Synthesis and evaluation of N8-acetylspermidine analogues as inhibitors of bacterial acetylpolyamine amidohydrolase

Article abstract of DOI:10.1016/j.bmc.2013.05.045

Polyamines are small essential polycations involved in many biological processes. Enzymes of polyamine metabolism have been extensively studied and are attractive drug targets. Nevertheless, the reversible acetylation of polyamines remains poorly understood. Although eukaryotic N⁸-acetylspermidine deacetylase activity has already been detected and studied, the specific enzyme responsible for this activity has not yet been identified. However, a zinc deacetylase from Mycoplana ramosa, acetylpolyamine amidohydrolase (APAH), has been reported to use various acetylpolyamines as substrates. The recently solved crystal structure of this polyamine deacetylase revealed the formation of an 'L'-shaped active site tunnel at the dimer interface, with ideal dimensions and electrostatic properties for accommodating narrow, flexible, cationic polyamine substrates. Here, we report the design, synthesis, and evaluation of N ⁸-acetylspermidine analogues bearing different zinc binding groups as potential inhibitors of APAH. Most of the synthesized compounds exhibit modest potency, with IC₅₀ values in the mid-micromolar range, but compounds bearing hydroxamate or trifluoromethylketone zinc binding groups exhibit enhanced inhibitory potency in the mid-nanomolar range. These inhibitors will enable future explorations of acetylpolyamine function in both prokaryotes and eukaryotes.

Full text of DOI:10.1016/j.bmc.2013.05.045

Bioorganic & Medicinal Chemistry 21 (2013) 4530–4540
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of N8-acetylspermidine analogues
as inhibitors of bacterial acetylpolyamine amidohydrolase
⇑
Christophe Decroos, Christine M. Bowman, David W. Christianson
Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104–6323, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Polyamines are small essential polycations involved in many biological processes. Enzymes of polyamine
metabolism have been extensively studied and are attractive drug targets. Nevertheless, the reversible
acetylation of polyamines remains poorly understood. Although eukaryotic N8-acetylspermidine deace-
tylase activity has already been detected and studied, the specific enzyme responsible for this activity
has not yet been identified. However, a zinc deacetylase from Mycoplana ramosa, acetylpolyamine ami-
dohydrolase (APAH), has been reported to use various acetylpolyamines as substrates. The recently
solved crystal structure of this polyamine deacetylase revealed the formation of an ‘L’-shaped active site
tunnel at the dimer interface, with ideal dimensions and electrostatic properties for accommodating nar-
row, flexible, cationic polyamine substrates. Here, we report the design, synthesis, and evaluation of N8-
acetylspermidine analogues bearing different zinc binding groups as potential inhibitors of APAH. Most of
the synthesized compounds exhibit modest potency, with IC50 values in the mid-micromolar range, but
compounds bearing hydroxamate or trifluoromethylketone zinc binding groups exhibit enhanced inhib-
itory potency in the mid-nanomolar range. These inhibitors will enable future explorations of acetylpoly-
amine function in both prokaryotes and eukaryotes.
Received 3 April 2013
Revised 14 May 2013
Accepted 18 May 2013
Available online 1 June 2013
Keywords:
Metalloenzyme
Polyamine deacetylase
Enzyme inhibitor
Polyamine analogues
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
of putrescine and spermidine was achieved in vitro by cell treat-
ment with the irreversible ODC inhibitor a-difluoromethylornithine
Polyamines such as putrescine, spermidine, and spermine are
ubiquitous in living organisms and implicated in numerous essen-
tial biological processes.1 For instance, polyamine concentrations
affect the cell cycle progression through tightly regulated biosyn-
thetic pathways.1,2 At the molecular level, since polyamines are
polycations, they can bind to nucleic acids and modulate DNA–pro-
tein interactions.2 Given the importance of polyamines in different
cellular processes, various enzymes of polyamine metabolism have
been studied as potential drug targets.3,4 For example, since upreg-
ulation of polyamine biosynthesis is a hallmark of certain cancers,5,6
inhibitors of ornithine decarboxylase (ODC), S-adenosylmethionine
decarboxylase, spermidine synthase, and spermine synthase have
been evaluated in approaches to cancer chemotherapy. Depletion
(DFMO),7 leading to inhibition of cell growth.8 Although DFMO
failed in clinical trials as a cancer chemotherapeutic agent, it was
approved by the FDA for the treatment of parasitic infections such
as African sleeping sickness.9
While most of the enzymes of polyamine metabolism have been
extensively studied, enzymes involved in the reversible acetylation
of polyamines are less well understood. The acetylation of poly-
amines decreases their overall charge, which is believed to regulate
their function in vivo. Indeed, acetylated polyamines destabilize
nucleosome structure, whereas the corresponding free polyamines
bind to DNA and facilitate condensation.10,11 In eukaryotes, sper-
midine can be either N1- or N8-acetylated by two distinct enzymes:
a cytoplasmic spermidine/spermine N1-acetyltransferase12 or a nu-
clear spermidine N8-acetyltransferase.13 Despite similar structures,
N1- and N8-acetylspermidine are metabolized differently once
formed: N1-acetylspermidine is catabolized to putrescine by a
cytosolic polyamine oxidase,14 and N8-acetylspermidine is hydro-
lyzed to generate spermidine by a specific cytosolic N8-acetylspe-
rmidine deacetylase that is unable to use N1-acetylspermidine as
a substrate.15
Abbreviations: APAH, acetylpolyamine amidohydrolase; dppm, bis(diphenyl-
phosphino)methane; 9-BBN, 9-borabicyclo[3.3.1]nonane; DMP, Dess–Martin peri-
odinane; DFMO,
a-difluoromethylornithine; Boc2O, di-tert-butyl pyrocarbonate;
ESI, electrospray ionization; HRMS, high-resolution mass spectrometry; HDAC,
histone deacetylase; m-CPBA, m-chloroperbenzoic acid; NMO, N-methylmorpholine
N-oxide; CDI, N,N0-carbonyldiimidazole; ODC, ornithine decarboxylase; rt, room
temperature; Boc, tert-butoxycarbonyl; TBAF, tetra-n-butylammonium fluoride;
TMSCF3, trifluoromethyltrimethylsilane; PPh3, triphenylphosphine.
Even though the N8-acetylspermidine deacetylase activity has
been characterized and studied in vivo and in subcellular fractions,
no eukaryotic polyamine deacetylase has been identified to date.
⇑
Corresponding author. Tel.: +1 215 898 5714.
E-mail address: chris@sas.upenn.edu (D.W. Christianson).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.05.045

C. Decroos et al. / Bioorg. Med. Chem. 21 (2013) 4530–4540
4531
H
N
H
N
the molecular details of substrate recognition in the enzyme active
site.18 Key interactions are made by the N4 secondary amino group
of N8-acetylspermidine, which donates a hydrogen bond to E117
H2N
H2N
O
O
A
B
and makes a cation–p interaction with F225; the N1 primary ami-
O
H
N
no group, which donates a hydrogen bond to E106 in the other
monomer of the homodimer; the amide NH group, which donates
a hydrogen bond to the backbone carbonyl of G167; and the amide
carbonyl group, which coordinates to the Zn2+ ion and accepts a
hydrogen bond from Y323. Based on the mechanism of catalysis
by the related HDACs,30–32 a nucleophilic Zn2+-bound water mole-
cule is activated by metal coordination and general base H159
(Fig. 2). Nucleophilic attack at the scissile carbonyl group of the
substrate results in the formation of a tetrahedral intermediate sta-
bilized by metal coordination and hydrogen bond interactions with
surrounding residues. The collapse of this intermediate is enabled
by H159, which serves as a general acid catalyst in this step of the
mechanism,31 leading to the formation of products spermidine and
acetate.25
Based on the structural features important for substrate recog-
nition and catalysis, including the tetrahedral structure of the tran-
sition state flanking the tetrahedral intermediate, we designed and
synthesized potential APAH inhibitors based on the N8-acetylsper-
midine substrate-like scaffold. As shown in Figure 3, compounds
I–X all share a common 1,3-diaminopropane moiety to preserve
key enzyme–substrate hydrogen bond interactions with the poly-
amine N1 and N4 groups (Fig. 2). However, each compound differs
in the nature of its head group designed to mimic substrate or tet-
rahedral transition state binding to the active site Zn2+ ion. Most of
these Zn2+-binding groups have been successfully incorporated
into effective inhibitors of HDACs29,33–39 and other metallohydro-
lases.40–43
NH2
N
H
N
H
C
H2N
N
H
O
D
O
H
N
NH2
N
N
H
H
E
Figure 1. Substrates of APAH from Mycoplana ramosa: (A) acetylputrescine, (B)
acetylcadaverine, (C) N1-acetylspermidine, (D) N8-acetylspermidine, and (E) N1-
acetylspermine.
However, a prokaryotic polyamine deacetylase has been reported:
acetylpolyamine amidohydrolase (APAH) from Mycoplana ramo-
sa.16 Notably, APAH has broader substrate specificity in compari-
son with the mammalian enzyme. As shown in Figure 1, APAH
substrates include both small and large acetylpolyamines such as
acetylputrescine, acetylcadaverine, N1- and N8-acetylspermidine,
and N1-acetylspermine.16,17 Bacterial APAH is a dimeric zinc-
dependent hydrolase16,17 as recently confirmed in crystal structure
determinations of APAH from M. ramosa18 and from B. pseudomal-
lei.19 As previously proposed,20 APAH adopts the
a/b fold first ob-
served for the binuclear manganese metalloenzyme arginase21
and also shared by the histone deacetylases (HDACs).22–24 Key ac-
tive site residues required for the chemical mechanism of deacety-
lation are conserved between APAH and HDACs.25 In contrast with
the HDACs, the dimerization of APAH results in the formation of a
narrow ‘L’-shaped active site at the dimer interface,18 conferring
specificity for slender, flexible substrates rather than the large pep-
tide substrates typically processed by HDACs. This structural fea-
ture now guides the design of specific inhibitors of APAH.
2.2. Chemistry
Syntheses of compounds I–X are summarized in Schemes 1–4.
Compounds I–X were each synthesized from key intermediates 3
or 4. As shown in Scheme 1, 3 and 4 were obtained in two steps.
