Polyaminohydroxamic acids and polyaminobenzamides as isoform selective histone deacetylase inhibitors

Article abstract of DOI:10.1021/jm701384x

A series of polyaminohydroxamic acids (PAHAs) and polyaminobenzamides (PABAs) were synthesized and evaluated as isoform-selective histone deacetylase (HDAC) inhibitors. These analogues contain a polyamine chain to increase affinity for chromatin and facil

Full text of DOI:10.1021/jm701384x

J. Med. Chem. 2008, 51, 2447–2456
2447
Polyaminohydroxamic Acids and Polyaminobenzamides as Isoform Selective Histone
Deacetylase Inhibitors§
Sheeba Varghese,† Thulani Senanayake,† Tracey Murray-Stewart,‡ Kim Doering,‡ Alison Fraser,‡ 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
ReceiVed NoVember 5, 2007
A series of polyaminohydroxamic acids (PAHAs) and polyaminobenzamides (PABAs) were synthesized
and evaluated as isoform-selective histone deacetylase (HDAC) inhibitors. These analogues contain a
polyamine chain to increase affinity for chromatin and facilitate cellular import. Seven PAHAs inhibited
HDAC >50% (1 µM), and two PABAs inhibited HDAC >50% (5 µM). Compound 17 increased acetylated
R-tubulin in HCT116 colon tumor cells 253-fold but only modestly increased p21waf1 and acetylated histones
3 and 4, suggesting that 17 selectively inhibits HDAC 6. PABA 22 alone minimally increased p21waf1 and
acetylated histones 3 and 4 but caused dose-dependent increases in p21waf1 in combination with 0.1 µM
5-azadeoxycytidine. Finally, 22 appeared to be a substrate for the polyamine transport system. None of
these compounds were cytotoxic at 100 µM. PAHAs and PABAs exhibit strikingly different cellular effects
from SAHA and have the potential for use in combination antitumor therapies with reduced toxicity.
Introduction
The dynamic status of acetylation on specific histone lysine
residues, mediated by histone acetyltransferases (HATs) and
histone deacetylases (HDACs),a plays a critical role in the
regulation of gene expression.1,2 In some tumor cell types,
hypoacetylation of histones caused by aberrant HDAC activity
results in the underexpression of growth regulatory factors such
as the cyclin dependent kinase inhibitor p21Waf1 (also known
as CDKN1A and CIP1) and thus contributes to the development
of cancer.1,2 Histone hyperacetylation caused by HDAC inhibi-
tors such as trichostatin A (TSA, 1), N-(2-aminophenyl)-4-[N-
(pyridin-3-ylmethoxycarbonyl)aminomethyl]benzamide 2 (MS-
275),3 and SAHA (3) (Figure 1) can cause growth arrest in a
wide range of transformed cells and can inhibit the growth of
human tumor xenografts.1,2,4,5 Both 2 and 3 are effective in the
clinic, especially when used in combination with DNA meth-
yltransferase inhibitors such as 5-aza-2′-deoxycytidine (5-aza-
dC).6 Although they are effective both in vitro and in vivo,
HDAC inhibitors typified by 1-3 inhibit multiple isoforms of
HDAC and produce side effects through activity in noncancer-
ous cells. Thus, it is desirable to identify potent HDAC inhibitors
that restore the expression of normal tumor suppressor genes
without producing significant dose-limiting toxicity.6 Current
structure-activity studies involving analogues of 1-3 have
focused largely on modifications to the aromatic ring moiety
and the aliphatic linker region present in these molecules.5,7
We recently reported a series of polyaminohydroxamic acid
(PAHA) derivatives that incorporate structural features of the
Figure 1. Structures of 1 (trichostatin A), 2, 3 (SAHA), 4 (spermidine),
and 5 (spermine).
polyamines spermidine and spermine (4 and 5, respectively,
Figure 1) and the linker and hydroxamic acid moieties found
in 1 and 3.8 This strategy is based on the observation that
polyamine analogues have high affinities for DNA9–12 and enter
cells using the cellular polyamine transport system.9,13 Thus,
PAHAs could be selectively directed to DNA and the associated
histones. It has been shown that HDAC isoforms have a high
degree of sequence homology in the binding pocket but differ
in primary sequence at residues in the rim region outside the
lysine residue binding site.4 Thus, it may be possible to produce
isoform specific inhibitors for individual HDACs by altering
the polyamine chain composition and its associated terminal
alkyl group, since these portions of the molecule would be
expected to interact with the rim region. Using these design
criteria, we successfully identified three lead compounds from
a library consisting of only 16 analogues.8 We now report 15
additional analogues in the PAHA series (compounds 6-20)
and have extended our studies to include polyaminobenzamides
(PABAs) 21-23, which incorporate the benzamide moiety of
2 and also possess HDAC inhibitory activity in vitro.
§
This manuscript is dedicated to Professor James K. Coward on the
occasion of his retirement.
* To whom correspondence should be addressed. Phone: 313-577-1523.
Fax: 313-577-2033. E-mail: pwoster@wayne.edu.
†
Wayne State University.
Johns Hopkins University.
‡
a Abbreviations: HAT, histone acetyltransferase; HDAC, histone deacety-
lase, SAHA, suberoylanilide hydroxamic acid; PAHA, polyaminohydrox-
amic acid; PABA, polyaminobenzamide; 5-aza-dC, 5-aza-2′-deoxycytidine;
AcTubulin, acetylated R-tubulin; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt.
Chemistry
PAHA analogues 6–20 (Table 1) were synthesized using
routes that were previously described.8 The synthetic routes to
10.1021/jm701384x CCC: $40.75
2008 American Chemical Society
Published on Web 03/19/2008

2448 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8
Varghese et al.
Table 1. Structures of PAHA Analogues 6-20 and Their Inhibitory Activity against HeLa Cell Extract Histone Deacetylase at 1.0 µM
PABA analogues 21-23 are shown in Schemes 1 and 2. Our
initial strategy involved N-Boc protection14 of 4-(amino)meth-
ylbenzoic acid 24, followed by coupling with o-nitroaniline
(EDCI, HOBT, TEA) to form 25.15,16 Compound 25 was then
appended to the protected carboxylate 268 as previously
described8 followed by reduction of the nitro group (SnCl2)3,16
and deprotection (30% HBr in AcOH)8 to afford target benza-
mide 21. Although the EDCI coupling to form 25 proceeded in
65% yield, the reaction resulted in multiple side products that
complicated purification. In addition, in our hands the reduction

Polyaminohydroxamic Acids and Polyaminobenzamides
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8 2449
Scheme 1
protecting groups (30% HBr in AcOH)18,19 then afforded to the
Scheme 2
desired benzamides 22 and 23 (average of 74% yield from 31).
Biological Evaluation
PAHAs 6-20 were evaluated for their ability to inhibit
isolated HDAC activity at 1 µM in the Fluor de Lys assay
system (Biomol International LP, Plymouth Meeting, PA),
employing 1.0 µM TSA (1) as a positive control.8 The results
of these studies are summarized in Table 1. PAHAs 6, 8, 9, 13,
17, 18, and 20 all produced greater than 50% inhibition, with
compound 18 producing the greatest inhibition (88.7%). Under
the same conditions, TSA produced 100% inhibition of the
enzyme. PABA analogues 21-23 were similarly evaluated at
concentrations of 5.0 µM, using 5.0 µM 2 as a positive control.
