in
SFRP2 expression that appeared to be dose dependent for 25
and 27 (Figure 4A). Compound 27 produced the largest
increase in SFRP2 expression at 10 μM (4.8-fold increase).
Compounds 25 and 26 did not produce significant increases in
GATA4 levels at 5 and 10 μM (Figure 4B), and compound 27
induced a 1.3-fold increase in GATA4 mRNA protein at
10 μM and had no significant effect at 5 μM (Figure 4B). The
increase in GATA4 mRNA caused by 10 μM 27 is repro-
ducible, but is not statistically significant (P > 0.05).
Figure 4. Effect of compounds 25-27 on the re-expression of
secreted frizzle-related protein 2 (SFRP2, (A)) and the transcription
factor GATA4 ((B)) mRNA. Calu-6 human anaplastic nonsmall cell
lung carcinoma cells were treated with either a 5 or 10 μM concen-
tration of 25, 26, or 27 for 24 h as described in the Experimental
Section. cDNA was then synthesized from mRNA, amplified and
measured by qPCR. Each data point is the average of three determi-
nations that differed in all cases by 5% or less.
of LSD1 were conducted as previously reported.10,25 Enzy-
matic activity of LSD1 in the presence of target compounds was
determined using luminol-dependent chemiluminescence to
measure the production of H2O2, as previously described.25,33
The results of these experiments are summarized in Table 1 and
graphically in Figure 2. As previously observed, compound 2d
at 10 μM reduced LSD1 activity by 82.9%. Among the 31 urea
and thiourea isosteres 3-33, six compounds were essentially
inactive (i.e., produced <20% inhibition), while 11 analogues
(ureas4 and 5, thioureas6, 10, 18, 25, 26, 27, 29, and 30 andthe
disubstituted carbamoylurea 31) reduced LSD1 activity by
50% or greater at 10 μM concentration (Figure 2). The three
most effective LSD1 inhibitors, compounds 25-27, were
chosen for additional studies as outlined below. Subsequent
experiments were conducted in the Calu-6 human anaplastic
nonsmall cell lung carcinoma line because it has a highly
reproducible responsetoepigenetic modulation and becauseit
is known that various tumor suppressor genes are silenced in
this line. For synthetic analogues to be effective at the cellular
level, any observed decreases in cellular LSD1 activity should
be accompanied by an increase in global H3K4me1 and
H3K4me2 content. Thus, the ability of compounds 25, 26,
and 27 to produce increases in global H3K4me1 and
H3K4me2 levels was measured as previously described.10
The results of these studies are shown in Figure 3. At 24 h,
analogues 25 and 27 produced significant increases in both
H3K4me1 (Figure 3A) and H3K4me2 (Figure 3B), while
analogue 26 induced a significant increase in H3K4me1 but
decreased the relative amount of H3K4me2. A similar pattern
The (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-
phenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) reduction
assay was used to determine the effects of compounds 25-27

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14 5205
on cell viability in the Calu-6 cell line. Cells were treated with
increasing concentrations of each test compound for 96 h
prior to measurement of cell viability, and growth inhibition
(GI50) values were then determined from the resulting dose-
response curve. As seen in Figure 5, compounds 25, 26, and 27
produced moderate reduction in cell viability, with GI50
values of 10.3, 38.3, and 9.4 μM, respectively.
expression at both 5 and 10 μM. The order of potency in this
regard was 27 > 26 > 25. Compound 27 produced a 1.3-fold
increase in GATA4 expression at 10 μM that was not statisti-
cally significant and 25-27 at all other concentrations produced
no effect on GATA4. The observed increases in SFRP2
re-expression following treatment with 25-27, and the increase
in GATA4 re-expression induced by 10 μM 27, are consistent
with the previously reported effects of the parent compounds 1c
and 2d.25 The disparity in the ability of 25-27 to induce SFRP2
expression, but not GATA4 expression, suggests that LSD1
inhibition may have variable effects at different gene promoters.
A more detailed account of the effects of 3-33 on these and
other transcription factors will be reported in a subsequent
manuscript.
As discussed above, compounds 25-27 proved to be only
moderately cytotoxic in the Calu-6 nonsmall cell lung carci-
noma line in vitro. Compounds 25 and 27 produced the most
prominent reduction in cell viability, exhibiting GI50 values of
10.3 and 9.4 μM, respectively. These values are comparable to
the GI50 value for other epigenetic modulators such as the
polyaminohydroxamic acid and polyaminobenzamide HDAC
inhibitors developed in our laboratory50,51 and the parent
compound 2d. In addition, these GI50 values are in the range
of the histone deacetylase (HDAC) inhibitor MS-275, as
measured in three colon tumor cell lines.52 Compound 26
was significantlty less cytotoxic, exhibiting a GI50 value of
38.3 μM. Our data suggests that decreases in H3K4me2 at 24
and 48 h and/or minimal effects on the re-expression of SFRP2
and GATA4 by 26 could account for this reduced cytotoxicity.
It is important to note that epigenetic modulators such as
those mentioned above are generally used in combination
with traditional cytotoxic agents and serve to restore the
ability of transformed cells to undergo apoptosis.53 As such,
cytotoxicity is less of an issue as long as the compound
produces epigenetic effects in tumor cells that can be exploited
by traditional cytotoxic agents. We have recently shown that
the LSD1 inhibitor 2d alone has little effect in vivo on tumor
cell growth in an HCT116 human colon carcinoma mouse
xenograft model but acts synergistically to limit tumor growth
in combination with the DNA methyltransferase inhibitor
5-azacytidine.54 Additional studies are now being conducted
to determine whether isosteres of 2d such as 25-27 also
produce a synergistic effect on tumor cell growth in vivo.
Additional biological studies, as well as the synthesis and
evaluation of additional LSD1 inhibitors in this and other
compound libraries, is an ongoing effort in our laboratory.
Discussion
The potential LSD1 inhibitors 3-33 were synthesized using
pathways that are facile and relatively inexpensive and that
can be used to introduce chemical diversity into the resulting
urea and thiourea analogues, thus making them suitable for
generation of a library of related ureas and thioureas. Our
initial series of guanidine and biguanide derivatives25 repre-
sented the first novel small molecule inhibitors of LSD1 with
potential for development as therapeutic agents. The current
studies suggest that replacement of the imine NH function-
ality of the terminal guanidine in 1c with oxygen or sulfur is an
allowable isosteric change, and active analogues in both the
urea and thiourea series were identified (Figure 2). However,
the sulfur isosteric replacement is likely more acceptable
because the six best LSD1 inhibitors (6, 10, 18, and 25-27)
were all thioureas. A more bulky aromatic substituent on the
terminal nitrogen, as in 25-27, appears to impart greater
activity than the smaller alkyl or benzyl substituents found in
6, 10, and 18. There did not appear to be predictable differ-
ences in activity between analogues with 3, 4, or 7 carbon
central chains, suggesting that this parameter may not have a
great influence on inhibition of the enzyme. This is especially
apparent among 25-27 (terminal N-substituent = 2,2-di-
phenylethyl), which have 7, 4, and 3 carbon central regions,
respectively, but vary in activity by less than 5%. However, by
contrast, among compounds 28-30 (terminal N-substituent =
1,1-diphenylmethyl), inhibitory potency did appear to be
proportional to the length of the internal carbon chain. In
addition to the urea and thiourea derivatives, the carbamoy-
lurea 31, designed as an analogue of 2d, also produces potent
inhibition of LSD1 (73.9% at 10 μM). Additional analogues
will need to be synthesized and evaluated to generate a more
accurate set of structure/activity relationships for this series of
compounds.
The inhibitory effects of 25-27 on LSD1 (Figure 2), com-
bined with the observed methylation levels at the H3K4
chromatin mark (Figure 3A-D), strongly suggest that LSD1
is inhibited in the Calu-6 tumor cell line, resulting in increases
in the substrates H3K4me1 and H3K4me2. The anomalous
reduction in H3K4me2 at 24 and 48 h caused by 26 are
unexpected and have yet to be explained. In future studies,
we will attempt to elucidate the mechanism of this effect. In
addition, the effects of 25-27 on other histone demethylases,
including LSD2,47 the Jumonji C (JmjC) domain-containing
demethylases,10,11,17,18 and the recently discovered JmjC de-
methylase PHF8,48,49 need to be determined. Additional ex-
periments are being undertaken to determine the inhibitor
selectivity of these analogues.
Experimental Section
All reagents and dry solvents were purchased from Aldrich
Chemical Co. (Milwaukee, WI), Sigma Chemical Co. (St. Louis,
MO), or Acros Chemical (Chicago, IL) and were used without
further purification except as noted below. Pyridine was dried by
passing it through an aluminum oxide column and then stored
over KOH. Triethylamine was distilled from potassium hydro-
xide and stored in a nitrogen atmosphere. Methanol was distilled
from magnesium and iodine under a nitrogen atmosphere and
stored over molecular sieves. Methylene chloride was distilled
from phosphorus pentoxide, and chloroform was distilled from
calcium sulfate. Tetrahydrofuran was purified by distillation
from sodium and benzophenone. Dimethyl formamide was dried
by distillation from anhydrous calcium sulfate and was stored
under nitrogen. Preparative scale chromatographic procedures
were carried out using E. Merck silica gel 60, 230-440 mesh. Thin
layer chromatography was conducted on Merck precoated silica
gel 60 F-254. Ion exchange chromatography was conducted on
Compounds 25-27 were next evaluated for the ability to
induce the re-expression of SFRP2 and GATA4 protein, as
determined by qPCR from treated Calu-6 human lung carci-
noma cells. In the case of SFRP2, all three analogues induced
increases of the corresponding mRNA between 1.3- and 4.8-
fold (Figure 4A). These increases appeared to be dose-dependent,
except in the case of 26, which induced same level of SFRP2

5206 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Sharma et al.
Dowex 1 ꢀ 8-200 anion exchange resin. Compounds 41a-c40,55
were synthesized as previously described.
chromatography on silica gel (eluted with CH2Cl2) to give
37c as a white solid (1.48 g, 53% based on 34c, TLC Rf: 0.45
(n-hexane/EtOAc, 9:1). 1H NMR (CDCl3): δ 7.32-7.19 (m,
10H, Ar-H), 4.08 (t, 1H, J = 8.0 Hz, CHPh2), 3.44 (t, 2H, J =
6.8 Hz, CH2NCS), 2.41 (m, 2H, CH2CH2). 13C NMR (CDCl3):
δ 143.17, 129.08, 127.97, 126.99 (Ar-C), 48.12, 43.66, 35.69 (CH
and CH2).
1,1-Diphenylmethylisothiocyanate (37a). Isothiocyanate 37a
was prepared from 1,1-diphenylethylamine 34a and carbon
disulfide using the procedure described above for the synthesis
of 37c. The product was isolated as a white solid in 70% yield.
TLC Rf: 0.90 (n-hexane/MeCO2Et, 4:1). 1H NMR (CDCl3):
δ 7.40-7.31 (m, 10H, Ar-H), 5.99 (s, 1H, CHPh2). 13C NMR
(CDCl3): δ 139.43, 129.18, 128.57, 126.85 (Ar-C), 64.82 (CH).
2,2-Diphenylethylisothiocyanate (37b). Isothiocyanate 37b
was prepared from 1,1-diphenylethylamine 34a and carbon
disulfide using the procedure described above for the synthesis
of 37c. The product was isolated as a white solid in 87% yield. 1H
NMR (DMSO-d6): δ 7.36-7.29 (m, 8H, Ar-H), 7.24-7.20 (t, 2H,
J = 7.2 Hz, Ar-H), 4.45 (t, 1H, J = 8.0 Hz, CHPh2), 4.34 (d, 2H,
J = 7.6 Hz, CH2NCS). 13C NMR (DMSO-d6): δ 1 41.64, 129.31,
128.53, 127.67 (Ar-C), 51.18, 48.95 (CH and CH2).
