Exploring Structural Determinants of Inhibitor Affinity and Selectivity in Complexes with Histone Deacetylase 6

Article abstract of DOI:10.1021/acs.jmedchem.9b01540

Inhibition of histone deacetylase 6 (HDAC6) has emerged as a promising therapeutic strategy for the treatment of cancer, chemotherapy-induced peripheral neuropathy, and neurodegenerative disease. The recent X-ray crystal structure determination of HDAC6 enables an understanding of structural features directing affinity and selectivity in the active site. Here, we present the X-ray crystal structures of five HDAC6-inhibitor complexes that illuminate key molecular features of the inhibitor linker and capping groups that facilitate and differentiate binding to HDAC6. In particular, aromatic and heteroaromatic linkers nestle within an aromatic cleft defined by F583 and F643, and different aromatic linkers direct the capping group toward shallow pockets defined by the L1 loop, the L2 loop, or somewhere in between these pockets. These results expand our understanding of factors contributing to the selective inhibition of HDAC6, particularly regarding interactions that can be targeted in the region of the L2 pocket.

Full text of DOI:10.1021/acs.jmedchem.9b01540

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Article
Exploring Structural Determinants of Inhibitor Affinity
and Selectivity in Complexes with Histone Deacetylase 6
Jeremy D. Osko, Nicholas J. Porter, Poli Adi Narayana Reddy, You-Cai Xiao, Johanna
Rokka, Manfred Jung, Jacob M. Hooker, Joseph M. Salvino, and David W. Christianson
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01540 • Publication Date (Web): 03 Dec 2019
Downloaded from pubs.acs.org on December 5, 2019
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Exploring Structural Determinants of Inhibitor Affinity and Selectivity
in Complexes with Histone Deacetylase 6
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Jeremy D. Osko,1 Nicholas J. Porter,1 Poli Adi Narayana Reddy,2 You-Cai Xiao,2 Johanna
Rokka,3 Manfred Jung,4 Jacob M. Hooker,3 Joseph M. Salvino,2 and David W. Christianson1,*
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1Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania,
231 South 34th Street, Philadelphia, PA 19104-6323, United States
2The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, United States
3Athinoula A. Martinos Center for Biomedical Imaging, Massachusetts General Hospital and
Harvard Medical School, Charlestown, Massachusetts 02129, United States
4Institute of Pharmaceutical Sciences, University of Freiburg, Albertstraße 25, 79104 Freiburg,
Germany
*author to whom correspondence should be sent: E-mail chris@sas.upenn.edu
Abstract: Inhibition of histone deacetylase 6 (HDAC6) has emerged as a promising therapeutic
strategy for the treatment of cancer, chemotherapy-induced peripheral neuropathy, and
neurodegenerative disease. The recent X-ray crystal structure determination of HDAC6 enables
an understanding of structural features directing affinity and selectivity in the active site. Here,
we present the X-ray crystal structures of five HDAC6-inhibitor complexes that illuminate key
molecular features of inhibitor linker and capping groups that facilitate and differentiate binding
to HDAC6. In particular, aromatic and heteroaromatic linkers nestle within an aromatic cleft
defined by F583 and F643, and different aromatic linkers direct the capping group toward
shallow pockets defined by the L1 loop, the L2 loop, or somewhere in between these pockets.
These results expand our understanding of factors contributing to the selective inhibition of
HDAC6, particularly regarding interactions that can be targeted in the region of the L2 pocket.
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Introduction
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The metal ion-dependent histone deacetylases (HDACs) require a single Zn2+ ion (or
possibly Fe2+) to catalyze the hydrolysis of acetyl-L-lysine side chains of protein substrates in
vivo or peptide substrates in vitro to yield L-lysine and acetate coproducts.1-5 While these
enzymes are named after their first reported substrates,1 thousands of non-histone proteins
have since been identified that contain acetyl-L-lysine residues.6 Non-histone proteins, too,
undergo strategic acetylation-deacetylation cycles in the regulation of diverse biological
functions.
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The metal-dependent HDACs consist of the class I isozymes, HDACs 1–3 and 8; the
class IIa isozymes, HDACs 4, 5, 7, and 9; the class IIb isozymes, HDACs 6 and 10; and the sole
class IV isozyme, HDAC11.7 Despite the common fold and catalytic mechanism shared by the
catalytic domain of each HDAC isozyme,5,8-10 the HDACs have varying cellular localizations,
expression patterns, client proteins, catalytic activities, and domain assemblies.11 For example,
HDAC612,13 is the only isozyme containing two catalytic domains, designated CD1 and CD2.14,15
HDAC6 serves as a tubulin deacetylase in the cell cytosol and helps maintain microtubule
acetylation levels that facilitate microtubule dynamics.16 The CD2 domain of HDAC6 is
responsible for catalyzing the tubulin deacetylation reaction;17 inhibition of this function
suppresses microtubule dynamics and can lead to cell cycle arrest and apoptosis.18 Selective
inhibition of HDAC6 CD2 is being explored for cancer chemotherapy, mitigation of
chemotherapy-induced peripheral neuropathy, and the treatment of neurodegenerative
diseases.19-26
Recent X-ray crystal structure determinations of HDAC6 CD2 from Homo sapiens
(human)27 and Danio rerio (zebrafish)27,28 (Figure 1a) have enabled the analysis of enzyme-
inhibitor complexes and the characterization of molecular features responsible for affinity and
selectivity in the HDAC6 CD2 active site.29-35 Typically, inhibitors consist of a metal-binding
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group, a linker, and a capping group that makes interactions in the outer active site cleft.
Binding interactions of the capping group contribute significantly to affinity and selectivity for
HDAC6 CD2. The active site cleft of HDAC6 is slightly wider than the active site clefts of class I
HDACs, which confers selectivity for the binding of inhibitors with sterically bulky aromatic
linkers; there is an associated entropic advantage that confers selectivity for HDAC6 binding as
well.30 The capping groups of these inhibitors generally bind in a pocket defined by the L1 loop
flanking the active site. Notably, sterically bulky inhibitors with bifurcated capping groups make
interactions in the L1 pocket as well as another shallow pocket defined by the L2 loop and
nearby residues G640–N645 (Figure 1b).33 To some degree, the molecular structure of the
linker moiety may help steer the capping group toward one pocket or the other.
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Figure 1. (a) Ribbon-plot of HDAC6 CD2 from D. rerio (PDB 6PZS). The catalytic Zn2+ ion appears as a
gray sphere. (b) Structure of the HDAC6 CD2 complex with the inhibitor RTS-V5, which contains a
bifurcated capping group (PDB 6CW8); the molecular surface of the active site illustrates the L1 and L2
pockets. (c) Inhibitors of HDAC6 CD2 (IC50 values are listed below compound numbers): 1, (E)-3-(1-((4-
((dimethylamino)methyl)phenyl)sulfonyl)-1H-pyrrol-3-yl)-N-hydroxyacrylamide (Resminostat); 2, 2-(4-
bromophenyl)-N-hydroxyoxazole-4-carboxamide; 3, N-(4-(hydroxycarbamoyl)benzyl)-2-
(trifluoromethyl)nicotinamide; 4, 4-(((6,6-dimethylbicyclo[3.1.1]heptan-2-yl)amino)methyl)-N-
hydroxybenzamide; 5, benzyl ((S)-1-(((S)-1-((4-(hydroxycarbamoyl)-benzyl)amino)-1-oxopropan-2-
yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate; 6, benzyl (2-((2-((4-(hydroxycarbamoyl)benzyl)amino)-2-
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oxoethyl)amino)-2-oxoethyl)carbamate; 7, benzyl ((S)-1-(((S)-1-((3-(hydroxycarbamoyl)benzyl)amino)-1-
oxopropan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate.