Compounds 21-23 produced 37.4%, 51.2%, and 66.7% inhibi-
tion, respectively (Figure 2), compared to 51.6% inhibition by
2 at the same concentration. Dose–response experiments (not
shown) revealed that 22 and 23 possess IC50 values of 4.9 and
3.8 µM, respectively, which compares favorably to the IC50
value of 4.8 µM reported for 2.3
of the nitro group produced an unacceptable yield (32%). We
thus adopted the synthetic strategy outlined in Scheme 2.
o-Aminoaniline 27 was mono-N-Boc protected14,16 to form 28
in 80% yield. Commercial 4-(methoxycarbonyl)benzylamine 29
was then coupled to the previously described intermediates 30a
and 30b8,17 to afford compounds 31a and 31b in 95.3% and
92.4% yield, respectively. Hydrolysis of the ester (1.0 N LiOH)
yielded the corresponding carboxylates 32a and 32b, which were
coupled to 288,17 to form 33a and 33b. Removal of the mesityl
Figure 2. Structures of PABA analogues 21-23 and their inhibitory
activity against histone deacetylase at 5.0 µM. Percent inhibition values
reported are the average of three determinations that in all cases differed
by 5% or less.

2450 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8
Varghese et al.
Table 2. Fold Increase in the Re-Expression of p21Waf1, Acetylated
Histones H3 (AcH3) and H4 (AcH4), and Acetylated R-tubulin
(AcTubulin) Proteins in HCT116 Colon Carcinoma Cellsa
compd
p21waf1
AcH3
AcH4
AcTubulin
9
0.41
0.46
3.01
0.86
2.01
1.64
2.56
1.98
2.33
0.61
0.49
2.55
1.13
0
0
0.95
1.36
14.22
4.74
17.4
7.82
12.06
11.68
20.84
3.25
1.95
1.12
1.97
6.76
75.7
11.98
89.01
36.3
10
11
12
13
14
15
16
17
18
20
22
SAHA
3.76
0
3.34
5.19
2.38
0
21.49
0
0
72.9
101.36
253.25
11.99
6.07
2.27
13.24
1.19
30.65
106.57
a Cells were treated for 24 h with a 10 µM concentration of compounds
9-18, 20, 22, and SAHA. Data points are single determinations derived
from infrared detection and quantification of representative Western blots.
Compounds 9-18, 20, and 22 were next evaluated for their
ability to promote in vitro re-expression of the cyclin-dependent
kinase inhibitor p21Waf1, in cultured HCT116 human colon
carcinoma cells, as shown in Table 2. These analogues were
also assayed for their ability to promote increased acetylated
histones H3 and H4 (AcH3, AcH4) and acetylated R-tubulin
(AcTubulin). Following treatment for 24 h, the effects of 9,
10, 12, 14, 18-20 were unremarkable. However, as summarized
in Figure 3, PAHAs 11, 13, 15-17 all promoted a >50-fold
increase in acetylated R-tubulin in the HCT116 cell line (Figure
3A). In the case of 17, AcTubulin levels increased more than
253-fold. By contrast, PABA 22 had no significant effect on
the acetylation status of this protein. Each compound also
produced significant effects on p21waf1 re-expression, and all
were more effective than SAHA (3) in this regard (Figure 3B).
These analogues also produced slight to moderate increases in
the levels of AcH3 and AcH4. Under these conditions, 3 caused
no increase in p21waf1, a modest increase (13.2-fold) in
AcTubulin, and 30.7- and 106-fold increases in AcH3 and
AcH4, respectively.
Figure 3. Fold increase in the re-expression of p21Waf1, acetylated
histones H3 (AcH3) and H4 (AcH4), and acetylated R-tubulin (AcTub)
proteins in HCT116 colon carcinoma cells. Cells were treated for 24 h
with 10 µM compounds 11, 13, 15, 16, 18, 22, and SAHA: (A) p21,
AcH3, AcH4, and AcTub values in the HCT166 cell line; (B)
amplification of p21 data from part A. Data points are single
determinations derived from infrared detection and quantification of
representative Western blots.
Benzamide 22, which exhibited an IC50 of 4.9 µM against
HDAC in the Fluor de Lys assay system, was evaluated in
combination with 0.1 µM of the DNA methyltransferase
inhibitor 5-aza-2′-deoxycytidine (5-aza-dC) for additive or
synergistic effects with respect to re-expression of p21waf1, as
well as p16, a tumor suppressor gene important for cell cycle
regulation (Figure 4). In the HCT116 cell line, no additive
effects were observed for the re-expression of p16, whose levels
appeared to decrease slightly in the presence of either 22 alone
or 22 plus pretreatment with 0.1 µM 5-aza-dC. By contrast,
dose-dependent additive effects were observed in the re-
expression of p21waf1 when treatment with increasing concentra-
tions of 22 was preceded by treatment with 0.1 µM 5-aza-dC.
proteinaceous transporter, the mammalian polyamine transport
system may operate by attracting positively charged substrates,
followed by endocytosis,20 in which case negatively charged
substrates would be less likely to bind to the membrane receptor.
However, the lack of transport of these molecules does not
formally preclude the two-step pathway proposed by Poulin and
colleagues.21 By contrast, benzamide 21 produced a noncom-
petitive inhibition of 14C-spermidine uptake (Ki ) 7.7 µM,
Figure 5), suggesting that it is readily accumulated through the
polyamine transporter.
A
14C-spermidine uptake competition assay20,21 was con-
ducted to determine whether PAHAs or PABAs are substrates
for the polyamine transport system in the ML-1 acute myelog-
enous leukemia cell line. In the case of all PAHA analogues
tested, no competition for the uptake of 14C-spermidine was
observed, suggesting that PAHAs are not taken up into cells
by the polyamine transporter. At physiological pH, the hydrox-
amic acid moiety (pKa ≈ 8.8) is less than 5% ionized. However,
in unrelated experiments, we have observed that polyamine
analogues with terminal, oxygen-containing substituents that can
ionize at physiological pH are poor substrates for the polyamine
transport system (unpublished observations). This is consistent
with the hypothesis that, rather than being a membrane-bound
The cytotoxicity of compounds 6-23 was determined in the
HCT116 colon carcinoma cell line using a standard 3-(4,5-
dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sul-
fophenyl)-2H-tetrazolium (MTS) reduction assay procedure after
96 h of treatment. The results of these studies are summarized
in Table 3. The traditional HDAC inhibitor SAHA (3) was found
to have an IC50 value of 1.8 µM in the HCT116 cell line. By
contrast, IC50 values for compounds 6–19 and 21–23 were all
found to be >100 µM under the same conditions. Compound
20, which inhibited HDAC activity by 58.6% at a concentration
of 1.0 µM, was initially found to have IC50 values of 38.6 and
47.2 µM against the HCT116 line. However, in the presence of

Polyaminohydroxamic Acids and Polyaminobenzamides
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8 2451
in the activity of PABAs 21-23 (Figure 2), although more
analogues will need to be examined to make any structure-
function correlations.
When the cellular effects of PAHAs 6-20 and PABA 22
are examined, distinct differences from the effects of the
traditional HDAC inhibitor SAHA are observed. Since the linker
and metal binding moieties of compounds 6-20 are identical
to or close homologues of SAHA, these differences in specificity
of cellular effects are most likely due to the polyamine portion
of the molecules. The compounds shown in Figure 3 produce
much greater effects on the levels of AcTubulin than SAHA,
3. These compounds possess either a four-carbon (as in 11, 13,
and 15), five-carbon (as in 17), or six-carbon (as in 16) linker
chain, which is comparable to the six-carbon linker chain of 3,
supporting the contention that the observed effect on AcTubulin
is mediated through the polyamine portion of the molecule.
