Pyrimidinone-peptoid hybrid molecules with distinct effects on molecular chaperone function and cell proliferation

Article abstract of DOI:10.1016/j.bmc.2007.12.014

The Hsp70 molecular chaperones are ATPases that play critical roles in the pathogenesis of many human diseases, including breast cancer. Hsp70 ATP hydrolysis is relatively weak but is stimulated by J domain-containing proteins. We identified pyrimidinone-

Full text of DOI:10.1016/j.bmc.2007.12.014

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 3291–3301
Pyrimidinone-peptoid hybrid molecules with distinct effects
on molecular chaperone function and cell proliferation
Christine M. Wright,a Raj J. Chovatiya,a Nora E. Jameson,a David M. Turner,b
Guangyu Zhu,c Stefan Werner,b Donna M. Huryn,b,d James M. Pipas,a
Billy W. Day,c,d Peter Wipfb,c,d and Jeffrey L. Brodskya,*
aDepartment of Biological Sciences, University of Pittsburgh, 274 Crawford Hall, Pittsburgh, PA 15260, USA
bCenter for Chemical Methodologies and Library Development, University of Pittsburgh, Pittsburgh, PA 15260, USA
cDepartment of Chemistry, University of Pittsburgh, Pittsburgh, PA 15260, USA
dDepartment of Pharmaceutical Sciences, University of Pittsburgh, Pittsburgh, PA 15213, USA
Received 16 August 2007; revised 4 December 2007; accepted 6 December 2007
Available online 14 December 2007
Abstract—The Hsp70 molecular chaperones are ATPases that play critical roles in the pathogenesis of many human diseases, includ-
ing breast cancer. Hsp70 ATP hydrolysis is relatively weak but is stimulated by J domain-containing proteins. We identified pyri-
midinone-peptoid hybrid molecules that inhibit cell proliferation with greater potency than previously described Hsp70 modulators.
In many cases, anti-proliferative activity correlated with inhibition of J domain stimulation of Hsp70.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
can stimulate Hsp70 ATP hydrolysis, an activity that
is required for maximal tumorigenesis.10 SV40 is highly
related to at least four human polyomaviruses,11,12 two
of which have been definitively linked to two disease
states—progressive multifocal leukoencephalopathy
(PML) and kidney transplant rejection—in immuno-
compromised patients.13
The heat shock protein Hsp70, and its constitutively
expressed counterpart Hsc70, play essential roles in
many diverse cellular processes, including protein fold-
ing and transport, and the rearrangement of multi-pro-
tein complexes.1–3 Hsp70 contains an N-terminal
ATPase domain and a C-terminal peptide binding
domain.4–6 Hsp70 substrates bind the peptide binding
domain and stimulate the ATPase activity of Hsp70.
However, Hsp70 is a weak ATPase and maximal
Hsp70 activity requires J domain-containing proteins,
such as the Hsp40 class of molecular chaperones.7 The
J domain interacts with the Hsp70 ATPase domain
through a conserved His-Pro-Asp sequence to stimulate
Hsp70 ATP hydrolysis.8,9 One example of a J domain-
containing protein is the large tumor antigen (TAg) of
the polyomavirus, Simian Virus 40 (SV40). SV40 was
a contaminant of the original polio vaccine and trans-
forms rodent cell lines in culture. The TAg J domain
Hsp70 has been reported to be upregulated in various
cancers including breast, lung, colon, and cervix.14–16
However, it is unclear if changes in expression lead to
tumor formation or if these changes are in response to
the tumor environment. In support of the first scenario,
the over-expression of Hsp70 is sufficient to transform
some cell lines, and transgenic mice constitutively
over-expressing Hsp70 develop T-cell lymphomas.17,18
Consistent with these data, reduction of Hsp70 levels
by antisense RNA is sufficient to induce apoptosis
in several breast cancer cell lines, and Hsp70 over-
expression in breast cancer tissues has been correlated
with increased resistance to chemotherapy and poor
prognosis.19–22
Keywords: Hsp70; Hsp40; Chaperone; ATP; SV40; T antigen; Ugi;
Biginelli; Dihydropyrimidinone; Tetrahydropyrimidinone; J domain;
Apoptosis; ATPase; SK-BR-3.
Until recently, 15-deoxysperagulin (DSG) was the only
Hsp70 modulator reported.23 DSG has been used clini-
cally as an immunosuppressant to prevent kidney rejec-
tion after transplantation, and ꢀ70% of kidney
*
Corresponding author. Tel.: +1 412 624 4831; fax: +1 412 624
4759; e-mail: jbrodsky@pitt.edu
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.12.014

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C. M. Wright et al. / Bioorg. Med. Chem. 16 (2008) 3291–3301
transplant patients who were treated with DSG showed
an excellent or good response.24 However, current stud-
ies show that other immunosuppressive regimens have a
lower rate of kidney rejection,25 and therefore DSG is
not widely used clinically. In in vitro experiments DSG
interacts with similar affinities (KD = ꢀ4–5 lM) with
both Hsp70 and Hsp90.26 Moreover, the mechanism
by which DSG affects chaperone function is unclear,
although the compound modestly stimulates Hsp70’s
ATPase activity27 and may bind to the C-terminus of
Hsp70,28 a region involved in co-chaperone interaction.