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To further explore the role of the linker in steering inhibitor capping groups toward these
pockets, and also to determine how the chemical nature of the capping group (i.e.,
hydrophobicity or hydrophilicity) influences binding in these pockets, we now present five new
X-ray crystal structures of zebrafish HDAC6 CD2 complexed with recently-designed inhibitors
1–5 (Figure 1c) determined at resolutions ranging 1.50–2.30 Å. To probe structure-affinity
relationships, inhibitors 6 and 7 were also prepared and evaluated but not used for
crystallographic studies. Zebrafish HDAC6 CD2 is used for these studies since it yields crystals
of far greater quality than the human enzyme; moreover, the active site structures of zebrafish
HDAC6 CD2 and human HDAC6 CD2 are essentially identical (with the exception of N530D
and N645M substitutions, respectively, at the mouth of the active site).27 These structures
provide new insight regarding inhibitor binding interactions in the L1 and L2 pockets, as well as
the role of aromatic or heteroaromatic linkers, including benzyl, oxazole, and 3-vinylpyrrole
moieties, as they position capping groups in the outer active site cleft.
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Results and Discussion
Chemistry. Newly synthesized compounds 3–7 were prepared as summarized in
Schemes 1–5. Detailed synthetic procedures are described in the Experimental Section. The
IC50 value of each inhibitor was determined as outlined in the Experimental Section and
recorded in Figure 1.
The synthesis of N-(4-(hydroxycarbamoyl)benzyl)-2-(trifluoromethyl)nicotinamide (3)
was accomplished as summarized in Scheme 1:
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Scheme 1
O
Cl
O
H2N
6
7
O
O
O
OH
O
Na2CO3, dioxane:H2O(1:1)
Over night
N
H
O
H
N
O
+
Fmoc NH
O
T3P, DiPEA, CH2Cl2
0 oC-RT
Fmoc
8
H2N
OH
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9
9
O
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OH
50% T3P in CH2Cl2, DiPEA,
CH2Cl2, 0 oC, 1h
O
O
O
N
CF3
N
N
O
30%Piperidine , Methanol
RT, 1h
H
H
H
H2N
O
N
N
CF3
O
O
10
11
O
4N HCl in Dioxane
MeOH, 0 oC-RT, 4h
N
H
H
N
N
CF3
OH
O
3
The synthesis of 4-(((6,6-dimethylbicyclo[3.1.1]heptan-2-yl)amino)methyl)-N-
hydroxybenzamide (4) was achieved in two steps as summarized in Scheme 2:
Scheme 2
The synthesis of benzyl ((S)-1-(((S)-1-((4-(hydroxycarbamoyl)benzyl)amino)-1-
oxopropan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (5) was accomplished as
summarized in Scheme 3:
Scheme 3
O
O
H
N
O
O
O
TFA/CH2Cl2
0 oC- RT, 2h
H
O
T3P, DiPEA, CH2Cl2
0 oC, 1h
N
O
N
OH
O
N
O
H
OH
O
O
O
N
H
+
O
H
NH2
O
13
14
O
O
O
O
H
H
4N HCl in Dioxane
0 oC-RT
N
N
T3P, DiPEA, CH2Cl2 , 0 oC-RT
O
N
N
O
N
N
H
H
H
H
H
H
O
N
O
N
OH
O
O
10
5
O
15
O
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The synthesis of benzyl (2-((2-((4-(hydroxycarbamoyl)benzyl)amino)-2-oxoethyl)amino)-
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2-oxoethyl)-carbamate (6) was accomplished as summarized in Scheme 4:
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Scheme 4
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O
O
O
O
H
O
H
OH
LiOH.H2
O
N
T3P, DiPEA, CH2Cl2
0 oC, 1h
N
O
N
O
N
O
H2N
HCl
+
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O
N
OH
H
O
H
H
O
O
O
MeOH:H2O (5:1), RT, 6h
17
16
O
O
O
O
O
O
O
H
H
H
N
O
N
N
N
4N HCl in Dioxane
0 oC-RT
N
OH
+
O
N
N
O
O
N
N
H
T3P, DiPEA, CH2Cl2
0 oC-RT
H
N
H
H
H
H
H2N
O
H
H
O
O
O
N
O
O
O
H
O
O
10
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6
The synthesis of benzyl ((S)-1-(((S)-1-((3-(hydroxycarbamoyl)benzyl)amino)-1-
oxopropan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (7) was accomplished using 14 as
prepared in the synthesis of 5, outlined below in Scheme 5:
Scheme 5
NH2
O
O
O
O
O
O
O
O
O
T3P, DiPEA, CH2Cl2
0 oC-RT
LiOH, THF:MeOH:H2O (5:5:1)
OH
RT, 6h
H
N
H
N
H
+
N
O
O
N
N
OH
O
N
N
O
N
OH
H
H
H
H
H
O
O
O
20
19
14
O
O
H2N
O
O
O
O
O
O
O
H
N
H
4N HCl in Dioxane, MeOH
0 oC-RT, 4h
O
O
N
OH
N
N
N
O
N
N
N
H
H
H
H
H
H
O
O
50% T3P in CH2Cl2, DiPEA,
CH2Cl2,0 oC, 1h
21
7
X-ray crystal structures of HDAC6 CD2–inhibitor complexes. The 2.30 Å-resolution
crystal structure of the HDAC6 CD2–1 complex contains 2 monomers in the asymmetric unit,
and the inhibitor binding mode is identical in each monomer. The inhibitor hydroxamate group
chelates the catalytic Zn2+ ion with bidentate coordination geometry (Figure 2). The Zn2+-bound
hydroxamate C=O group also accepts a hydrogen bond from Y745, and the Zn2+-bound
hydroxamate N–O– group accepts a hydrogen bond from H573. The hydroxamate NH group
donates a hydrogen bond to H574.
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Figure 2. Polder omit map of the HDAC6 CD2-1 complex (PDB 6PZR; monomer A, contoured at 3.0 σ).
Atoms are color-coded as follows: C = light blue (monomer A) or wheat (inhibitor), N = blue, O = red, S =
yellow, Zn2+ =gray sphere, and I– = larger sand spheres. Metal coordination and hydrogen bond
interactions are indicated by solid and dashed black lines, respectively. Locations of the L1 and L2
pockets are indicated.
The 3-vinylpyrrole linker is sandwiched between F583 and F643, making favorable offset
aromatic  interactions. To achieve this interaction, the  systems of the hydroxamate and the
3-vinylpyrrole moiety of 1 are rotated on average 85° away from planarity and hence not in
resonance with each other.
The inhibitor capping group is oriented toward the L2 pocket instead of the L1 pocket. To
date, the only other inhibitor with a capping group that binds in the L2 pocket is one containing a
bifurcated capping group, such that one substituent binds in the L1 pocket and the other
substituent binds in the L2 pocket (Figure 1b).33 Polar residues in the L2 pocket include N530
and N645, as well as S531 near the interface of the L2 and L1 pockets, but these residues do
not form hydrogen bonds with the inhibitor capping group.
Unexpectedly, two iodide ions from the crystallization buffer bind in the active site and
flank the bound inhibitor (Figure 2). Each iodide ion is quite large (effective ionic radius = 2.20
Å) and makes a number of contacts with the protein and inhibitor. One iodide ion accepts a
hydrogen bond from H614 (average I---N separation = 3.7 Å) and makes van der Waals
contacts with F643, L712, and the phenylsulfone moiety in the capping group of the inhibitor.
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The other iodide ion accepts a hydrogen bond from H463 (average I---N separation = 3.7 Å) and
makes van der Waals contacts with S531, I532, F533, and F583. These interactions are
consistent with the intermolecular interactions of iodide ions classified in a survey of more than
350 iodide binding sites in refined protein structures – negatively charged iodide ions often bind
in hydrophobic patches, adjacent to aromatic residues, while also capable of making hydrogen
bond interactions.36
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The 1.75 Å-resolution crystal structure of the HDAC6 CD2–2 complex contains 2
monomers in the asymmetric unit, and the inhibitor binding mode is identical in each monomer.