Compound 17, with a five-carbon linker, produced the greatest
increase in the expression of AcTubulin (>253-fold). It is
noteworthy that none of the analogues that increased the levels
of AcTubulin have the so-called “cap” group that is present in
linker chain of 3 and that is thought to bind to the surface
binding area of the HDAC active site. The ability of PAHAs to
increase the levels of AcTubulin does not correlate with their
ability to inhibit HDAC in the commercial assay containing all
of the HDAC isoforms. However, such a correlation may exist
with HDAC 6 activity alone, or the increase in AcTubulin may
be due to interaction of these PAHAs with another site.
Nonetheless, the data suggest that compounds such as 17
selectively inhibit HDAC 6. As was mentioned above, com-
pound 17 produced 51.5% inhibition of HDAC isoforms in a
HeLa cell lysate HDAC at 1 µM. In a preliminary study
involving recombinant HDAC isoforms 3 and 6, it was
determined that 17 produced 99.1% and 99.7% inhibition of
HDACs 3 and 6, respectively. This inhibition profile is consistent
with the high degree of induction of AcTubulin re-expression
by 17. The inhibition of recombinant HDAC 3 and 6 is now
being determined for all of the analogues reported in this
manuscript, and the results of these experiments are forthcoming.
As shown in Table 2 and Figure 3, 10.0 µM PABA 22 caused
a 2.55-fold increase in the expression of p21waf1 in the HCT116
cell line. This compound is structurally similar to 17 except
that it has a six-carbon (rather than five-carbon) linker and has
a benzamide in place of the hydroxamic acid metal binding
moiety. However, unlike 17, PABA 22 did not increase the
expression of AcH3 and AcH4 and only increased AcTubulin
expression by 2-fold. Compound 22 was also evaluated as an
inhibitor of recombinant HDACs 3 and 6, and produced a 1.3%
and 11.7% inhibition of these isoforms, respectively. This
inhibition profile is consistent with the absence of induction of
AcTubulin re-expression by 22. The data shown in Figure 4
demonstrate that 22 reduced the expression of p16 and had only
a moderate effect on p21waf1 levels (1.63-fold at 25 µM). When
22 was combined with the known p16/p21waf1 inducer 5-aza-
dC, no re-expression of p16 was observed. However, the
combination of 22 and 5-aza-dC produced an additive, dose
dependent increase in p21waf1 levels.
Figure 4. Dose–response data for the re-expression of p16 and p21waf1
proteins in HCT116 human colon carcinoma cells following 24 h of
treatment with PABA 22, +/- 5-aza-dC pretreatment. Data points are
single determinations derived from infrared detection-based quantifica-
tion of representative Western blots.
1.0 mM aminoguanidine, a diamine oxidase inhibitor, this effect
was completely abolished. Compound 20 possesses a terminal
primary amine moiety, and thus, the observed cytotoxicity is
likely the result of the production of toxic metabolites by serum
amine oxidases.
Discussion
Clinical studies indicate that HDAC inhibitors such as 2 are
effective therapies for human cancer,22 but dose-limiting toxicity
remains a problem.6,23 HDAC inhibitors partially restore normal
cell cycle and apoptotic functions in human tumor cells. In vitro
models have demonstrated that synergistic re-expression of
genes silenced by promoter hypermethylation can be achieved
by the sequential application of deoxynucleotide methyltrans-
ferase inhibitors and HDAC inhibitors.24 Phase I trials sequenc-
ing 2′-deoxy-5-azacytidine (5-aza-dC) with HDAC inhibitors
in patients with acute myelocytic leukemia demonstrate a
correlation between chromatin remodeling and clinical efficacy,
and robust clinical responses have developed at rates higher
than treatment with either demethylating agents or HDAC
inhibitors alone.24,25 However, step-down dosing of the HDAC
inhibitors in these combinations is often required to minimize
toxicity. Consequently, effective HDAC inhibitors having
minimal inherent toxicity on their own may provide excellent
candidates for combination with agents like 5-aza-dC. Thus,
compounds such as those described in this manuscript could
be used as part of a combination therapy approach with a lower
overall incidence of adverse side effects.
Not surprisingly, in the PAHA series, the length of the linker
chain adjacent to the hydroxamic acid metal binding group
appears to have a dramatic effect on HDAC inhibitory activity
(Table 1). For example, HDAC inhibition increases for com-
pounds 7-9 as the length of the chain increases from five to
eight carbons. Compound 18, the most effective inhibitor in
this series, has a seven-carbon linker chain. However, the
inhibitory activity of 13 cannot be explained on the basis of
linker length, suggesting that other factors can contribute to the
activity of PAHAs. It is noteworthy that the most active
inhibitors, compounds 9, 13, and 18, are significantly more
active that compounds 19 and 20, which contain a traditional
“cap” group that is thought to bind to a surface region in the
HDAC active site.1,2,4 Linker length also appears to play a role
The polyamine transport system, which is known to import
the natural polyamines and a variety of their analogues, is up-
regulated in most tumor cell types.26 As discussed above, none
of the PAHA analogues tested were taken up by the polyamine
transport system. However, PABA 21 was shown to be an
effective noncompetitive inhibitor of the uptake of 14C-
spermidine, suggesting that it is a substrate for the polyamine
transport system. Because the polyamine transport system is

2452 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8
Varghese et al.
Figure 5. Inhibition of 14C-spermidine uptake by compound 21. The concentration-dependent uptake of 14C-spermidine was measured over a
range of concentrations in the presence of 0.0, 10.0, 25.0, and 50.0 µM concentrations of compound 21. Each data point is an average of two
determinations that differed by <1% in all cases.
Table 3. Cytotoxicity Data for PAHA Analogues 6-20 and PABA
Analogues 21-23 in HCT116 Human Colon Cancer Cellsa
analogues described herein are now being evaluated as inhibitors
of individually expressed HDAC isoforms, which are now
available commercially. The results of these studies, as well as
in vivo efficacy studies of 17 and 22 in a mouse xenograft
model, will be reported in a future manuscript.
% HDAC inhibition at
IC50 in wild type
compd
SAHA
1.0 µM
HCT 116 cells (µM)
100
1.8
6
7
8
9
10
11
12
13
14
15
16
17
18
19
55.2
46.3
54.6
73.9
46.7
15.9
41.4
71.3
38.6
6.7
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
ND
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 hydroxide 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. Dimethylformamide was dried by distillation from
anhydrous calcium sulfate and was stored under nitrogen. Prepara-
tive 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 Dowex 1X8-200 anion
exchange resin. Compounds 26, 30a, and 30b (Schemes 1 and 2)
were synthesized as previously described.8
16.5
51.5
88.7
2.0
20
58.6
58.6
28.0
30.8
66.7 (5 µM)
38.6
20 + 1.0 mM AG
>100
>100
>100
>100
21
22
23
a Cells were treated for 96 h with a range of concentrations of each test
compound, and cell viability was measured using a standard MTS reduction
assay. Each data point is the average of two separate experiments that
differed by <5% in every case, with each data point within an experiment
performed in triplicate. AG ) aminoguanidine. ND ) not determined.
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 Nicolet 5DXB FT-IR spectrophotometer and are referenced
highly active in tumor cells, PABAs related to 21 have the
potential to be preferentially imported into tumor cells in high
concentrations.
1
to polystyrene. In all cases, H NMR, 13C NMR, and IR spectra
As shown in Table 3, PAHAs 6-20 and PABAs 21-23
exhibited IC50 values greater than 100 µM in the HCT116 colon
carcinoma cell line. By contrast, 3 is significantly more
cytotoxic, with an IC50 value of 1.8 µM in the HCT116 line.