In the first step, 1,3-diaminopropane was N-alkylated with an
alkylbromide, either 5-bromopent-1-ene or 7-bromohept-1-ene,
to yield alkylamines 1 and 2, respectively. This reaction was per-
formed with an excess of 1,3-diaminopropane (10 equiv, neat) to
favor the monoalkylation of the unprotected diamine over poly-
alkylation. Monoalkylamines 1 and 2 were then quantitatively
N-protected with tert-butoxycarbonyl (Boc) groups using di-tert-
butyl pyrocarbonate (Boc2O).
We developed an alternative route for the synthesis of com-
pounds I, II, and III compared with that previously published.26,27
The new synthetic route allows for more flexibility in generating
additional compounds from intermediate 4. As depicted in
Scheme 2, compounds I, II, and III were synthesized from carbox-
ylic acid 5. Oxidative cleavage of alkene 4 into 5 was performed by
the Sharpless method with ruthenium chloride as catalyst and so-
dium periodate as oxidant44,45 in solvent system H2O/AcOEt/MeCN
(3/2/2).46 N,N0-Carbonyldiimidazole (CDI) mediated coupling of
carboxylic acid 5 with N-O-dimethylamine quantitatively led to
Weinreb amide 6. N-Methoxy-N-methylamides are well-known
Inhibitors of APAH will be helpful tools for the exploration of
both prokaryotic and eukaryotic acetylpolyamine function
in vivo. For instance, inhibitors of the mammalian N8-acetylsper-
midine deacetylase have been described earlier, some of which
exhibit low nanomolar inhibitory activity.26,27 So far, the HDAC
inhibitor M34428 and the trifluoromethylketone analogue of
L-argi-
nine29 are the only compounds reported to inhibit the bacterial
polyamine deacetylase in the low and mid-micromolar range,
respectively.18,29 Here, we report the synthesis of new polyamine
derivatives as well as new synthetic routes for some of the previ-
ously described mammalian N8-acetylspermidine deacetylase
inhibitors.26,27 All compounds synthesized in the current study
are analogues of N8-acetylspermidine bearing different functional
groups targeting Zn2+ coordination. We also report the inhibitory
potency of these compounds against M. ramosa APAH.
2. Results and discussion
2.1. Inhibitor design
The X-ray crystal structure of inactive H159A APAH complexed
with N8-acetylspermidine (PDB accession code 3Q9C) illustrates
R
R
R
+
NH
NH
NH
+
NH3
N
HN
N
OH
NH
OH
NH
OH
+
H
Y323
Y323
Y323
-
H159
NH
H159
NH
H159
NH
transfer
-
O
O
O
O
OH
2+
HO
2+
H
+
+
+
2+
HN
HN
HN
Zn
D284
Zn
D284
Zn
H197
D195
H197
D195
H197
D195
D284
H158
H158
H158
Figure 2. Mechanism of APAH.

4532
C. Decroos et al. / Bioorg. Med. Chem. 21 (2013) 4530–4540
H
N
H2N
H2N
H2N
N
H
I
COOH
H2N
H2N
H2N
H2N
N
H
II
H2N
H2N
H2N
N
H
III
OH
O
O
O
CF3
O
N
H
IV
N
H
V
SAc
N
H
VI
O
O
S
N
H
VII
SAc
N
H
VIII
SH
N
H
IX
O
O
OH
N
H
X
B
OH
Figure 3. Structures of target compounds as potential APAH inhibitors. These compounds are analogues of the substrate N8-acetylspermidine (Fig. 1D).
zoic acid (m-CPBA). Regioselective ring opening of unsymmetrically
a
b
n
n
n
substituted epoxide 12 was achieved using lithium bromide and
acetic acid yielding
-bromohydrin 13.55 Subsequent alcohol oxida-
tion of 13 with DMP led to the formation of
-bromoketone 14.53
Br
H2N
N
H
BocHN
N
Boc
a
n = 3 or 5
1 n = 3 (94%)
2 n = 5 (95%)
3 n = 3 (quant.)
4 n = 5 (quant.)
a
Bromide 14 was then treated with potassium thioacetate to afford
Scheme 1. Synthesis of intermediates 3 and 4. Reagents and conditions: (a) 1,3-
diaminopropane (neat, 10 equiv), 0 °C (1 h) then room temperature (RT) (2 h); (b)
Boc2O (3 equiv) in CH2Cl2, 0 °C then rt (overnight).
thioester 15. Oxirane 12 also served as a precursor for the synthesis
of
a-methoxyketone derivative VI. Ring opening of its epoxide moi-
ety with sodium methoxide in MeOH regioselectively formed the
corresponding
DMP afforded
a
a
-methoxyalcohol 16, after which oxidation with
reagents for the synthesis of ketones from carboxylic acids in the
presence of Grignard or organolithium reagents.47 Using this strat-
egy, methylketone 7 was synthesized from Weinreb amide 6 with
an excess of methylmagnesium bromide (5 equiv). CDI-activated
carboxylic acid 5 was reacted with unprotected hydroxylamine
to form the corresponding hydroxamic acid 8.48 Deprotection of
compounds 5, 7, and 8 with anhydrous HCl (4 N) in dioxane at
room temperature led to target compounds I, II, and III as dihydro-
chloride salts.
-methoxyketone 17.53 Deprotection of compounds
11, 15, and 17 with anhydrous HCl (4 N in dioxane, or 1 N in AcOEt)
at room temperature led to target compounds IV, V, and VI as dihy-
drochloride salts. Synthesis of the a-mercaptoketone from thioester
15 was attempted. Successful thioacetate group alcoholysis in
MeOH under basic conditions (sodium methoxide) gave the corre-
sponding N-Boc protected
ted -mercaptoketone could be isolated after deprotection under
acidic conditions.
a-mercaptoketone, but no pure deprotec-
a
Alkene 4 also served as a key common intermediate for the syn-
thesis of compounds IV, V, and VI as shown in Scheme 3. As for the
synthesis of carboxylic acid 5, introduction of the aldehyde function-
ality was achieved by an oxidative cleavage of alkene 4. This was
done sequentially by first oxidizing 4 to the corresponding cis-diol
with osmium tetroxide as catalyst in the presence of N-methylmor-
pholine N-oxide (NMO) in dioxane/H2O (4/1) as solvent49 until com-
pletion of the reaction (3 h). The cis-diol derived from 4 was not
isolated, but directly cleaved to aldehyde 9 by reaction with sodium
periodate. Nucleophilic trifluoromethylation of aldehyde 9 was
achieved using Ruppert’s reagent (trifluoromethyltrimethylsilane,
TMSCF3)50 and a source of fluoride, tetra-n-butylammonium fluo-
ride (TBAF), as initiator under usual conditions.51,52 Trifluoromethyl
carbinol 10 was then easily oxidized to the corresponding trifluoro-
methyl ketone 11 by Dess–Martin periodinane (DMP).53,54 Alkene 4
was quantitatively oxidized to epoxide 12 using m-chloroperben-
As shown in Scheme 4, alkene 3 was used as precursor for
compounds VII–X. Alkene 3 hydroboration with 9-borabicyclo
[3.3.1]nonane (9-BBN)56 followed by oxidation with H2O2 under ba-
sic conditions (NaOH) quantitatively afforded alcohol 18. Subse-
quent bromination of 18 was achieved using carbon tetrabromide
(CBr4) in the presence of triphenylphosphine (PPh3). Corresponding
alkyl bromide 19 was then treated with potassium thioacetate to
afford 20, as for the synthesis of thioesther 15. Thioacetate group
alcoholysis in MeOH under basic conditions (sodium methoxide)
gave thiol 21. Alkyl bromide 19 also served a precursor for the syn-
thesis of sulfone derivative IX. Nucleophilic substitution of bromine
by sodium thiomethoxide in EtOH afforded thioether 22, after
which oxidation with m-CPBA led to sulfone 23. Hydroboration of
alkene 3 with pinacolborane using [Ir(cod)Cl]2 as catalyst and 1,1-
bis(diphenylphosphino)methane (dppm) as ligand under usual
conditions selectively gave terminal boronic ester 24.57 Complete
5
a
b
BocHN
5
N
COOH
H2N
N
H
COOH
2HCl
4
Boc
5 (98%)
I (93%)
5
c
d
b
N
5
BocHN
N
O
BocHN
N
H2N
N
H
2HCl
Boc
Boc
O
O
O
6 (quant.)
7 (92%)
II (94%)
H
N
H
N
5
e
b
5
BocHN
N
OH
H2N
N
H
OH
2HCl
Boc
O
O
8 (88%)
III (96%)
Scheme 2. Synthesis of compounds I–III. Reagents and conditions: (a) RuCl3 (4 mol %), NaIO4 (4.5 equiv) in H2O/AcOEt/MeCN (3/2/2), rt (3 h); (b) anhydrous HCl (4 N) in
dioxane, rt (2 h); (c) CDI (2 equiv) in CH2Cl2, rt (1 h), then MeNHOMeꢀHCl (2 equiv), rt (overnight); (d) MeMgBr (5 equiv) in THF, 0 °C (2 h) then rt (1 h); (e) CDI (1.5 equiv) in
THF, rt (1 h), then NH2OHꢀHCl (2 equiv), rt (overnight).

C. Decroos et al. / Bioorg. Med. Chem. 21 (2013) 4530–4540
4533
5
5
5
a
b
c
H
CF3
CF3
4
BocHN
N
BocHN
N
BocHN
N
Boc
Boc
Boc
O
OH
O
9 (97%)
10 (82%)
11 (95%)
d
CF3
H2N
N
H
2HCl
g
O
IV (94%)
5
5
5
e
f
4
BocHN
h
N
BocHN
d
N
Br
BocHN
N
Br
O
Boc
Boc
Boc
OH
O
12 (quant.)
13 (91%)
14 (97%)
5
BocHN
5
N
SAc
H2N
N
SAc
2HCl
Boc
H
O
O
15 (97%)
V (97%)
5
i
j
k
12
BocHN
N
O
BocHN
N
O
H2N
N
H
O
2HCl
Boc
Boc
OH
O
O
16 (93%)
17 (91%)
VI (91%)
Scheme 3. Synthesis of compounds IV–VI. Reagents and conditions: (a) NMO (2.5 equiv), OsO4 (2.5 mol %) in dioxane/H2O (4/1), rt (3 h), then NaIO4 (2.5 equiv), rt (20 min);
(b) TMSCF3 (3 equiv), TBAF (10 mol %) in THF, rt (2 h), then TBAF (in THF containing ca. 5% H2O, 1.5 equiv), rt (45 min); (c) DMP (4 equiv) in CH2Cl2, rt (overnight); (d)
anhydrous HCl (4 N) in dioxane, rt (2 h); (e) m-CPBA (2 equiv) in CH2Cl2, 0 °C then rt (21 h); (f) LiBr (3.2 equiv), AcOH (3 equiv) in THF, rt (overnight); (g) DMP (3 equiv), rt
(3 h); (h) KSAc (6 equiv) in MeCN, rt (overnight); (i) NaOMe (6 equiv) in MeOH, rt (24 h); (j) DMP (6 equiv) in CH2Cl2, rt (24 h); (k) anhydrous HCl (1 N) in AcOEt, rt (6 h).