The inhibitor hydroxamate group chelates the catalytic Zn2+ ion with bidentate coordination
geometry in similar fashion to that observed for the binding of 1, making identical metal
coordination and hydrogen bond interactions (Figure 3). Unique to 2 is the heterocyclic oxazole
linker, which makes favorable offset aromatic  interactions with F583 and F643. Because of
the geometry and orientation of the 5-membered ring of the oxazole moiety, the bromophenyl
capping group is oriented toward solution and does not fully interact with either the L1 or the L2
pocket. Accordingly, a 5-membered heteroaromatic ring linker connected directly to the Zn2+-
bound hydroxamate does not appear to be ideal for targeting capping group binding in these
pockets.
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Figure 3. Polder omit map of the HDAC6 CD2–2 complex (PDB 6Q0Z; monomer A, contoured at 4.0 σ).
Atoms are color-coded as follows: C = light blue (monomer A), dark gray (symmetry mate) or wheat
(inhibitor), N = blue, O = red, Br = magenta, and Zn2+ = gray sphere. Metal coordination and hydrogen
bond interactions are indicated by solid and dashed black lines, respectively. Locations of the L1 and L2
pockets are indicated.
The  systems of the hydroxamate moiety and the oxazole moiety of 2 are not coplanar,
and hence not in resonance with each other: the corresponding average N-C-C-C dihedral
angle is 50°. However, the oxazole ring and the bromophenyl rings are, on average, within 14°
of planarity. Thus, these aromatic rings are in resonance with one another and bind as a nearly
rigid unit. The bromine atom of the bromophenyl capping group is oriented toward solvent but
does not contact symmetry mate residues T780 or N784.
The 1.50 Å-resolution crystal structure of the HDAC6 CD2–3 complex contains 2
monomers in the asymmetric unit. Unexpectedly, the inhibitor binding mode is different in
monomers A and B. Hydroxamate-Zn2+ coordination is monodentate in monomer A: the
hydroxamate N–O– group coordinates to Zn2+ and accepts a hydrogen bond from Y745, and the
hydroxamate C=O group accepts a hydrogen bond from the Zn2+-bound water molecule, which
in turn hydrogen bonds to H573 and H574 (Figure 4a). In monomer B, hydroxamate-Zn2+
coordination is bidentate, with the hydroxamate C=O and N–O– groups chelating the metal ion
(Figure 4b).
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Figure 4. (a) Polder omit map of the HDAC6 CD2–3 complex (PDB 6PZO; monomer A, contoured at 3.5
σ). Atoms are color-coded as follows: C = light blue (monomer A), dark gray (symmetry mate) or wheat
(inhibitor), N = blue, O = red, F = magenta, Zn2+ =gray sphere, and solvent = small red spheres. Metal
coordination and hydrogen bond interactions are indicated by solid and dashed black lines, respectively.
A red dashed line indicates a possible n→* interaction between a C-F group and the amide carbonyl. (b)
Polder omit map of the HDAC6 CD2–3 complex (PDB 6PZO; monomer B, contoured at 3.5 σ). Atoms are
color-coded as follows: C = light blue (monomer B), dark gray (symmetry mate) or wheat (inhibitor), N =
blue, O = red, F = magenta, Zn2+ = gray sphere, and solvent = small red spheres. Metal coordination and
hydrogen bond interactions are indicated by solid and dashed black lines, respectively. Locations of the
L1 and L2 pockets are indicated.
In both monomers, the benzyl linker is sandwiched between F583 and F643, making
favorable offset aromatic  interactions. However, the  systems of the hydroxamate and the
benzyl moieties of 3 are oriented differently in each monomer. In monomer A, they are within 8°
of planarity. In monomer B, they are rotated 36° away from planarity. These conformational
differences may arise from the different Zn2+ coordination modes of the inhibitor in each
monomer.
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Also different in each monomer is the orientation of the amide connecting the linker to
the trifluoromethylpyridine capping group, although the capping group occupies essentially the
same location in the L1 pocket. There, the nitrogen of the pyridine ring makes a water-mediated
hydrogen bond with R660 of a symmetry mate. The fluorine atoms of the trifluoromethyl moiety
make no hydrogen bond interactions with protein residues, but they do make van der Waals
interactions with H463, P464, and L712 in the L1 pocket. Despite the ability of electronegative
aromatic ring substituents to influence intermolecular interactions,37,38 the trifluoromethylpyridine
ring of 3 does not appear to make any interactions that would benefit from such electrostatic
modulation.
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Interestingly, one of the fluorine atoms of the trifluoromethyl moiety appears to make an
n→* interaction with the amide carbonyl group of the inhibitor. The F---C distance is 2.7 Å and
the F---C=O angle is 86° in monomer A; the corresponding values are 2.8 Å and 118° in
monomer B. While this interaction is likely to be weak, it may contribute to the orientation of the
trifluoromethylpyridine capping group.
The 1.79 Å-resolution crystal structure of the HDAC6 CD2–4 complex contains one
monomer in the asymmetric unit. Unique to the structure of this inhibitor is the monoterpene
capping group, a pinene moiety. This is the first crystal structure of an HDAC–inhibitor complex
containing a terpene derivative. The structural and stereochemical diversity of terpenoid natural
products (which currently number more than 80,000)39 points toward vast chemodiversity readily
incorporated in the design of new inhibitor capping groups. The hydroxamate group of 4
coordinates to Zn2+ in monodentate fashion, and the benzyl linker makes favorable offset 
interactions with F643 and F583 (Figure 5). The secondary amino group of the linker engages in
a hydrogen bond network with S531 through two intervening water molecules, and the nonpolar
pinene moiety sits in the L1 pocket and makes van der Waals interactions with H463, P464, and
L712.
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Figure 5. Polder omit map of the HDAC6 CD2–4 complex (PDB 6PZS; contoured at 4.0 σ). Atoms are
color-coded as follows: C = light blue (HDAC6 CD2) or wheat (inhibitor), N = blue, O = red, Zn2+ = gray
sphere, and solvent = small red spheres. Metal coordination and hydrogen bond interactions are indicated
by solid and dashed black lines, respectively. Locations of the L1 and L2 pockets are indicated.
The 1.74 Å-resolution crystal structure of the HDAC6 CD2–5 complex contains 2
monomers in the asymmetric unit. Electron density corresponding to bidentate hydroxamate-
Zn2+ coordination interactions is observed in both monomers, although the average Zn2+---O
separation of 2.6 Å for the hydroxamate C=O group is somewhat long to be considered inner
sphere coordination. The benzyl linker of 5 makes favorable offset aromatic  interactions
with F643 and F583.
Inhibitor 5 is one of the largest inhibitors studied in complex with HDAC6 CD2 and
highlights the feasibility of designing a peptide to bind to a new protein landscape. The capping
group of 5 is the N-protected dipeptide Cbz-Leu-Ala (Cbz = carbobenzyloxy), and this peptide
sequence maps out three different conformations as it interacts with the L1 pocket.
In monomer A, the alanine residue of the Cbz-Leu-Ala moiety adopts a single
conformation, but the Cbz-Leu segment adopts two different conformations as indicated by
somewhat noisy but nonetheless interpretable electron density (Figure 6a). In one conformation,
the Cbz phenyl group makes a weakly polar C–H hydrogen bond with D460. In the alternate
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conformation, inhibitor binding is stabilized by water-mediated hydrogen bonds with H614 and
the backbone NH group of F643.