Based on the above-noted limitations of DSG, there is a
need for more specific Hsp70 modulators with defined
modes of action that can be used as biological probes
and also, potentially, as improved therapeutic agents.
reactions (see Table 1). Substituents on the pyrimidi-
none at R1 are in Class 3, whereas modifications of
R2 and R3 of the peptoid are in Class 4 and 5, respec-
tively. An additional 15 compounds, precursors to the
MAL3-101 analogs, were isolated from the Biginelli
reaction without a subsequent Ugi reaction34 and were
placed in Class 1 and 2 (Table 1; Class 2 compounds
are methyl ester derivatives of distinct compounds in
Class 1). The free diacid of MAL3-101 (Class 6) and a
derivative of a related compound, MAL3-39 (Class 7),
were also synthesized. The specific chemical modifica-
tions of each compound are shown in Table 1 and the
structures of select compounds are shown in Figure 1.
Initially, all compounds were screened for inhibition of
SK-BR-3 breast cancer cell growth in an established cell
proliferation assay.35,36 The concentration of compound
at which cellular growth is inhibited to 50% of the
DMSO control (GI50) was calculated. In this assay,
the GI50 for MAL3-101 was 27 2 lM (Table 2), which
agrees well with the effect of this compound on induc-
tion of the apoptotic pathway in SK-BR-3 cells.33
MAL3-51 (Fig. 1), a structurally related compound with
little effect on chaperone function,29 had a significantly
less pronounced activity (Table 2). Paclitaxel, a small
molecule stabilizer of microtubules that stimulates cellu-
lar apoptosis,37,38 was used as a positive control and
inhibited breast cancer cell proliferation with a GI50 of
6.1 1.2 nM (‘PTX’, Table 2). Notably, a number of
MAL3-101 derivatives inhibited SK-BR-3 cell growth;
GI50s for sixteen compounds ranged from 6.0 0.4 to
42.6 6 lM, with nine compounds yielding GI50 values
<10 lM. We then examined the effects of select com-
pounds with activity in this assay on the growth of
two other cell lines: MCF7 breast cancer cells and
HT29 colon cancer cells. DMT003088 and
DMT003132 (Class 3) and DMT003052 (Class 5) exhib-
ited GI50s in SK-BR-3 cells at a range from 6.2 to
8.8 lM, and similarly inhibited MCF7 and HT29
growth with GI50s ranging from 2.4 to 7.8 lM (Table
3). Of interest, MAL3-101 also inhibited MCF7 growth
but had no effect on HT29 cells. This result suggests that
MAL3-101 exhibits some specificity of action with re-
gard to different cancer cell lines, as previously
proposed.33
To this end, we sought and previously reported on struc-
tural analogs of DSG that affect Hsp70 ATPase activ-
ity.29,30 These efforts led to the identification of
compounds such as MAL3-101 (Fig. 1) that interfere
with TAg stimulation of Hsp70’s ATPase activity.29
Compounds that interfere with J domain stimulation
of Hsp70 may also be anti-tumorigenic since Hsp70
must interact with specific J domain-containing proteins
to perform each of its many functions.31,32 In fact,
MAL3-101 induces apoptosis in the SK-BR-3 breast
cancer cell line with an apparent ED50 of <10 lM.33
Unfortunately, MAL3-101 has a high molecular weight,
low solubility, and high lipophilicity. In contrast, chem-
ical derivatives of MAL3-101 might exhibit improved
physical characteristics while still maintaining or pos-
sessing improved anti-proliferative and chaperone mod-
ulating activities.
We now report on the synthesis and characterization of
new MAL3-101 derivatives. These compounds, and pre-
viously described compounds with similar characteris-
tics,34 were screened for their effects on SK-BR-3
breast cancer cell proliferation, on endogenous Hsp70
ATPase activity, and on TAg (J domain)-stimulated
ATPase activity. We discovered that members of two
derivative classes inhibited breast cancer cell growth in
the low micromolar range, some at lower concentrations
than MAL3-101. In many cases, these effects correlated
with inhibition of TAg-stimulated Hsp70 ATP hydroly-
sis. Our study is the first to relate distinct small molecule
compounds to Hsp70 modulation and reduced breast
cancer cell proliferation.
The majority of the compounds with effects on cell
growth had improved physicochemical attributes rela-
tive to MAL3-101, including reduced molecular weight,
increased solubility, and decreased lipophilicity. For
example, the predicted water solubility (logS) of
MAL3-101 is À11.7 and the predicted lipophilicity
(clogP) is 10.3 (QikProp, version 2.1). By comparison,
for DMT003086 these values improved to À10.9 and
8.0, respectively, and for DMT003132 even greater
improvements to À9.7 and 7.8, respectively, were noted.