As was pointed out above, there may be advantages to
administration of an HDAC inhibitor that effectively promotes
the re-expression of aberrantly silenced genes with minimal
inherent toxicity. In tumor therapy, such agents could be used
in place of HDAC inhibitors with dose limiting side effects, in
order to decrease the overall toxicity to normal tissue during
combination chemotherapy. In addition, such agents may prove
useful in disease states such as diabetes, where HDACs play a
role, but cytotoxicity is not an acceptable end point. The
synthesis of additional analogues in the PAHA and PABA series
is an ongoing concern in our laboratories. In addition, the
were consistent with assigned structures. Microanalyses were
performed by Galbraith Laboratories, Knoxville, TN, and were
within 0.4% of calculated values.
Compounds 6-20 were produced using a synthetic scheme that
was previously described.7 Spectral data for each of these analogues
are given below.
20-{N-[(2,2-Diphenyl)propyl]amino}-8-oxo-9,13,17-triazae-
icosanohydroxamic Acid Trihydrobromide (6). 1H NMR (CDCl3)
δ 1.37 (m, 4H), 1.40 (m, 4H), 1.71 (m, 2H), 1.88 (m, 4H), 1.97 (t,
J ) 7.2 Hz, 2H), 2.07 (t, J ) 7.2 Hz, 2H), 2.30 (q, J ) 7.6 Hz,
2H), 2.85 (m, 12H), 3.10 (t, J ) 6.8 Hz, 2H), 3.93 (t, J ) 8 Hz,
1H), 7.11 (m, 2H), 7.21 (m, 8H). Anal. (C32H54Br3N5O3) C, H, N.
16-{N-[(2,2-Diphenyl)propyl]amino}-7-oxo-8,12-diazahexade-
1
canohydroxamic Acid Dihydrobromide (7). H NMR (CDCl3)
δ 1.10 (m, 2H), 1.40 (m, 2H), 1.69 (m, 4H), 1.71 (m, 2H), 1.97 (t,
J ) 6.8 Hz, 2H), 2.06 (m, 2H), 2.31 (m, 2H), 2.84 (m, 8H), 3.09

Polyaminohydroxamic Acids and Polyaminobenzamides
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8 2453
(t, J ) 6.4 Hz, 2H), 3.94 (t, J ) 7.6 Hz, 1H), 7.11 (m, 2H), 7.21
(m, 8H). 13C NMR (400 MHz CDCl3) 13.38, 20.66, 22.79, 22.89,
24.63, 25.02, 25.78, 27.55, 30.88, 35.57, 36.00, 45.19, 46.47, 46.79,
46.96, 48.11, 61.85, 127.15, 127.64, 129.20, 143.68, 174.84, 177.58.
IR (cm-1): 3376.6, 2929.7, 2780.5, 1634.5, 1557.5, 1452.4. Anal.
(C29H46Br2N4O3) C, H, N.
18-{N-[(2,2-Diphenyl)propyl]amino}-9-oxo-10,14-diazaocta-
decanohydroxamic Acid Dihydrobromide (8). 1H NMR (CDCl3)
δ 1.11 (m, 6H), 1.38 (m, 4H), 1.54 (m, 4H), 1.70 (m, 2H), 1.96 (t,
J ) 7.6 Hz, 2H), 2.06 (t, J ) 7.2 Hz, 2H), 2.32 (m, 2H), 2.84 (m,
8H), 3.10 (t, J ) 6.8 Hz, 2H), 3.98 (t, J ) 7.6 Hz, 1H), 7.12 (m,
2H), 7.22 (m, 8H). 13C NMR (CDCl3) δ 13.38, 20.65, 22.78, 22.88,
24.91, 25.33, 25.77, 27.93, 27.99, 30.88, 32.46, 35.77, 35.96, 61.85,
127.15, 127.63, 129.20, 143.68, 174.85, 177.93. IR (cm-1) 3420.8,
2936.6, 2845.4, 2793.5, 1637.5, 1554.5, 1450.8. Anal. (C31H50-
Br2N4O3) C, H, N.
19-N-[(2,2-Diphenyl)propyl]amino-10-oxo-11,15-diazanona-
decanohydroxamic Acid Dihydrobromide (9). 1H NMR (CDCl3)
δ 1.08 (m, 8H), 1.39 (m, 4H), 1.54 (m, 4H), 1.70 (m, 2H), 1.96 (t,
J ) 7.2 Hz, 2H), 2.06 (t, J ) 7.2 Hz, 2H), 2.31 (q, J ) 8.0 Hz,
2H), 2.81 (m, 8H), 3.10 (t, J ) 6.8 Hz, 1H), 7.11 (m, 2H), 7.14–7.24
(m, 8H). 13C NMR (CDCl3) δ 13.38, 20.65, 22.79, 22.88, 24.96,
25.39, 25.77, 27.99, 28.19, 30.87, 32.42, 35.80, 35.96, 38.75, 45.14,
61.85, 71.23, 109.99, 127.15, 127.63, 129.20, 130.12, 143.67,
166.48, 173.72, 174.86, 178.00. Anal. (C32H52Br2N4O3) C, H, N.
17-N-{4-[(N,N-Dimethylamino)benzyl]amino}-8-oxo-9,13-di-
azaheptadecanohydroxamic Acid Dihydrobromide (10). 1H
NMR (CDCl3) δ 1.12 (m, 4H), 1.40 (m, 4H), 1.60 (m, 4H), 1.72
(m, 2H), 1.98 (t, J ) 7.2 Hz, 2H), 2.08 (t, J ) 6.8 Hz, 2H), 2.89
(m, 8H), 3.11 (s, 6H), 3.18 (m, 2H), 7.36 (d, J ) 8.4 Hz, 2H), 7.43
(d, J ) 8.4 Hz, 2H). 13C NMR (CDCl3) δ 13.38, 20.67, 22.83,
22.95, 24.05, 24.88, 25.23, 25.52, 25.77, 27.76, 27.91, 28.09, 31.32,
35.72, 36.01, 61.85, 115.48, 117.49, 120.96, 121.74, 130.02, 131.00,
132.62, 138.98, 141.18, 141.88, 168.77, 177.83. Anal. (C25H47-
Br2N5O3) C, H, N.
15-N-[4-(Isopropyl)benzyl]amino-6-oxo-7,11-diazapentade-
canohydroxamic Acid Dihydrobromide (11). 1H NMR (CDCl3)
δ 1.08 (d, J ) 7.2 Hz, 6H), 1.43 (m, 4H), 1.60 (m, 4H), 1.72 (m,
2H), 2.02 (m, 2H), 2.11 (m,2H), 2.89 (m, 6H), 3.12 (m, 2H), 4.05
(s, 2H), 7.26 (s, 4H). IR (cm-1): 3397.7, 2936.2, 2787.0, 1637.0,
1612.9, 1548.1, 1424.8. Anal. (C24H46Br2N4O3) C, H, N.
17-N-[4-(Isopropyl)benzyl]amino-8-oxo-9,13-diazaheptade-
canohydroxamic Acid Dihydrobromide (12). 1H NMR (D2O) δ
1.08 (d, J ) 7.2 Hz, 6H), 1.14 (m, 4H), 1.24 (m, 4H), 1.61 (m,
4H), 1.75 (m, 2H), 2.01 (t, 2H), 2.12 (t, 2H), 2.89 (m, 6H), 3.12 (t,
2H), 4.12 (s, 2H), 7.27 (s, 4H). IR (cm-1): 3539.4, 3468.1, 3402.4,
2941.3, 2787.0, 1634.5, 1612.9, 1483.2, 1431.3. Anal. (C25H46-
Br2N4O3) C, H, N.