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Figure 6. (a) Polder omit map of the HDAC6 CD2–5 complex (PDB 6PZU; monomer A, contoured at 3.0
σ). Atoms are color-coded as follows: C = light blue (monomer A) or wheat (inhibitor), N = blue, O = red,
Zn2+ = gray sphere, and solvent = small red spheres. Metal coordination and hydrogen bond interactions
are indicated by solid and dashed black lines, respectively. (b) Polder omit map of the HDAC6 CD2–5
complex (PDB 6PZU; monomer B, contoured at 3.0 σ). Atoms are color-coded as follows: C = light blue
(monomer B) or wheat (inhibitor), N = blue, O = red, Zn2+ = gray sphere, and solvent = small red spheres.
Metal coordination and hydrogen bond interactions are indicated by solid and dashed black lines,
respectively. Locations of the L1 and L2 pockets are indicated.
In monomer B, the inhibitor conformation is such that the Cbz-Leu-Ala moiety extends
somewhat further into the L1 pocket, although electron density for the Cbz protecting group is
noisy (Figure 6b). This presumably signifies molecular disorder. Inhibitor binding is stabilized by
water-mediated hydrogen bonds with H614 and the backbone NH group of F643. No other
hydrogen bond interactions are observed between the inhibitor and enzyme residues. It is
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interesting that the Cbz-Leu-Ala capping group is reasonably ordered despite the lack of direct
enzyme-inhibitor hydrogen bonds in this complex.
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To further explore structure-affinity relationships for inhibitors bearing dipeptide capping
groups related to that of inhibitor 5, compounds 6 and 7 were synthesized and compared to
compound 5 for inhibitory activity (Figure 1). Compound 6 contained a Cbz-Gly-Gly capping
group and hence lacked substitution at the amino acid Cα atoms, but was otherwise isosteric
with compound 5. Notably, compound 6 was essentially equipotent to 5 with respect to HDAC6
CD2 inhibitory activity. Thus, the amino acid side chains of 5 do not contribute substantially to
inhibitory potency. In compound 7, the Cbz-Leu-Ala capping group is identical to that of 5, but it
is substituted at the meta position of the phenyl ring. This structural variation resulted in more
than a 10-fold loss of inhibitory potency against HDAC6 CD2, thereby confirming the strict steric
requirements for entry into the zinc binding group pocket with a para-substituted aromatic linker
as found in compounds 5 and 6.
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Structural determinants of enzyme-Inhibitor affinity. The binding interactions
observed for all five enzyme-inhibitor complexes in this study are highly informative regarding
intermolecular interactions of the linker segment and the capping group of the inhibitor in the
active site of HDAC6 CD2. It is notable that without exception, the aromatic ring linker of each
inhibitor binds in an aromatic crevice formed by the side chains of F583 and F643. Regardless
of the ring size (5- or 6-membered) or composition (phenyl, oxazole, 3-vinylpyrrole), the
aromatic linker of the inhibitor makes favorable offset  interactions in the aromatic crevice.
The linker moiety of each inhibitor plays an important role in orienting the capping group
in the outer active site cleft. As such, it is intriguing that the 3-vinylpyrrole linker of inhibitor 1
directs the capping group toward the L2 pocket, where it displaces solvent and makes van der
Waals interactions with various residues lining the pocket. Inhibitor capping groups rarely bind in
this region in crystal structures reported to date, and until now has only been observed for
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inhibitors with bifurcated, sterically bulky capping groups.33 There are some polar residues in or
near this pocket that could be targeted for hydrogen bond interactions, such as S531. The side
chain of this residue is at the edge of the L2 pocket and hydrogen bonds to the backbone NH
group of acetyllysine substrates.27 S531 is unique to HDAC6 CD2, and inhibitors that engage in
hydrogen bond interactions with this residue tend to exhibit selectivity for binding to HDAC6
CD2.29,31,33,34 The lack of such an interaction in the binding of 1, also known as the cancer
chemotherapy drug Resminostat, is consistent with the broad isozyme selectivity observed for
this inhibitor, i.e., it is a pan-HDAC inhibitor.40
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The binding of two iodide ions along with inhibitor 1 in the HDAC6 CD2 active site results
from the crystallization conditions used to prepare crystals of the enzyme-inhibitor complex. It is
not clear whether iodide binding is a cause or a consequence of the binding conformation of 1.
However, in the structure of the HDAC6 CD1–1 complex (PDB 6UOB), recently determined in
the absence of iodide ions, the inhibitor capping group adopts an alternate conformation that
places it in the L1 pocket. This could suggest that iodide binding influences the binding
conformation of 1 as observed in the HDAC6 CD2 active site.
In the HDAC6 CD2–1 complex, each iodide ion is stabilized by a single hydrogen bond
and an array of van der Waals interactions with the steric bulk of surrounding protein residues
and inhibitor atoms (Figure 2). It is interesting to speculate that iodide ions could play some role
in facilitating the binding of inhibitors with suitable steric bulk in their linker segments and
capping groups. If so, this might enhance HDAC6 CD2 inhibition in iodide-rich tissues such as
the thyroid. The pan-HDAC inhibitor Vorinostat does not exhibit a therapeutic effect in patients
diagnosed with radioiodine-refractory thyroid cancer,41 but then again Vorinostat contains a
slender hydrocarbon linker segment and a smaller capping group in comparison with inhibitor 1.
From the array of HDAC6 CD2–inhibitor binding conformations observed to date, it
appears that the L1 pocket is preferentially occupied by the capping groups of
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phenylhydroxamate inhibitors, unless the binding of other species, e.g., iodide ions, leads to the
binding of the capping group in the L2 pocket. Even so, inhibitor capping groups can make
favorable van der Waals interactions in either pocket. Interactions in both pockets are
simultaneously exploited by the phenylhydroxamate inhibitor RTS-V5, which bears a bifurcated
capping group such that one substituent binds in the L2 pocket and the other substituent binds
in the L1 pocket (Figure 1b).33 Since the three-dimensional contours of the L1 and L2 pockets at
the mouth of the active site can vary from one HDAC isozyme to another, the incorporation of
bifurcated capping groups in phenylhydroxamate inhibitors may serve as a useful molecular
strategy for the improvement of isozyme selectivity as well as affinity.
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In addition to alternative capping group binding conformations in the HDAC6 CD2 active
site, it is interesting to consider the utility of halogen atoms as incorporated into various inhibitor
designs, such as 2 and 3. For instance, the para-bromophenyl capping group of 2 contributes to
selectivity for HDAC6 CD2 inhibition over other HDAC isozymes, and the substitution of the
para-bromine atom with a chlorine or a fluorine atom enhances selectivity for HDAC6 CD2 over
HDAC8; substitution with a hydrogen atom decreases selectivity in isosteres of 2.42 However,
the crystal structure of the HDAC6 CD2–2 complex (Figure 3) reveals that the para-bromo
group is oriented out toward solvent, making no specific interactions in the outer active site cleft.
This could suggest that the effect of the para-substituted halogen atom on the capping group of
2 and its derivatives arises from electrostatic inductive effects, perhaps enhanced by the near
planarity of the  systems of the para-bromophenyl ring and the oxazole ring. The more
electron-withdrawing this linker-capping group combination becomes by substitution of
increasingly electronegative halogen atoms for the para-bromo group, the more the anionic
Zn2+-bound hydroxamate moiety will be stabilized.
Consider, too, the trifluoromethyl group of inhibitor 3. Here, the fluorine atoms make van
der Waals interactions with residues in the L1 pocket, and these interactions presumably
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contribute to potent inhibitory activity. Additionally, the trifluoromethyl group serves as a steric
protrusion that locks the trifluoropyridyl ring in a conformation that is 125° on average out of the
plane of the adjacent amide linker (Figure 4). Consequently, the  systems of the amide group
and the pyridyl ring are not in resonance with each other, so the ability of the trifluoromethyl
group to inductively stabilize the Zn2+-bound hydroxamate moiety will be minimal. However, the
trifluoromethyl group is perfectly pre-oriented for making van der Waals interactions in the L1
pocket.