General Procedure for Preparation of N-Boc Protected (Bis)-
thioureas. 1,12-Bis-{3-[1-(benzyl)thioureado]}-4,9-[N-(tert-butyl)-
oxycarbonyl)]-4,9-diazadodecane (43d). In a 100 mL round-
bottom flask, a 0.3 g portion of 4,9-[N-(tert-butyl)oxycarbonyl)]-
4,9-diaza-1,12-diaminododecane 41b (0.0008 mol) was dissolved
in 20 mL of HPLC grade CH2Cl2 under a nitrogen atmosphere
and the mixture was cooled to 0 °C. A solution of benzylisothio-
cyanate (240 mg, 0.0016 mol) in 5 mL of CH2Cl2 was then added
dropwise with stirring, and the reaction mixture was allowed to
stir at room temperature for 5 h. During this time, the formation
of product was monitored by TLC (CH2Cl2/MeOH/NH4OH
89:10:1). After completion of the reaction, the CH2Cl2 was
removed under reduced pressure to produce a viscous colorless
oil. The crude product was purified by flash chromatography on
silica gel eluted with CH2Cl2/MeOH/NH4OH (94.5:5:0.5 fol-
lowed by 89:10:1) to furnish pure 43d (0.46 g, 88% yield) as
viscous oil. Rf: 0.46 (CH2Cl2/MeOH/NH4OH, 89:10:1). 1H
NMR (CDCl3): δ 7.31 (m, 10H, Ar-H), 6.31 (b, 2H, NH), 4.55
(bs, 4H, NCH2), 3.54 (bs, 4H, NCH2), 3.20 (bs, 4H, NCH2), 3.10
(bs, 4H, NCH2), 1.65 (bs, 4H, CH2CH2), 1.46 (bs, 4H, CH2CH2),
1.38 (s, 18H, C[CH3]3).
All 1H and 13C NMR spectra were recorded on a Varian
Mercury 400 mHz spectrometer, and all chemical shifts are
reported as δ values referenced to TMS or DSS. Infrared spectra
were recorded on a Jasco FT-IR spectrophotometer and are
referenced to polystyrene. In all cases, 1H NMR, 13C NMR,
and IR spectra were consistent with assigned structures. Mass
spectra were recorded on a Kratos MS 80 RFA (EI and CI) or
Kratos MS 50 TC (FAB) mass spectrometer. Prior to biological
testing, target molecules 3-33 were determined to be 95% pure
or greater by HPLC chromatography using an Agilent series
1100 high-performance liquid chromatograph fitted with a C18
reversed-phase column.
Synthetic H3K4me2 peptides were purchased from Millipore
(Billerica, MA). Calu-6 cells were maintained in RPMI medium,
supplemented with 10% fetal bovine serum (Gemini Bio-
Products, Woodland, CA) and grown at 37 °C in 5% CO2
atmosphere.
3,3-Diphenylpropylisocyanate (35c). A 4.24 g (0.020 mol)
portion of 3,3-diphenylpropylamine was dissolved in 90 mL of
dry toluene in a 250 mL round-bottomed flask under a nitrogen
atmosphere, and triphosgene (2.98 g, 0.010 mol) was added to
the reaction mixture. The reaction mixture was heated under
reflux for 5 h and then cooled to room temperature, at which
time an additional 0.5 g of triphosgene was added. The reaction
was then stirred for an additional 18 h at room temperature.
During this time, the formation of product was monitored by
TLC using hexane:ethyl acetate (3:1). When the reaction was
complete, activated charcoal (0.50 g) was carefully added into
reaction mixture to decolorize the solution, which was stirred for
30 min and filtered. The filtrate was concentrated under reduced
pressure to give a light pale-yellow semisolid. A 100 mL portion
of n-hexane/ethyl ether(1:1 ratio) was then added, and the
mixture was stirred for 15 min. The solution was filtered and
concentrated to afford 4.23 g of viscous material. The crude
product was purified by flash chromatography on silica gel
eluted with dichloromethane to furnish 3,3-diphenylpropyliso-
cyanate 35c as a white solid (1.31 g, 28% yield). 1H NMR
(CDCl3): δ 7.38-7.10 (m, 10H, Ar-H), 4.09 (t, 1H, J = 7.2 Hz,
CHPh2), 3.27 (t, 2H, J = 6.4 Hz, CH2NCS), 2.36 (m, 2H,
CH2CH2). 13C NMR (CDCl3): δ 143.69, 128.94, 128.01, 126.85
(Ar-C), 48.14, 41.51, 36.87 (CH and CH2).
General Procedure for Preparation of Isothiocyanates
37a-c. 3,3-Diphenylpropylisothiocyanate (37c). In a 250 mL
round-bottomed flask under a nitrogen atmosphere, 3,3-diphenyl-
propylamine 34c (2.10 g, 0.010 mol) was dissolved in 40 mL of
freshly distilled THF, 3.64 g (5.0 mL, 0.036 mol) of triethylamine
was added, and the mixture was cooled to 5 °C in an ice bath.
Carbon disulfide (0.76 g, 0.96 mL, 0.10 mol) was then added to
the reaction mixture via syringe over 20 min. Following addition
of carbon disulfide, the mixture was stirred an additional 30 min,
warmed to room temperature, and allowed to stir a further 2 h. A
1H NMR of an aliquot (after removing the solvent in vacuo)
indicated that conversion to the dithiocarbamate salt 36c was
complete. 1H NMR (DMSO-d6): δ 8.46 (t, 1H, NH), 7.34-7.12
(m, 8H, Ar-H), 7.06 (t, 2H, Ar-H), 3.94 (t, 1H, CHPh2), 3.34 (m,
2H, CH2NCS), 3.04 (q, 6H, NCH2CH3), 2.24 (m, 2H, CH2CH2),
1.20 (t, 6H, NCH2CH3).
The reaction mixture from above was recooled in an ice bath,
2.38 g of tosyl chloride (0.012 mol) was added, and the reaction
mixture was allowed to stir for 30 min at 5 °C. It was then
warmed to room temperature and stirred for an additional 3 h.
The solvent was removed in vacuo, the reaction was partitioned
between 40 mL of 1.0 N HCl and 150 mL of Et2O, and the two-
phased mixture was stirred for 10 min. The organic layer was
separated and the aqueous layer was extracted with a 100 mL
portion of Et2O. The combined organic layers was dried over
Na2SO4 and concentrated to produce a viscous oil that solidi-
fied during vacuum drying. The product was purified by flash
1,12-Bis-{3-[1-(ethyl)thioureado]}-4,9-[N-(tert-butyl)oxy-
carbonyl)]-4,9-diazadodecane (43g). Compound 43g was prepared
from 375 mg of 41b (375 mg, 0.0009 mol) and ethylisothiocyanate
according to procedure described above for the synthesis of 43d
to afford 43g (512 mg, 95%) as viscous oil. Rf: 0.52 (CH2Cl2/
MeOH/NH4OH, 89:10:1). 1H NMR(CDCl3):δ7.40 (b, 2H, NH),
6.00 (b, 2H, NH), 3.56 (m, 4H, NCH2), 3.34 (b, 4H, NCH2), 3.26
(b, 4H, NCH2), 3.12 (b, 4H, NCH2), 1.71 (b, 4H, CH2CH2),
1.50 (bs, 4H, CH2CH2), 1.40 (s, 18H, C(CH3)3), 1.20 (t, 6H, J =
7.2 Hz, CH3). 13C NMR (CDCl3): δ 80.36 ([CH3]3C), 46.95, 43.34,
41.19, 38.12, 28.63, 27.31, 26.16 (CH2), 14.28 (CH3).
1,12-Bis-{3-[1-(propyl)thioureado]}-4,9-[N-(tert-butyl)oxy-
carbonyl)]-4,9-diazadodecane (43j). Compound 43j was pre-
pared from 260 mg of 41b (0.0007 mol) and n-propylisothiocya-
nate according to procedure described above for the synthesis of
43d to afford 43j (380 mg, 96%) as viscous oil. Rf: 0.51 (CH2Cl2/
1
MeOH/NH4OH, 89:10:1). H NMR (CDCl3): δ3.50-3.36 (b,
8H, NCH2), 3.28-3.20 (m, 8H, NCH2), 3.26 (b, 4H, NCH2) 1.78
(b, 4H, CH2CH2), 1.52 (bs, 4H, CH2CH2), 1.46 (s, 18H, C-
[CH3]3), 0.73 (t, 6H, J = 7.2 Hz, CH3). 13C NMR (CDCl3):
δ80.36 ([CH3]3C), 46.95, 43.34, 41.19, 38.12, 28.63, 27.31, 26.16
(CH2), 14.28 (CH3).
1,15-Bis-{3-[1-(benzyl)thioureado]}-4,12-[N-(tert-butyl)oxy-
carbonyl)]-4,12-diazapentadecane (43k). Compound 43k was
prepared from 220 mg of 41c (0.0005 mol) and benzylisothio-
cyanate according to procedure described above for the synth-
esis of 43d to afford 43k (360 mg, 96%) as viscous oil. 1H NMR

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14 5207
(CDCl3): δ 7.39-7.30 (m, 10H, Ar-H), 4.76 (b, 4H, CH2Ph),
3.46 (b, 4H, NCH2), 3.18 (m, 8H, NCH2), 1.52 (b, 4H,
CH2CH2), 1.54 (b, 4H, CH2CH2), 1.44 (s, 18H, C(CH3)3),
1.28 (b, 6H, CH2CH2).
1,11-Bis-{3-[1-(benzyl)thioureado]}-4,8-[N-(tert-butyl)oxy-
carbonyl)]-4,8-diazaundecane (43n). Compound 43n was pre-
pared from 291 mg of 41a (0.0008 mol) and benzylisothio-
cyanate according to the procedure described above for 43d
to afford 43n (373 mg, 73%) as viscous oil. Rf: 0.87 (CH2Cl2/
MeOH/NH4OH, 89:10:1). 1H NMR (CDCl3): δ 7.35 (m, 10H,
Ar-H), 4.58 (bs, 4H, N-CH2), 3.58 (bs, 4H, N-CH2), 3.21 (b,
4H, N-CH2), 3.10 (b, 4H, N-CH2), 1.72 (b, 6H, CH2CH2),
1.40 (s, 18H, C[CH3]3).
1,11-Bis-{3-[1-(propyl)thioureado]}-4,8-[N-(tert-butyl)oxy-
carbonyl)]-4,8-diazaundecane (43r). Compound 43r was pre-
pared from 291 mg of 41a (0.0008 mol) and n-propylisothio-
cyanate according to the procedure described above for 43d
to afford 43r (379 mg, 86%) as viscous oil. Rf: 0.57 (CH2Cl2/
MeOH/NH4OH, 89:10:1). 1H NMR (CDCl3): δ 7.29 (bs, 1H,
NH), 6.44 (s, 2H, NH), 3.43 (bs, 4H, N-CH2), 3.01-3.15 (b,
12H, N-CH2), 1.61 (bs, 6H, CH2CH2), 1.47 (m, J = 7.2 Hz,
4H, CH2CH3), 1.31 (s, 18H, C[CH3]3), 0.81 (t, J = 7.2 Hz, 6H,
CH2CH3).
1,11-Bis-{3-[1-(n-ethyl)thioureado]}-4,8-[N-(tert-butyl)oxy-
carbonyl)]-4,8-diazaundecane (43s). Compound 43s was pre-
pared from 291 mg of 41a (0.0008 mol) and ethylisothiocyanate
according to the procedure described above for 43d to afford 43s
(347 mg, 83%) as viscous oil. Rf: 0.72 (CH2Cl2/MeOH/NH4OH,
89:10:1). 1H NMR (CDCl3): δ 7.24 (bs, 2H, NH), 6.22 (bs, 2H,
NH), 3.49 (bs, 4H, CH2N), 3.29 (bs, 4H, N-CH2), 3.20 (b, 4H,
N-CH2), 3.07 (b, 4H, N-CH2), 1.62-1.74 (b, 6H, CH2CH2), 1.37
(s, 18H, C[CH3]3), 1.14 (t, 6H, CH2CH3).
J = 7.2 Hz, NCH2), 1.69 (bs, 4H, CH2CH2), 1.49 (b, 4H,
CH2CH2), 1.40 (bs, 18H, C[CH3]3), 1.24 (m, 6H, CH2CH2).