15-N-[2-(Phenyl)benzyl]amino-6-oxo-7,11-diazapentadecano-
hydroxamic Acid Dihydrobromide (13). 1H NMR (CDCl3) δ 1.14
(m, 4H), 1.33 (m, 2H), 1.41 (m, 6H), 1.71 (m, 2H), 1.99 (t, J )
7.2 Hz, 2H), 2.09 (t, J ) 7.6 Hz, 2H), 2.65 (t, J ) 7.6 Hz, 2H),
2.76 (t, J ) 8 Hz, 2H), 3.12 (t, J ) 6.8 Hz, 2H), 4.17 (s, 2H), 7.28
(m, 3H), 7.36–7.45 (m, 6H). IR (cm-1): 3539.4, 3461.6, 3414.9,
2953.9, 2851.8, 1636.3, 1612.9, 1561.0, 1457.3, 1431.3. Anal.
(C26H40Br2N4O3) C, H, N.
17-N-{2-[(Phenyl)thio]ethyl]amino-8-oxo-9,13-diazaheptade-
canohydroxamic Acid Dihydrobromide (16). 1H NMR (CDCl3)
δ 1.15 (m, 4H), 1.41 (m, 4H), 1.57 (m, 4H), 1.72 (m, 2H), 1.99 (t,
J ) 7.6 Hz, 2H), 2.08 (t, J ) 7.2 Hz, 2H), 2.87 (m, 6H), 3.11 (m,
6H), 7.13–7.36 (m, 4H). 13C NMR (CDCl3) δ 24.88, 25.24, 25.78,
27.77, 27.93, 29.39, 35.72, 36.01, 45.20, 46.17, 46.99, 127.80,
129.71, 129.74, 130.60, 130.63. Anal. (C23H42Br2N4O3S) C, H, N.
16-N-[4-(tert-Butyl)benzyl]amino-7-oxo-8,12-diazahexade-
canohydroxamic Acid Dihydrobromide (17). 1H NMR (CDCl3)
δ 1.14 (s, 9H), 1.42 (m, 4H), 1.59 (m, 4H), 1.70 (m, 2H), 1.99 (m,
2H), 2.08 (t, J ) 7.2 Hz, 2H), 2.87 (m, 8H), 3.10 (t, J ) 7.2 Hz,
2H), 4.04 (s, 2H), 7.26 (d, J ) 8.4 Hz, 2H), 7.42 (d, J ) 8.4 Hz,
2H). 13C NMR (CDCl3) δ 30.48, 30.52, 35.51, 126.41, 126.43,
129.88, 129.90, 168.31, and 175.60. Anal. (C25H46Br2N4O3) C,
H, N.
16-N-[4-(tert-Butyl)benzyl]amino-9-oxo-10,14-diazaoctade-
canohydroxamic Acid Dihydrobromide (18). 1H NMR (CDCl3)
δ 1.14 (m, 13H), 1.58 (m, 5H), 1.59 (m, 5H), 1.70 (m, 2H), 1.99
(m, 2H), 2.08 (m, 2H), 2.89 (m, 8H), 3.11 (m, 2H), 4.05 (s, 2H),
7.27 (d, J ) 8.4 Hz, 2H), 7.42 (d, J ) 8.4 Hz, 2H). 13C NMR
(CDCl3) δ 22.80, 24.88, 28.02, 30.50, 35.96, 45.17, 47.05, 126.42,
129.89. Anal. (C27H50Br2N4O3) C, H, N.
15-N-[4-(tert-Butyl)benzyl]amino-7-N-{[4-N,N-(dimethyl)ami-
nobenzyl]amino}-6-oxo-7,11-diazapentadecanohydroxamic Acid
Dihydrobromide (19). 1H NMR (CDCl3) δ 1.27 (s, 9H), 1.33 (m,
8H), 1.62 (m, 6H), 2.22 (m, 4H), 2.30 (m, 6H), 2.57 (m, 12H),
3.01 (m, 4H), 3.19 (m, 4H), 4.07 (s, 2H), 4.68 (d, J ) 5.6 Hz, 2H),
6.87 (m, 2H), 6.95 (d, J ) 5.6 Hz, 4H), 7.25 (m, 4H), 7.40 (d, J )
8.0 Hz, 2H), 7.79 (d, J ) 8.0 Hz, 2H), 7.99 (d, J ) 7.6 Hz, 4H).
Anal. (C33H55Br2N5O3) C, H, N.
17-Amino-9-N-{[4-N,N-(dimethyl)aminobenzyl]amino}-8-oxo-
9,13-diazaheptadecanohydroxamic Acid Dihydrobromide (20).
1H NMR (CDCl3) δ 1.11 (m, 6H), 1.26 (m, 4H), 1.52 (m, 4H),
1.73 (m, 1H), 1.82 (m, 1H), 2.19 (m, 2H), 2.81 m, 6H), 3.01 (s,
6H), 3.22 (m, 2H), 7.22 (m, 2H), 7.39 (m, 2H). 13C NMR (CDCl3)
δ 22.91, 24.02, 25.12, 27.22, 28.29, 32.4, 32.55, 38.89, 46.51,
115.11, 120.99, 121.00, 128.51, 128.67, 130.12, 139.25, 142.11,
179.23. IR (cm-1): 3407.6, 2940.8, 2860.3, 1712.1, 1615.3, 1514.0.
Anal. (C24H45Br2N5O3) C, H, N.
4-{[N-(tert-Butyloxycarbonyl)amino]methyl}benzoic Acid
(25). A mixture of 2.0 g (0.013 mol) of 24 in 100 mL of dioxane/
1.0 N sodium hydroxide (2:1) was cooled in an ice bath with
stirring. To this mixture a 3.16 g (0.0015 mol) portion of di-tert-
butyl dicarbonate was added, and the mixture was allowed to stir
for 12 h at room temperature. The dioxane was removed, and the
pH of the resulting aqueous solution was adjusted to 3.0 using 1.0
N HCl. The aqueous layer was extracted with three 50 mL portions
of ethyl acetate, and the organic layers were combined, washed
with brine, and dried over anhydrous magnesium sulfate. Filtration
and removal of the solvent under reduced pressure afforded the
crude N-Boc protected intermediate, which was purified on a silica
gel column using ethyl acetate/hexane (3:4) followed by ethyl
acetate/hexane (3:1). The pure compound was obtained as fine white
powder (2.12 g) in 63.8% yield. 1H NMR (CDCl3) δ 1.44 (s, 9H),
4.40 (s, 2H) 7.39 (d, J ) 8.4 Hz, 2H), 8.08 (d, J ) 8.0 Hz, 2H).
13C NMR (CDCl3) 28.59, 44.59, 127.45, 128.56, 130.76, 135.37,
171.41.
17-N-[2-(Phenyl)benzyl]amino-8-oxo-9,13-diazaheptadecano-
hydroxamic Acid Dihydrobromide (14). 1H NMR (CDCl3) δ 1.31
(m, 2H), 1.41 (m, 6H), 1.70 (m, 2H), 2.01 (m, 2H), 2.11 (m, 2H),
2.64 (m, 2H), 2.75 (m, 2H), 2.83 (m, 2H), 3.12 (m, 2H), 4.16 (s,
2H), 7.21(m, 1H), 7.42(m, 9H). IR (cm-1): 3552.4, 3474.5, 3414.0,
3228.1, 2955.6, 2806.4, 1637.9, 1612.9, 1554.5, 1450.8, 1431.3.
Anal. (C28H44Br2N4O3) C, H, N.
15-N-{2-[(Phenyl)thio]ethyl]amino-6-oxo-7,11-diazapentade-
canohydroxamic Acid Dihydrobromide (15). 1H NMR (CDCl3)
δ 1.42 (m, 4H), 1.58 (m, 4H), 1.73 (m, 2H), 2.03 (m, 2H), 2.11
(m, 2H), 2.91 (m, 6H), 3.12 (m, 6H), 7.2–7.3 (m, 4H). 13C NMR
(CDCl3) δ 22.73, 22.90, 24.52, 24.78, 25.78, 29.39, 35.44, 36.06,
45.22, 47.01, 127.79, 129.73, 130.13, 130.63, 132.78, 177.18. Anal.