2. Results and discussion
Since MAL3-101 induces apoptosis in breast cancer
cells,33 a library of structurally related compounds was
prepared in an effort to identify those with improved
physiochemical, biochemical, and cellular activities. A
total of 27 analogs of MAL3-10134 were synthesized,
analogous to MAL3-101, through successive three-com-
ponent Biginelli and four-component Ugi condensation
reactions to create pyrimidinone-peptoid hybrids
(referred to as dihydropyrimidinones in Ref. 29)
(Fig. 1). These compounds were placed in three distinct
classes depending on the specific reagents utilized in the
Interestingly, inhibition of breast cancer growth was
limited to two classes of MAL3-101 derivatives, Class
3 and 5. Members of these classes are pyrimidinone-pep-
toid hybrid molecules that have variations in either the
aldehyde-derived group of the dihydropyrimidinone or
the amine group of the peptoid strand. The difference

C. M. Wright et al. / Bioorg. Med. Chem. 16 (2008) 3291–3301
3293
Figure 1. Synthetic sequence and representative products.
between molecules in Class 1 and 3 is the presence of the
peptoid component, which suggests that this segment is
essential for inhibiting cell proliferation. Compounds in
Class 1 are acids and probably cross the cell membrane
inefficiently. It is possible that the presence of the
peptoid increases lipophilicity and subsequent entry into
the cell. However, the presence of the peptoid compo-
nent alone is insufficient for anti-proliferative activity,
as compounds in Class 4, which contain a peptoid,
had no effect on breast cancer cell growth (Table 2). In
addition, our results indicate that specific aldehyde- or
amine-derived substituents differentially affect breast
cancer cell proliferation. For example, compounds
in Class 4 had various substituents at R2 in place of
the 5-formyl-2-methoxycarbonylmethoxybenzoic acid
methyl ester present in MAL3-101. Replacement of the

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C. M. Wright et al. / Bioorg. Med. Chem. 16 (2008) 3291–3301
Table 1. Substituents in pyrimidinone-peptoid hybrid molecule
Class
N/A
Name
R1
R2
R3
R4
MAL3-101
p-Biphenyl
n-Hexyl
n-Butyl
CO2Me
O
CO2Me
N
N/A
MAL3-51
Different scaffold
p-Iodophenyl
tert-Butyl
O
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
DMT002272
DMT003034
DMT003036
DMT003038
DMT003116
MAL1-274
o-Biphenyl
c-Hexyl
Different scaffold
Different scaffold
Different scaffold
Different scaffold
Different scaffold
Different scaffold
Different scaffold
Different scaffold
Different scaffold
Different scaffold
Different scaffold
Different scaffold
Different scaffold
Different scaffold
Different scaffold
p-(tert-Butyl)phenyl
c-Propyl
p-(c-Hexyl)phenyl
p-Chlorophenyl
2-Naphthyl
Phenyl
MAL2-06A
MAL2-10A
MAL2-116-17
MAL2-116-20
MAL2-11B
MAL2-13
DMT003042*
DMT003044*
DMT003047*
m-Nitrophenyl
p-Trifluoromethylphenyl
p-Biphenyl
p-Nitrophenyl
p-Biphenyl
Phenyl
c-Propyl
3
3
3
3
3
DMT003082
DMT003084
DMT003086
DMT003088
DMT003090
p-Trifluoromethylphenyl
p-(tert-Butyl)phenyl
m-Nitrophenyl
n-Hexyl
n-Hexyl
n-Hexyl
n-Hexyl
n-Hexyl
n-Butyl
n-Butyl
n-Butyl
n-Butyl
n-Butyl
CO2Me
O
CO2Me
CO2Me
O
CO2Me
CO2Me
O
CO2Me
Phenyl
CO2Me
O
CO2Me
p-Chlorophenyl
CO2Me
O
CO2Me

C. M. Wright et al. / Bioorg. Med. Chem. 16 (2008) 3291–3301
3295
Table 1 (continued)
Class
Name
R1
R2
R3
R4
3
3
3
3
3
DMT003092
p-Nitrophenyl
n-Hexyl
n-Butyl
n-Butyl
n-Butyl
n-Butyl
n-Butyl
CO2Me
O
CO2Me
DMT003094
DMT003096
DMT003100
DMT003132
2-Naphthyl
o-Biphenyl
c-Hexyl
n-Hexyl
n-Hexyl
n-Hexyl
n-Hexyl
CO2Me
O
O
O
O
CO2Me
CO2Me
CO2Me
CO2Me
CO2Me
c-Propyl
CO2Me
CO2Me
3
DMT003134
p-(c-Hexyl)phenyl
n-Hexyl
n-Butyl