1,12-Bis-{3-[1-(2,2-diphenylethyl)thioureado]}-4,9-[N-(tert-
butyl)oxycarbonyl)]-4,9-diazadodecane (43aa). Compound 43aa
was prepared from 161 mg of 41b (0.0004 mol) and 37b according
to procedure described above for the synthesis of 43d to afford
43aa (295 mg, 84%) as a white solid. Rf: 0.60 (CH2Cl2/MeOH/
NH4OH, 89:10:1). 1H NMR (CDCl3): δ 7.32-7.20 (m, 20H,
Ar-H), 5.77 (b, 2H, NH), 4.29 (b, 2H, CHPh2), 4.02 (b, 4H,
NCH2), 3.56 (bs, 4H, NCH2), 3.26 (bs, 4H, NCH2), 3.12 (bs, 4H,
NCH2), 1.70 (b, 4H, CH2CH2), 1.48 (b, 4H, CH2CH2), 1.41 (s,
18H, C[CH3]3).
1,11-Bis-{3-[1-(2,2-diphenylethyl)thioureado]}-4,9-[N-(tert-
butyl)oxycarbonyl)]-4,9-diazadodecane (43bb). Compound 43bb
was prepared from 193 mg (0.0005 mol) of 41b and 37b according
to procedure described above for the synthesis of 43d to afford
43bb (350 mg, 80%) as a white solid. Rf: 0.63 (CH2Cl2/MeOH/
NH4OH, 89:10:1). 1H NMR (CDCl3): δ 7.32-7.20 (m, 20H,
Ar-H), 5.77 (bs, 2H, NH), 4.29 (bs, 2H, CHPh2), 4.02 (bs, 4H,
NCH2), 3.56 (bs, 4H, NCH2), 3.26 (bs, 4H, NCH2), 3.12 (t, 4H,
J = 7.2 Hz, NCH2), 1.71 (b, 4H, CH2CH2), 1.41 (b, 20H, CH2
and C[CH3]3).
1,11-Bis-{3-[1-(1,1-diphenylmethyl)thioureado]}-4,8-[N-(tert-
butyl)oxycarbonyl)]-4,8-diazaundecane (43cc). Compound 43cc
was prepared from 192 mg of 41a (0.0005 mol) and 37a according
to procedure described above for the synthesis of 43d to afford
43cc as a white solid (350 mg, 83%), Rf: 0.63 (CH2Cl2/MeOH/
NH4OH, 89:10:1). 1H NMR (CDCl3): δ 7.34-7.27 (m, 20H,
Ar-H), 6.43 (d, 2H, J = 5.2 Hz, NCH), 6.02 (b, 2H, NH), 3.52
(d, 4H, J = 5.2 Hz, NCH2), 3.06 (m, 8H, NCH2), 1.66 (bs, 6H,
CH2CH2), 1.36 (bs, 18H, C[CH3]3).
1,12-Bis-{3-[1-(1,1-diphenylmethyl)thioureado]}-4,9-[N-(tert-
butyl)oxycarbonyl)]-4,9-diazadodecane (43dd). Compound 43dd
was prepared from 201 mg of 41b (0.0005 mol) and 37a according
to procedure described above for the synthesis of 43d to afford
43dd (380 mg, 89%) as white solid. Rf: 0.60 (CH2Cl2/MeOH/
NH4OH, 89:10:1). 1H NMR (CDCl3): δ 7.40 (b, 2H, NH), 7.34-
7.27 (m, 20H, Ar-H), 6.43 (d, 2H, J = 5.2 Hz, NCH), 6.02 (b, 2H,
NH), 3.52 (d, 4H, J = 5.2 Hz, NCH2), 3.06 (bs, 8H, NCH2),
1.63 (m, 4H, CH2CH2), 1.42 (bs, 4H, CH2CH2), 1.36 (s, 18H,
C[CH3]3).
1,11-Bis-{3-[1-(3,3-diphenylpropyl)thioureado]}-4,8-[N-(tert-
butyl)oxycarbonyl)]-4,8-diazaundecane (43u). Compound 43u
was prepared from 155 mg of 41a (0.0004 mol) and 37c according
to procedure described above for the synthesis of 43d to afford 43u
(290 mg, 81%) as a white solid. Rf: 0.44 (CH2Cl2/MeOH/NH4OH,
1
89:10:1). H NMR (CDCl3): δ 7.29-7.15 (m, 22H, Ar-H, and
NH), 5.88 (b, 2H, NH), 4.04 (t, 2H, J = 7.6 Hz, CHPh2), 3.53 (b,
4H, NCH2), 3.28 (b, 4H, NCH2), 3.23 (b, 4H, NCH2), 3.12 (b, 8H,
NCH2), 2.36 (q, 4H, J = 8.0 Hz, NCH2), 1.70 (m, 2H, CH2CH2),
1.47 (b, 4H, CH2CH2), 1.40 (s, 20H, C[CH3]3).
1,15-Bis-{3-[1-(1,1-diphenylmethyl)thioureado]}-4,12-[N-(tert-
butyl)oxycarbonyl)]-4,12-diazapentadecane (43ee). Compound 43ee
was prepared from 223 mg of 41c and 37a according to procedure
described above for the synthesis of 43d to afford 43ee (408 mg,
91%) as a white solid. Rf: 0.77 (CH2Cl2/MeOH/NH4OH, 89:10:1).
1H NMR (CDCl3): δ 7.45 (b, 2H, NH), 7.33-7.26 (m, 20H, Ar-H),
6.41 (d, 2H, J = 2.8 Hz, NCH), 6.03 (b, 2H, NH), 3.51 (m, 4H,
NCH2), 3.04 (m, 8H, NCH2), 1.54 (bs, 4H, CH2CH2), 1.45 (b, 4H,
CH2CH2), 1.35 (bs, 18H, C[CH3]3), 1.23 (m, 6H, CH2CH2).
General Procedure for Preparation of N-Boc Protected (Bis)-
ureas. 1,12-Bis-{3-[1-(benzyl)ureado]}-4,9-[N-(tert-butyl)oxy-
carbonyl)]-4,9-diazadodecane (43e). In a 100 mL round-bottom
flask, a 0.35 g portion of 4,9-[N-(tert-butyl)oxycarbonyl)]-4,9-
1,12-diamino-diazadodecane 41b (0.0009 mol) was dissolved in
20 mL of HPLC grade CH2Cl2 under a nitrogen atmosphere
and the mixture was cooled to 0 °C. A solution of benzylisocya-
nate (0.235 g, 0.0018 mol) in 5 mL of CH2Cl2 was then added
dropwise with stirring, and the reaction mixture was allowed to
stir at room temperature for 24 h. During this time, the formation
of product was monitored by TLC (CH2Cl2:MeOH:NH4OH
89:10:1). When the starting material had been consumed, the
CH2Cl2 was removed under reduced pressure to afford a viscous
colorless material. The crude product was purified by flash chro-
matography on silica gel eluted with CH2Cl2:MeOH:NH4OH
(97:2.5:0.5 followed by 94.5:5.0:0.5) to furnish pure 43e (0.50 g,
86% yield) as viscous oil. Rf: 0.54 (CH2Cl2/MeOH/NH4OH,
89:10:1). 1H NMR (CDCl3): δ 3.20-3.02 (m, 16H, NCH2), 1.64
(b, 4H, CH2CH2), 1.48 (b, 4H, CH2CH2), 1.43 (s, 18H, C[CH3]3),
1.11 (t, 6H, J = 6.4 Hz, CH3).
1,12-Bis-{3-[1-(3,3-diphenylpropyl)thioureado]}-4,9-[N-(tert-
butyl)oxycarbonyl)]-4,9-diazadodecane (43w). Compound 43w
was prepared from 161 mg of 41b (0.0004 mol) and 37c according
to procedure described above for the synthesis of 43d to afford 43w
(322 mg, 89%) as a white solid. Rf: 0.52 (CH2Cl2/MeOH/NH4OH,
1
89:10:1). H NMR (CDCl3): δ 7.25-7.16 (m, 22H, Ar-H, and
NH), 5.88 (b, 2H, NH), 4.02 (t, 2H, J = 8.0 Hz, CHPh2), 3.17
(b, 8H, NCH2), 3.09 (b, 4H, NCH2), 2.37 (q, 4H, J = 7.6 Hz,
CH2CH), 1.76-1.65 (m, 8H, CH2CH2), 1.41 (s, 18H, C[CH3]3).
1,15-Bis-{3-[1-(3,3-diphenylpropyl)thioureado]}-4,12-[N-(tert-
butyl)oxycarbonyl)]-4,12-diazapentadecane (43y). Compound
43y was prepared from 178 mg of 41c (0.0004 mol) and 37c
according to procedure described above for the synthesis of 43d
to afford 43y (305 mg, 80%) as a white solid. Rf: 0.57 (CH2Cl2/
MeOH/NH4OH, 89:10:1). 1H NMR (CDCl3): δ 7.28-7.15 (m,
20H, Ar-H), 5.88 (b, 2H, NH), 4.02 (t, 2H, J = 8.0 Hz, CHPh2),
3.54 (b, 4H, NCH2), 3.28 (b, 4H, NCH2), 3.23 (b, 4H, NCH2), 3.08
(t, 4H, J = 7.2Hz, NCH2), 2.36 (q, 4H, J = 7.6Hz, CH2CH), 1.69
(bs, 4H, CH2CH2), 1.50 (b, 4H, CH2CH2), 1.40 (s, 18H, C[CH3]3),
1.28 (m, 6H, CH2CH2).
1,15-Bis-{3-[1-(2,2-diphenylethyl)thioureado]}-4,12-[N-(tert-
butyl)oxycarbonyl)]-4,12-diazapentadecane (43z). Compound
43z was prepared from 223 mg of 41c (0.0005 mol) and 37b
according toproceduredescribed above for the synthesis of43d to
afford 43z (288 mg, 79%) as a white solid. Rf: 0.68 (CH2Cl2/
1
MeOH/NH4OH, 89:10:1). H NMR (CDCl3): δ 7.31-7.19 (m,
20H, Ar-H), 5.75 (b, 2H, NH), 4.28 (b, 2H, CHPh2), 4.02 (b, 4H,
NCH2), 3.54 (b, 4H, NCH2), 3.25 (b, 4H, NCH2), 3.09 (t, 4H,

5208 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Sharma et al.
1,12-Bis-{3-[1-(ethyl)ureado]}-4,9-[N-(tert-butyl)oxycarbonyl)]-
4,9-diazadodecane (43f). Compound 43f was prepared from 368 mg
of 41b (0.0009 mol) and ethylisocyanate according to the procedure
described above for 43e to afford 43f (480 mg, 96%) as viscous oil.
Rf: 0.54 (CH2Cl2/MeOH/NH4OH, 89:10:1). 1H NMR (CDCl3): δ
3.20-3.02 (m, 16H, NCH2), 1.64 (b, 4H, CH2CH2), 1.48 (b, 4H,
CH2CH2), 1.43 (s, 18H, C[CH3]3), 1.11 (t, 6H, J = 6.4 Hz, CH3).
1,15-Bis-{3-[1-(benzyl)ureado]}-4,12-[N-(tert-butyl)oxycarbonyl)]-
4,12-diazapentadecane (43h). Compound 43h was prepared from
230 mg of 41b (0.0005mol) and benzylisocyanate according to the
procedure described above for 43e to afford 43h (350 mg, 96%) as
viscous oil. Rf: 0.50 (CH2Cl2/MeOH/NH4OH, 89:10:1). 1H
NMR (CD3OD): δ 7.30 (m, 10H, Ar-H), 4.29 (s, 4H, CH2Ph),
3.21-3.15 (m, 8H, NCH2), 3.12 (t, 4H, J = 7.2 Hz, NCH2), 1.70
(bs, 4H, CH2CH2), 1.50 (bs, 4H, CH2CH2), 1.44 (s, 18H, C-
[CH3]3), 1.32 (bs, 6H, CH2CH2).
procedure described above for 43e to afford 43t (420 mg, 98%)
as a viscous oil. Rf: 0.58 (CH2Cl2/MeOH/NH4OH, 89:10:1). 1H
NMR (CDCl3): δ 7.29-7.15 (m, 20H, Ar-H), 5.50 (b, 2H, NH),
3.96 (t, 2H, J = 8.0 Hz, CHPh2), 3.25 (t, 4H, J = 6.4 Hz, NCH2),
3.10 (b, 12H, NCH2), 2.23(q, 4H, J=7.2Hz, NCH2), 1.72 (b, 2H,
CH2CH2), 1.61 (b, 4H, CH2CH2), 1.42 (s, 18H, C[CH3]3).