(C21H38Br2N4O3S) C, H, N.
A 0.60 g (0.0024 mol) portion of the N-Boc protected intermedi-
ate above was dissolved in 10.0 mL of dichloromethane and cooled
to 0 °C, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hy-
drochloride (EDCI, 0.503 g, 0.0026 mol) and N-hydroxybenzot-
riazole (HOBt, 0.35 g, 0.0026 mol) were added with stirring. The
mixture was allowed to stir for 15 min at 0 °C, after which time
0.26 mL (0.19 mL, 0.0026 mol) of triethylamine was added. The
reaction mixture was stirred for an additional 15 min at 0 °C, and
then a 0.360 g portion (0.0026 mol) of o-nitroaniline dissolved in
2 mL of dichloromethane was added, followed by stirring for 8 h
at room temperature. The reaction mixture was then concentrated
in vacuo to yield a dark-yellow semisolid. The semisolid was
dissolved in 50 mL of water and extracted with three 50 mL portions
of chloroform, and the combined organic layers were dried over

2454 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8
Varghese et al.
anhydrous magnesium sulfate. Filtration and removal of the solvent
then afforded crude 25, which was purified by column chromatog-
raphy (hexane/ethyl acetate 3:1) to yield pure 25 as yellow crystals
(0.68 g, 76.7%). H NMR (CDCl3) δ 1.47(s, 9H) 4.43 (d, d, J )
6.0 Hz, 2H), 5.56 (s, 1H), 7.48 (m, 5H), 8.06 (d, J ) 8.4 Hz, 1H),
8.19 (d, J ) 8.0 Hz, 2H).
(s, 3H), 2.31 (s, 3H), 2.43 (s, 6H), 2.56 (s, 6H), 2.90 (q, J ) 6 Hz,
2H), 3.05, (m, 2H), 3.15–3.22 (m, 6H), 3.66 (m, 1H), 3.906 (s,
3H), 4.14–4.46 (m, 2H), 5.96 (t, 1H), 6.32 (t, 1H), 6.90 (m, 4H),
7.00 (m, 4H), 7.12–7.21 (m, 6H), 7.29–7.36 (m, 2H), 7.96–8.00
(m, 2H). 13C NMR (CDCl3) δ 21.16, 22.91, 23.08, 24.46, 24.72,
25.67, 28.82, 28.91, 29.91, 32.74, 36.42, 36.59, 36.71, 43.26, 44.09,
45.29, 45.79, 48.86, 52.32, 126.65, 127.63, 127.70, 128.79, 130.10,
130.16, 132.25, 132.31, 133.35, 140.22, 142.64, 142.83, 143.83,
144.13, 167.08, 173.46. IR (cm-1) 3386, 3295, 2969, 2843, 1721,
1652, 1543, 1313, 1147, 913, 730.
1
17-N-[4-(tert-Butyl)benzyl]amino-1-N-[4-(N-(2-aminophenyl)
benzamido)methyl]amino-1,5-dioxo-6,10-diazatetradecana-
mide Dihydrobromide (21). Compound 21 was synthesized from
25 and 26 using methodology described by our laboratory7 and a
previously published benzamide synthesis.14,15 In our hands, these
syntheses provided 21 but in poor overall yield. Thus, the complete
experimental details are not described in this manuscript. An
improved synthesis used to produce 22 and 23 is described below.
1H NMR (D2O) δ (ppm) 1.14 (s, 9H), 1.59–1.90 (comlex m, 10H),
2.99 (m, 10H), 3.53 (s, 1H), 3.61 (s, 1H), 4.04 (s, 2H), 6.81 (m,
2H), 7.23 (m, 4H), 7.41 (m, 2H), 7.66 (m, 4H). Anal.
(C37H54Br2N6O3) C, H, N.
2-[N-(tert-Butyloxycarbonyl)amino]aniline (28). A 1.0 g por-
tion of benzene-1,2-diamine 27 (0.009 mol) was dissolved in 30
mL of chloroform, and the reaction mixture was cooled to 0 °C.
To this mixture was added an aqueous solution of sodium
bicarbonate (0.78 g, 0.0054 mol) and sodium chloride (0.54 g,
0.000 92mol), and the mixture was allowed to stir at 0 °C for 30
min. Di-tert-butyl dicarbonate (2.018, 0.0092 mol) was dissolved
in 20 mL of chloroform, and the solution was slowly added to the
mixture. The mixture was allowed to stir at 0 °C for an additional
10 min, warmed to room temperature, and then refluxed for 12 h.
The mixture was then cooled to room temperature and extracted
with three 30 mL portions of chloroform. The combined organic
layers were washed with 50 mL of saturated sodium bicarbonate
and 50 mL of saturated sodium chloride solution and then dried
over anhydrous magnesium sulfate. The combined organic layers
were then filtered, and the solvent was removed in vacuo to yield
crude 28. The crude compound was purified on a silica gel column
eluted with hexane/ethyl acetate (3:1, then 2:1) to yield compound
28 as an off-white solid (1.55 g, 80.1%). 1H NMR (CDCl3) δ 1.51
(s, 9H), 3.73 (br, 2H), 6.21 (br, 1H), 6.78 (m, 2H), 7.01 (m, 1H),
7.28 (d, J ) 9.2 Hz, 1H). 13C NMR (CDCl3) δ 28.0, 29.9 116,
124, 127, 129.4, 129.9, 158. IR (cm-1) 3435, 1735.
17-{N-[4-(tert-Butyl)benzyl]-N-[2-(mesitylene)sulfonyl]}amino-
13-N-[2-(mesitylene)sulfonyl]-1-N-[4-(carboxy)benzyl]amino-1,8-
dioxo-9,13-diazaheptadecanamide Methyl Ester (31a). A 0.341
g (0.000 42 mol) of 30a was dissolved in 3 mL of dichloromethane,
and to this mixture was added 2 mL of dichloromethane containing
29 (0.08 g, 0.0005 mol), HOBT (0.067 g, 0.0005 mol), and
triethylamine (0.050 g, 0.0007 mol). The reaction mixture was
cooled to 0 °C, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
(EDCI) (0.163 g, 0.000 85 mol) was added in 5 mL of dichlo-
romethane. Following the addition of EDCI, the resulting mixture
was stirred at 0 °C for 10 additional min, and the mixture was
allowed to warm to room temperature and stirred for 12 h. The
resulting dichloromethane solution was washed with 20 mL of 0.5
M HCl, 20 mL of water, and 20 mL of saturated NaHCO3. The
organic layer was dried over anhydrous magnesium sulfate and
filtered, and the dichloromethane was removed under reduced
pressure. The crude product was purified by column chromatog-
raphy (hexane/ethyl acetate 2:1, then 1:2) to yield 31a as a white
solid (0.383 g, 95.3%) 1H NMR (CDCl3) δ 1.27 (m, 4H), 1.32 (s,
9H), 1.37 (m, 4H), 1.53 (m, 4H), 1.70 (m, 2H), 2.19–2.25 (m, 4H),
2.37 (s, 3H), 2.61 (s, 6H), 3.13–3.24 (m, 6H), 3.44 (t, 2H), 3.76 (s,
3H), 4.29 (s, 2H), 4.83 (s, 2H), 7.14–7.37 (m, 8H), 7.74–8.0 (m,
3H), 8.39 (m, 1H). 13C NMR (CDCl3) δ 21.9, 22.8, 25.2, 28.2,
31.9, 37.1, 43.9, 47.7, 48.2, 48.7, 52.5, 53.7, 128.8, 129.7, 133.4,
137.6, 142.9, 144.6, 172.0, 172.7.