CO2Me
O
CO2Me
4
4
4
DMT002218
DMT002220
DMT002222
p-Biphenyl
p-Biphenyl
p-Biphenyl
Phenyl
p-Hydroxyphenyl
p-Biphenyl
n-Hexyl
n-Hexyl
n-Hexyl
n-Butyl
n-Butyl
n-Butyl
4
DMT002260
p-Biphenyl
n-Hexyl
n-Butyl
CO2Me
4
4
4
DMT002264
DMT002264
DMT003058
p-Biphenyl
p-Biphenyl
p-Biphenyl
Methyl
c-Hexyl
2-Pyridyl
n-Hexyl
n-Hexyl
n-Hexyl
n-Butyl
n-Butyl
n-Butyl
4
DMT003112
p-Biphenyl
n-Hexyl
n-Butyl
CO2Me
OH
(continued on next page)

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C. M. Wright et al. / Bioorg. Med. Chem. 16 (2008) 3291–3301
Table 1 (continued)
Class
Name
R1
R2
R3
R4
4
5
5
5
5
5
5
5
6
DMT003114
p-Biphenyl
n-Hexyl
n-Butyl
O
CO2Me
DMT002286
DMT003052
DMT003102
DMT003104
DMT003106
DMT003108
DMT003110
DMT003020
p-Biphenyl
p-Biphenyl
p-Biphenyl
p-Biphenyl
p-Biphenyl
p-Biphenyl
p-Biphenyl
p-Biphenyl
n-Butyl
n-Butyl
n-Butyl
n-Butyl
n-Butyl
n-Butyl
n-Butyl
n-Butyl
CO2Me
O
O
O
O
O
O
O
O
CO2Me
CO2Me
O
CO2Me
Benzyl
CO2Me
CO2Me
2-phenethyl
CO2Me
CO2Me
CO2Me
N
CO2Me
CO2Me
N
CO2Me
NH
CO2Me
CO2Me
n-Hexyl
CO2H
CO2H

C. M. Wright et al. / Bioorg. Med. Chem. 16 (2008) 3291–3301
3297
Table 1 (continued)
Class
Name
R1
R2
R3
R4
N
7
DMT003024
p-Nitrophenyl
p-Biphenyl
n-Butyl
O
N/A
PTX (nM)
Different scaffold
*Denotes a methyl ester.
5-formyl-2-methoxycarbonylmethoxybenzoic
acid
DMT003036 (Table 2). Since the only difference be-
tween members of Class 1 and Class 3, many of which
inhibit SK-BR-3 proliferation, is the presence of the
peptoid component, this suggests that the pyrimidinone
is sufficient for inhibition of J protein stimulation of
Hsp70. In contrast, the peptoid component is required
for anti-proliferative activity, potentially by altering
the lipophilicity and cell permeability of the molecules,
but is insufficient on its own to confer this activity (also
see above). In addition, the aldehyde-derived substituent
attached to the pyrimidinone affects the biochemical and
anti-proliferative activity of the compound. For exam-
ple, the presence of the p-tert-butylphenyl group in
either DMT003036 (Class 1) or DMT003084 (Class 3)
greatly enhanced co-chaperone stimulation of Hsp70
(Fig. 2A–B and Tables 1 and 2), whereas the p-nitro-
phenyl group in both MAL2-13 and DMT003092
(Tables 1 and 2) had little effect. Moreover, the magni-
tude of the anti-proliferative activity was sensitive to
the 4-substituents in the dihydropyrimidinones.
The compounds with phenyl (DMT003088) or p-nitro-
phenyl (DMT003086 and DMT003092) groups at this
position were among the strongest inhibitors of breast
cancer cell proliferation (Table 2). Also, the addition
of aliphatic groups derived from cyclohexylcarboxalde-
hyde (DMT003100) and cyclopropylcarboxaldehyde
(DMT003132) was important for the inhibition of breast
cancer cell proliferation. These results suggest that the
future development of a structure–activity relationship
for Hsp70 modulation and reduced breast cancer cell
proliferation is feasible.
methyl ester aldehyde utilized in the synthesis of
MAL3-101 with any one of 10 distinct aldehydes re-
sulted in compounds devoid of anti-proliferative
activity. In contrast, GI50 values were sensitive to varia-
tions in the aldehyde-derived component of the pyrimid-
inone (Class 3) or the amine (Class 5) of the peptoid.
This suggests that the p-biphenylcarboxaldehyde and
n-hexylamine groups found in MAL3-101 are not criti-
cal for inhibition of breast cancer cell growth. Indeed,
certain modifications of these groups increased anti-pro-
liferative
activity
significantly.
For
example,
DMT003086 (Class 3) inhibited breast cancer prolifera-
tion with a GI50 of 6.0 lM and DMT003106 (Class 5)
had a GI50 of 7.1 lM (Table 1).