1,12-Bis-{3-[1-(3,3-diphenylpropyl)ureado]}-4,9-[N-(tert-butyl)-
oxycarbonyl)]-4,9-diazadodecane (43v). Compound 43v was pre-
pared from 193 mg of 41b (0.0005 mol) and 35c according to the
procedure described above for 43e to afford 43v (386 mg, 92%) as
1
viscous oil. H NMR (CDCl3): δ 7.29-7.13 (m, 22H, Ar-H, and
NH),5.50(b,2H,NH),3.96(t,2H,J=8.0Hz,CHPh2), 3.25 (t, 4H,
J = 6.4 Hz, NCH2), 3.10 (m, 12H, NCH2), 2.24 (b, 4H, CH2CH2),
1.60 (b, 4H, CH2CH2), 1.42 (s, 22H, CH2CH2 and C[CH3]3).
1,15-Bis-{3-[1-(3,3-diphenylpropyl)ureado]}-4,12-[N-(tert-
butyl)oxycarbonyl)]-4,12-diazapentadecane (43x). Compound 43x
was prepared from 158 mg of 41c (0.0004 mol) and 35c according
to the procedure described above for 43e to afford 43x (310 mg,
95%) as viscous oil. Rf: 0.50 (CH2Cl2/MeOH/NH4OH, 89:10:1).
1H NMR (CDCl3): δ 7.30-7.12 (m, 20H, Ar-H), 5.50 (b, 2H,
NH), 4.40 (b, 2H, NH), 3.97 (t, 2H, J = 7.2 Hz, CHPh2), 3.25
(t, 4H, J = 6.4 Hz, NCH2), 3.10 (bs, 12H, NCH2), 2.26 (q, 4H,
J = 8.0 Hz, CH2CH2), 1.60 (bs, 4H, CH2CH2), 1.42 ((s, 18H,
C[CH3]3), 1.24 (bs, 6H, CH2CH2).
1,12-Bis-{3-[1-(propyl)ureado]}-4,9-[N-(tert-butyl)oxycarbonyl)]-
4,9-diazadodecane (43i). Compound 43i was prepared from 260 mg
of 41c (0.0005 mol) and n-propylisocyanate according to the
procedure described above for 43e to afford 43i (356 mg, 94%)
1
as viscous oil. Rf: 0.54 (CH2Cl2/MeOH/NH4OH, 89:10:1). H
NMR (CD3OD): δ 3.22 (m, 8H, NCH2), 3.09 (t, 4H, J = 6.4 Hz,
NCH2), 3.05 (t, 4H, J = 7.6 Hz, NCH2), 1.70 (b, 4H, CH2CH2),
1.50 (m, 8H, CH2CH2), 1.45 (s, 18H, C[CH3]3), 0.90 (t, 6H, J =
7.6 Hz, CH3).
General Procedure for Preparation of N-Boc Protected (Bis)-
carbamylureas 51-53. 1,12-Bis-{5-[1-(N,N-diphenyl)carbamyl]-
ureado}-4,9-[N-(tert-butyl)oxycarbonyl)]-4,9-diazadodecane (51).
A 0.34 g portion of N,N-diphenylamine 48 (0.34 g, 0.0002 mol) in
5 mL of dry CH2Cl2 was added dropwise into a cold solution of
N-chlorocarbonylisocyanate 45 (0.22 g, 0.0002 mol) in 5.0 mL of
CH2Cl2, and the reaction was stirred for 30 min under N2
atmosphere. A solution of 41b (0.3 g, 0.0008 mol) and NEt3
(0.3 g, 0.0003 mol) in 10 mL of CH2Cl2 was then added via
syringe, and the reaction mixture was allowed to stir at room
temperature for 18h. Duringthis time, the progress for formation
of product was monitored by TLC (CH2Cl2/MeOH/NH4OH,
89:10:1). The dichloromethane was removed under reduced
pressure to produce a viscous material, which was purified by
flash chromatography on silica gel eluted with CH2Cl2/MeOH/
NH4OH (89:10:1) to furnish pure 51 as a white solid (140 mg,
21%). 1H NMR (CDCl3): δ 8.44 (s, 2H, NH), 7.41-7.39 (m, 8H,
Ar-H), 7.29-7.23 (m, 12H, Ar-H), 6.75 (s, 2H, NH), 3.25-3.08
(m, 12H, NCH2), 1.78-1.69 (m, 6H, CH2CH2), 1.43 (s, 18H,
C[CH3]3).
1,15-Bis-{5-[1-(N,N-diphenyl)carbamyl]ureado}-4,12-[N-(tert-
butyl)oxycarbonyl)]-4,12-diazapentadecane (52). Compound 52
was made from 48, 45, and 41c according to the procedure
described above for the synthesis of 51 to afford pure 52 (140 mg,
21%) as a viscous material. Rf: 0.88 (CH2Cl2/MeOH/NH4OH
89:10:1). 1H NMR (CDCl3): δ 8.40 (s, 2H, NH), 7.37-7.28 (m,
8H, Ar-H), 7.20-7.12 (m, 12H, Ar-H), 6.76 (s, 2H, NH),
3.23-3.06 (m, 12H, NCH2), 1.72 (m, 4H, CH2CH2), 1.39 (bs,
22H, CH2CH2 and C[CH3]3). HRMS (CSI-MS m/z) calcd for
C48H62N8O8 [Mþ] = 878.47; found 879.40 [MþH].
1,11-Bis-{5-[1-(N,N-diphenyl)carbamyl]ureado}-4,8-[N-(tert-
butyl)oxycarbonyl)]-4,8-diazaundecane (53). Compound 55 was
made from 48, 45 and 41a according to the procedure described
above for the synthesis of 51 to afford pure 53 (115 mg, 18%) as a
white solid. Rf: 0.90(CH2Cl2/MeOH/NH4OH, 89:10:1). 1HNMR
(CDCl3): δ 8.44 (s, 2H, NH), 7.41-7.37 (m, 8H, Ar-H), 7.31-7.27
(m, 12H, Ar-H), 6.77 (s, 2H, NH), 3.30-3.12 (m, 12H, NCH2),
1.75 (t, 4H, J = 6.8 Hz, CH2CH2), 1.47 (b, 4H, CH2CH2), 1.43 (s,
18H, C[CH3]3), 1.27 (b, 6H, CH2CH2).
General Procedure for Cleavage of N-Boc Protecting Group. 1,12-
Bis-{3-[1-(benzyl)thioureado]}-4,9-diazadodecane (3). In a 100 mL
round-bottom flask, a 0.4 g portion of 43d (402 mg, 0.0006 mol) was
dissolved in 30 mL of HPLC grade EtOAc under a nitrogen
atmosphere, and 4.0 mL of a 1.0 M solution of HCl in EtOAc
was added. The reaction mixture was allowed to stir at room
temperature for 48 h, during which time the formation of product
1,15-Bis-{3-[1-(benzyl)ureado]}-4,12-[N-(tert-butyl)oxycarbonyl)]-
4,12-diazapentadecane (43l). Compound 43l was prepared from
225 mg of 41c (0.0005 mol) and ethylisocyanate according to the
procedure described above for 43e to afford 43l (280 mg, 94%) as
viscous oil. Rf: 0.37 (CH2Cl2/MeOH/NH4OH, 89:10:1). 1H
NMR (CD3OD): δ 3.28-3.12 (m, 8H, NCH2), 3.10-3.06 (m,
8H, NCH2), 1.68 (b, 4H, CH2CH2), 1.54 (b, 4H, CH2CH2), 1.44
(s, 18H, C[CH3]3), 1.30 (b, 6H, CH2CH2), 1.08 (t, 6H, J = 7.2 Hz,
CH2CH2).
1,15-Bis-{3-[1-(propyl)ureado]}-4,12-[N-(tert-butyl)oxycarbonyl)]-
4,12-diazapentadecane (43m). Compound 43m was prepared from
225 mg of 41c (0.0005 mol) and propylisocyanate according to the
procedure described above for 43e to afford 43m (280 mg, 92%) as
viscous oil. Rf: 0.35 (CH2Cl2/MeOH/NH4OH, 89:10:1). 1H NMR
(CD3OD): δ 3.25-3.16 (m, 8H, NCH2), 3.09-3.02 (m, 8H, NCH2),
1.68 (b, 4H, CH2CH2), 1.52 (b, 8H, CH2CH2), 1.44 (s, 18H,
C[CH3]3), 1.30 (b, 6H, CH2CH2),0.90(t, 6H, J=7.2Hz, CH2CH2).
1,11-Bis-{3-[1-(ethyl)ureado]}-4,8-[N-(tert-butyl)oxycarbonyl)]-
4,8-diazaundecane (43o).Compound 43o was prepared from 287 mg
of 41a (0.0007 mol) and ethylisothiocyanate according to the
procedure described above for 43e to afford 43o (245 mg, 62%) as
viscous oil, Rf: 0.63 (CH2Cl2/MeOH/NH4OH, 89:10:1). 1H NMR
(CDCl3): δ 5.59 (bs, 1H, NH), 4.60 (bs, 1H, NH), 3.08-3.31
(m, 16H, N-CH2), 1.58-1.78 (m, 6H, CH2CH2), 1.43 (s, 18H,
C[CH3]3), 1.10 (t, J = 7.2 Hz, 6H, CH2CH3).
1,11-Bis-{3-[1-(benzyl)ureado]}-4,8-[N-(tert-butyl)oxycarbonyl)]-
4,8-diazaundecane (43p). Compound 43p was prepared from
302 mg of 41a (0.0008 mol) and benzylisothiocyanate according
to the procedure described above for 43e to afford 43p (485 mg,
95%) as viscous oil, Rf: 0.63 (CH2Cl2/MeOH/NH4OH, 89:10:1).
1H NMR (CDCl3): δ 7.10-7.30 (m, 10H, Ar-H), 4.20 (bs, 4H,
PhCH2), 2.98-3.20 (m, 12H, N-CH2), 1.65 (p, 2H, CH2CH2),
1.55 (p, J = 6.4 Hz, 4H, CH2CH2), 1.39 (s, 18H, C[CH3]3).
1,11-Bis-{3-[1-(n-propyl)ureado]}-4,8-[N-(tert-butyl)oxycar-
bonyl)]-4,8-diazaundecane (43q). Compound 43q was prepared
from 291 mg of 41a (0.0008 mol) and n-propylisothiocyanate
according to the procedure described above for 43e to afford
43q (359 mg, 91%) as viscous oil. Rf: 0.63 (CH2Cl2/MeOH/
1
NH4OH, 89:10:1). H NMR (CDCl3): δ 5.60 (bs, 1H, NH),
4.70 (bs, 1H, NH), 3.05-3.28 (m, 16H, N-CH2), 1.60-1.78 (m,
6H, CH2CH2), 1.47 (m, J = 7.2, 4H, CH2CH3), 1.42 (s, 18H,
C[CH3]3), 0.88 (t, J = 7.2 Hz, 6H, CH2CH3).
1,11-Bis-{3-[1-(3,3-diphenylpropyl)ureado]}-4,8-[N-(tert-butyl)-
oxycarbonyl)]-4,8-diazaundecane (43t). Compound 43t was pre-
pared from 194 mg (0.0005 mol) of 41a and 35c according to the

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14 5209
was monitored by TLC (CH2Cl2/MeOH/NH4OH 89:10:1 or
78:20:2). The product precipitated as a white crystalline solid during
the course of the reaction. When completion of the reaction was
confirmed by TLC, the solvent was removed under reduced pressure
to produce a white powder. The solid product was stirred with
30 mL of fresh EtOAc, and the solvent was decanted. The solid so
obtained was vacuum-dried to give pure 3 as a white solid (315 mg,
95% yield). An analytical sample was obtained by purification on
silica gel (CH2Cl2:MeOH:NH4OH 89:10:1). 1H NMR (CD3OD):
δ 7.32-7.10 (m, 10H, Ar-H), 4.67 (s, 4H, CH2Ph), 3.71 (t, 4H, J =
5.6 Hz, NCH2), 3.01 (bs, 8H, NCH2), 1.95 (m, 4H, CH2CH2), 1.78
(bs, 4H, CH2CH2). MS (CI m/z) calcd for C26H40N6S2 [Mþ.] =
500.28; found 501.4 [MþH].