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The pinene capping group of inhibitor 4 binds in the L1 pocket, which appears to be the
preferred binding conformation for a phenylhydroxamate inhibitor bearing a single capping
group. The pinene capping group is a unique feature of this inhibitor and to our knowledge
represents the first use of a terpene as an inhibitor capping group. Although the inhibitory
potency of 4 is very good (although relatively modest with IC50 = 149 nM), the impressive
chemodiversity represented by tens of thousands of terpene natural products suggests that
there is vast untapped potential for molecular diversification of HDAC inhibitor capping groups
using terpene derivatives for the improvement of inhibitor affinity and selectivity.39
The peptide-based capping group of inhibitor 5 exhibits some variability in its
conformation as it binds in the L1 pocket, but it is notable that the Cbz-Leu-Ala moiety is
reasonably ordered even though the dipeptide did not naturally evolve to bind to this protein
surface. In a study of para-substituted phenylsulfonamide inhibitors bound in the active site of
carbonic anhydrase II, the use of flexible glycine residues resulted in mainly disordered binding
of the para-substituted Gly-Gly-Gly-Bz moiety (Bz = benzyl).43 Thus, it is feasible for a de novo
designed peptide to achieve binding when anchored to a protein surface of choice, but the
peptide is more likely to bind in ordered fashion if its glycine content is limited. It is interesting
that despite lacking amino acid side chains in the Cbz-Gly-Gly capping group, compound 6 is
essentially equipotent to inhibitor 5 (Figure 1). This is consistent with the crystal structure of the
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HDAC6 CD2–5 complex, which shows that the amino acid side chains of the Cbz-Leu-Ala
capping group do not make substantial interactions in the active site. The 20-fold loss of
inhibitory potency against HDAC6 CD2 measured for compound 7 indicates that the steric
requirements for optimal binding in the HDAC6 CD2 active site favor a para-substituted over a
meta-substituted phenyl linker group.
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Conclusions
The current study demonstrates how a variety of aromatic and heteroaromatic linkers
direct inhibitor capping group interactions toward different regions of the HDAC6 CD2 active site
spanning the L1 and the L2 pockets. Ideal aromatic linkers for binding to HDAC6 CD2 are para-
substituted phenylhydroxamates, i.e., inhibitors in which the capping group extends out away
from the hydroxamate group in linear fashion. Inhibitors in which the capping group extends out
at an angle, such as that found in the oxazole inhibitor 2 or the meta-substituted
phenylhydroxamate inhibitor 7, appear to be less well-suited for capturing affinity interactions at
the mouth of the active site in the L1 or L2 pockets.
Phenylhydroxamate inhibitors generally bind with their capping groups oriented into the
L1 pocket, but occasional examples exist where capping groups bind in the L2 pocket, e.g., as
observed for inhibitor 1 in the presence of iodide ions. Both the L1 and L2 pockets can be
simultaneously occupied by the substituents of bifurcated capping groups to enhance affinity
and selectivity, as exemplified by the binding of RTS-V5 (Figure 1b).33 Intermolecular
interactions of inhibitor capping groups provide a means of structural diversification that can be
tailored to a specific HDAC isozyme, since the three-dimensional contour of the mouth of the
active site tends to vary from one isozyme to another. Structural studies of HDAC6 CD2-
inhibitor complexes continue to reveal fundamental structural features underlying enzyme-
inhibitor affinity and selectivity, and thus promise to inform current efforts in medicinal chemistry
to optimize the properties of HDAC inhibitors for use in biology and medicine.
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Experimental Section
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Reagents. All buffers and chemicals were purchased from Fisher, Millipore Sigma, or
Hampton Research and used without further purification. The purity of each final tested
compound is as follows: compound 1, as purchased from APExBIO, purity = 98%; compound 2,
as previously reported,42 purity > 95%; compounds 3–7, as determined using LC-MS, purity >
95% (Supporting Information, Figure S1).
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N-(4-(hydroxycarbamoyl)benzyl)-2-(trifluoromethyl)nicotinamide (3). To a stirred
solution of 11 (50 mg; 0.12 mmol) in 4 mL of methanol at 0 oC was added dropwise 0.5 mL of 4
N HCl in dioxane. The reaction mixture was slowly warmed to room temperature and stirred for
an additional 4 h. Completion of the reaction was confirmed by LC-MS. The solvent was then
evaporated and dried under high vacuum to afford compound 3 as a light-yellow solid (32 mg;
0.094 mmol, 80%). Product was confirmed by 1H NMR and MS.
NMR: 1H NMR (400 MHz, MeOD-D4): δ 8.75 (d, J = 4.5 Hz, 1H), 8.00 (d, J = 7.8 Hz, 1H), 7.81 –
7.65 (m, 3H), 7.48 (d, J = 8.1 Hz, 2H), 4.60 (s, 2H).
Mass: m/z calc’d for C15H13F3N3O3 [M+H]+; 340.1; found 340.1.
4-(((6,6-dimethylbicyclo[3.1.1]heptan-2-yl)amino)methyl)-N-hydroxybenzamide (4).
To a solution of 12 (73 mg; 0.24 mmol, 1 eq) in 2 mL 1:1 THF/MeOH was added 0.50 mL
hydroxylamine (50% aq, 0.25 g; 3.6 mmol, 15 eq) and aqueous NaOH (5.0 M, 0.10 mL; 0.50
mmol, 2 eq). The reaction mixture was stirred for 3 h at room temperature. The product was
purified by preparative reversed-phase HPLC with a gradient of 0.025% TFA in water and
methanol. Upon drying in vacuo product 4 (29 mg; 0.096 mmol, 40%) was obtained as a white
solid. Product was confirmed by 1H NMR and MS.
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1H NMR (DMSO-d6): δ 11.3 (s, 1H), 9.1 (s, 1H), 9.0 (s, 2H), 7.78 (d, 2H), 7.54 (d, 2H), 4.2 (m,
2H), 3.34 (s, 2H), 2.91 (s, 2H), 2.58-2.23 (m, 7H), 2.03-1.71 (m, 5H), 1.73-1.38 (m, 4H), 1.59-
1.37 (m, 1H), 1.23-1.07 (m, 3H), 0.99-0.72 (m, 4H).
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Mass: m/z calc’d for C18H26N2O2 [M+H]+, 303.2; found 303.2.
Benzyl ((S)-1-(((S)-1-((4-(hydroxycarbamoyl)benzyl)amino)-1-oxopropan-2-
yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (5). To a stirred solution of 15 (80 mg; 0.12
mmol) in 4 mL of methanol at 0 oC was added 0.5 mL of 4 N HCl in dioxane dropwise slowly.
The reaction mixture was warmed to room temperature and stirred for an additional 4 h.
Completion of the reaction was confirmed by LC-MS. The solvent was evaporated to yield the
crude product, which was purified by preparative reverse phase HPLC to afford 5 (30 mg; 0.061
mmol, 51%). Product was confirmed by 1H NMR and MS.
1H NMR (400 MHz, DMSO): δ 11.59 (s, 1H), 11.17 (s, 1H), 8.40 (t, J = 6.2 Hz, 1H), 8.03 (d, J =
7.3 Hz, 1H), 7.70 (t, J = 7.7 Hz, 2H), 7.44 (d, J = 8.3 Hz, 1H), 7.41 – 7.17 (m, 7H), 5.13 – 4.88
(m,2H), 4.42 – 4.22 (m, 2H), 4.14 – 3.94 (m, 1H), 3.83 – 3.63 (m, 1H), 3.63 – 3.34 (m, 1H), 1.80
– 1.34 (m, 1H), 1.31 – 1.15 (m, 4H), 0.89 – 0.70 (m, 6H).