Based on the known impact of Hsp70 function on breast
cancer cell survival (see above), each compound was
then examined for its effects on the single turnover ATP-
ase activity of Hsp70. The single turnover assay utilizes
Hsp70 that is pre-bound to ATP, thus ensuring that the
rate-limiting ATP hydrolytic step (KCAT) is monitored
independent of ATP binding and ATP release. For these
assays, we employed the yeast Hsp70, Ssa1p, which can
be purified in high quantities with few contaminants.39
We also examined the effects of the compounds on the
ability of a model J domain-containing protein, the
SV40 TAg, to activate Hsp70’s ATPase activity. TAg
can be purified in large quantities and selective inhibi-
tors of TAg may be useful against polyomavirus infec-
tions. Furthermore, we previously showed that TAg
and endogenous yeast J domain-containing proteins
stimulate Ssa1p to a similar extent.29
It is interesting to note that inhibition of breast cancer
cell proliferation was not always directly related to mod-
ulation of co-chaperone activity. For example,
DMT003088 and DMT003092 were two of the strongest
inhibitors of breast cancer cell growth, but had little ef-
fect on TAg stimulation of Hsp70 (Table 2). There are
several explanations for these data: first, these com-
pounds might differentially affect the properties of dis-
tinct J domain-containing proteins, only one of which
may interact with Hsp70 and promote cell survival. In
fact, MAL3-101 exerts different effects on the abilities
of TAg and the yeast J domain-containing protein,
Ydj1p, to enhance Hsp70 ATPase activity.29 Second,
the compounds might be metabolized within the cell to
generate anti-proliferative compounds. Third, the com-
pound’s anti-proliferative effect might be through an
alternate mechanism. Conversely, some compounds that
had strong effects on chaperone function had little im-
pact on SK-BR-3 proliferation. Here too, several scenar-
ios can be envisaged: First, the compounds may not be
cell permeable. While this may be likely for the acids
As shown in Table 2 and in Figure 2, the chaperone
modulators fell into several groups, and in some but
not all cases their attributes correlated with the chemical
classifications presented in Table 1. Interestingly, many
compounds inhibited J domain stimulation of Hsp70
but had no effect on the endogenous ATPase activity
of Hsp70. For example, in Class 1, DMT003036 had
no effect on Hsp70 ATP hydrolysis, but inhibited
TAg-stimulated ATP hydrolysis by 3.5-fold (Table 2
and Fig. 2A). Some molecules in Class 3 and 5, which
inhibited breast cancer cell growth, had similar bio-
chemical activities: DMT003084, in Class 3, inhibited
TAg stimulation of Hsp70 by 5.2-fold (Table 2 and
Fig. 2B), and DMT003102, in Class 5, inhibited TAg
stimulation by 1.9-fold (Table 2 and Fig. 2D).
Despite being able to inhibit the TAg stimulation of
Hsp70, compounds in Class 1 had no effect on breast
cancer cell proliferation, as exemplified by

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C. M. Wright et al. / Bioorg. Med. Chem. 16 (2008) 3291–3301
Table 2. In vivo and in vitro effects of the pyrimidinone-peptoid
compounds
in Class 1, it is an unlikely explanation for the inactive
pyrimidinone-peptoid hybrids in Class 3 and 5 since
they have similar solubilities and lipophilicities as the ac-
tive compounds (e.g., DMT003086 and DMT003106).
Second, compounds that are cell permeable could be
metabolized once inside the cell. Finally, the TAg/Ssa1p
chaperone interaction may not be the most appropriate
indicator for anti-proliferative effects on SK-BR-3
growth.
Name
SK-BR-3 Fold change Fold change
GI50
Hsp70 ATP TAg-stimulated
hydrolysis
Hsp70 ATP hydrolysis
MAL3-101
MAL3-51
27
>50
2
1.7
1.1
1.0
1.0
1.0
1.1
1.1
1.3
À1.1
1.5
À1.1
1.2
1.1
1.2
2.6
2.3
1.9
1.0
1.2
1.1
1.1
1.3
1.0
1.0
À1.1
1.2
1.2
2.2
3.2
11.8
9.9
3.7
1.6
3.2
4.2
19.1
1.1
6.1
1.5
1.5
12.3
1.0
À2.0
À2.0
À5.0
1.5
DMT002272 >50
DMT003034 >50
DMT003036 >50
DMT003038 >50
DMT003116 >50
À3.5
1.0
Our data also indicate the existence of new classes of
Hsp70 modulators. For example, the pyrimidinone mol-
ecules containing a methyl ester (Class 2) moderately
stimulated Ssa1p ATP hydrolysis, but had a limited ef-
fect on TAg induced ATP hydrolysis in single turnover
ATPase assays (data not shown). Some compounds in
Class 4 represent another new class of chaperone modu-