1,12-Bis-{3-[1-(benzyl)ureado]}-4,9-diazadodecane (4). Com-
pound 4 was prepared from 480 mg (0.0007 mol) of 43e accord-
ing to procedure described above for the synthesis of 3 to afford
370 mg (94%) of 4 as a white solid. 1H NMR (D2O): δ 7.32 (m,
4H, Ar-H), 7.26 (m, 6H, Ar-H), 4.22 (s, 4H, CH2Ph), 3.16 (t, 4H,
J = 6.4 Hz, NCH2), 2.88 (t, 4H, J = 7.2 Hz, NCH2), 2.81 (bs,
4H, NCH2), 1.25 (p, 4H, J = 6.4 and 7.2 Hz, CH2CH2), 1.57 (m,
4H, CH2CH2). 13C NMR (D2O): δ 160.89 (CdO), 139.83,
129.00, 127.45, 126.97 (Ar-C), 46.96, 45.12, 43.66, 36.44,
26.71, 22.92 (CH2).
1,12-Bis-{3-[1-(ethyl)ureado]}-4,9-diazadodecane (5). Com-
pound 5 was prepared from 448 mg (0.0008 mol) of 43f according
to procedure described above for the synthesis of 3 to afford 330
mg (96%) of5 asa white solid. 1H NMR (D2O):δ 3.15(t, 4H, J =
5.6 Hz, N-CH2), 3.05-2.98 (m, 12H, NCH2), 1.79 (p, 4H, J = 7.2
Hz, CH2CH2), 1.71 (bs, 4H, CH2CH2), 1.01 (t, 6H, J = 7.2 Hz,
CH3). 13C NMR (D2O): δ 160.88 (CdO), 47.06, 45.31, 36.67,
35.27, 26.70, 23.06 (CH2), 14.63 (CH3).
1,12-Bis-{3-[1-(ethyl)thioureado]}-4,9-diazadodecane(6). Com-
pound 6 was prepared from 470 mg (0.0008 mol) of43g according
to procedure described above for the synthesis of 3 to afford
314 mg (87%) of 6 as a white solid. 1H NMR (D2O): δ 3.51 (bs,
4H, NCH2), 3.31 (bs, 4H, NCH2), 3.06 (bs, 4H, NCH2), 1.93 (p,
4H, J = 6.4 Hz, CH2CH2), 1.75 (bs, 4H, CH2CH2), 1.12 (t, 6H,
J=6.0Hz, CH3).13C NMR (DMSO-d6):δ154.38, 153.98 (CdO),
47.13, 45.00, 40.92, 26.13, 23.15 (CH2), 13.49 (CH3).
NCH2), 3.10-2.80 (m, 8H, NCH2), 1.85 (b, 4H, CH2CH2), 1.59
(b, 4H, CH2CH2), 1.32 (b, 6H, CH2CH2).
1,15-Bis-{3-[1-(ethyl)ureado]}-4,12-diazapentadecane (11). Com-
pound 11 was prepared from 255 mg (0.0004 mol) of 43l according
to procedure described above for the synthesis of 3 to afford 178 mg
(89%) of 11 as a white solid. 1H NMR (D2O): δ 3.16 (t, 4H, J =
7.2 Hz, NCH2), 3.08 (q, 4H, J = 7.6 Hz, NCH2), 2.99 (m, 8H,
NCH2), 1.79 (p, 4H, J=7.2Hz, CH2CH2), 1.62 (bs, 4H, CH2CH2),
1.32 (s, 6H, CH2CH2), 1.02 (t, 6H, J = 7.2 Hz, CH3). 13C NMR
(D2O): δ 160.92 (CdO), 47.079, 45.15, 36.67, 35.25, 27.89, 26.69,
25.66 (CH2), 14.65 (CH3).
1,15-Bis-{3-[1-(n-propyl)ureado]}-4,12-diazapentadecane (12).
Compound 12 was prepared from 255 mg (0.0004 mol) of 43m
according to procedure described above for the synthesis of 3 to
afford 180 mg (89%) of 12 as a white solid. 1H NMR (D2O): δ
3.16 (t, 4H, J = 5.6 Hz, NCH2), 2.99 (m, 12H, NCH2), 1.79 (p,
4H, J = 7.2 Hz, CH2CH2), 1.62 (m, 4H, CH2CH2), 1.42 (q, 4H,
J = 6.4 Hz, CH2CH2), 1.32 (bs, 6H, CH2CH2), 0.81 (t, 6H, J =
7.2 Hz, CH3)). 13C NMR (D2O): δ 161.03 (CdO), 47.79, 45.14,
42.00, 36.67, 27.89, 26.71, 25.65, 22.86 (CH2), 10.77 (CH3).
1,11-Bis-{3-[1-(benzyl)thioureado]}-4,8-diazaundecane (13).
Compound 13 was prepared from 373 mg (0.0005 mol) of 43n
according to procedure described above for the synthesis of 3 to
afford 302 mg (99%) of 13 as white solid. 1H NMR (DMSO-d6):
δ 9.09 (bs, 2H, NH), 8.21 (t, 2H, NH), 8.00 (bs, 2H, NH),
7.20-7.32 (m, 10H, Ar-H), 4.64 (bs, 4H, N-CH2), 3.48 (bs, 4H,
N-CH2), 2.97 (bs, 4H, N-CH2), 2.87 (bs, 4H, N-CH2), 2.02 (p,
2H, CH2CH2), 1.86 (p, 4H, CH2CH2). 13C NMR (DMSO-d6): δ
128.92, 127.91, 127.45 (Ar-C), 47.43, 45.30, 44.60, 41.33, 26.37,
23.01 (CH2).
1,11-Bis-{3-[1-(ethyl)ureado]}-4,8-diazaundecane (14). Com-
pound 14 was prepared from 245 mg (0.0005 mol) of 43o
according to procedure described above for the synthesis of 3
to afford 178 mg (96%) of 14 as white solid. 1H NMR (DMSO-
d6): δ 9.14 (bs, 2H, NH), 6.00 (bs, 4H, NH), 2.88-3.08 (m, 12H,
CH2N), 2.82 (bs, 4H, CH2N), 2.02 (bs, 2H, CH2CH2), 1.71 (bs,
4H, CH2CH2), 0.95 (t, J = 7.2 Hz, 6H, CH2CH3). 13C NMR
(DMSO-d6): δ 159.16 (CdO), 45.25, 44.51, 36.84, 34.80, 27.51,
22.98 (CH2), 16.34 (CH3).
1,15-Bis-{3-[1-(benzyl)ureado]}-4,12-diazapentadecane (7). Com-
pound 7 was prepared from 320 mg (0.0005 mol) of 43h according
to procedure described above for the synthesis of 3 to afford 250 mg
(95%) of 7 as a white solid. 1H NMR (D2O): δ 7.35 (m, 4H, Ar-H),
7.28 (m, 6H, Ar-H), 4.25 (s, 4H, CH2Ph), 3.18 (t, 4H, J = 5.6 Hz,
NCH2), 2.88 (t, 4H, J = 7.2 Hz, NCH2), 2.81 (t, 4H, J = 8.0 Hz,
NCH2), 1.76 (p, 4H, J=7.2Hz,CH2CH2), 1.53 (m, 4H, CH2CH2),
1.26 (bs, 6H, CH2CH2). 13C NMR (D2O): δ 160.91 (CdO), 139.83,
129.01, 127.48, 126.99 (Ar-C), 47.78, 44.97, 43.68, 36.47, 27.87,
26.71, 25.64, 25.59 (CH2).
1,12-Bis-{3-[1-(n-propyl)ureado]}-4,9-diazadodecane (8). Com-
pound 8 was prepared from 330 mg (0.0006 mol) of 43i according
to procedure described above for the synthesis of 3 to afford
228 mg (90%) of 8 as a white solid. 1H NMR (D2O): δ 3.14 (t, 4H,
J = 6.4 Hz, NCH2), 3.00-2.95 (m, 12H, NCH2), 1.79 (p, 4H, J =
6.4 Hz, CH2CH2), 1.70 (bs, 4H, CH2CH2), 1.40 (q, 4H, J =
6.4 Hz, CH2CH2), 0.79 (t, 6H, J = 7.2 Hz, CH3). 13C NMR
(D2O): δ 160.98 (CdO), 47.05, 45.31, 42.03, 36.67, 26.70, 23.05,
22.82 (CH2), 10.77 (CH3).
1,12-Bis-{3-[1-(n-propyl)thioureado]}-4,9-diazadodecane (9).
Compound 9 was prepared from 350 mg (0.0006 mol) of 43j
according to procedure described above for the synthesis of 3 to
afford 240 mg (86%) of 9 as a white solid. 1H NMR (D2O): δ 3.59
(b, 4H, NCH2), 3.23 (b, 4H, NCH2), 3.07-3.00 (m, 8H, NCH2),
1.92 (p, 4H, J = 7.2 and 6.4 Hz, CH2CH2), 1.75 (b, 4H, CH2CH2),
1.57-1.48 (m, 4H, CH2CH2), 0.85 (t, 6H, J = 7.2 Hz, CH3).
1,15-Bis-{3-[1-(benzyl)thioureado]}-4,12-diazapentadecane (10).
Compound 10 was prepared from 340 mg (0.0005 mol) of 43k
according to procedure described above for the synthesis of 3 to
afford 214 mg (77%) of 10 as a white solid. 1H NMR (D2O): δ
7.37-7.30 (m, 10H, Ar-H), 4.58 (b, 4H, CH2Ph), 3.58 (b, 4H,
1,11-bis-{3-[1-(benzyl)ureado]}-4,8-diazaundecane (15). Com-
pound 15 was prepared from 485 mg (0.0007 mol) of 43p
according to procedure described above for the synthesis of 3
to afford 364 mg (99%) of 15 as white solid. 1H NMR (DMSO-
d6): δ 9.21 (bs, 6H, NH), 7.17-7.30 (m, 10H, Ar-H), 4.19 (s, 4H,
N-CH2), 3.08 (bs, 4H, N-CH2), 2.94 (bs, 2H, N-CH2), 2.82 (bs,
4H, N-CH2), 2.02 (b, 2H, CH2CH2), 1.74 (b, 4H, CH2CH2). 13
C
NMR (DMSO-d6): δ 159.24 (CdO), 141.49, 128.89, 127.63,
127.20 (Ar-C), 45.27, 44.53, 43.57, 37.02, 27.45, 22.93 (CH2).
1,11-Bis-{3-[1-(n-propyl)ureado]}-4,8-diazaundecane (16).
Compound 16 was prepared from 359 mg (0.0006 mol) of 43q
according to procedure described above for the synthesis of 3 to
afford 303 mg (99%) of 16 as a white solid. 1H NMR (DMSO-
d6): δ 9.24 (bs, 6H, NH), 2.82-3.06 (m, 16H, N-CH2), 2.04
(b, 2H, CH2CH2), 1.73 (b, 4H, CH2CH2), 1.33 (m, J = 7.2 Hz,
4H, CH2CH3), 0.80 (t, J = 7.2 Hz, 4H, CH2CH3). 13C NMR
(DMSO-d6): δ 159.29 (CdO), 45.22, 44.52, 41.88, 36.97, 27.39,
23.78, 22.92 (CH2), 12.04 (CH3).
1,11-Bis-{3-[1-(n-propyl)ureado]}-4,8-diazaundecane (17). Com-
pound 17 was prepared from 379 mg (0.0006 mol) of 43r according
to procedure described above for the synthesis of 3 to afford
317 mg (99%) of 17 as white solid. 1H NMR (DMSO-d6): δ 9.46
(b, 2H, NH), 9.16 (b, 2H, NH), 7.82 (b, 2H, NH), 2.85-3.90 (b,
16H, N-CH2), 1.84 (b, 2H, CH2CH2), 1.59 (b, 4H, CH2CH2), 1.44
(m, 4H, CH2CH3), 0.85 (t, 6H, CH2CH3). 13C NMR (DMSO-d6):
δ 45.30, 44.58, 26.37, 22.92, 22.71, 22.00 (CH2), 12.10 (CH3).