5
5
5
a
b
c
3
BocHN
N
OH
BocHN
N
Br
BocHN
N
SAc
Boc
Boc
Boc
18 (quant.)
19 (96%)
20 (94%)
d
H2N
N
H
SAc
2HCl
VII (94%)
5
SH
e
f
20
BocHN
N
H2N
N
H
SH
2HCl
Boc
21 (98%)
VIII (93%)
5
S
5
g
h
d
19
BocHN
N
BocHN
N
S
H2N
N
S
O O
2HCl
Boc
22 (98%)
Boc
H
O
O
23 (97%)
IX (97%)
5
i
j
OH
O
3
BocHN
N
B
O
H2N
N
B
2HCl
Boc
H
OH
24 (82%)
X (89%)
Scheme 4. Synthesis of compounds VII–X. Reagents and conditions: (a) 9-BBN (2.5 equiv) in THF, 0 °C then rt (20 h), then NaOH, H2O2, 0 °C then rt (30 min); (b) CBr4
(2 equiv), PPh3 (2 equiv) in THF, 0 °C then rt (overnight); (c) KSAc (6 equiv) in MeCN, rt (overnight); (d) anhydrous HCl (4 N) in dioxane, rt (2 h); (e) NaOMe (2 equiv) in MeOH,
rt (2 h); (f) anhydrous HCl (1 N) in AcOEt, rt (5 h); (g) NaSMe (6 equiv) in EtOH, 60 °C (overnight); (h) m-CPBA (3 equiv) in CH2Cl2, 0 °C then rt (2 h); (i) [Ir(cod)Cl]2 (5 mol %),
dppm (10 mol %), pinacolborane (1.9 equiv), rt (24 h); (j) aqueous HCl (6 N), reflux (24 h).
Table 1
deprotection of 24 was achieved in aqueous HCl (6 N) under reflux
Inhibitory potency of compounds I–X against M. ramosa APAH
affording compound X as a dihydrochloride salt. Deprotection of
compounds 20, 21, and 23 with anhydrous HCl (4 N in dioxane, or
1 N in AcOEt) at room temperature led to target compounds VII,
VIII, and IX as dihydrochloride salts.
Compound
Head-group
IC50 (lM)
I
II
III
IV
–COOH
1800 200
160 10
0.39 0.03
0.27 0.03
–COCH3
–CONHOH
–COCF3
2.3. Enzyme inhibition
38
6
V
–COCH2SAc
39 10
All N8-acetylspermidine analogues were tested in vitro for
APAH inhibition. Results are summarized in Table 1. Three com-
pounds exhibit very poor inhibitory potency against APAH: carbox-
ylic acid I, thioester VII, and sulfone IX, with IC50 values in the
millimolar range. In contrast, sulfone and thioester analogues of
SAHA are effective inhibitors of HDAC in the mid- or low micromo-
lar range, respectively,33,58 and carboxylic acid I was previously
shown to be a potent inhibitor of the mammalian N8-acetylspermi-
dine deacetylase in the low micromolar range.27
4000 1000
380 50
1900 200
VI
–COCH2OCH3
–SAc
–SH
–SO2CH3
–B(OH)2
VII
VIII
IX
26
3
10000 3000
230 40
X
der of potency, these compounds are:
a
-methoxyketone VI, boro-
nic acid X, ketone II, thioester V, and thiol VIII. Ketone II is a
modest inhibitor of the bacterial polyamine deacetylase but a
A second set of compounds are modest inhibitors of APAH, with
inhibitory potencies in the mid-micromolar range. In increasing or-

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C. Decroos et al. / Bioorg. Med. Chem. 21 (2013) 4530–4540
mid-nanomolar selective inhibitor of the mammalian N8-acetyl-
spermidine deacetylase.26 The efficacy and specificity of ketone II
have also been demonstrated in vivo, and this compound was used
to probe the function of the mammalian polyamine deacetylase.59
In general, the incorporation of each of these functional groups in
the design of HDAC inhibitors resulted in the generation of highly
potent compounds with inhibitory potency in the nanomolar
range.33,35–39 However, in contrast to our N8-acetylspermidine ana-
logues, HDAC inhibitors are designed based on the combination of
a metal-binding group, a linker, and an active site-capping group.
For a given Zn2+-binding group, optimization of the linker and
the capping group to optimize interactions with the mouth of the
active site cleft is usually required to achieve exceptional inhibi-
tory potency.
3. Conclusions
In summary, we have designed and synthesized a series of N8-
acetylspermidine analogues bearing different functional groups
targeting Zn2+ coordination interactions in the active site of APAH.
Most analogues studied are modest inhibitors, but two—com-
pounds III and IV, bearing hydroxamate and trifluoromethylketone
groups, respectively—exhibit nanomolar inhibitory potency. Future
work on the optimization of these leads may facilitate the develop-
ment of even better APAH inhibitors. Moreover, compounds III and
IV may also be useful tools for probing the function of acetylpoly-
amines in both eukaryotes and prokaryotes, and in searching for
the as-yet unidentified mammalian N8-acetylspermidine
deacetylase.
The best two APAH inhibitors showing potency in the nanomo-
lar range are hydroxamate III and trifluoromethylketone IV
(Table 1). Hydroxamates have been extensively studied as metal-
coordinating groups for the design of metalloenzyme inhibitors,
such as the FDA-approved HDAC inhibitor SAHA for anti-cancer
chemotherapy.60 Hydroxamates form a stable bidentate five-mem-
bered ring complex with the catalytic Zn2+ ion that contributes to
high affinity. Hydroxamate III exhibits an IC50 value of 390 nM
against APAH. This compound is also a potent inhibitor of mamma-
lian N8-acetylspermidine deacetylase activity, with an apparent Ki
of 1 nM measured with subcellular extracts.27
Trifluoromethylketones are well-known to exist as gem-diol
hydrate in aqueous solution. This was further demonstrated for
compound IV with 13C and 19F NMR in D2O. Indeed, a single peak
at ꢁ85.1 ppm for 19F NMR and a quadruplet at 93.6 ppm for 13C
NMR clearly indicate that the trifluoromethylketone group is hy-
drated in water. This gem-diol form mimics the tetrahedral inter-
mediate in zinc metalloenzyme-catalyzed hydrolytic reactions, as
first demonstrated for carboxypeptidase A.61,62 Surprisingly, as
shown in Figure 4, the data obtained for compound IV fit better
with a two-site binding model, whereas the concentration-re-
sponse curve of most of the other compounds were typical of a
one-site binding model as for hydroxamate III. The biphasic curve
obtained for inhibition by trifluoromethylketone IV yields IC50 val-
4. Experimental section
4.1. Chemistry
4.1.1. General procedures
All reagents were of at least 95% purity and purchased from
Fisher Scientific, Alfa Aesar or Sigma Aldrich. All solvents were of
HPLC grade and purchased from Fisher Scientific or Sigma Aldrich.
For reactions requiring anhydrous conditions, solvents (THF,
MeCN, and MeOH) were purchased as anhydrous grade from Fisher
Scientific (except CH2Cl2, which was freshly distilled under N2 from
P2O5). Reactions were monitored by TLC with Sigma Aldrich alumi-
num plates (silica gel with fluorescent indicator, 60 Å, 200 lm) and
visualized by staining with a ninhydrin solution or under UV light
when necessary. Flash column chromatography was performed
using Fisher Scientific silica gel 60 (230–400 mesh). Melting points
were determined using a Mel-Temp Electrothermal apparatus and
were uncorrected. High-resolution mass spectrometry (HRMS) was
performed on a Waters LC-TOF mass spectrometer (model LCT-XE
Premier) using electrospray ionization (ESI) in positive mode. For
compound X, the boronic acid moiety was derivatized by adding
(+)-pinanediol to enable analysis by mass spectrometry.
1H and 13C NMR spectra were recorded on Bruker DMX 360 and
DRX 500 spectrometers operating at 360 and 500 MHz, respec-
tively, for 1H NMR and at 90.6 and 125.6 MHz, respectively, for
13C NMR. 19F NMR spectra were recorded at 282.4 MHz on a Bruker
DMX 360 spectrometer, and 11B NMR spectra at 128 MHz on a Bru-
ker DMX 400 spectrometer. 1H and 13C NMR chemical shifts (d) are
reported in ppm relative to the residual solvent peak. NMR cou-
pling constants (J) are reported in Hz, and multiplicities are de-
noted as follows: s, singlet; br s, broad singlet; d, doublet; t,
triplet; q, quadruplet; and m, multiplet.
ues of 270 nM and 38
compound V, with IC50 values of 39
l
M. Similar behavior is also observed for
M and 4 mM. While such bi-
l
phasic inhibition is also observed in other systems, for example,
the inhibition of smooth muscle endothelin-converting enzyme
by the metalloprotease inhibitor phosphoramidon,63 the reason
for this uncommon inhibition mode against APAH remains unclear.
This type of dose–response curve is usually observed when a ligand
binds to a receptor existing in two different affinity states,64 or to a
receptor or a transporter in two different sites.65 However, the
X-ray crystal structure of the APAH–IV complex reveals the bind-
ing of the gem-diol form of the inhibitor solely in the enzyme ac-
tive site (study in progress), and we have not observed any other
notable features, such as time-dependent inhibition, in our mea-
surements. Thus, it is possible that monomers A and B of APAH ex-
ist in two different affinity states with regard to the binding of IV.
4.1.2. N1-(Pent-4-enyl)propane-1,3-diamine (1)
To 35.2 mL of 1,3-diaminopropane (422 mmol) at 0 °C and un-
der argon was added dropwise 5-bromopent-1-ene (5.0 mL,
42.2 mmol). The solution was stirred at 0 °C one hour, and two
additional hours at room temperature. The reaction mixture was
Figure 4. Inhibition of APAH by: (A) hydroxamate III and (B) trifluoromethylketone IV.