17-{N-[4-(tert-Butyl)benzyl]-N-[2-(mesitylene)sulfonyl]}amino-
13-N-[2-(mesitylene)sulfonyl]-1-N-[4-(carboxy)benzyl]amino-1,8-
dioxo-9,13-diazaheptadecanamide (32a). A 0.383 g (0.0004 mol)
portion of 31a was dissolved in 12 mL of tetrahydrofuran:water
(4:2) and cooled to 0 °C, and 6 mL of 1.0 N LiOH was added to
the mixture dropwise. The solution was warmed to room temper-
ature and allowed to stir for 16 h, during which time the reaction
was monitored by TLC. The mixture was again cooled to 0 °C,
neutralized by the dropwise addition of 2.0 N HCl, and extracted
with three 20 mL portions of ethyl acetate. The ethyl acetate layers
were combined, washed with brine, and dried over anhydrous
magnesium sulfate. Removal of the solvent in vacuo yielded
compound 32a as a cloudy yellow oil (0.372 g, 98.4%) that was of
sufficient purity to be used in the next reaction without further
1
purification. H NMR (CDCl3) δ 1.22 (m, 4H), 1.33 (s, 9H), 1.41
(m, 4 h), 1.49 (m, 4H), 1.72 (q, 2H), 2.20 (m, 4H), 2.34 (s, 6H),
2.66 (s, 12H), 3.08 (m, 6H), 3.39 (t, 2H), 4.29 (s, 2H), 4.37 (s,
2H), 7.09 (m, 2H), 7.19 (m, 4H), 7.37 (m, 2H), 7.54 (m, 2H), 7.68
(m, 1H), 7.99 (m, 2H), 8.27 (m, 1H).
17-{N-[3,3-(Diphenyl)propyl]-N-[2-(mesitylene)sulfonyl]}amino-
13-N-[2-(mesitylene)sulfonyl]-1-N-[4-(carboxy)benzyl]amino-1,8-
dioxo-9,13-diazaheptadecanamide (32b). Compound 32b was
made from 31b exactly as described for the synthesis of 32a in
1
98.1% yield. H NMR (CDCl3) δ 1.28–1.33 (m, 10H), 1.56–1.64
(m, 6H), 2.01 (q, J ) 7.6 Hz, 2H), 2.10 (t, J ) 7.6 Hz, 2H), 2.25
(s, 3H), 2.32 (s, 3H), 2.43 (s, 6H), 2.53 (s, 6H), 2.91 (t, J ) 8 Hz,
2H), 3.08 (m, 4H), 3.06–3.16 (m, 4H), 3.72 (t, J ) 7.6 Hz,1H),
4.40 (s, 2H), 6.0 (br, 1H), 6.97 (d, J ) 8.0 Hz, 4H), 7.04 (d, J )
8.0 Hz, 4H), 7.14 (m, 2H), 7.18 (m, 4H), 7.33 (m, 2H), 7.68 (t,
1H), 7.94 (m, 2H). 13C NMR (CDCl3) δ 21.00, 21.16, 22.83, 23.05,
25.43, 25.58, 28.59, 29.91, 32.80, 36.47, 43.37, 43.60, 48.68,
102.29, 126.65, 127.60, 127.76, 128.79, 130.61, 132.29, 132.36,
133.07, 140.25, 142.94, 143.73, 174.07, 174.29, 176.96.
17-{N-[4-(tert-Butyl)benzyl]-N-[2-(mesitylene)sulfonyl]}amino-
13-N-[2-(mesitylene)sulfonyl]-1-N-[4-(N-(2-aminophenyl)benza-
mido)methyl]amino-1,8-dioxo-9,13-diazaheptadecanamide (33a).
Compound 33a was synthesized from 32a and 28 using the
1
procedure described for the synthesis of 31a in 80.7% yield. H
NMR (CDCl3) δ 1.27 (s, 9H), 1.33 (m, 8H), 1.62 (m, 6H), 2.22
(m, 4H), 2.30 (m, 6H) 2.57 (m, 12H), 3.01 (m, 4H), 3.19 (m, 4H),
4.07 (s, 2H), 4.18 (d, J ) 5.6 Hz, 2H), 6.87 (m, 2H), 6.95 (d, J )
5.6 Hz, 4H), 7.25 (m, 4H), 7.40 (J ) 7.8 Hz, 2H), 7.99 (d, J ) 7.6
Hz, 4H).
17-{N-[3,3-(Diphenyl)propyl]-N-[2-(mesitylene)sulfonyl]}amino-
13-N-[2-(mesitylene)sulfonyl]-1-N-[4-(N-(2-aminophenyl)benza-
mido)methyl]amino-1,8-dioxo-9,13-diazaheptadecanamide (33b).
Compound 33b was synthesized from 32b and 28 using the
1
procedure described for the synthesis of 31a in 84.2% yield. H
NMR (CDCl3) δ 1.37 (m, 8H), 1.49–1.53 (m, 11H), 1.64 (m, 4H),
2.04 (m, 6H), 2.21 (s, 3H), 2.31 (s, 3H), 2.43 (s, 6H), 2.54 (s, 6H),
2.90 (t, J ) 8 Hz, 2H), 2.92 (t, J ) 8 Hz, 2H), 3.13–3.20 (m, 6H),
3.64 (t, J ) 7.6 Hz,1H), 4.45 (d, J ) 6 Hz, 2H), 6.05 (broad s,
1H), 2.65 (broad s, 1H), 6.90 (d, J ) 5.6 Hz,2H), 6.98 (d, J ) 5.6
Hz, 4H), 7.14–7.31 (m, 12H), 7.31 (m1H), 7.65 (m, 1H), 7.89 (d,
J ) 8 Hz, 2H), 9.4 (br, 1H). 13C NMR (400 MHz CDCl3) δ 14.14,
21.19, 22.91, 23.08, 24.42, 25.72, 28.52, 28.86, 36.54, 43.19, 44.07,
45.17, 48.84, 125.70, 126.65, 127.63, 127.89, 128.03, 128.79,
132.27, 132.32, 140.21, 140.19, 142.66, 143.12, 143.78, 173.69,
183.70. IR (cm-1) 3439, 3021, 2921, 2843, 1652, 1526, 1313, 1213,
1095, 850.
17-{N-[3,3-(Diphenyl)propyl]-N-[2-(mesitylene)sulfonyl]}amino-
13-N-[2-(mesitylene)sulfonyl]-1-N-[4-(carboxy)benzyl]amino-1,8-
dioxo-9,13-diazaheptadecanamide Methyl Ester (31b). Com-
pound 31b was synthesized from 30b and 29 exactly as described
for the synthesis of 31a in 92.4% yield. 1H NMR (CDCl3) δ
1.25–1.37 (m, 10H), 1.55–1.67 (m, 6H), 1.98–2.08 (m, 4H), 2.20

Polyaminohydroxamic Acids and Polyaminobenzamides
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8 2455
17-N-[4-(tert-Butyl)benzyl]amino-1-N-[4-(N-(2-aminophenyl)
benzamido)methyl]amino-1,8-dioxo-9,13-diazaheptadecana-
mide Dihydrobromide (22). A 4.2 g portion of phenol (0.44 mol)
was dissolved in 20 mL of 30% HBr in acetic acid in a stoppered
flask. Compound 33a (0.200 g, 0.0002 mol) in 15 mL of ethyl
acetate was then added in three portions over a period of 3 h. After
the addition was complete, the reaction mixture was stirred for an
additional 15 h at room temperature, then cooled to 0 °C and diluted
with 30 mL of water. The aqueous phase was washed with two 30
mL portions of ethyl acetate before being lyophylized to give the
crude product as yellow solid. This crude product was washed with
methanol and filtered to yield the tetrahydrobromide salt of 22
(0.099 g, 74.2%) as an off-white solid. An analytical sample of 22
at 14 000 rpm for 15 min. The pellet was resuspended once with
250 µL of ice-cold lysis buffer B (10 mM Tris, pH 7.6, 0.25 M
sucrose, 3 mM CaCl2, and 5 mM butyric acid). Sulfuric acid was
added to a concentration of 0.4 N, and the tubes were incubated at
4 °C for overnight. Debris was pelleted by centrifugation, and the
supernatant was collected. Histones were precipitated by addition
of 10 volumes of acetone and incubated at –20 °C overnight. Pellets
were collected by centrifugation, briefly dried under vacuum, and
resuspended in doubly distilled H2O.