1,11-Bis-{3-[1-(ethyl)thioureado]}-4,8-diazaundecane (18). Com-
pound 18 was prepared from 347 mg (0.0006 mol) of 43s according
to procedure described above for the synthesis of 3 to afford
282 mg (99%) of 18 as white solid. 1H NMR (DMSO-d6): δ 9.10
(bs, 2H, NH), 7.78 (bs, 2H, NH), 7.70 (bs, 2H, NH), 3.43 (bs, 4H,

5210 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Sharma et al.
N-CH2), 3.32 (bs, 4H, N-CH2), 2.97 (bs, 4H, N-CH2), 2.86 (bs, 4H,
N-CH2), 2.02 (b, 2H, CH2CH2), 1.83 (b, 4H, CH2CH2), 1.02 (t,
J = 7.2 Hz, 6H, CH2CH3). 13C NMR (DMSO-d6): δ 45.27, 44.58,
38.83, 31.99, 26.38, 22.98 (CH2), 15.12 (CH3).
145.37, 129.11, 128.30, 126.70 (Ar-C), 48.61, 47.30, 45.15, 41.42,
34.93, 28.58, 26.41, 25.94 (CH and CH2).
1,15-Bis-{3-[1-(2,2-diphenylethyl)thioureado]}-4,12-diazapenta-
decane (25). Compound 25 was prepared from 260 mg (0.0003
mol) of 43z according to procedure described above for the
synthesis of 3 to afford 201 mg (90%) of 25 as a white solid. 1H
NMR (DMSO-d6): δ 8.91 (bs, 3H, NH), 7.70 (b, 1H, NH), 7.52
(b, 1H, NH), 7.26 (bs, 16H, Ar-H), 7.16 (bs, 4H, Ar-H), 4.36 (b,
2H, CHPh2), 4.04 (b, 4H, NCH2), 3.45 (b, 4H, NCH2), 2.78 (b,
8H, NCH2), 1.78 (b, 4H, CH2CH2), 1.58 (b, 4H, CH2CH2), 1.25
(b, 6H, CH2CH2). 13C NMR (DMSO-d6): δ 181.50 (CdS),
143.36, 129.16, 128.62, 127.07 (Ar-C), 50.44, 48.78, 47.30, 45.09,
28.59, 26.40, 26.25, 25.91(CH2).
1,12-Bis-{3-[1-(2,2-diphenylethyl)thioureado]}-4,9-diazado-
decane (26). Compound 26 was prepared from 280 mg (0.0003
mol) of 43aa according to procedure described above for the
synthesis of 3 to afford 214 mg (89%) of 26 as a white solid. 1H
NMR (DMSO-d6): δ 9.05 (b, 4H, NH), 7.79 (b, 2H, NH), 7.53
(bs, 2H, NH), 7.28 (bs, 16H, Ar-H), 7.14 (m, 4H, Ar-H), 4.36
(bs, 2H, CHPh2), 4.02 (bs, 4H, NCH2), 3.42 (bs, 4H, NCH2),
2.81 (b, 8H, NCH2), 1.80 (bs, 4H, CH2CH2), 1.66 (bs, 4H,
CH2CH2). 13C NMR (DMSO-d6): δ 183.29 (CdS), 143.39,
129.16, 128.63, 127.07 (Ar-C), 50.46, 48.76, 46.57, 45.09,
41.21, 26.25, 23.16 (CH and CH2).
1,11-Bis-{3-[1-(2,2-diphenylethyl)thioureado]}-4,8-diazaun-
decane (27). Compound 27 was prepared from 330 mg (0.0004
mol) of 43bb according to procedure described above for the
synthesis of 3 to afford 220 mg (79%) of 27 as a white solid. 1H
NMR (DMSO-d6): δ 9.13 (b, 4H, NH), 7.77 (bs, 2H, NH), 7.50
(bs, 2H, NH), 7.27 (bs, 16H, Ar-H), 7.16 (bs, 4H, Ar-H), 4.35 (bs,
2H, CHPh2), 4.04 (b, 4H, NCH2), 3.66 (bs, 4H, NCH2), 3.42 (bs,
4H, NCH2), 2.94 (bs, 4H, NCH2), 2.80 (bs, 4H, NCH2), 2.01 (b,
2H, CH2CH2), 1.79 (bs, 4H, CH2CH2). 13C NMR (DMSO-d6): δ
183.20(CdS), 143.38, 129.17, 128.63, 127.07 (Ar-C), 50.45, 48.68,
46.24, 44.57, 41.05, 26.28, 22.98 (CH and CH2).
1,11-Bis-{3-[1-(1,1-diphenylmethyl)thioureado]}-4,8-diazaun-
decane (28). Compound 28 was prepared from 335 mg (0.0004
mol) of 43cc according to procedure described above for the
synthesis of 3 to afford 227 mg (80%) of 28 as a white solid. 1H
NMR (DMSO-d6): δ 8.90 (b, 4H, NH), 8.29 (b, 2H, NH),
7.40-7.22 (m, 20H, Ar-H), 6.72 (b, 2H, CH), 4.56 (b, NH), 3.52
(b, 4H, NCH2), 2.97 (m, 8H, NCH2), 2.02 (b, 2H, CH2), 1.87 (b,
4H, CH2CH2). 13C NMR (DMSO-d6): δ 183.22 (CdS), 143.43,
129.08, 127.89, 127.56 (Ar-C), 61.28 (CH), 45.37, 44.59, 41.34,
26.33, 23.00 (CH2).
1,11-Bis-{3-[1-(3,3-diphenylpropyl)ureado]}-4,8-diazaundecane
(19). Compound 19 was prepared from 400 mg (0.0005 mol) of 43t
according to procedure described above for the synthesis of 3 to
afford 290 mg (86%) of 19 as a white solid. 1H NMR (DMSO-d6):
δ9.10 (bs, 4H, NH), 7.27-7.21 (m, 16H, Ar-H), 7.18-7.10 (m, 4H,
Ar-H), 3.96 (t, 2H, J = 7.2 Hz, CHPh2), 3.02 (t, 4H, J = 6.4 Hz,
NCH2), 2.92 (b, 4H, NCH2), 2.84 (t, 4H, J = 7.2 Hz, NCH2), 2.79
(bs, 4H, NCH2), 2.09 (q, 4H, J = 8.0 Hz, CH2CH2), 1.99 (m,
2H, CH2CH2), 1.69 (m, 4H, CH2CH2). 13C NMR (DMSO-d6): δ
159.22 (CO), 145.50, 129.08, 128.28, 126.72 (Ar-C), 48.51, 45.25,
44.49, 38.77, 36.88, 36.07, 27.45, 22.95 (CH and CH2).
1,11-Bis-{3-[1-(3,3-diphenylpropyl)thioureado]}-4,8-diazaun-
decane (20). Compound 20 was prepared from 260 mg (0.0003
mol) of 43u according to procedure described above for the
synthesis of 3 to afford 205 mg (92%) of 20 as a white solid. 1H
NMR (DMSO-d6): δ 9.10 (b, 4H, NH), 7.91 (b, 2H, NH),
7.32-7.14 (m, 20H, Ar-H), 6.10 (b, 2H, NH), 4.04 (t, 2H, J =
7.6 Hz, CHPh2), 3.45 (b, 4H, NCH2), 3.24 (b, 4H, NCH2), 2.98
(b, 4H, NCH2), 2.88 (b, 4H, NCH2), 2.61 (m, 4H, CH2CH2),
2.04 (m, 2H, CH2CH2), 1.85 (m, 4H, CH2CH2). 13C NMR
(DMSO-d6): δ 145.36, 129.11, 128.31, 126.78 (Ar-C), 48.62,
45.29, 44.60, 42.80, 41.02, 34.97, 26.34, 22.96 (CH and CH2).
1,12-Bis-{3-[1-(3,3-diphenylpropyl)ureado]}-4,9-diazadodecane
(21). Compound21 was preparedfrom 370 mg(0.42mmol)of43v
according to procedure described above for the synthesis of 3 to
afford285 mg (90%) of21 as a white solid. 1H NMR (DMSO-d6):
δ 9.00 (bs, 4H, NH), 7.21-7.12 (m, 20H, Ar-H, and NH), 3.96 (t,
2H, J = 7.2 Hz, CHPh2), 3.02 (t, 4H, J = 6.4 Hz, NCH2), 2.84 (t,
4H, J = 6.4 Hz, NCH2), 2.79 (b, 12H, NCH2), 2.09 (q, 4H, J =
7.2 Hz, CH2CH2), 1.69 (t, 4H, J = 6.4 Hz, CH2CH2), 1.63 (b, 4H,
CH2CH2). 13C NMR (DMSO-d6): δ 159.27 (CdO), 145.49,
129.08, 128.28, 126.73 (Ar-C), 48.49, 46.50, 45.10, 38.76, 36.88,
36.07, 27.46, 23.23 (CH and CH2). MS (EI m/z) calculated for
C42H56N6O2 [Mþ 3 ] = 676.45; found 677.40 [MþH].
1,12-Bis-{3-[1-(3,3-diphenylpropyl)thioureado]}-4,9-diazado-
decane (22). Compound 22 was prepared from 260 mg (0.0003
mol) of 43w according to procedure described above for the
synthesis of 3 to afford 205 mg (92%) of 22 as a white solid. 1H
NMR (DMSO-d6): δ 9.02 (bs, 4H, NH), 8.02 (b, 2H, NH),
7.30-7.12 (m, 22H, Ar-H, and NH), 4.03 (t, 2H, J = 7.6 Hz,
CHPh2), 3.43 (bs, 4H, NCH2), 3.23 (bs, 4H, NCH2), 2.85 (b,
8H, NCH2), 2.24 (m, 4H, CH2CH2), 1.84 (b, 4H, CH2CH2),
1.67 (b, 4H, CH2CH2). 13C NMR (DMSO-d6): δ 145.34, 129.14,
128.31, 126.79 (Ar-C), 48.58, 46.59, 45.13, 41.18, 34.93, 26.29,
23.26 (CH and CH2).
1,15-Bis-{3-[1-(3,3-diphenylpropyl)ureado]}-4,12-diazapenta-
decane (23). Compound 23 was prepared from 290 mg (0.0003
mol) of 43x according to procedure described above for the
synthesis of 3 to afford 225 mg (88%) of 23 as a white solid. 1H
NMR (DMSO-d6): δ 8.94 (bs, 4H, NH), 7.27-7.21 (m, 16H, Ar-
H), 7.13-7.10 (m, 4H, Ar-H), 3.96 (t, 2H, J = 7.2 Hz, CHPh2),
3.02 (t, 4H, J = 6.9 Hz, NCH2), 2.84 (t, 4H, J = 7.2 Hz, NCH2),
2.77 (bs, 8H, NCH2), 2.09 (d, 4H, J = 7.2 Hz, CH2CH2), 1.69 (t,
4H, J = 6.4 Hz, CH2CH2), 1.56 (bs, 4H, CH2CH2), 1.21 (bs,
6H, CH2CH2). 13C NMR (DMSO-d6): δ 159.35 (CdO), 145.49,
129.07, 128.28, 126.72 (Ar-C), 48.49, 47.24, 45.09, 38.75, 36.82,
36.07, 28.55, 27.48, 26.37, 25.91 (CH and CH2).
1,12-Bis-{3-[1-(1,1-diphenylmethyl)thioureado]}-4,9-diazado-
decane (29). Compound 29 was prepared from 354 mg (0.0004
mmol) of 43dd according to procedure described above for the
synthesis of 3 to afford 262 mg (87%) of 29 as a white solid. 1H
NMR (DMSO-d6): δ 8.95 (b, 4H, NH), 8.30 (bs, 2H, NH), 7.30
(m, 20H, Ar-H), 6.72 (b, 2H, CHPh2), 3.51 (b, 4H, NCH2), 2.88
(b, 8H, NCH2), 1.87 (b, 4H, CH2CH2), 1.66 (b, 4H, CH2CH2). 13
C
NMR(DMSO-d6):δ183.26 (CdS), 143.42, 129.08, 127.89, 127.57
(Ar-C), 61.30 (CH), 46.60, 45.22, 41.42, 26.38, 23.28 (CH2).