C. Decroos et al. / Bioorg. Med. Chem. 21 (2013) 4530–4540
4535
then partitioned between AcOEt (250 mL), brine (40 mL), saturated
aqueous NaHCO3 (40 mL), and H2O (40 mL). The aqueous layer was
extracted with AcOEt and the combined organic extracts were
dried over Na2SO4, filtered, and concentrated in vacuo. Purification
by flash column chromatography on SiO2 with CHCl3/MeOH/
NH4OH gradients afforded pure alkyl diamine 1 (5.64 g, 94%) as a
slightly yellow oil. 1H NMR (360 MHz, CDCl3) d: 5.81–5.70 (m,
1H), 4.96 (dd, J = 1.8 Hz, J = 17.3 Hz, 1H), 4.90 (dd, J = 1.8 Hz,
J = 10.1 Hz, 1H), 2.72 (t, J = 6.6 Hz, 2H), 2.63 (t, J = 6.8 Hz, 2H),
2.56 (t, J = 7.2 Hz, 2H), 2.06–2.00 (m, 2H), 1.63–1.41 (m, 7H). 13C
NMR (125.6 MHz, CDCl3) d: 138.2, 114.4, 49.3, 47.7, 40.3, 33.5,
31.3, 29.0. HRMS (ESI) calcd for C8H19N2 [M+H]+ 143.1548, found
143.1549.
for 3 h, the reaction mixture was quenched by the addition of iso-
propanol (80 mL), the suspension filtered over a celite pad, and the
filtrate concentrated in vacuo. The aqueous phase was extracted
with AcOEt and the combined organic extracts were washed with
brine, dried over Na2SO4 and concentrated in vacuo. The residue
was further purified by flash column chromatography on SiO2 with
hexanes/AcOEt gradients to afford carboxylic acid 5 as a colorless
oil (4.11 g, 98%). 1H NMR (500 MHz, CDCl3) d: 8.79 (br s, 1H),
5.37 (br s, 1H), 3.21 (t, J = 6.3 Hz, 2H), 3.12 (t, J = 6.6 Hz, 2H), 3.07
(t, J = 6.0 Hz, 2H), 2.32 (t, J = 7.4 Hz, 2H), 1.66–1.60 (m, 4H), 1.54–
1.48 (m, 2H), 1.43 (s, 9H), 1.42 (s, 9H), 1.33–1.27 (m, 2H). 13C
NMR (125.6 MHz, CDCl3) d: 178.6, 156.5 (2C), 79.7, 79.4, 46.9,
44.2, 38.0, 34.0, 28.6 (7C), 28.2, 26.5, 24.5. HRMS (ESI) calcd for
C
19H36N2NaO6 [M+Na]+ 411.2471, found 411.2473.
4.1.3. N1-(Hept-6-enyl)propane-1,3-diamine (2)
Alkylation of 1,3-diaminopropane (27.4 mL, 328 mmol) with 7-
bromohept-1-ene (5 mL, 32.8 mmol) was performed under the
same conditions as for 1, and afforded after purification alkyl dia-
mine 2 as a slightly yellow oil (5.31 g, 95%). 1H NMR (360 MHz,
CDCl3) d: 5.79–5.68 (m, 1H), 4.93 (dd, J = 1.1 Hz, J = 17.3 Hz, 1H),
4.87 (dd, J = 1.1 Hz, J = 10.1 Hz, 1H), 2.70 (t, J = 6.8 Hz, 2H), 2.60
(t, J = 6.8 Hz, 2H), 2.53 (t, J = 6.8 Hz, 2H), 1.99 (m, 2H), 1.57 (m,
2H), 1.45 (m, 2H), 1.36–1.24 (m, 4H), 1.15 (s, 3H). 13C NMR
(90.6 MHz, CDCl3) d: 139.0, 114.3, 50.2, 48.0, 40.7, 34.0, 33.7,
30.1, 28.9, 26.9. HRMS (ESI) calcd for C10H23N2 [M+H]+ 171.1861,
found 171.1853.
4.1.7. N0,N00-Bis(tert-butoxycarbonyl)-6-[(3-
aminopropyl)amino]-N-methoxy-N-methylhexanamide (6)
To a solution of carboxylic acid 5 (2.08 g, 5.35 mmol) in dry
CH2Cl2 (80 mL) under argon was added CDI (1.74 g, 10.7 mmol).
After one hour at room temperature, N,O-dimethylhydroxylamine
hydrochloride (1.04 g, 10.7 mmol) was added to the solution. After
stirring overnight, the reaction mixture was diluted CH2Cl2
(100 mL), washed with 0.1 M HCl (3 ꢂ 50 mL), water, and brine.
The organic layer was dried over Na2SO4, filtered and concentrated
in vacuo. The residue was further purified by flash column chroma-
tography on SiO2 with hexanes/AcOEt gradients to afford Weinreb
amide 6 (2.31 g, quantitative) as a colorless oil. 1H NMR (360 MHz,
CDCl3) d: 5.21 (br s, 1H), 3.65 (s, 3H), 3.22 (t, J = 6.1 Hz, 2H), 3.15 (s,
3H), 3.14–3.03 (m, 4H), 2.39 (t, J = 7.2 Hz, 2H), 1.67–1.58 (m, 4H),
1.56–1.46 (m, 2H), 1.43 (s, 9H), 1.41 (s, 9H), 1.34–1.26 (m, 2H).
13C NMR (90.6 MHz, CDCl3) d: 174.6, 156.1 (2C), 79.6, 79.1, 61.3,
46.9, 44.1, 37.8, 32.3, 31.9, 28.6 (7C), 28.3, 26.8, 24.5. HRMS (ESI)
calcd for C21H42N3O6 [M+H]+ 432.3074, found 432.3091.
4.1.4. N1,N3-Bis(tert-butoxycarbonyl)-N1-(pent-4-enyl)propane-
1,3-diamine (3)
To a solution of alkyl diamine 1 (5.40 g, 38.0 mmol) in dry
CH2Cl2 (150 mL) at 0 °C and under argon was added dropwise
Boc2O (26.2 mL, 114 mmol). The solution was allowed to warm
gradually to room temperature and was stirred overnight. The
reaction mixture was concentrated in vacuo and the resulting res-
idue was purified by flash column chromatography on SiO2 with
hexanes/AcOEt gradients to afford pure 3 as a colorless oil
(12.7 g, quantitative). 1H NMR (500 MHz, CDCl3) d: 5.78–5.70 (m,
1H), 5.10 (br s, 1H), 4.96 (dd, J = 2.0 Hz, J = 17.3 Hz, 1H), 4.91 (dd,
J = 2.0 Hz, J = 10.0 Hz, 1H), 3.19 (t, J = 6.3 Hz, 2H), 3.08 (t,
J = 7.5 Hz, 2H), 3.04 (t, J = 6.5 Hz, 2H), 1.97 (m, 2H), 1.62–1.52 (m,
4H), 1.40 (s, 9H), 1.38 (s, 9H). 13C NMR (125.6 MHz, CDCl3) d:
156.1 (2C), 137.9, 115.0, 79.5, 79.0, 46.5, 44.0, 37.7, 31.0, 28.5
(7C), 27.6. HRMS (ESI) calcd for C18H34N2NaO4 [M+Na]+ 365.2416,
found 365.2410.
4.1.8. N,N0-Bis(tert-butoxycarbonyl)-7-[(3-
aminopropyl)amino]heptan-2-one (7)
To a solution of Weinreb amide 6 (1.36 g, 3.15 mmol) in dry THF
(25 mL) at 0 °C and under argon was added dropwise methylmag-
nesium bromide (1.0 M in THF, 15.8 mL, 15.8 mmol). The solution
was stirred 2 h at 0 °C and then 1 h at room temperature. The reac-
tion mixture was cooled down to 0 °C, quenched by the addition of
saturated aqueous NH4Cl (20 mL), diluted with AcOEt (100 mL),
and the aqueous phase was extracted with AcOEt. Combined or-
ganic extracts were washed with brine, dried over Na2SO4, and
concentrated in vacuo. The residue was further purified by flash
column chromatography with hexanes/AcOEt gradients to afford
ketone 7 as a colorless oil (1.12 g, 92%). 1H NMR (500 MHz, CDCl3)
d: 5.21 (br s, 1H), 3.17 (t, J = 6.5 Hz, 2H), 3.06 (t, J = 6.5 Hz, 2H), 3.03
(t, J = 6.5 Hz, 2H), 2.36 (t, J = 7.5 Hz, 2H), 2.06 (s, 3H), 1.61–1.56 (m,
2H), 1.55–1.49 (m, 2H), 1.48–1.41 (m, 2H), 1.39 (s, 9H), 1.37 (s, 9H),
1.22–1.16 (m, 2H). 13C NMR (125.6 MHz, CDCl3) d: 208.9, 156.1
(2C), 79.5, 79.0, 46.8, 43.9, 43.6, 37.7, 29.9, 28.5 (7C), 28.3, 26.4,
23.5. HRMS (ESI) calcd for C20H38N2O5Na [M+Na]+ 409.2678, found
409.2670.
4.1.5. N1,N3-Bis(tert-butoxycarbonyl)-N1-(hept-6-enyl)propane-
1,3-diamine (4)
Boc-protection of alkyl diamine 2 (5.1 g, 29.9 mmol) with Boc2O
(20.6 mL, 89.7 mmol) was performed under the same conditions as
for 3, and afforded 4 as a colorless oil (11.1 g, quantitative). 1H
NMR (500 MHz, CDCl3) d: 5.63 (m, 1H), 5.26 (br s, 1H), 4.84 (dd,
J = 1.6 Hz, J = 17.2 Hz, 1H), 4.79 (dd, J = 1.6 Hz, J = 10.1 Hz, 1H),
3.11 (t, J = 6.0 Hz, 2H), 3.00 (t, J = 6.0 Hz, 2H), 2.95 (t, J = 6.3 Hz,
2H), 1.91 (apparent q (dt), J = 7.2 Hz, 2H), 1.55–1.50 (m, 2H),
1.42–1.33 (m, 2H), 1.32 (s, 9H), 1.30 (s, 9H), 1.29–1.24 (m, 2H),
1.19–1.11 (m, 2H). 13C NMR (125.6 MHz, CDCl3) d: 155.9 (2C),
138.5, 114.3, 79.2, 78.6, 46.8, 43.7, 37.6, 33.5, 28.4, 28.8 (7C),
4.1.9. N0,N00-Bis(tert-butoxycarbonyl)-6-[(3-
aminopropyl)amino]-N-hydroxyhexanamide (8)
28.2, 26.2. HRMS (ESI) calcd for
393.2729, found 393.2727.