lators. For example, DMT002220 dramatically stimu-
lated both Ssa1p ATP hydrolysis and TAg stimulated
Ssa1p ATP hydrolysis (Fig. 2C and Table 2). Although
compounds in Class 2 and 4 had no effect on breast can-
cer cell proliferation, they may serve as modifiers for
protein conformational diseases: The Gestwicki lab re-
cently described an Hsp70 agonist which inhibited the
in vitro aggregation of the b amyloid protein linked to
Alzheimer’s disease.40
À2.4
À1.6
À1.4
À1.1
1.1
MAL1-274
MAL2-06A
MAL2-10A
>50
>50
>50
MAL2-116-17 >50
MAL2-116-20 >50
1.1
MAL2-11B
MAL2-13
>50
>50
À3.5
1.1
DMT003042 >50
DMT003044 >50
DMT003046 >50
DMT003082 >50
DMT003084 29
À1.2
À1.7
1.0
À4.8
À5.2
À1.5
À1.1
À5.1
À1.1
À1.1
À4.0
À2.1
À2.6
À1.6
1.1
0
DMT003086 6.0 0.4
DMT003088 6.5 0.7
DMT003090 16
DMT003092 6.9 0.4
6
DMT003094 10
DMT003096 17
1
3
In conclusion, we have identified several classes of
Hsp70 modulators that inhibit cell proliferation. Based
on the fact that Hsp70 exhibits a strong anti-apoptotic
effect at several distinct nodes in the apoptotic path-
way,14–16 we believe that the inhibitors of cell prolifera-
tion act directly on Hsp70 and induce pre-apoptotic
phenomena. This, in turn, slows and ultimately arrests
cell growth. This model is consistent with the fact that
the GI50s for MAL3-101 in SK-BR-3 and MCF7 cells
reported here (14–27 lM) matches well with the ED50
for this compound to induce apoptosis in SK-BR-3
cells33 (ꢀ8 lM). Many of these compounds also have
improved physicochemical characteristics, such as de-
creased molecular weight and lipophilicity and increased
solubility. Continued efforts will seek to improve the po-
tency of these compounds and to examine their effects in
other cellular models for human diseases.
DMT003100 6.3 1.0
DMT003132 6.2 0.4
DMT003134 42
DMT002218 >50
DMT002220 >50
DMT002222 >50
DMT002260 >50
DMT002262 >50
DMT002264 >50
DMT003058 >50
DMT003112 >50
DMT003114 48
DMT002286 39
6
9.2
6.0
À2.0
1.0
1.1
5.5
2.1
1.2
5
6
À1.2
À1.8
À1.9
1.3
DMT003052 8.8 0.3
DMT003102 14
DMT003104 32
2
2
DMT003106 7.1 0.4
À1.1
À1.1
À1.3
À2.2
À5.0
N/A
DMT003108 7.5 0.8 À1.1
DMT003110 >50
DMT003020 >50
DMT003024 >50
10.3
1.1
3. Experimental methods
2.3
PTX (nM)
6.1 1.2 N/A
3.1. General method for the synthesis of compounds in
Class 1–5
The GI50 for each compound (in lM) was assayed as described in
Section 3 as was the fold change for the endogenous and TAg-stimu-
lated Hsp70 ATPase activities.
The synthesis of MAL3-101 was previously reported29
employing successive Biginelli41 and Ugi reactions to
create a pyrimidinone-peptoid hybrid (Fig. 1). In brief,
benzyl acetoacetate and 6-ureidohexanoic acid were
combined with p-biphenylcarboxaldehyde and reacted
in a solution of tetrahydrofuran (THF) and HCl. The
product, MAL2-11B, was combined with n-butylisocya-
nide, 5-formyl-2-methoxycarbonylmethoxybenzoic acid
methyl ester, and n-hexylamine in an Ugi condensation
reaction to create MAL3-101. The MAL3-101 deriva-
tives were synthesized in similar reactions, but one reac-
tant was altered to create an indexed library.34 The
Table 3. Effects of select compounds on the growth of MCF7 breast
cancer cells and HT29 colon cancer cells
MCF7 GI50
HT29 GI50
MAL3-101
MAL3-51
14.7 0.7
>50
>50
>50
DMT003088
DMT003132
DMT003052
2.4 0.8
6.3 0.2
7.8 0.3
4.8 0.3
5.3
3.1 0.4
2
The GI50 for the indicated compounds (in lM) was obtained as
described in Section 3.

C. M. Wright et al. / Bioorg. Med. Chem. 16 (2008) 3291–3301
3299
A
B
100
80
100
80
60
40
60
40
20
0
20
0
0
100
200
300
400
500
600
700
0
100
200
300
400
500
600
700
time (sec)
time (sec)
100
80
100
80
C
D
60
60
40
40
20
0
20
0
0
100
200
300
400
500
600
700
0
100
200
300
400
500
600
700
time (sec)
time (sec)
Figure 2. The MAL3-101 derivatives have distinct effects on chaperone activity. The endogenous and TAg-stimulated ATPase activities of Hsp70 for
prototypical members of (A) Class 1 (e.g., DMT003036), (B) Class 3 (e.g., DMT003084), (C) Class 4 (e.g., DMT002220), and (D) Class 5 (e.g.,
DMT003102) are shown. Filled circles, DMSO; filled squares 0.4 lM TAg and DMSO; open circles, 300 lM of the indicated compound; open
squares, 0.4 lM TAg and 300 lM of the indicated compound.
purities of the compounds were determined using a vari-
ety of methods (evaporative light scattering, total ion
count/MS, and UV spectroscopy) and the data for each
compound are shown in Supplemental Table 1.