Mass: m/z calc’d for C25H33N4O6 [M+H]+; 285.2; found 285.3.
Benzyl (2-((2-((4-(hydroxycarbamoyl)benzyl)amino)-2-oxoethyl)amino)-2-oxoethyl)-
carbamate (6). To a stirred solution of 18 (120 mg; 0.24 mmol) in 3 mL of methanol at 0 oC was
added 0.5 mL of 4 N HCl in dioxane dropwise. The reaction mixture was slowly warmed to room
temperature and stirred for 4 h. Completion of the reaction was confirmed by LC-MS. Solvent
was evaporated to yield crude product, which was purified by reverse phase preparative HPLC
to afford 6 (40 mg, 0.096, 40%). Product was confirmed by 1H NMR and MS.
1H NMR (400 MHz, MeOD): δ 7.71 (t, J = 9.0 Hz, 2H), 7.38 (d, J = 8.1 Hz, 2H), 7.34 – 7.25 (m,
5H), 5.03 (s, 2H), 4.44 (s, 2H), 3.92 (s, 2H), 3.81 (s, 2H).
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Mass: m/z calc’d for C20H22N4NaO6 [M+Na]+; 437.1; found 437.2.
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Benzyl ((S)-1-(((S)-1-((3-(hydroxycarbamoyl)benzyl)amino)-1-oxopropan-2-
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yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (7). To a stirred solution of 21 (150 mg; 0.26
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mmol) in 4 mL of methanol at 0 oC was added 0.5 mL of 4 N HCl in dioxane dropwise. The
reaction mixture was slowly warmed to room temperature and stirred for 4 h. Completion of the
reaction was confirmed by LC-MS. Solvent was evaporated to yield crude product, which was
purified by reverse phase preparative HPLC to afford 7 (45 mg; 0.092 mmol, 35%). Product was
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confirmed by 1H NMR and MS.
1H NMR (400 MHz, DMSO): δ 11.18 (s, 1H), 8.40 (t, J = 5.9 Hz, 1H), 8.03 (d, J = 7.2 Hz, 1H),
7.66 (s, 1H), 7.60 (d, J = 4.0 Hz, 1H), 7.44 – 7.27 (m, 7H), 5.08 – 4.94 (m, 2H), 4.30 (dd, J =
12.7, 6.3 Hz, 3H), 4.05 (dd, J = 15.1, 8.1 Hz, 1H), 1.70 – 1.54 (m, 1H), 1.44 (dd, J = 18.0, 10.4
Hz, 2H), 1.25 (d, J = 6.9 Hz, 3H), 0.84 (dd, J = 12.5, 6.4 Hz, 6H).
Mass: m/z calc’d for C25H33N4O6 [M+H]+; 485.2; found 485.3.
4-((((9H-Fluoren-9-yl)methoxy)carbonylamino)methyl)benzoic acid (8). To a stirred
solution of 4-(aminomethyl) benzoic acid (2 g; 13.23 mmol) in 10 mL (1:1 dioxane-H2O) at 0 oC
were added Na2CO3 (4.19 g; 39.7 mmol) and Fmoc-Cl (3.59 g; 13.23 mmol) simultaneously.
The reaction mixture was slowly brought to room temperature and stirred for 12 h. Completion
of the reaction was confirmed by liquid chromatography-mass spectrometry (LC-MS). The
reaction mixture was then cooled to 0 oC in an ice bath and acidified with 1 N HCl to pH 5. The
precipitated solid was filtered and washed with H2O and hexane, and then dried under high
vacuum to afford 8 (4.4 g; 11.78 mmol, 90%) as a white solid. Product was confirmed by 1H
NMR and MS.
1H NMR (400 MHz, DMSO): δ 12.87 (s, 1H), 8.03 – 7.78 (m, 3H), 7.69 (t, J = 10.3 Hz, 2H), 7.49
– 7.37 (m, 2H), 7.33 (t, J = 8.0 Hz, 3H), 4.38 (d, J = 6.8 Hz, 2H), 4.24 (m, 3H).
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Mass: m/z calc’d for C23H18NO4 [M-H]+; 372.1; found 372.3.
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(9H-Fluoren-9-yl) methyl 4-(tetrahydro-2H-pyran-2-yloxycarbamoyl)-
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benzylcarbamate (9). To a stirred solution of 8 (1.0 g; 2.55 mmol) and O-(tetrahydro-2H-pyran-
2-yl)hydroxylamine (328 mg; 2.8 mmol) in 20 mL of dry CH2Cl2 at 0 oC were added diisopropyl
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ethyl amine (0.58 mL; 3.31 mmol) and T3P 50% solution in CH2Cl2 by weight (2.10 g; 3.31
mmol) dropwise simultaneously. The reaction mixture was allowed to stir for 30 min at 0 oC.
Completion of the reaction was confirmed by LC-MS. The reaction mixture was quenched with
cold water and the product was extracted with CH2Cl2 and dried over anhydrous Na2SO4. The
solvent was evaporated under reduced pressure and the crude product was purified by flash
column chromatography to afford 9 (1.02 g; 2.16 mmol, 85%). Product was confirmed by 1H
NMR and MS.
1H NMR (400 MHz, DMSO): δ 11.59 (s, 1H), 7.99 – 7.84 (m, 3H), 7.71 (dd, J = 7.4, 5.6 Hz, 4H),
7.42 (t, J = 7.4 Hz, 2H), 7.30 (dt, J = 20.7, 10.0 Hz, 3H), 4.99 (s, 1H), 4.35 (dd, J = 10.7, 6.0 Hz,
2H), 4.23 (t, J = 7.0 Hz, 3H), 4.06 (s, 1H), 3.51 (t, J = 12.3 Hz, 1H), 1.67 (d, J = 42.9 Hz, 3H),
1.55 (s, 3H).
Mass: m/z calc’d for C28H28N2NaO5 [M+Na]+; 495.2; found 495.3.
4-(Aminomethyl)-N-(tetrahydro-2H-pyran-2-yloxy)benzamide (10). To a stirred
solution of 9 (700 mg; 1.48 mmol) in 20 mL of MeOH at room temperature was added 10 mL of
piperidine all at once and the reaction mixture was stirred for 1 h. Completion of the reaction
was confirmed by LC-MS. The volatiles were evaporated under reduced pressure to yield the
crude product, which was purified by flash column chromatography to afford the desired amine
10 (343 mg; 1.37 mmol, 92%) as a white solid. Product was confirmed by 1H NMR and MS.
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1H NMR (400 MHz, CDCl3): δ 7.73 (d, J = 8.2 Hz, 2H), 7.38 (d, J = 8.1 Hz, 2H), 5.08 (d, J = 2.9
Hz, 1H), 4.07 – 3.95 (m, 1H), 3.93 (s, 2H), 3.71 – 3.62 (m, 1H), 1.99 – 1.76 (m, 3H), 1.76 – 1.52
(m, 3H).
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Mass: m/z calc’d for C13H18N2NaO3 [M+Na]+; 273.1; found 273.2.
N-(4-(tetrahydro-2H-pyran-2-yloxycarbamoyl)benzyl)-2-(trifluoromethyl)-
nicotinamide (11). To a stirred solution of 2-(trifluoromethyl)nicotinic acid (50 mg; 0.2 mmol)
and 10 (38 mg; 0.2 mmol) in 5 mL of dry CH2Cl2 at 0 oC was added diisopropyl ethyl amine (103
mg; 0.8 mmol) and T3P 50% solution in CH2Cl2 by weight (82 mg; 0.26 mmol) simultaneously
dropwise. The reaction mixture was stirred for 1 h at 0 oC. Completion of the reaction was
confirmed by LC-MS. The reaction mixture was quenched with cold water and the product was
extracted with CH2Cl2 and then dried over anhydrous Na2SO4. The solvent was evaporated
under reduced pressure to yield the crude product, which was purified by flash column
chromatography to afford compound 11 (94 mg; 0.22 mmol, 85%). Product was confirmed by 1H
NMR and MS.