C
20H38N2NaO4 [M+Na]+
To a solution of carboxylic acid 5 (600 mg, 1.54 mmol) in dry
THF (20 mL) under argon was added CDI (376 mg, 2.32 mmol).
After 1 h at room temperature, hydroxylamine hydrochloride
(215 mg, 3.09 mmol) was added to the solution. After stirring over-
night, the reaction mixture was diluted with a 5% KHSO4 aqueous
solution (60 mL) and extracted with AcOEt. Combined organic ex-
tracts were washed with brine, dried over Na2SO4, filtered and con-
centrated in vacuo. The residue was further purified by flash
column chromatography on SiO2 with AcOEt/MeOH gradients to
4.1.6. N,N0-Bis(tert-butoxycarbonyl)-6-[(3-
aminopropyl)amino]hexanoic acid (5)
To a solution of alkene 4 (4.00 g, 10.8 mmol) in 200 mL of a H2O/
AcOEt/MeCN (3/2/2) mixture were added successively ruthe-
nium(III) chloride hydrate (35%, 256 mg, 432
lmol) and sodium
periodate (10.4 g, 48.6 mmol). After stirring at room temperature

4536
C. Decroos et al. / Bioorg. Med. Chem. 21 (2013) 4530–4540
afford hydroxamic acid 8 (550 mg, 88%) as a slightly yellow oil. 1
H
washed with brine, dried over Na2SO4, and concentrated in vacuo.
The residue was purified by flash column chromatography on SiO2
with hexanes/AcOEt gradients to afford trifluoromethylketone 11
as a slightly yellow oil (1.32 g, 95%). 1H NMR (500 MHz, CDCl3) (ke-
tone/hydrate : 1/0) d: 5.26 (br s, 1H), 3.11 (t, J = 6.7 Hz, 2H), 3.03 (t,
J = 6.5 Hz, 2H), 2.96 (t, J = 6.0 Hz, 2H), 2.60 (t, J = 7.1 Hz, 2H), 1.60–
1.53 (m, 4H), 1.45–1.39 (m, 2H), 1.33 (s, 9H), 1.30 (s, 9H), 1.23–1.17
(m, 2H). 13C NMR (500 MHz, CDCl3) d: 191.2 (q, J = 35.2 Hz), 156.1
(2C), 115.5 (q, J = 291.4 Hz), 79.5, 78.8, 46.6, 43.4, 37.6, 36.1, 28.3
(7C), 28.1, 25.9, 22.0. 19F NMR (338.8 MHz) d: ꢁ79.4. HRMS (ESI)
calcd for C20H35N2O5F3Na [M+Na]+ 463.2396, found 463.2397.
NMR (500 MHz, DMSO-d6) d: 10.29 (br s, 1H), 8.61 (br s, 1H), 6.72
(br s, 1H), 3.08 (apparent q (2t), J = 7.5 Hz, 4H), 2.88 (apparent q
(dt), J = 6.5 Hz, 2H), 1.93 (t, J = 7.4 Hz, 2H), 1.58–1.54 (m, 2H),
1.52–1.46 (m, 2H), 1.44–1.39 (m, 2H), 1.38 (s, 9H), 1.37 (s, 9H),
1.21–1.15 (m, 2H). 13C NMR (125.6 MHz, DMSO-d6) d: 169.0,
155.6, 154.6, 78.2, 77.4, 46.3, 44.3, 37.6, 32.2, 28.2, 28.1 (6C),
27.6, 25.9, 24.9. HRMS (ESI) calcd for C19H37N3O6Na [M+Na]+
426.2580, found 426.2576.
4.1.10. N,N0-Bis(tert-butoxycarbonyl)-6-[(3-
aminopropyl)amino]hexanal (9)
To a solution of alkene 4 (2.71 g, 7.31 mmol) in 150 mL of a
dioxane/H2O (4/1) mixture were successively added NMO mono-
hydrate (2.47 g, 18.3 mmol) dissolved in 6 mL of water, and os-
4.1.13. N1,N3-Bis(tert-butoxycarbonyl)-N1-[5-(oxiran-2-
yl)pentyl]propane-1,3-diamine (12)
To a solution of alkene 4 (2.89 g, 7.80 mmol) in dry CH2Cl2
(150 mL) under argon at 0 °C was added dropwise m-CPBA (77%,
3.50 g, 15.6 mmol) in 40 mL dry CH2Cl2. The solution was allowed
to reach room temperature and stirred until completion of the
reaction (21 h) as shown by TLC. Saturated aqueous NaHCO3
(50 mL) was added to the reaction mixture and the aqueous layer
was extracted with AcOEt. Combined organic extracts were dried
over Na2SO4, filtered and concentrated in vacuo. The residue was
purified by flash column chromatography on SiO2 with hexanes/
AcOEt gradients to afford epoxide 12 (3.01 g, quantitative) as a col-
orless oil. 1H NMR (360 MHz, CDCl3) d: 5.28 (br s, 1H), 3.05 (t,
J = 7.2 Hz, 2H), 2.97–2.88 (m, 4H), 2.71–2.67 (m, 1H), 2.54–2.52
(m, 1H), 2.26–2.24 (m, 1H), 1.51–1.46 (m, 2H), 1.38–1.26 (m,
6H), 1.27 (s, 9H), 1.24 (s, 9H), 1.18–1.14 (m, 2H) . 13C NMR
(90.6 MHz, CDCl3) d: 155.8 (2C), 79.0, 78.4, 51.8, 46.6, 46.5, 43.6,
37.3, 32.1, 28.3, 28.2 (7C), 26.4, 25.5. HRMS (ESI) calcd for
mium tetroxide (47 mg, 185 lmol). TLC monitoring showed
completion after 3 h at room temperature. Sodium periodate
(3.91 g, 18.3 mmol) was added to the solution. After stirring for
20 min, the reaction mixture was quenched with isopropanol
(50 mL), the suspension filtered over a celite pad, and the filtrate
concentrated in vacuo. The aqueous phase was extracted with
AcOEt and the combined organic extracts were washed with brine,
dried over Na2SO4 and concentrated in vacuo. The residue was fur-
ther purified by flash column chromatography on SiO2 with hex-
anes/AcOEt gradients to afford aldehyde 9 as a colorless oil
(2.64 g, 97%). 1H NMR (500 MHz, CDCl3) d: 9.63 (s, 1H), 5.25 (br
s, 1H), 3.11 (t, J = 7.0 Hz, 2H), 3.02 (t, J = 6.7 Hz, 2H), 2.96 (t,
J = 7.4 Hz, 2H), 2.31 (t, J = 6.9 Hz, 2H), 1.55–1.49 (m, 4H), 1.42–
1.37 (m, 2H), 1.32 (s, 9H), 1.30 (s, 9H), 1.21–1.15 (m, 2H). 13C
NMR (125.6 MHz, CDCl3) d: 202.1, 155.9 (2C), 79.3, 78.7, 46.6,
43.8, 43.6, 37.5, 28.3 (7C), 27.8, 26.2, 21.6. HRMS (ESI) calcd for
C
20H39N2O5 [M+H]+ 387.2859, found 387.2854.
C
19H36N2O5Na [M+Na]+ 395.2522, found 395.2509.
4.1.14. N,N’-Bis(tert-butoxycarbonyl)-7-[(3-
aminopropyl)amino]-1-bromoheptan-2-ol (13)
4.1.11. N,N0-Bis(tert-butoxycarbonyl)-7-[(3-
aminopropyl)amino]-1,1,1-trifluoroheptan-2-ol (10)
To a solution of aldehyde 9 (1.53 g, 4.11 mmol) in dry THF
(20 mL) at room temperature and under argon were added succes-
sively TMSCF3 (1.82 mL, 12.3 mmol) and anhydrous TBAF (1.0 M in
To a solution of epoxide 12 (2.10 g, 5.43 mmol) in dry THF
(25 mL) under argon were added successively lithium bromide
(1.51 g, 17.4 mmol) and glacial acetic acid (930 lL, 16.2 mmol)
dropwise. After stirring at room temperature overnight, saturated
aqueous NaHCO3 (30 mL) was added to the reaction mixture. The
aqueous layer was extracted with AcOEt and the combined organic
extracts were dried over Na2SO4, filtered and concentrated in va-
cuo. Purification by flash column chromatography on SiO2 with
THF, 410 lL, 410 lmol). After 2 h at room temperature, TBAF
(1.0 M in THF containing ca. 5% H2O, 6.2 mL, 6.20 mmol) was added
dropwise and the solution was stirred for 45 min and concentrated
in vacuo. The corresponding residue was partitioned between
AcOEt (100 mL) and saturated aqueous NaHCO3 (50 mL) and the
aqueous phase was extracted with AcOEt. Combined organic ex-
tracts were washed with brine, dried over Na2SO4, and concen-
trated in vacuo. Purification by flash column chromatography on
SiO2 with hexanes/AcOEt gradients afforded trifluoromethylalco-
hol 10 as a colorless oil (1.49 g, 82%). 1H NMR (360 MHz, CDCl3)
d: 5.32 (br s, 1H), 4.46 (br s, 1H), 3.79–3.73 (m, 1H), 3.11 (t,
J = 6.5 Hz, 2H), 3.03 (t, J = 6.5 Hz, 2H), 2.97 (t, J = 6.5 Hz, 2H),
1.58–1.48 (m, 6H), 1.46–1.38 (m, 2H), 1.34 (s, 9H), 1.32 (s, 9H),
1.25–1.14 (m, 2H). 13C NMR (90.6 MHz, CDCl3) d: 156.3 (2C),
125.5 (q, J = 281.8 Hz), 79.6, 79.1, 69.7 (q, J = 29.0 Hz), 46.9, 43.9,
37.6, 29.5, 28.3 (7C), 28.1, 26.4, 24.6. 19F NMR (338.8 MHz) d:
ꢁ79.8. HRMS (ESI) calcd for C20H37N2NaO5F3 [M+Na]+ 465.2552,
found 465.2553.
hexanes/AcOEt gradients afforded
a-bromohydrin 13 (2.31 g,
91%) as a colorless oil. 1H NMR (360 MHz, CDCl3) d: 5.26 (br s,
1H), 3.71–3.64 (m, 1H), 3.38 (dd, J = 4.0 Hz, J = 10.1 Hz, 1H), 3.30
(dd, J = 6.5 Hz, J = 10.1 Hz, 1H), 3.13 (t, J = 6.5 Hz, 2H), 3.05–2.96
(m, 5H), 1.58–1.53 (m, 2H), 1.48–1.39 (m, 4H), 1.37–1.31 (m,
2H), 1.36 (s, 9H), 1.34 (s, 9H), 1.22–1.15 (m, 2H). 13C NMR
(90.6 MHz, CDCl3) d: 156.0 (2C), 79.4, 78.9, 70.7, 46.9, 43.9, 39.8,
37.6, 34.9, 28.4 (7C), 27.9, 26.6, 25.2. HRMS (ESI) calcd for
C
20H39N2O5BrNa [M+Na]+ 489.1940, found 489.1931.