1,15-Bis-{3-[1-(1,1-diphenylmethyl)thioureado]}-4,12-diazapenta-
decane (30). Compound 30 was prepared from 390 mg (0.0004 mol)
of 43ee according to procedure described above for the synthesis
1
of 3 to afford 298 mg (89%) of 30 as a white solid. H NMR
(DMSO-d6): δ 8.90 (b, 4H, NH), 8.35 (b, 2H, NH), 7.30 (bs, 20H,
Ar-H), 6.73 (bs, 2H, CHPh2), 3.51 (bs, 4H, NCH2), 2.89 (bs, 4H,
NCH2), 2.81 (bs, 4H, NCH2), 1.87 (bs, 4H, CH2CH2), 1.60 (bs,
4H, CH2CH2), 1.26 (b, 6H, CH2CH2). 13C NMR (DMSO-d6): δ
183.29 (CdS), 143.45, 129.06, 127.89, 127.55 (Ar-C), 61.30 (CH),
47.32, 45.23, 41.42, 28.59, 26.41, 25.93 (CH2).
1,12-Bis-{5-[1-(N,N-diphenyl)carbamyl]ureado}-4,9-diazado-
decane (31). Compound 31 was prepared from 51 (130 mg,
0.0002 mol) according to procedure described above for the
synthesis of 3 to afford 85 mg of 31 (75%) as a white solid. 1H
NMR (DMSO-d6): δ 9.08 (bs, 4H, NH), 8.31 (t, 2H, J = 5.6 Hz,
NH), 7.86 (s, 2H, NH), 7.41-7.37 (m, 8H, Ar-H), 7.30-7.26
1,15-Bis-{3-[1-(3,3-diphenylpropyl)thioureado]}-4,12-diazapenta-
decane (24). Compound 24 was prepared from 287 mg (0.0003 mol)
of 43y according to procedure described above for the synthesis of 3
to afford 230 mg (92%) of 24 as a white solid. 1H NMR (DMSO-
d6): δ 8.87 (bs, 4H, NH), 7.89 (bs, 4H, NH), 7.32-7.25 (m, 16H,
Ar-H), 7.18-7.14 (m, 4H, Ar-H), 4.10 (b, 2H, CHPh2), 3.44(b, 4H,
NCH2), 3.23 (b, 4H, NCH2), 2.87 (m, 8H, NCH2), 2.25 (d, 4H, J =
7.6 Hz, CH2CH2), 1.83 (t, 4H, J = 7.2 Hz, CH2CH2), 1.68 (m, 4H,
CH2CH2), 1.28 (b, 6H, CH2CH2). 13C NMR (DMSO-d6): δ

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14 5211
(m, 12H, Ar-H), 3.21 (m, 4H, NCH2), 2.84 (bs, 8H, NCH2), 1.81
(m, 4H, CH2CH2), 1.67 (bs, 4H, CH2CH2). 13C NMR (DMSO-
d6): δ 154.36, 153.97 (CdO), 142.41, 130.19, 128.45, 127.77
(Ar-C), 46.52, 45.13, 37.08, 26.70, 23.26 (CH2). MS (EI m/z)
calcd for C38H46N8O4 [Mþ 3 ] = 678.36; found 679.32 [MþH].
1,15-Bis-{5-[1-(N,N-diphenyl)carbamyl]ureado}-4,12-diazapenta-
decane (32). Compound 32 was prepared from 52 (90 mg, 0.0001
mol) according to procedure described above for the synthesis of 3
to afford 42 mg of 32 (55%) as a white solid. 1H NMR (DMSO-d6):
δ 8.92 (b, 4H, NH), 8.32 (bs, 2H, NH), 7.88 (bs, 2H, NH), 7.40-
7.31 (m, 20H, Ar-H), 3.21 (bs, 4H, NCH2), 2.83 (bs, 8H, NCH2),
1.81 (bs, 4H, CH2CH2), 1.60 (bs, 4H, CH2CH2), 1.27 (bs, 6H,
CH2CH2). 13C NMR (DMSO-d6):δ 154.39, 154.00 (CdO), 142.42,
130.01, 128.44, 127.79 (Ar-C), 45.27, 45.15, 37.04, 28.60, 26.73,
26.42, 25.96(CH2).
were seeded in 100 μL medium in a 96-well plate and the cells
were allowed to attach at 37 °C in 5% CO2 for one day. The
medium was aspirated and cells were treated with 100 μL
of fresh medium containing appropriate concentrations of
each test compound. The cells were incubated for 4 days at
37 °C in 5% CO2. After 4 days, 20 μL of the MTS reagent
solution (Promega CellTiter 96 Aqueous One Solution cell
proliferation assay) was added to the medium. The cells were
incubated for another 2 h at 37 °C under 5% CO2 environment.
Absorbance was measured at 490 nm on a microplate reader
equipped with SOFTmax PRO 4.0 software to determine the
cell viability.
Acknowledgment. The excellent technical assistance of Amy
Hacker-Prietz is gratefully acknowledged. The research described
in this manuscript was supported by NIH grant 1R01CA149095
(P.M.W.), 1RO1CA98454 (R.A.C.), and 1RO1CA51085
(R.A.C.) and by grants from the Samuel Waxman Cancer
Research Foundation (R.A.C., P.M.W.) and the Susan B.
Komen for the Cure Foundation (R.A.C.). In addition, a
portion of this work was supported by a generous grant
from Progen Pharmaceuticals, Ltd., Brisbane, Queensland,
Australia (P.M.W., R.A.C.). Professor Casero also serves
as a consultant to Progen Pharmaceuticals.
1,11-Bis-{5-[1-(N,N-diphenyl)carbamyl]ureado}-4,8-diazaun-
decane (33). Compound 33 was prepared from 53 (110 mg, 0.0001
mol) according to procedure described above for the synthesis
1
of 3 to afford 66 mg of 33 (75%) as a white solid. H NMR
(DMSO-d6): δ 9.14 (bs, 4H, NH), 8.32 (t, 2H, J = 5.6 Hz, NH),
7.87 (s, 2H, NH), 7.42-7.38 (m, 8H, Ar-H), 7.31-7.27 (m, 12H,
Ar-H), 3.20 (m, 4H, NCH2), 2.97 (bs, 4H, NCH2), 2.84 (bs, 4H,
NCH2), 2.02 (m, 2H, CH2CH2), 1.81 (m, 4H, CH2CH2). 13C
NMR (DMSO-d6): δ 154.38, 153.98 (CdO), 142.42, 130.21,
128.46, 127.79 (Ar-C), 45.29, 44.56, 37.04, 26.74, 23.03(CH2).
Expression, Purification, and Demethylase Assay of Recombi-
nant Proteins. Full-length human LSD1 cDNA was subcloned
into the pET15b bacterial expression vector (Novagen, Madison,
WI) in frame with an N-terminal 6ꢀ HIS-tag and transformed
into the BL21(DE3) strain of Escherichia coli. Following selection,
expression and purification of recombinant LSD1 protein were
performed as previously described.10 Briefly, expressionofLSD1-
HIS protein was induced by 1 mM IPTG for 6 h at 25 °C. The
HIS-tagged protein was purified using Ni-NTA affinity purifica-
tion resin and column as recommended by the manufacturer
(Qiagen, Valencia, CA). Bound protein was eluted by imidazole
and the eluate was dialyzed in PBS at 4 °C. Enzymatic activity of
LSD1 was examined using luminol-dependent chemilumines-
cence to measure the production of H2O2, as previously described.33
In brief, LSD1 activity was assayed in 50 mM Tris, pH 8.5, 50 mM
KCl, 5 mM MgCl, 5 nmol luminol, and 20 μg/mL horseradish
peroxidase with the indicated concentrations of H3K4me2 (1-21 aa)
peptide as substrate. The integral values were calibrated against
standards containing known concentrations of H2O2, and the
activities expressed as pmols H2O2/mg protein/min.
Note Added after ASAP Publication. This paper published
on the web on June 22, 2010 with several missed corrections. The
revised version published on June 25, 2010.
References
(1) Marks, P. A.; Richon, V. M.; Breslow, R.; Rifkind, R. A. Histone
deacetylase inhibitors as new cancer drugs. Curr. Opin. Oncol.
2001, 13 (6), 477–483.
(2) Luger, K.; Mader, A. W.; Richmond, R. K.; Sargent, D. F.;
Richmond, T. J. Crystal structure of the nucleosome core particle
at 2.8 A resolution. Nature 1997, 389 (6648), 251–260.
(3) Jenuwein, T.; Allis, C. D. Translating the histone code. Science
2001, 293 (5532), 1074–1080.
(4) Johnstone, R. W. Histone-deacetylase inhibitors: novel drugs for
the treatment of cancer. Nature Rev. Drug Discovery 2002, 1 (4),
287–299.
(5) Herman, J. G.; Baylin, S. B. Gene silencing in cancer in association
with promoter hypermethylation. N. Engl. J. Med. 2003, 349 (21),
2042–2054.
(6) Robertson, K. D. DNA methylation, methyltransferases, and
cancer. Oncogene 2001, 20 (24), 3139–3155.
Western Blotting. Cytoplasmic and nuclear fractions were
prepared for Western blot analysis using the NE-PER nuclear
and cytoplasmic extraction kit (Pierce, Rockford, IL). Primary
antibodies against H3K4me1 and H3K4me2 were from Millipore.
The pCNA monoclonal antibody was purchased from Oncogene
Research Products (Cambridge, MA). Dye-conjugated secondary
antibodies were used for quantification of Western blot results
using the Odyssey infrared detection system and software
(LI-COR Biosciences, Lincoln, NE).
RNA Isolation and qPCR. RNA was extracted using TRIzol
reagents (Invitrogen, Carlsbad, CA). First-strand cDNA was
synthesized using SuperScript III reverse transcriptase with an
oligo(dT) primer (Invitrogen). qPCR was performed using the
following primers: SFRP2 sense, 50AAG CCT GCA AAA ATA
AAA ATG ATG; SFRP2 antisense, 50TGT AAA TGG TCT
TGC TCT TGG TCT (annealing at 57.4 °C); GATA4 sense,
50GGC CGC CCG ACA CCC CAA TCT; GATA4 antisense,
50 ATA GTG ACC CGT CCC ATC TCG (annealing at 64 °C).
qPCR was performed in a MyiQ single color real-time PCR machine
(Bio-Rad, Hercules, CA) with GAPDH as an internal control.
Determination of Cell Viability. Calu-6 human anaplastic
nonsmall cell lung carcinoma cells were maintained in culture
using RPMI medium plus 10% fetal bovine serum. For the
(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-
sulfophenyl)-2H-tetrazolium) (MTS) reduction assay, 4000 cells/well
(7) Shogren-Knaak, M.; Ishii, H.; Sun, J.-M.; Pazin, M. J.; Davie,
J. R.; Peterson, C. L. Histone H4-K16 Acetylation Controls
Chromatin Structure and Protein Interactions. Science 2006,
311 (5762), 844–847.
(8) Baylin, S. B.; Ohm, J. E. Epigenetic gene silencing in cancer
a
;
mechanism for early oncogenic pathway addiction? Nature Rev.
Cancer 2006, 6 (2), 107–116.
(9) Jones, P. A.; Baylin, S. B. The epigenomics of cancer. Cell 2007,
128 (4), 683–692.
(10) Shi, Y.; Lan, F.; Matson, C.; Mulligan, P.; Whetstine, J. R.; Cole,
P. A.; Casero, R. A.; Shi, Y. Histone demethylation mediated
by the nuclear amine oxidase homolog LSD1. Cell 2004, 119 (7),
941–953.
(11) Whetstine, J. R.; Nottke, A.; Lan, F.; Huarte, M.; Smolikov, S.;
Chen, Z.; Spooner, E.; Li, E.; Zhang, G.; Colaiacovo, M.; Shi, Y.
Reversal of histone lysine trimethylation by the JMJD2 family of
histone demethylases. Cell 2006, 125 (3), 467–481.
(12) Bannister, A. J.; Kouzarides, T. Reversing histone methylation.
Nature 2005, 436 (7054), 1103–1106.
(13) Kouzarides, T. Histone methylation in transcriptional control.
Curr. Opin. Genet. Dev. 2002, 12 (2), 198–209.
(14) Martin, C.; Zhang, Y. The diverse functions of histone lysine
methylation. Nature Rev. Mol. Cell Biol. 2005, 6 (11), 838–849.