1H NMR (400 MHz, CDCl3): δ 8.90 (s, 1H), 8.75 (d, J = 4.6 Hz, 1H), 7.91 (d, J = 7.8 Hz, 1H),
7.70 (d, J = 7.7 Hz, 2H), 7.55 (dd, J = 7.8, 4.8 Hz, 1H), 7.39 (d, J = 7.8 Hz, 2H), 6.42 (s, 1H),
5.06 (s, 1H), 4.66 (d, J = 5.8 Hz, 2H), 4.05 – 3.91 (m, 1H), 3.69 – 3.59 (m, 1H), 1.86 (dd, J =
18.5, 11.2 Hz, 3H), 1.74 – 1.52 (m, 3H).
Mass: m/z calc’d for C20H20F3N3NaO4 [M+Na]+; 446.1; found 446.2.
Methyl 4-(((6,6-dimethylbicyclo[3.1.1]heptan-2-yl)amino)methyl)benzoate (12). A
solution of methyl 4-formylbenzoate (0.306 mg; 1.86 mmol, mole 1.1 eq.) and (–)-cis-
myrtanylamine (247 mg; 1.62 mmol, 1 eq.) in 5 mL methanol was stirred for 1.5 h at room
temperature. Then, 175 mg sodium borohydride was added portion-wise and the reaction
mixture was stirred until no starting material remained as determined by thin layer
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chromatography (approximately 4 h). The mixture was concentrated in vacuo and partitioned
between ethyl acetate and water. The aqueous layer was extracted two more times with ethyl
acetate and the combined organic phases were dried over sodium sulfate. Solvent was
removed under reduced pressure and product 12 was purified by column chromatography,
affording 463 mg (1.54 mmol, 94%) of a yellowish oil. Product was confirmed by 1H NMR and
MS.
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1H NMR (chloroform-d): δ 8.0 (d, 2H), 7.6 (d, 2H), 4.17-3.77(m, 3H), 3.98 (s, 2H), 2.77-1.65 (m,
11H), 1.65-0.43 (m, 11H).
Mass: m/z calc’d for C19H27NO2 [M+H]+, 302.2; found 302.7.
(S)-tert-butyl 2-((S)-2-(benzyloxycarbonylamino)-4-methylpentanamido)propanoate
(13). To a stirred solution of (S)-2-(benzyloxycarbonylamino)-4-methylpentanoic acid (1.46 g;
5.5 mmol) and (S)-tert-butyl 2-aminopropanoate hydrochloride (1.0 g; 5.5 mmol) in 20 mL dry
CH2Cl2 at 0 oC were added diisopropyl ethyl amine (3.8 mL, 2.2 mmol) and T3P 50% solution in
CH2Cl2 by weight (5.45 g; 7.15 mmol) dropwise simultaneously. The reaction mixture was stirred
for an additional 1 h at 0 oC, and completion of the reaction was confirmed by LC-MS. The
reaction mixture was quenched with cold water and the product was extracted with CH2Cl2 and
dried over anhydrous Na2SO4. The solvent was removed under reduced pressure to provide the
crude reaction product, which was purified by flash column chromatography to afford 13 (1.94
mg; 4.94 mmol, 90%) as a white solid. Product was confirmed by 1H NMR and MS.
1H NMR (400 MHz, CDCl3): δ 7.43 – 7.28 (m, 5H), 6.46 (d, J = 6.6 Hz, 1H), 5.30 (s, 1H), 5.19 (t,
J = 13.3 Hz, 1H), 5.11 (s, 2H), 4.42 (p, J = 7.1 Hz, 1H), 4.18 (t, J = 14.8 Hz, 1H), 1.76 – 1.54 (m,
3H), 1.46 (s, 9H), 1.38 – 1.25 (m, 3H), 0.96 (t, J = 15.3 Hz, 6H).
Mass: m/z calc’d for C21H32N2NaO5 [M+Na]+; 415.2; found 415.2.
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(S)-2-((S)-2-(benzyloxycarbonylamino)-4-methylpentanamido)propanoic acid (14).
To a stirred solution of 13 (1.8 g; 4.59 mmol) in 30 mL CH2Cl2 at 0 oC was added 10 mL of TFA.
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The reaction mixture was then warmed to room temperature and stirred for 2 h. Completion of
the reaction was confirmed by LC-MS. Volatiles were evaporated under reduced pressure to
yield crude product which was co-distilled with 20 mL of toluene to afford 14 (1.5 g; 4.45 mmol,
98%) as a viscous liquid. Product was confirmed by 1H NMR and MS.
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1H NMR (400 MHz, CDCl3): δ 7.41 – 7.28 (m, 5H), 7.11 (s, 3H), 5.70 (d, J = 8.3 Hz, 1H), 5.19 –
5.01 (m, 2H), 4.62 – 4.46 (m, 1H), 4.32 (d, J = 5.9 Hz, 1H), 1.76 – 1.48 (m, 3H), 1.48 – 1.31 (m,
3H), 0.99 – 0.82 (m, 6H).
Mass: m/z calc’d for C17H24N2NaO5 [M+Na]+; 359.2; found 359.3.
Benzyl (2S)-4-methyl-1-oxo-1-((2S)-1-oxo-1-(4-(tetrahydro-2H-pyran-2-
yloxycarbamoyl)benzylamino)propan-2-ylamino)pentan-2-yl carbamate (15). To a stirred
solution of 14 (67 mg; 0.2 mmol) and 10 (50 mg; 2.0 mmol) in 3 mL of dry CH2Cl2 at 0 oC were
added diisopropyl ethyl amine (0.14 mL; 0.8 mmol) and T3P 50% solution in CH2Cl2 by weight
(165 mg; 2.6 mmol) simultaneously dropwise. The reaction mixture was stirred for an additional
1 h at 0 oC. Completion of the reaction was confirmed by LC-MS. The reaction mixture was
quenched with cold water. The product was extracted with CH2Cl2 and dried over anhydrous
Na2SO4. The solvent was evaporated under reduced pressure to yield the crude product, which
was purified by flash column chromatography to afford 15 (91 mg; 0.160 mmol, 80%) as
confirmed by 1H NMR and MS.
1H NMR (400 MHz, DMSO): δ 11.59 (s, 1H), 8.41 (t, J = 5.7 Hz, 1H), 8.03 (d, J = 7.3 Hz, 1H),
7.71 (d, J = 8.2 Hz, 2H), 7.44 (d, J = 8.2 Hz, 1H), 7.39 – 7.23 (m, 7H), 5.13 – 4.88 (m, 3H), 4.39
– 4.19 (m, 3H), 4.05 (dd, J = 14.1, 8.8 Hz, 2H), 3.69 – 3.45 (m, 2H), 1.68 (d, J = 30.2 Hz, 3H),
1.45 (dd, J = 11.1, 6.5 Hz, 2H), 1.24 (d, J = 6.9 Hz, 6H), 0.85 (t, J = 6.4 Hz, 6H).
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Mass: m/z calc’d for C30H40N4NaO7 [M+Na]+; 591.3; found 591.5.
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Methyl 2-(2-(benzyloxycarbonylamino)acetamido)acetate (16). To a stirred solution
of 2-(benzyloxycarbonylamino)acetic acid (1.0 g; 4.78 mmol) and methyl 2-aminoacetate
hydrochloride (600 mg; 4.78 mmol) in 20 mL dry CH2Cl2 at 0 oC were added diisopropyl ethyl
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amine (3.43 mL; 19.12 mmol) and T3P 50% solution in CH2Cl2 by weight (3.95 g; 6.21 mmol)
dropwise simultaneously. The reaction mixture was stirred for an additional 1 h at 0 oC, and
completion of the reaction was confirmed LC-MS. The reaction mixture was quenched with cold
water, and the product was extracted with CH2Cl2 and dried over anhydrous Na2SO4. The
solvent was removed under reduced pressure to provide the crude reaction product, which was
purified by flash column chromatography to afford 16 (1.18 g; 4.21 mmol, 88%) as a white solid.