4.1.15. N,N’-Bis(tert-butoxycarbonyl)-7-[(3-
aminopropyl)amino]-1-bromoheptan-2-one (14)
To a solution of
a-bromohydrin 13 (2.10 g, 4.49 mmol) in dry
CH2Cl2 (50 mL) at room temperature and under argon was added
DMP (5.72 g, 13.5 mmol). After 3 h, the reaction mixture was
cooled down to 0 °C, quenched with the addition of an aqueous so-
dium thiosulfate solution (0.5 M) saturated with NaHCO3 (100 mL),
and the aqueous layer was extracted with AcOEt. Combined organ-
ic extracts were washed with brine, dried over Na2SO4, and con-
centrated in vacuo. The residue was purified by flash column
chromatography on SiO2 with hexanes/AcOEt gradients to afford
4.1.12. N,N0-Bis(tert-butoxycarbonyl)-7-[(3-
aminopropyl)amino]-1,1,1-trifluoroheptan-2-one (11)
To a solution of trifluoromethylalcohol 10 (1.40 g, 3.16 mmol)
in dry CH2Cl2 (50 mL) at room temperature and under argon was
added DMP (5.37 g, 12.7 mmol). The reaction mixture was stirred
overnight at room temperature, then cooled down to 0 °C,
quenched with the addition of an aqueous sodium thiosulfate solu-
tion (0.5 M) saturated with NaHCO3 (150 mL), and the aqueous
layer was extracted with AcOEt. Combined organic extracts were
a
-bromoketone 14 as a colorless oil (2.03 g, 97%). 1H NMR
(360 MHz, CDCl3) d: 5.21 (br s, 1H), 3.78 (s, 2H), 3.12 (t,
J = 6.5 Hz, 2H), 3.04–2.95 (m, 4H), 2.54 (t, J = 7.2 Hz, 2H), 1.56–

C. Decroos et al. / Bioorg. Med. Chem. 21 (2013) 4530–4540
4537
1.47 (m, 4H), 1.45–1.36 (m, 2H), 1.33 (s, 9H), 1.31 (s, 9H), 1.21–1.12
(m, 2H). 13C NMR (90.6 MHz, CDCl3) d: 201.7, 155.9 (2C), 79.3, 78.7,
46.6, 43.8, 39.5, 37.6, 34.2, 28.3, 27.8 (7C), 26.1, 23.4. HRMS (ESI)
calcd for C20H37N2O5BrNa [M+Na]+ 487.1784, found 487.1794.
(0.5 M in THF, 43.8 mL, 21.9 mmol). The solution was allowed to
warm up to room temperature and stirred for 20 h. The reaction
mixture was then cooled down to 0 °C, and aqueous NaOH (6 M,
30 mL) was added, followed by the dropwise addition of H2O2
(30%, 15 mL). After stirring 30 min at room temperature, the solu-
tion was concentrated in vacuo. The aqueous phase was diluted
with 50 mL of water, and extracted with AcOEt. Combined organic
extracts were washed with brine, dried over Na2SO4, and concen-
trated in vacuo. The residue was purified by flash column chroma-
tography on SiO2 with hexanes/AcOEt gradients to afford alcohol
18 as a colorless oil (3.15 g, quantitative). 1H NMR (500 MHz,
CDCl3) d: 5.35 (br s, 1H), 3.54 (t, J = 6.5 Hz, 2H), 3.16 (t, J = 6.5 Hz,
2H), 3.06 (t, J = 6.5 Hz, 2H), 3.01 (t, J = 6.5 Hz, 2H), 2.74 (br s, 1H),
1.61–1.55 (m, 2H), 1.53–1.43 (m, 4H), 1.38 (s, 9H), 1.36 (s, 9H),
1.29–1.23 (m, 2H). 13C NMR (125.6 MHz, CDCl3) d:156.2 (2C),
79.5, 79.0, 62.4, 47.0, 43.9, 37.6, 32.3, 28.4 (7C), 27.8, 23.0.
HRMS (ESI) calcd for C18H36N2O5Na [M+Na]+ 383.2522, found
383.2508.
4.1.16. S-{N,N’-Bis(tert-butoxycarbonyl)-7-[(3-
aminopropyl)amino]-2-oxoheptyl}thioacetate (15)
To a solution of
a-bromoketone 14 (1.72 g, 3.70 mmol) in dry
MeCN (25 mL) at room temperature under argon was added potas-
sium thioacetate (2.54 g, 22.2 mmol). After stirring overnight, the
reaction mixture was diluted with water and the aqueous layer
was extracted with AcOEt. Combined organic extracts were
washed with brine, dried over Na2SO4, filtered, and concentrated
in vacuo. Purification by flash column chromatography on SiO2
with hexanes/AcOEt gradients afforded thioester 15 (1.65 g, 97%)
as a colorless oil. 1H NMR (500 MHz, CDCl3) d: 5.21 (br s, 1H),
3.68 (s, 2H), 3.18 (t, J = 6.5 Hz, 2H), 3.08–3.02 (m, 4H), 2.50 (t,
J = 7.5 Hz, 2H), 2.32 (s, 3H), 1.62–1.54 (m, 4H), 1.49–1.43 (m, 2H),
1.40 (s, 9H), 1.38 (s, 9H), 1.25–1.18 (m, 2H). 13C NMR
(125.6 MHz, CDCl3) d: 203.8, 194.3, 156.1 (2C), 79.5, 79.0, 46.7,
43.9, 41.5, 39.0, 37.7, 30.2, 28.5 (7C), 28.4, 26.3, 23.4. HRMS (ESI)
calcd for C22H40N2O6SNa [M+Na]+ 483.2505, found 483.2517.
4.1.20. N1,N3-Bis(tert-butoxycarbonyl)-N1-(5-
bromopentyl)propane-1,3-diamine (19)
To a solution of alcohol 18 (3.00 g, 8.32 mmol) in dry THF
(100 mL) at 0 °C were added successively CBr4 (5.52 g, 16.6 mmol)
and PPh3 (4.37 g, 16.7 mmol). The reaction mixture was allowed to
reach room temperature and stirred overnight. After removal of
the solvent in vacuo, purification of the residue by flash column
chromatography on SiO2 with hexanes/AcOEt gradients afforded
4.1.17. N,N’-Bis(tert-butoxycarbonyl)-7-[(3-
aminopropyl)amino]-1-methoxyheptan-2-ol (16)
Epoxide 12 (1.33 g, 3.44 mmol) was dissolved in a 0.5 M solu-
tion of sodium methoxide in MeOH (41.2 mL, 20.6 mmol). The
reaction mixture was stirred under argon at room temperature un-
til completion of the reaction (24 h) as shown by TLC. After re-
moval of the solvent in vacuo, the residue was partitioned
between AcOEt (100 mL) and water (25 mL), and the aqueous layer
was extracted with AcOEt. Combined organic extracts were
washed with brine, dried over Na2SO4, filtered and concentrated
in vacuo. Purification by flash column chromatography on SiO2
with hexanes/AcOEt gradients afforded alcohol 16 (1.34 g, 93%)
as a colorless oil. 1H NMR (500 MHz, CDCl3) d: 5.33 (br s, 1H),
3.66–3.60 (m, 1H), 3.27–3.24 (m, 4H), 3.15–3.10 (m, 3H), 3.02–
2.96 (m, 4H), 2.72 (br s, 1H), 1.55–1.50 (m, 2H), 1.43–1.35 (m,
4H), 1.33 (s, 9H), 1.31 (s, 9H), 1.29–1.23 (m, 2H), 1.20–1.14 (m,
2H). 13C NMR (125.6 MHz, CDCl3) d: 156.0 (2C), 79.2, 78.7, 77.1,
69.9, 58.8, 46.9, 43.7, 37.4, 33.1, 28.3 (7C), 28.2, 26.8, 25.2. HRMS
(ESI) calcd for C21H43N2O6 [M+H]+ 419.3121, found 419.3124.
alkyl bromide 19 (3.38 g, 96%) as
a
colorless oil. 1H NMR
(500 MHz, CDCl3) d: 5.21 (br s, 1H), 3.23 (t, J = 6.5 Hz, 2H), 3.17
(t, J = 6.5 Hz, 2H), 3.08 (t, J = 6.5 Hz, 2H), 3.02 (t, J = 6.5 Hz, 2H),
1.83–1.77 (m, 2H), 1.61–1.56 (m, 2H), 1.51–1.44 (m, 2H), 1.39 (s,
9H), 1.37 (s, 9H), 1.38–1.31 (m, 2H). 13C NMR (125.6 MHz, CDCl3)
d: 156.0 (2C), 79.5, 78.9, 46.7, 43.8, 37.7, 33.6, 32.4, 28.4 (7C),
27.7, 25.4. HRMS (ESI) calcd for C18H36N2O4Br [M+H]+ 423.1858,
found 423.1866.