Protein Expression Analysis. Following treatment, HCT116
cells were lysed in buffer M (25 mM HEPES, pH 7.9, 150 mM
NaCl, 0.5 mM EDTA, 0.1% Triton-X, 10% glycerol, 0.1 mg/ml
BSA, 1 mM DTT) containing an EDTA-free protease inhibitor
cocktail, at 4 °C for 20 min. Lysate was clarified by centrifugation
at 14000g for 15 min at 4 °C, and the resulting supernatant was
used for analysis. Total protein concentration was determined using
the BioRad DC assay (Hercules, CA), with absorbance measured
using a spectrophotometer at a wavelength of 750 nm. Absorbance
was converted to protein content using a bovine serum albumin
standard curve. Total cellular proteins (30 µg per lane) were
separated on 10% Bis-Tris NOVEX gels (Invitrogen, Carlsbad, CA),
transferred to Immunoblot PVDF membrane (Bio-Rad), and proteins
of interest were visualized by Western blot analysis using the
following primary antibodies: acetylhistone H3 (06-599) (diluted
1:1000) and acetylhistone H4 (06-866) (diluted 1:1000), from
Upstate Biotechnologies (Billerica, MA); p21Waf1 (556431) (diluted
1:500) from BD Pharmingen (San Jose, CA); p16 (36-5600) (1:
1000) from Zymed (San Francisco, CA); acetylated R-tubulin
(Sigma No. T-6793) (1:2000); and ꢀ-actin (sc-1615) (diluted
1:1500) from Santa Cruz (Santa Cruz, CA). Following washes, blots
were incubated with species-specific, fluorophore-conjugated sec-
ondary antibodies to allow visualization and quantification of
immunoreactive proteins using the Odyssey infrared detection
system and software (LI-COR, Lincoln, NE).
PAHA and PABA uptake by the polyamine transporter was
determined by measuring the ability of each compound to compete
with the uptake of 14C-spermidine, as previously described.29
Cytoproliferative Responses to PAHA and PABA Exposure.
Cell proliferation was quantified using the CellTiter 96 One Solution
MTS assay (Promega, Madison, WI). Cells were seeded in triplicate
in 96-well plates at a density of 3000 cells per well and allowed to
attach overnight. Medium was aspirated and replaced with 100 µL
of fresh medium containing the appropriate concentration of HDAC
inhibitor, PAHA, or PABA. Following incubation at 37 °C, 5%
CO2, 20 µL of CellTiter 96 One Solution was added per well and
incubated 1.5 h at 37 °C and absorbance measured at 490 nm.
1
was prepared by recrystallization from aqueous ethanol. H NMR
(D2O) δ 1.15 (s, 9H), 1.21 (m, 4H), 1.34–1.71 (m, 10H), 1.97 (t,
1H), 2.11 (m, 3H), 2.85 (m, 6H), 3.07 (m, 2H), 3.97 (s, 1H), 4.05
(s, 1H), 4.23 (m, 2H), 7.19 (d, J ) 8 Hz, 2H), 7.24–7.34 (m, 4H),
7.44 (m, 2H), 7.61 (d, J ) 8 Hz, 2H), 7.84 (d, J ) 7.6 Hz, 2H).
Anal. (C40H60Br2N6O3) C, H, N.
17-N-[3,3-(Diphenyl)propyl]amino-1-N-[4-(N-(2-aminophenyl)
benzamido)methyl]amino-1,8-dioxo-9,13-diazaheptadecana-
mide Dihydrobromide (23). Compound 23 was prepared from 33b
exactly as described for the synthesis of 22 in 76.7% yield. An
analytical sample of 23 was prepared by recrystallization from
1
aqueous ethanol. H NMR (D2O) δ 1.12 (m, 4H), 1.20 (m, 4H),
1.53 (m, 4H), 1.69 (m, 2H), 2.01 (t, J ) 7.2 Hz, 2H), 2.14 (m,
2H), 2.32 (m, 2H), 2.70–2.84 (m, 8H), 3.09 (t, J ) 7.2 Hz, 2H),
3.95 (t, J ) 7.2 Hz, 1H), 4.29 (s, 2H), 6.64 (d, J ) 8.8 Hz,1H),
6.83 (d, J ) 8.8 Hz, 1H), 6.80 (m, 1H), 6.98 (m, 1H), 7.13 (m,
2H), 7.26 (m, 6H), 7.34 (m, 4H), 7.77 (d, J ) 8 Hz, 2H). 13C
NMR (D2O) δ 16.92, 20.52, 22.73, 22.83, 25.35, 25.75, 27.95,
28.07, 30.86, 35.71, 35.79, 35.89, 42.79, 45.06, 46.37, 46.72, 46.90,
48.06, 57.58, 113.83, 115.49, 117.48, 121.42, 126.64, 127.12,
127.58, 128.19, 128.65, 129.16, 129.22, 130.13, 130.31, 132.61,
143.58, 144.54, 176.85, 177.44, 177.82. IR (cm-1) 3408, 2934,
2847, 1469, 1378. Anal. (C44H60Br2N6O3) C, H, N.
Histone Deacetylase Activity Assay. Compounds 6-23 were
evaluated for their ability to inhibit isolated HDAC in a com-
mercially available assay (Fluor de Lys assay system, Biomol
International LP, Plymouth Meeting, PA), employing 1 and 2 as
positive controls.8 The reaction mixture contains a HeLa cell nuclear
extract and a commercial substrate containing acetylated lysine side
chains. The substrate and extract are incubated in the presence of
the appropriate concentration of the inhibitor. Deacetylation of the
substrate followed by mixing with the provided developer generates
a fluorophore, and comparison of inhibited vs control relative
fluorescence using a standard plate reader was employed to
determine percent HDAC activity remaining. For studies involving
individually expressed HDACs 3 and 6, the appropriate HDAC
protein was purchased from BPS Bioscience (San Diego, CA), and
a comparable solution of the pure isoform was substituted for the
HeLa cell assay in the Fluor de Lys kit. All determinations were
carried out in triplicate, and reported values are the average of these
determinations, which in no case varied by more than 3%.
Cell Lines and Drug Treatment. HCT116 colon carcinoma cells
were provided by Dr. Bert Vogelstein.27 Cells were maintained in
McCoy’s 5A medium supplemented with 10% fetal calf serum, at
37 °C, 5% CO2. Seeded cells were allowed to adhere overnight,
then treated with 1 (Wako Pure Chemicals, Richmond, VA), 2
(Mitsui Pharmaceuticals, Chiba, Japan), SAHA (BioVision, Moun-
tain View, CA), or the desired test compound for the concentration
and time indicated in the figure captions. For combination studies,
attached cells were incubated with 0.1 µM 5-aza-dC (Sigma) for
24 h, after which medium was removed and replaced with fresh
medium containing the appropriate concentration of PABA 22 for
an additional 24 h.
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