3.1.3. Synthesis of pyrimidinone-peptoid hybrids (Class 3–
5). The synthesis of these compounds was previously re-
ported.34 Class 3 compounds utilize the compounds syn-
thesized in Class 1 as the pyrimidinone building block,
which is further reacted in the Ugi multicomponent con-
densation. Compounds in Class 4 and 5 utilize p-biphen-
ylcarboxaldehyde as the aldehyde reagent in the Biginelli
reaction and an assortment of aldehydes (Class 4) or es-
ters (Class 5) in the Ugi reaction.
3.1.1. Synthesis of Class 1 pyrimidinones. Compounds in
this class were synthesized as described previously34 and
contain only the heterocyclic pyrimidinone product of
the Biginelli reaction. The aldehyde component utilized
in each reaction is shown in Table 1.
3.2. Synthesis of the MAL3-101 diacid (Class 6) and
DMT003024 (Class 7)
3.1.2. Synthesis of methyl 6-ureidohexanoate (Class 2). A
solution of 6-ureidohexanoic acid (0.996 g, 5.72 mmol,
1.0 equiv) in methanol/benzene (2:7, 60 mL) was cooled
to 0 ꢁC, and a solution of trimethylsilyldiazomethane in
hexanes (2.0 M, 3.43 mL, 6.86 mmol, 1.2 equiv) was
added. The reaction mixture was warmed to room tem-
perature and stirred for 1 h prior to removing all volatile
components in vacuo. The crude methyl ester (1.06 g,
5.61 mmol, 98%) was used without further purification
in the Biginelli reaction as previously described34
(Scheme 1).
The MAL3-101 diacid was synthesized by reacting
MAL3-101 with NaOH as described previously.34
DMT003024 (benzyl 1-(6-((1-(biphenyl-4-yl)-2-(butyla-
mino)-2-oxoethyl)(2-morpholinoethyl)amino)-6-oxohex-
yl)-6-methyl-4-(4-nitrophenyl)-2-oxo-1,2,3,4-tetrahydro-
pyrimidine-5-carboxylate) was prepared as follows:
4-phenylbenzylaldehyde (1.0 equiv) and 4-(2-amino-
ethyl)morpholine (1.0 equiv) were added at room temper-
ature to a suspension of dihydropyrimidinone (1.0 equiv)
O
O
TMSCHN2,
MeOH/benzene 2/7, r.t.
H
N
H
NH2
N
NH2
HO
MeO
O
O
6-ureidohexanoic acid
methyl 6-ureidohexanoate
Scheme 1.

3300
C. M. Wright et al. / Bioorg. Med. Chem. 16 (2008) 3291–3301
in methanol. After stirring the reaction mixture for
10 min, n-butylisocyanide (1.0 equiv) was added and the
reaction mixture was heated to reflux for 24 h. After cool-
ing to room temperature and concentration in vacuo, the
residue was extracted with EtOAc, washed twice with an
aqueous 10% NaOH solution, once with brine, and dried
(MgSO4) prior to purification by chromatography on
SiO2 on an ISCO Companion chromatography system.
The product was obtained as a 2:1 mixture of diastereo-
mers based on 1H NMR integration of triplets at d 0.88
and 0.94 ppm, respectively. Major diastereomer: 1H
NMR (300 MHz, CDCl3) d 8.06 (d, 2H, J = 8.7 Hz),
7.62–7.25 (m, 14 H), 7.17–7.14 (m, 2H), 6.56 (br s,
0.8H), 5.90 (d, 0.4H, J = 2.3 Hz), 5.44 (br s, 0.9H), 5.13
(d, 1H, J = 12.2 Hz), 5.01 (d, 1H, J = 12.2 Hz), 3.83–
3.16 (m, 10H), 2.54 (s, 3H), 2.44–1.99 (m, 9H), 1.69–
1.25 (m, 10H), 0.88 (t, 2H, J = 7.2 Hz); 13C NMR
(75 MHz, CDCl3) d 173.6, 169.6, 165.3, 153.0, 150.3,
147.3, 141.3, 140.1, 135.5, 134.3, 133.2, 129.8, 129.4,
128.8, 128.4, 128.2, 127.6, 127.3, 127.2, 126.9, 123.8,
102.7, 66.5, 66.2, 53.5, 53.3, 39.3, 33.1, 31.3, 29.6, 26.4,
24.8, 19.9, 16.2, 13.6; MS (ESI) m/z (rel intensity) 859
([M+H]+, 100); HRMS (ESI) m/z calcd for C49H59N6O8
859.4316, found 859.4377. Characteristic signals of minor
diastereomer: 1H NMR (300 MHz, CDCl3) d 8.80 (br s,
0.2H), 6.27 (m, 0.4H), 5.53 (br s, 0.3H), 0.94 (t, 1H,
J = 7.2 Hz); 13C NMR (75 MHz, CDCl3) d 174.3, 170.1,
65.3, 62.4, 57.6, 56.8, 43.7, 39.5, 33.8, 32.1, 26.1, 20.2,
13.7.