Product was confirmed by 1H NMR and MS.
1H NMR (400 MHz, CDCl3): δ 7.45 – 7.29 (m, 5H), 6.61 (s, 1H), 5.51 (s, 1H), 5.13 (s, 2H), 4.05
(d, J = 5.2 Hz, 2H), 3.92 (d, J = 5.6 Hz, 2H).
Mass: m/z calc’d for C13H16N2NaO5 [M+Na]+; 303.1; found 303.1.
2-(2-(Benzyloxycarbonylamino)acetamido)acetic acid (17). To a stirred solution of 16
(1.18 g; 4.21 mmol) in 30 mL of MeOH and H2O (5:1) at room temperature was added
LiOH•H2O (707 mg; 16.84 mmol). The reaction mixture was stirred for 6 h. Completion of the
reaction was confirmed by thin layer chromatography. Volatiles were evaporated by reduced
pressure, and the crude product was acidified with 1 N HCl at 0 oC to obtain a white precipitate
which was filtered and dried under high vacuum to afford 17 (890 mg; 3.34 mmol, 80%). Product
was confirmed by 1H NMR and MS.
1H NMR (400 MHz, DMSO): δ 12.56 (s, 1H), 8.15 (t, J = 5.7 Hz, 1H), 7.48 (t, J = 6.1 Hz, 1H),
7.41 – 7.25 (m, 5H), 5.03 (s, 2H), 3.76 (d, J = 5.8 Hz, 2H), 3.65 (d, J = 6.2 Hz, 2H).
Mass: m/z calc’d for C12H14N2NaO5 [M+Na]+; 289.1; found 289.1.
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Benzyl 2-oxo-2-(2-oxo-2-(4-(tetrahydro-2H-pyran-2-yloxycarbamoyl)benzylamino)
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ethylamino)ethylcarbamate (18). To a stirred solution of 17 (70 mg; 0.26 mmol) and 10 (65
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mg; 0.26 mmol) in 5 mL dry CH2Cl2 at 0 oC were added diisopropyl ethyl amine (0.19 mL; 1.05
mmol) and T3P 50% solution in CH2Cl2 by weight (217 mg; 0.34 mmol) dropwise. The reaction
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mixture was stirred for 1 h at 0 oC. Completion of the reaction was confirmed by LC-MS. The
reaction was quenched with cold water, and the product was extracted with CH2Cl2 and dried
over anhydrous Na2SO4. Solvent was evaporated under reduced pressure to provide crude
product, which was purified by flash column chromatography to afford 18 (120 mg; 0.24 mmol,
91%). The product was confirmed by 1H NMR and MS.
1H NMR (400 MHz, DMSO): δ 11.62 (s, 1H), 8.41 (t, J = 5.5 Hz, 1H), 8.23 (d, J = 5.6 Hz, 1H),
7.72 (d, J = 8.1 Hz, 2H), 7.53 (t, J = 5.5 Hz, 1H), 7.34 (d, J = 9.6 Hz, 7H), 5.13 – 4.87 (m, 3H),
4.33 (d, J = 5.8 Hz, 2H), 3.99 (d, J = 52.6 Hz, 1H), 3.76 (d, J = 5.7 Hz, 2H), 3.68 (d, J = 5.9 Hz,
2H), 3.51 (d, J = 11.1 Hz, 1H), 1.72 (m, 3H), 1.54 (m, 3H).
Mass: m/z calc’d for C25H30N4NaO7 [M+Na]+; 521.2; found 521.2.
Methyl 3-((5S,8S)-5-isobutyl-8-methyl-3,6,9-trioxo-1-phenyl-2-oxa-4,7,10-
triazaundecan-11-yl)benzoate (19). To a stirred solution of 14 (500 mg; 1.5 mmol) and methyl
3-(aminomethyl)benzoate (245 mg; 1.5 mmol) in 10 mL dry CH2Cl2 at 0 oC were added
diisopropyl ethyl amine (1.05 mL; 5.95 mmol) and T3P 50% solution in CH2Cl2 by weight (1.225
g, 1.95 mmol) simultaneously dropwise. The reaction mixture was stirred for 30 min at 0 oC.
Completion of the reaction was confirmed by LC-MS. The reaction was quenched with cold
water, and the product was extracted with CH2Cl2 and then dried over anhydrous Na2SO4.
Solvent was evaporated under reduced pressure to provide crude product, which was purified
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by flash column chromatography to afford 19 (610 mg; 1.26 mmol, 85%). Product was
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confirmed by 1H NMR and MS.
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1H NMR (400 MHz, CDCl3): δ 7.91 (dd, J = 10.4, 9.2 Hz, 2H), 7.53 – 7.42 (m, 1H), 7.42 – 7.28
(m, 6H), 6.88 (s, 1H), 6.56 (d, J = 7.4 Hz, 1H), 5.18 (d, J = 6.4 Hz, 1H), 5.02 (q, J = 12.2 Hz,
2H), 4.57 – 4.35 (m, 3H), 4.10 (d, J = 29.3 Hz, 1H), 3.88 (s, 3H), 1.71 – 1.55 (m, 4H), 1.52 –
1.43 (m, 1H), 1.39 (d, J = 6.8 Hz, 3H), 0.90 (d, J = 6.1 Hz, 6H).
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Mass: m/z calc’d for C26H34N3O6 [M+H]+; 484.2; found 484.3.
3-((5S,8S)-5-Isobutyl-8-methyl-3,6,9-trioxo-1-phenyl-2-oxa-4,7,10-triazaundecan-11-
yl)benzoic acid (20). To a stirred solution of 19 (450 mg; 0.93 mmol) in 10 mL of
(THF:MeOH:H2O) (5:5:1) at 0 oC was added LiOH•H2O (117 mg; 2.79 mmol). The reaction
mixture was warmed to room temperature and stirred for 6 h. Completion of the reaction was
confirmed by LC-MS. Volatiles were evaporated under reduced pressure to provide crude
product, which was neutralized with 1 N HCl, washed with water, and dried under vacuum to
afford 20 (380 mg; 0.809 mmol, 87%). Product was confirmed by 1H NMR and MS.
1H NMR (400 MHz, DMSO): δ 12.91 (s, 1H), 8.44 (t, J = 5.8 Hz, 1H), 8.01 (d, J = 7.4 Hz, 1H),
7.88 – 7.75 (m, 2H), 7.53 – 7.39 (m, 3H), 7.39 – 7.25 (m, 5H), 5.10 – 4.93 (m, 2H), 4.42 – 4.20
(m, 3H), 4.04 (dd, J = 14.7, 8.4 Hz, 1H), 1.66 – 1.54 (m, 1H), 1.49 – 1.35 (m, 2H), 1.32 – 1.19
(m, 3H), 0.83 (dd, J = 19.0, 13.3 Hz, 6H).
Mass: m/z calc’d for C25H32N3O6 [M+H]+; 470.2; found 470.3.
Benzyl (2S)-4-methyl-1-oxo-1-((2S)-1-oxo-1-(3-(tetrahydro-2H-pyran-2-
yloxycarbamoyl)-benzylamino)propan-2-ylamino)pentan-2-yl carbamate (21). To a stirred
solution of 20 (300 mg; 0.63 mmol) and O-(tetrahydro-2H-pyran-2-yl)hydroxylamine (74 mg;
0.63 mmol) in 20 mL dry CH2Cl2 at 0 oC were added diisopropyl ethyl amine (0.44 ml; 2.56
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