4.1.21. S-{N,N’-Bis(tert-butoxycarbonyl)-5-[(3-
aminopropyl)amino]pentyl} thioacetate (20)
Reaction of alkyl bromide 19 (1.70 g, 4.02 mmol) and potassium
thioacetate (2.75 g, 24.1 mmol) under the same conditions as for
compound 15 afforded after purification thioester 20 (1.58 g,
94%) as a colorless oil. 1H NMR (500 MHz, CDCl3) d: 5.21 (br s,
1H), 3.07 (t, J = 6.5 Hz, 2H), 2.96 (t, J = 6.5 Hz, 2H), 2.91 (t,
J = 6.0 Hz, 2H), 2.68 (t, J = 7.0 Hz, 2H), 2.14 (s, 3H), 1.51–1.47 (m,
2H), 1.44–1.38 (m, 2H), 1.38–1.32 (m, 2H), 1.28 (s, 9H), 1.26 (s,
9H), 1.19–1.13 (m, 2H). 13C NMR (125.6 MHz, CDCl3) d: 195.3,
155.8 (2C), 79.1, 78.5, 46.5, 43.6, 37.5, 30.3, 29.0, 28.7, 28.2 (7C),
4.1.18. N,N’-Bis(tert-butoxycarbonyl)-7-[(3-
aminopropyl)amino]-1-methoxyheptan-2-one (17)
To a solution of alcohol 16 (1.19 g, 2.84 mmol) in dry CH2Cl2
(50 mL) at room temperature and under argon was added DMP
(7.24 g, 17.1 mmol). After 24 h, the reaction mixture was cooled
down to 0 °C, quenched with the addition of an aqueous sodium
thiosulfate solution (0.5 M) saturated with NaHCO3 (150 mL), and
the aqueous layer was extracted with AcOEt. Combined organic ex-
tracts were washed with brine, dried over Na2SO4, and concentrated
in vacuo. The residue was purified by flash column chromatography
27.8, 25.7. HRMS (ESI) calcd for
441.2399, found 441.2406.
C
20H38N2O5SNa [M+Na]+
4.1.22. N,N’-Bis(tert-butoxycarbonyl)-5-[(3-
aminopropyl)amino]pentane-1-thiol (21)
To a solution of thioester 20 (550 mg, 1.31 mmol) in dry MeOH
(10 mL) under argon was added sodium methoxide (0.5 M solution
in MeOH, 5.3 mL, 2.65 mml). The solution was stirred at room tem-
perature for 2 h, quenched by the addition of 10% aqueous citric
acid (100 mL), and the aqueous phase was extracted with AcOEt.
Combined organic extracts were washed with brine, dried over
Na2SO4, and concentrated in vacuo. The residue was purified by
flash column chromatography on SiO2 with hexanes/AcOEt gradi-
ents to afford thiol 21 as a colorless oil (485 mg, 98%). 1H NMR
(500 MHz, CDCl3) d: 5.19 (br s, 1H), 3.07 (t, J = 6.0 Hz, 2H), 2.98
(t, J = 6.3 Hz, 2H), 2.92 (t, J = 6.3 Hz, 2H), 2.35 (apparent q (dt),
J = 7.3 Hz, 2H), 1.51–1.41 (m, 4H), 1.37–1.32 (m, 2H), 1.29 (s, 9H),
1.26 (s, 9H), 1.23–1.17 (m, 2H), 1.16 (t, J = 7.3 Hz, 1H). 13C NMR
(125.6 MHz, CDCl3) d: 155.8 (2C), 79.1, 78.5, 46.6, 43.7, 37.5,
on SiO2 with hexanes/AcOEt gradients to afford a-methoxyketone
17 as a colorless oil (1.08 g, 91%). 1H NMR (500 MHz, CDCl3) d:
5.21 (br s, 1H), 3.94 (s, 2H), 3.35 (s, 3H), 3.18 (t, J = 6.5 Hz, 2H),
3.08–3.02 (m, 4H), 2.38 (t. J = 7.5 Hz, 2H), 1.61–1.52 (m, 4H),
1.49–1.43 (m, 2H), 1.39 (s, 9H), 1.37 (s, 9H), 1.29–1.23 (m, 2H).
13C NMR (125.6 MHz, CDCl3) d: 208.5, 156.24 (2C), 79.5, 79.0,
77.6, 59.3, 46.8, 43.9, 38.6, 37.7, 28.4 (7C), 28.3, 26.4, 23.0. HRMS
(ESI) calcd for C21H40N2O6Na [M+Na]+ 439.2784, found 439.2772.
4.1.19. N,N’-Bis(tert-butoxycarbonyl)-5-[(3-
aminopropyl)amino]pentan-1-ol (18)
To a solution of alkene 3 (3.00 g, 8.76 mmol) in dry THF
(150 mL) at 0 °C and under argon was added dropwise 9-BBN

4538
C. Decroos et al. / Bioorg. Med. Chem. 21 (2013) 4530–4540
33.4, 28.2 (7C), 27.7, 25.3, 24.2. HRMS (ESI) calcd for C18H36N2O4S-
Na [M+Na]+ 399.2293, found 399.2293.
argon for 2 h. The reaction mixture was cooled down to 0 °C and,
diluted with Et2O (20 mL). The precipitate was filtered, washed
with cold Et2O, and dried in vacuo to afford carboxylic acid I (dihy-
drochloride salt) as a white solid (188 mg, 93%). mp: 172–174 °C.
1H NMR (500 MHz, D2O) d: 3.20 (t, J = 8.0 Hz, 2H), 3.17–3.11 (m,
4H), 2.45 (t, J = 7.4 Hz, 2H), 2.17–2.11 (m, 2H), 1.79–1.72 (m, 2H),
1.71–1.65 (m, 2H), 1.49–1.43 (m, 2H). 13C NMR (125.6 MHz, D2O)
d: 178.8, 47.6, 44.4, 36.6, 33.5, 25.2, 25.1, 23.8, 23.7. HRMS (ESI)
calcd for C9H21N2O2 [M+H]+ 189.1603, found 189.1604.
4.1.23. N1,N3-Bis(tert-butoxycarbonyl)-N1-[5-
(methylthio)pentyl]propane-1,3-diamine (22)
To a solution of alkyl bromide 19 (1.22 g, 2.88 mmol) in EtOH
(15 mL) was added sodium thiomethoxide (1.21 g, 17.3 mmol).
The reaction mixture was stirred overnight at 60 °C under argon.
After removal of the solvent under vacuo, the residue was parti-
tioned between CH2Cl2 (50 mL) and saturated aqueous NaHCO3
(25 mL), and the aqueous layer was extracted with CH2Cl2. Com-
bined organic extracts were washed with water, brine, dried over
Na2SO4, filtered, and concentrated in vacuo. Purification by flash
column chromatography on SiO2 with hexanes/AcOEt gradients
afforded thioether 22 (1.10 g, 98%) as a colorless oil. 1H NMR
(360 MHz, CDCl3) d: 5.21 (br s, 1H), 3.20 (t, J = 6.5 Hz, 2H), 3.13–
3.05 (m, 4H), 2.46 (t, J = 7.4 Hz, 2H), 2.06 (s, 3H), 1.66–1.54 (m,
4H), 1.53–1.46 (m, 2H), 1.43 (s, 9H), 1.41 (s, 9H), 1.38–1.29 (m,
2H). 13C NMR (90.6 MHz, CDCl3) d: 156.1 (2C), 79.6, 79.1, 46.9,
44.1, 37.8, 34.3, 29.0, 28.5 (7C), 28.2, 26.1, 15.6. HRMS (ESI) calcd
for C19H39N2O4S [M+H]+ 391.2631, found 391.2633.
4.1.27. 7-[(3-Aminopropyl)amino]heptan-2-one
dihydrochloride (II)
Deprotection of compound 7 (320 mg, 828 lmol) was per-
formed under the same conditions as for I to afford ketone II (dihy-
drochloride salt) as a white powder (202 mg, 94%). mp: 206–209 °C
(dec.). 1H NMR (500 MHz, D2O) d: 3.14 (t, J = 8.0 Hz, 2H), 3.10 (t,
J = 8.0 Hz, 2H), 3.06 (t, J = 7.8 Hz, 2H), 2.58 (t, J = 7.5 Hz, 2H), 2.19
(s, 3H), 2.11–2.05 (m, 2H), 1.71–1.65 (m, 2H), 1.60–1.54 (m, 2H),
1.38–1.31 (m, 2H). 13C NMR (125.6 MHz, D2O) d: 216.9, 47.5,
44.3, 42.7, 36.5, 29.2, 25.2, 25.0, 23.6, 22.5. HRMS (ESI) calcd for
C
10H23N2O [M+H]+ 187.1810, found 187.1813.
4.1.24. N1,N3-Bis(tert-butoxycarbonyl)-N1-[5-
(methylsulfonyl)pentyl]propane-1,3-diamine (23)
4.1.28. 6-[(3-Aminopropyl)amino]-N-hydroxyhexanamide
dihydrochloride (III)
To a solution of thioether 22 (500 mg, 1.28 mmol) in dry CH2Cl2
(10 mL) under argon at 0 °C was added dropwise m-CPBA (77%,
861 mg, 3.84 mmol) in 10 mL dry CH2Cl2. The solution was allowed
to reach room temperature and stirred until completion of the
reaction (2 h) as shown by TLC. Saturated aqueous NaHCO3
(10 mL) was added to the reaction mixture and the aqueous layer
was extracted with AcOEt. Combined organic extracts were dried
over Na2SO4, filtered and concentrated in vacuo. The residue was
purified by flash column chromatography on SiO2 with hexanes/
AcOEt gradients to afford sulfone 23 (524 mg, 97%) as a colorless
oil. 1H NMR (500 MHz, CDCl3) d: 5.14 (br s, 1H), 3.07 (t,
J = 7.0 Hz, 2H), 3.01 (t, J = 6.5 Hz, 2H), 2.92 (t, J = 6.5 Hz, 2H), 2.87
(t, J = 7.0 Hz, 2H), 2.75 (s, 3H), 1.73–1.67 (m, 2H), 1.53–1.48 (m,
2H), 1.44–1.40 (m, 2H), 1.31–1.26 (m, 2H), 1.30 (s, 9H), 1.27 (s,
9H). 13C NMR (125.6 MHz, CDCl3) d: 155.9 (2C), 79.3, 78.6, 54.3,
46.3, 43.6, 40.2, 37.5, 28.2 (7C), 28.0, 25.4, 22.0. HRMS (ESI) calcd
for C19H38N2O6SNa [M+Na]+ 445.2348, found 445.2344.