for 48 h. One plate of cells was used for a time zero cell
number determination (N = 16), and cells in other plates
were treated for 72 h or 96 h with either DMSO (1% v/v;
N = 8 for each plate) or a range of concentrations, in qua-
druplicate, of test agents. Cell number was determined
spectrophotometrically at 490 nm minus the absorbance
at 630 nm (background absorbance) after exposure to
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-
2-(4-sulfophenyl)-2H-tetrazolium (MTS) and N-methyl-
phenazine methylsulfate. The 50% growth inhibitory con-
centrations (GI50) of test agents were calculated over the
72 h or 96 h period. If the concentration required to ob-
tain a GI50 was higher than 50 lM, this is reported as
>50 in Table 2.
3.5. Hsp70 single turnover ATPase assays
Yeast Hsp70 (Ssa1p) and SV40 TAg were purified as de-
scribed previously39,42 and single turnover ATPase as-
says were performed as published.29 Endogenous
Hsp70 ATPase assays had 300 lM test compound or
DMSO added at 0 s, whereas the TAg stimulation
experiments had the compounds added at 60 s. For each
experiment, the timepoint at which compound was
added was set to zero, results from multiple experiments
were averaged, and kinetic data were obtained using
KaleidaGraph. The results were fit to a single exponen-
tial using the equation: % Product = A · (1 À exp(Àkt)),
where A is the amplitude of the reaction, k is the rate of
ATP hydrolysis, and t is time. The slope of the line for
each compound was compared to the slope of the
DMSO control and is reported as the fold change. All
reported data represent means of two or more indepen-
dent reactions and error bars represent standard devia-
tions of the data.
3.3. Synthesis of N-(3-((tert-butylcarbamoyl-(4-iodophen-
yl)methyl)-(2-morpholin-4-ylethyl)carbamoylpropylben-
zamide (MAL3-51)
According to the general protocol B reported in Ref. 29,
4-iodobenzaldehyde (0.982 g, 4.23 mmol), 4-(2-amino-
ethyl)morpholine (0.547 g, 0.552 mL, 0.423 mmol), 4-
benzoylaminobutyric acid (0.877 g, 0.423 mmol) , and
tert-butylisocyanide (0.349 g, 0.474 lL, 0.423 mmol) in
MeOH (10 mL) afforded MAL3-51 (2.02 g, 3.19 mmol,
75%) as colorless crystals after crystallization from ethyl
acetate/hexanes (1:1): mp 109 ꢁC; IR (film) 3261, 2966,
Acknowledgments
The authors thank Paul Cantalupo for purified TAg,
Susan Gilbert for valuable discussions, and Claire Cole-
man and Raghavan Balachandran for data analysis and
technical assistance. This work was supported by a Part-
nership for Cures and Goldman Philanthropic Partner-
ships Grant to J.L.B. C.M.W. was supported by a
Renal Epithelial Biology training Grant (DK61296)
and a Mellon Graduate Fellowship, and R.J.C. was sup-
ported by a fellowship from the Beckman Foundation.
P.W., D.M.T., S.W., and D.M.H. thank the NIH-NIG-
MS for Grant P50-GM067082 for support of the
CMLD program at the University of Pittsburgh.
1712, 1677, 1552, 1119 cmÀ1 1H NMR (300.1 MHz,
;
CDCl3) d 82 (d, 2H, J = 8.3 Hz), 7.65 (d, 2H,
J = 8.3 Hz), 7.51–7.39 (m, 3H), 7.25 (t, 1H,
J = 4.8 Hz), 7.04 (d, 2H, J = 6.1 Hz), 5.79 (s, 1H), 5.68
(s, 1H), 3.65–3.31 (m, 8H), 2.64–1.81 (m, 10H), 1.31
(s, 9H); 13C NMR (CDCl3) d 180.2, 174.2, 173.1,
141.8, 138.9, 137.9, 134.7, 131.7, 130.1, 95.4, 66.0,
61.8, 56.3, 52.1, 50.1, 42.0, 37.3, 28.0, 25.5, 20.9; MS
(EI) m/z (rel intensity) 634 (M+, 10), 522 (10), 113
(40), 100 (100); HRMS (EI) m/z calculated for
C29H39IN4O4 634.2016, found 634.2022.
Supplementary data
3.4. Cell proliferation assay
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmc.2007.12.014.
The cell proliferation assays were performed as previously
described35,36 using the breast cancer cell lines, SK-BR-3
and MCF-7, and the colon cancer cell line, HT29, where
indicated. In brief, cells were maintained as exponentially
growing cultures in DMEM with 10% FBS, 1% penicillin,
and 1% glutamine. The cells were seeded (4000 cells per
well) into 96-well plates and allowed to attach and grow
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