(15) Zhang, Y.; Reinberg, D. Transcription regulation by histone
methylation: interplay between different covalent modifications
of the core histone tails. Genes Dev. 2001, 15 (18), 2343–2360.
(16) Schneider, R.; Bannister, A. J.; Kouzarides, T. Unsafe SETs:
histone lysine methyltransferases and cancer. Trends Biochem.
Sci. 2002, 27 (8), 396–402.

5212 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Sharma et al.
(17) Tsukada, Y.; Zhang, Y. Purification of histone demethylases from
HeLa cells. Methods 2006, 40 (4), 318–326.
(18) Huarte, M.; Lan, F.; Kim, T.; Vaughn, M. W.; Zaratiegui, M.;
Martienssen, R. A.; Buratowski, S.; Shi, Y. The fission yeast JMJ2
reverses histone H3 lysine 4 tri-methylation. J. Biol. Chem. 2007,
282, 21662–21670.
(19) Liang, G.; Klose, R. J.; Gardner, K. E.; Zhang, Y. Yeast Jhd2p is a
histone H3 Lys4 trimethyl demethylase. Nature Struct. Mol. Biol.
2007, 14 (3), 243–245.
(20) Secombe, J.; Li, L.; Carlos, L.; Eisenman, R. N. The trithorax
group protein Lid is a trimethyl histone H3K4 demethylase re-
quired for dMyc-induced cell growth. Genes Dev. 2007, 21 (5),
537–551.
(21) Wang, J.; Hevi, S.; Kurash, J. K.; Lei, H.; Gay, F.; Bajko, J.; Su, H.;
Sun, W.; Chang, H.; Xu, G.; Gaudet, F.; Li, E.; Chen, T. The lysine
demethylase LSD1 (KDM1) is required for maintenance of global
DNA methylation. Nature Genet. 2009, 41 (1), 125–129.
(22) Liang, G.; Lin, J. C.; Wei, V.; Yoo, C.; Cheng, J. C.; Nguyen, C. T.;
Weisenberger, D. J.; Egger, G.; Takai, D.; Gonzales, F. A.; Jones,
P. A. Distinct localization of histone H3 acetylation and H3-K4
methylation to the transcription start sites in the human genome.
Proc. Natl. Acad. Sci. U.S.A 2004, 101 (19), 7357–7362.
(23) Schneider, R.; Bannister, A. J.; Myers, F. A.; Thorne, A. W.;
Crane-Robinson, C.; Kouzarides, T. Histone H3 lysine 4 methyla-
tion patterns in higher eukaryotic genes. Nature Cell Biol. 2004, 6
(1), 73–77.
(36) Bellelli, A.; Cavallo, S.; Nicolini, L.; Cervelli, M.; Bianchi, M.;
Mariottini, P.; Zelli, M.; Federico, R. Mouse spermine oxidase: a
model of the catalytic cycle and its inhibition by N,N1-bis(2,3-
butadienyl)-1,4-butanediamine. Biochem. Biophys. Res. Commun.
2004, 322 (1), 1–8.
(37) Wang, Y.; Hacker, A.; Murray-Stewart, T.; Frydman, B.; Valasinas,
A.; Fraser, A. V.; Woster, P. M.; Casero, R. A., Jr. Properties of
recombinant human N1-acetylpolyamine oxidase (hPAO): potential
role in determining drug sensitivity. Cancer Chemother. Pharmacol.
2005, 56 (1), 83–90.
(38) Cona, A.; Manetti, F.; Leone, R.; Corelli, F.; Tavladoraki, P.;
Polticelli, F.; Botta, M. Molecular basis for the binding of compe-
titive inhibitors of maize polyamine oxidase. Biochemistry 2004,
43 (12), 3426–3435.
(39) Stranska, J.; Sebela, M.; Tarkowski, P.; Rehulka, P.; Chmelik, J.;
Popa, I.; Pec, P. Inhibition of plant amine oxidases by a novel series
of diamine derivatives. Biochimie 2007, 89 (1), 135–144.
(40) Bi, X.; Lopez, C.; Bacchi, C. J.; Rattendi, D.; Woster, P. M. Novel
alkylpolyaminoguanidines and alkylpolyaminobiguanides with
potent antitrypanosomal activity. Bioorg. Med. Chem. Lett. 2006,
16 (12), 3229–3232.
(41) Gorbatenko, V. I. Chemistry of chlorocarbonyl isocyanate. Tetra-
hedron 1993, 49 (16), 3227–3257.
(42) DePrez, P.; Lively, S. E. Derivatives of urea and related diamines,
methods for their manufacture, and uses therefor. U.S. Patent
Appl. 20080125424, 2008.
(24) Lee, M. G.; Wynder, C.; Schmidt, D. M.; McCafferty, D. G.;
Shiekhattar, R. Histone H3 lysine 4 demethylation is a target of
nonselective antidepressive medications. Chem. Biol. 2006, 13 (6),
563–567.
(25) Huang, Y.; Greene, E.; Murray Stewart, T.; Goodwin, A. C.;
Baylin, S. B.; Woster, P. M.; Casero, R. A., Jr. Inhibition of
lysine-specific demethylase 1 by polyamine analogues results in
reexpression of aberrantly silenced genes. Proc. Natl. Acad. Sci.
U.S.A 2007, 104 (19), 8023–8028.
(26) Stavropoulos, P.; Hoelz, A. Lysine-specific demethylase 1 as a
potential therapeutic target. Expert Opin. Ther. Targets 2007,
11 (6), 809–820.
(27) Schmidt, D. M.; McCafferty, D. G. trans-2-Phenylcyclopropyl-
amine is a mechanism-based inactivator of the histone demethylase
LSD1. Biochemistry 2007, 46 (14), 4408–4416.
(28) Culhane, J. C.; Szewczuk, L. M.; Liu, X.; Da, G.; Marmorstein, R.;
Cole, P. A. A mechanism-based inactivator for histone demethyl-
ase LSD1. J. Am. Chem. Soc. 2006, 128 (14), 4536–4537.
(29) Szewczuk, L. M.; Culhane, J. C.; Yang, M.; Majumdar, A.; Yu, H.;
Cole, P. A. Mechanistic Analysis of a Suicide Inactivator of
Histone Demethylase LSD1. Biochemistry 2007, 46, 6892–6902.
(30) Gooden, D. M.; Schmidt, D. M.; Pollock, J. A.; Kabadi, A. M.;
McCafferty, D. G. Facile synthesis of substituted trans-2-arylcy-
clopropylamine inhibitors of the human histone demethylase
LSD1 and monoamine oxidases A and B. Bioorg. Med. Chem.
Lett. 2008, 18 (10), 3047–3051.
(31) Ueda, R.; Suzuki, T.; Mino, K.; Tsumoto, H.; Nakagawa, H.;
Hasegawa, M.; Sasaki, R.; Mizukami, T.; Miyata, N. Identification
of cell-active lysine specific demethylase 1-selective inhibitors.
J. Am. Chem. Soc. 2009, 131 (48), 17536–17537.
(32) Binda, C.; Valente, S.; Romanenghi, M.; Pilotto, S.; Cirilli, R.;
Karytinos, A.; Ciossani, G.; Botrugno, O. A.; Forneris, F.; Tardugno,
M.; Edmondson, D. E.; Minucci, S.; Mattevi, A.; Mai, A. Bio-
chemical, Structural, and Biological Evaluation of Tranylcypromine
Derivatives as Inhibitors of Histone Demethylases LSD1 and LSD2.
J. Am. Chem. Soc. 2010, 132, 6827–6833.
(43) Wong, R.; Dolman, S. J. Isothiocyanates from tosyl chloride
mediated decomposition of in situ generated dithiocarbamic acid
salts. J. Org. Chem. 2007, 72 (10), 3969–3971.
(44) Keller, O.; Keller, W. E.; van Look, G.; Wersin, G. tert-Butoxy-
carbonylation of Amino Acids and Their Derivatives: N-tert-
Butoxycarbonyl-L-Phenylalanine. Org. Synth. 1985, 63, 160–171.
(45) Bellevue,F.H.,III;Boahbedason,M.;Wu, R.;Woster, P.M.;Casero,
J. R. A.; Rattendi, D.; Lane, S.; Bacchi, C. J. Structural comparison of
alkylpolyamine analogues with potent in vitro antitumor or antipar-
asitic activity. Bioorg. Med. Chem. Lett. 1996, 6 (22), 2765.
(46) Hagemann, H. Synthesis and reactions of N-chlorocarbonyl iso-
cyanate. Angew. Chem., Intl. Ed. Engl. 1977, 16, 743–750.
(47) Karytinos, A.; Forneris, F.; Profumo, A.; Ciossani, G.; Battaglioli,
E.; Binda, C.; Mattevi, A. A novel mammalian flavin-dependent
histone demethylase. J. Biol. Chem. 2009, 284 (26), 17775–17782.
(48) Loenarz, C.; Ge, W.; Coleman, M. L.; Rose, N. R.; Cooper, C. D.;
Klose, R. J.; Ratcliffe, P. J.; Schofield, C. J. PHF8, a gene
associated with cleft lip/palate and mental retardation, encodes
for an Nepsilon-dimethyl lysine demethylase. Hum. Mol. Genet.
2010, 19 (2), 217–222.
(49) Yue, W. W.; Hozjan, V.; Ge, W.; Loenarz, C.; Cooper, C. D.;
Schofield, C. J.; Kavanagh, K. L.; Oppermann, U.; McDonough,
M. A. Crystal structure of the PHF8 Jumonji domain, an Nepsilon-
methyl lysine demethylase. FEBS Lett. 2010, 584 (4), 825–830.
(50) Varghese, S.; Gupta, D.; Baran, T.; Jiemjit, A.; Gore, S. D.; Casero,
R. A., Jr.; Woster, P. M. Alkyl-substituted polyaminohydroxamic
acids: a novel class of targeted histone deacetylase inhibitors.
J. Med. Chem. 2005, 48 (20), 6350–6365.
(51) Varghese, S.; Senanayake, T.; Murray-Stewart, T.; Doering, K.;
Fraser, A.; Casero, R. A.; Woster, P. M. Polyaminohydroxamic
Acids and Polyaminobenzamides as Isoform Selective Histone
Deacetylase Inhibitors. J. Med. Chem. 2008, 51 (8), 2447–2456.
(52) Flis, S.; Gnyszka, A.; Flis, K.; Splawinski, J. MS275 enhances
cytotoxicity induced by 5-fluorouracil in the colorectal cancer cells.
Eur. J. Pharmacol. 2010, 627 (1-3), 26–32.
(53) Fouladi, M. Histone deacetylase inhibitors in cancer therapy.
Cancer Invest. 2006, 24 (5), 521–527.
(54) Huang, Y.; Stewart, T. M.; Wu, Y.; Baylin, S. B.; Marton, L. J.;
Perkins, B.; Jones, R. J.; Woster, P. M.; Casero, R. A., Jr. Novel
Oligoamine Analogues Inhibit Lysine-Specific Demethylase 1 and
Induce Reexpression of Epigenetically Silenced Genes. Clin. Cancer
Res. 2009, 15, 7217–7228.
(33) Wang, Y.; Murray-Stewart, T.; Devereux, W.; Hacker, A.; Frydman,
B.; Woster, P. M.; Casero, R. A., Jr. Properties of purified recombi-
nant human polyamine oxidase, PAOh1/SMO. Biochem. Biophys.
Res. Commun. 2003, 304 (4), 605–611.
(34) Ferioli, M. E.; Berselli, D.; Caimi, S. Effect of mitoguazone on
polyamine oxidase activity in rat liver. Toxicol. Appl. Pharmacol.
2004, 201 (2), 105–111.
(35) Casara, P.; Jund, K.; Bey, P. General synthetic access to alpha-
allenyl amines and alpha-allenyl-alpha-amino acids as potential
enzyme activated, irreversible inhibitors of PLP-dependent en-
zymes. Tetrahedron Lett. 1984, 25, 1891–1894.
(55) Bellevue, F. H., III; Boahbedason, M.; Wu, R.; Woster, P. M.;
Casero, J. R. A.; Rattendi, D.; Lane, S.; Bacchi, C. J. Structural
comparison of alkylpolyamine analogues with potent in vitro anti-
tumor or antiparasitic activity. Bioorg. Med. Chem. Lett. 1996,
6 (22), 2765–2770.