Deprotection of compound 8 (350 mg, 867 lmol) was per-
formed under the same conditions as for I to afford hydroxamic
acid III (dihydrochloride salt) as a white powder (230 mg, 96%).
mp: 135–139 °C. 1H NMR (500 MHz, D2O) d: 3.19 (t, J = 8.0 Hz,
2H), 3.16–3.10 (m, 4H), 2.23 (t, J = 7.3 Hz, 2H), 2.16–2.10 (m, 2H),
1.77–1.71 (m, 2H), 1.70–1.64 (m, 2H), 1.45–1.39 (m, 2H). 13C
NMR (125.6 MHz, D2O) d: 173.1, 47.6, 44.4, 36.6, 32.0, 25.2, 25.0,
24.3, 23.7. HRMS (ESI) calcd for C9H22N3O2 [M+H]+ 204.1712, found
204.1722.
4.1.29. 7-[(3-Aminopropyl)amino]-1,1,1-trifluoroheptan-2-one
dihydrochloride (IV)
Deprotection of compound 11 (350 mg, 795 lmol) was per-
formed under the same conditions as for I to afford trifluorometh-
ylketone IV (dihydrochloride salt) as a white powder (234 mg,
94%). mp: 228–231 °C (dec.). 1H NMR (500 MHz, D2O) (ketone/hy-
drate : 0/1) d: 3.20 (t, J = 8.0 Hz, 2H), 3.17–3.12 (m, 4H), 2.18–2.11
(m, 2H), 1.88 (t, J = 8.2 Hz, 2H), 1.80–1.74 (m, 2H), 1.62–1.55 (m,
2H), 1.51–1.45 (m, 2H). 13C NMR (125.6 MHz, D2O) d: 123.6 (q,
J = 286.4 Hz), 93.6 (q, J = 31.4 Hz), 47.7, 44.4, 36.6, 33.7, 25.7,
25.3, 23.7, 20.7. 19F NMR (338.8 MHz) d: ꢁ85.1. HRMS (ESI) calcd
for C10H20N2OF3 [M+H]+ 241.1528, found 241.1534.
4.1.25. N1,N3-Bis(tert-butoxycarbonyl)-N1-[5-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)pentyl]propane-1,3-
diamine (24)
To a solution of [Ir(cod)Cl]2 (196 mg, 292
(225 mg, 585 mol) in dry CH2Cl2 (150 mL) under argon was added
pinacolborane (1.65 mL, 11.3 mmol) and alkene (2.00 g,
lmol) and dppm
l
3
4.1.30. 7-{[(3-Aminopropyl)amino]-2-oxoheptyl} thioacetate
dihydrochloride (V)
5.84 mmol) in 30 mL dry CH2Cl2. After stirring 24 h at room tem-
perature, the reaction mixture was cooled to 0 °C, quenched with
the addition of 60 mL of water, and the aqueous phase extracted
with Et2O. Combined organic extracts were dried over Na2SO4,
and concentrated in vacuo. The residue was purified by flash col-
umn chromatography on SiO2 with hexanes/AcOEt gradients to af-
ford 24 as a colorless oil (2.25 g, 82%). 1H NMR (500 MHz, CDCl3) d:
5.24 ppm (br s, 1H), 3.16 (t, J = 6.6 Hz, 2H), 3.04–2.99 (m, 4H),
1.59–1.54 (m, 2H), 1.45–1.39 (m, 2H), 1.37 (s, 9H), 1.35 (s, 9H),
1.35–1.30 (m, 2H), 1.21–1.15 (m, 2H), 1.15 (s, 12H), 0.68 (t,
J = 7.8 Hz, 2H). 13C NMR (125.6 MHz, CDCl3) d: 156.0 (2C), 82.6
(2C), 79.3, 78.8, 47.0, 43.8, 37.7, 29.5, 28.5 (7C), 28.3, 24.8 (4C),
23.8, 11.2 (br s). 11B NMR (128 MHz, CDCl3) d: 33.0 ppm. HRMS
(ESI) calcd for C24H48N2O5B [M+H]+ 471.3605, found 471.3615.
Deprotection of compound 15 (300 mg, 651 lmol) was per-
formed under the same conditions as for I to afford thioacetate
derivative V (dihydrochloride salt) as a white powder (210 mg,
97%). mp: 211–213 °C (dec.). 1H NMR (500 MHz, D2O) d: 4.00 (s,
2H), 3.23 (t, J = 8.0 Hz, 2H), 3.19 (t, J = 7.8 Hz, 2H), 3.16 (t,
J = 7.8 Hz, 2H), 2.81 (t, J = 7.2 Hz, 2H), 2.49 (s, 3H), 2.21–2.15 (m,
2H), 1.82–1.76 (m, 2H), 1.74–1.68 (m, 2H), 1.50–1.43 (m, 2H). 13C
NMR (125.6 MHz, D2O) d: 209.8, 200.1, 47.7, 44.5, 41.1, 39.3,
36.7, 29.5, 25.3, 25.1, 23.8, 22.7. HRMS (ESI) calcd for C12H25N2O2S
[M+H]+ 261.1637, found 261.1626.
4.1.31. 7-[(3-Aminopropyl)amino]-1-methoxyheptan-2-one
dihydrochloride (VI)
4.1.26. 6-[(3-Aminopropyl)amino]hexanoic acid
dihydrochloride (I)
A solution of compound 17 (240 mg, 576 lmol) in anhydrous
1 N HCl in AcOEt (30 mL) was stirred at room temperature under
argon for 6 h. The suspension was diluted with hexanes (20 mL).
The precipitate was filtered, washed with AcOEt and hexanes,
A solution of compound 5 (300 mg, 772
lmol) in anhydrous 4 N
HCl in dioxane (20 mL) was stirred at room temperature under

C. Decroos et al. / Bioorg. Med. Chem. 21 (2013) 4530–4540
4539
and dried in vacuo to afford
a
-methoxyketone VI (dihydrochloride
APAH by the synthesized diamine derivatives was analyzed using
a fluorimetric assay, as previously described.18 Activity was mea-
sured using the commercially available Fluor-de-Lys deacetylase
fluorogenic substrate (BML-KI104, Enzo Life Sciences). Briefly,
deacetylation of the substrate molecule allows a protease devel-
oper to cleave the peptide bond linking the C-terminal fluorophore,
resulting in a fluorescence shift. Assays were run at 25 °C and con-
salt) as an off-white solid (151 mg, 91%). mp: 216–218 °C (dec.). 1H
NMR (500 MHz, D2O) d: 4.35 (s, 2H), 3.44 (s, 3H), 3.19 (t, J = 8.0 Hz,
2H), 3.15 (t, J = 7.9 Hz, 2H), 3.11 (t, J = 7.8 Hz, 2H), 2.55 (t, J = 7.3 Hz,
2H), 2.16–2.10 (m, 2H), 1.77–1.71 (m, 2H), 1.68–1.62 (m, 2H),
1.45–1.39 (m, 2H). 13C NMR (125.6 MHz, D2O) d: 212.8, 76.5,
58.7, 47.6, 44.4, 37.8, 36.6, 25.3, 25.2, 23.7, 22.3. HRMS (ESI) calcd
for C11H25N2O2 [M+H]+ 217.1916, found 217.1925.
tained 250 nM enzyme, 150
in assay buffer (25 mM Tris (pH 8.2), 137 mM NaCl, 2.7 mM KCl
and 1 mM MgCl2 (100 M tris-(2-carboxyethyl)phosphine was
added only for the assay of thiol compound VIII) in a final volume
of 50 L. After 30 min, reactions were quenched by adding 100
M344 (Sigma Aldrich) and the appropriate Fluor-de-Lys developer
(BML-KI105, Enzo Life Sciences, 50 L). Fluorescence was mea-
lM substrate, and 0–100 mM inhibitor
4.1.32. S-{5-[(3-Aminopropyl)amino]pentyl} thioacetate
dihydrochloride (VII)
l
Deprotection of compound 20 (300 mg, 717
l
mol) was per-
l
lM
formed under the same conditions as for I to afford thioacetate
derivative VII (dihydrochloride salt) as a white powder (196 mg,
94%). mp: 260–262 °C (dec.). 1H NMR (500 MHz, D2O) d: 3.19 (t,
J = 8.0 Hz, 2H), 3.15 (t, J = 7.9 Hz, 2H), 3.11 (t, J = 7.8 Hz, 2H), 2.94
(t, J = 7.2 Hz, 2H), 2.41 (s, 3H), 2.16–2.10 (m, 2H), 1.78–1.72 (m,
2H), 1.69–1.64 (m, 2H), 1.52–1.46 (m, 2H). 13C NMR (125.6 MHz,
D2O) d: 202.2, 47.6, 44.4, 36.6, 30.1, 28.4, 28.1, 25.0, 24.7, 23.7.
l
sured after 45 min using a Fluoroskan II plate reader (excita-
tion = 355 nm, emission = 460 nm). Assays for each concentration
of inhibitor were performed in triplicate in separate experiments.
IC50 values for each compound were determined using the soft-
ware Graphpad Prism (2008).
HRMS (ESI) calcd for
219.1532.
C
10H23N2OS [M+H]+ 219.1531, found
Acknowledgments
4.1.33. 5-[(3-Aminopropyl)amino]pentane-1-thiol
dihydrochloride (VIII)
This work was supported by the National Institutes of Health
Grant GM49758. The Roy and Diana Vagelos Scholars Program in
Molecular Life Sciences supports the research of undergraduate
student Christine M. Bowman. Finally, we thank Dr. Rakesh Kohli
(University of Pennsylvania, Department of Chemistry) for record-
ing the high resolution mass spectra.
A solution of compound 21 (208 mg, 552 lmol) in anhydrous
1 N HCl in AcOEt (40 mL) was stirred at room temperature under ar-
gon for 5 h. The reaction mixture was concentrated in vacuo and
resuspended in hexanes (20 mL). The precipitate was filtered,
washed with hexanes, and dried in vacuo to afford thiol derivative
VIII (dihydrochloride salt) as a white solid (129 mg, 93%). mp: 284–
286 °C (dec.). 1H NMR (500 MHz, D2O) d: 3.24 (t, J = 8.0 Hz, 2H),
3.21–3.15 (m, 4H), 2.65 (t, J = 7.1 Hz, 2H), 2.21–2.15 (m, 2H),
1.82–1.76 (m, 2H), 1.76–1.70 (m, 2H), 1.59–1.52 (m, 2H). 13C NMR
(125.6 MHz, D2O) d: 47.8, 44.5, 36.7, 32.4; 25.0, 24.5, 23.8, 23.5.
HRMS (ESI) calcd for C8H21N2S [M+H]+ 177.1425, found 177.1424.
Supplementary data
Supplementary data (NMR spectra for compounds I–X) associ-
ated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.bmc.2013.05.045.
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