New matrix metalloproteinase inhibitors based on γ-fluorinated α-aminocarboxylic and α-aminohydroxamic acids

Article abstract of DOI:10.1016/j.bmc.2015.03.078

Abstract Matrix metalloproteinases (MMPs) are involved in a number of physiological as well as pathological processes such as atherosclerosis and tumorigenesis, where an up-regulation of MMPs is predominant. Fluorinated analogues of the hydroxamate-based

Full text of DOI:10.1016/j.bmc.2015.03.078

Bioorganic & Medicinal Chemistry 23 (2015) 3809–3818
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
New matrix metalloproteinase inhibitors based on
-aminocarboxylic and -aminohydroxamic acids
c-fluorinated
a
a
Malte Behrends a, Stefan Wagner b, Klaus Kopka b, , Otmar Schober b, Michael Schäfers b,c,d
,
Sadhana Kumbhar a, Mark Waller a,d, Günter Haufe a,d,
⇑
a Organisch-Chemisches Institut, Westfälische Wilhelms-Universität Münster, Corrensstraße 40, D-48149 Münster, Germany
b Klinik für Nuklearmedizin, Universitätsklinikum Münster, Albert-Schweitzer-Campus 1, D-48149 Münster, Germany
c European Institute for Molecular Imaging, Westfälische Wilhelms-Universität Münster, Waldeyerstraße 15, D-48149 Münster, Germany
d Cells-in-Motion Cluster of Excellence (EXC 1003-CiM), University of Münster, Waldeyerstraße 15, D-48149 Münster, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Matrix metalloproteinases (MMPs) are involved in a number of physiological as well as pathological pro-
cesses such as atherosclerosis and tumorigenesis, where an up-regulation of MMPs is predominant.
Fluorinated analogues of the hydroxamate-based non-peptidic broad-spectrum MMP inhibitor (MMPI)
CGS 27023A were synthesized and inhibition potencies for MMP-2 and MMP-9 in the nanomolar range
were measured using fluorimetric in vitro assays. The inhibition potencies of the herein reported
fluorinated MMPIs were comparable or even superior in some cases to their non-fluorinated analogues.
In contrast to the lead structure, both enantiomers of fluorinated MMPs were almost equally potent.
Received 19 November 2014
Revised 20 March 2015
Accepted 28 March 2015
Available online 6 April 2015
Keywords:
Matrix metalloproteinase inhibitors
CGS 27023A analogues
In vitro assay
Amino hydroxamic acid
Fluorine
Modelling studies suggest that the core
a-amino hydroxamic acid residues appear to influence the
relative potencies via specific inhibitor-peptidase interactions, including short fluorine–hydrogen con-
tacts, within the enzyme’s pockets. The binding of the essential hydroxamate group to the zinc ion is
rather unaffected by the rest of the molecule. In contrast, the corresponding
derivatives are 103 times less potent or were even inactive.
a-aminocarboxylic acid
Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction
site.17 Carboxylic or hydroxamic acid-moieties are predominantly
used as zinc chelators.18–21 Among the synthetic MMPIs there
are sulfone-based hydroxamic acids (e.g., Prinomastat or RS-
130830)11 as well as hydroxamate-based pseudopeptides (e.g.,
Batimastat or Marimastat).22,23 Over the last decade, two other
hydroxamic acids based MMPIs, CGS 25966 and CGS 27023A
(Fig. 1), have been investigated intensively.24–27 From both com-
pounds radiolabelled analogues have been synthesized bearing
the radionuclide [123/125I], [18F] or [11C], respectively, that showed
promising results in molecular imaging via SPECT and PET stud-
ies.4,25,28–32 All of the existing fluorinated analogues of such inhibi-
tors carry the fluorine-containing substituent in an aromatic
position.
In our group another CGS 27023A analogue has been synthe-
sized, where the p-methoxy group was replaced by a p-methyl
group and the isopropyl group of valin by a 2-fluoroallyl group.
This compound showed high inhibition potencies towards MMP-
2 and MMP-9.33 This result led to further studies on fluorinated
CGS 27023A analogues carrying the fluorine in an aliphatic
position, since it was believed that either the fluorine atom or
Matrix metalloproteinases (MMPs) belong to a class of endopep-
tidases that are zinc- and calcium-dependent and take over numer-
ous tasks in the organism,1 including degradation of protein
components within the extracellular matrix (ECM).2 MMPs are
divided into several subclasses with overlapping substrate specifici-
ties.3,4 In a healthy organism the activity of the MMPs is regulated
by their endogenous tissue inhibitors of metalloproteinases
(TIMPs).5,6 MMPs and TIMPs are normally balanced in physiological
processes,7,8 such as apoptosis, wound healing or ovulation.9
Elevated MMP expression is associated with numerous pathologies
like inflammation, atherosclerosis, and tumor progression and dis-
semination.10–12 Due to the important role of MMPs in a number
of diseases13 many different matrix metalloproteinase inhibitors
(MMPIs) have been developed to date.14–16 MMPIs are able to block
the activated enzymes by chelating the respective zinc(II) active
⇑
Corresponding author. Tel.: +49 (0)251 83 33281; fax: +49 (0)251 83 39772.
E-mail address: haufe@uni-muenster.de (G. Haufe).
the
c
-double bond would change the interaction between the
New address: Abteilung Radiopharmazeutische Chemie, Deutsches Krebs-
forschungszentrum, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany.
active site and the inhibitor.34,35
a
-Trifluoromethylated36 and
http://dx.doi.org/10.1016/j.bmc.2015.03.078
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

3810
M. Behrends et al. / Bioorg. Med. Chem. 23 (2015) 3809–3818
X
N
furnished the pure carboxylic acids 17–20 in yields between 85%
and 96% (f). A classical amide coupling between the acids and
O-tert-butyl hydroxylamine with EDC, HOBT and N-methyl mor-
pholine gave the pure tert-butyl hydroxamates 21–24 in 61–92%
yields (g). Finally, the hydrolysis to the final products with TFA at
elevated temperatures led to compounds 1–4 in 30–66% yields (h).
The fluorinated and non-fluorinated, saturated and unsaturated
amino acid derivatives (17–20) as well as the analogous hydrox-
amic acids (1–4) were then studied regarding their activity as inhi-
bitors of MMP-2 and MMP-9. Originally, we planned to prepare
O
O
O
S
O
S
N
OH
N
OH
*
N
H
N
H
O
O
R
MeO
CGS 27023A (X = N)
CGS 25966
MeO
1 (R = CH2CH3)
2 (R = CH2CH2F)
3 (R = CH2CH=CH2)
4 (R = CH2CF=CH2)
(X = CH)
[
18F]-labelled compound 2 for application in PET. Unfortunately,
all of our attempts to replace leaving groups such as tosyl or mesyl
by fluoride failed due to cyclization incorporating the carbonyl
oxygen to form mixtures of fluorine-free tetrahydrofurane and
other derivatives.
Figure 1. CGS compounds and analogues.
further perfluoroalkylated25
a
-aminohydroxamates have also been
synthesized and their potency as MMP inhibitors has been mea-
sured to be in the nanomolar scale. Fluorine substituents are
known to possess a strong impact on the properties of biologically
active compounds and influence their solubility, liability to meta-
bolism and enzyme inhibition potency.37,38
The aim of this work is to synthesize new fluorinated MMP inhi-
bitors, bearing a single fluorine substituent in an aliphatic position
of the molecule and to determine their potencies to target the dis-
ease relevant gelatinases A and B also called MMP-2 and MMP-9
inhibitors. Molecular modeling studies based on the known crystal
structure of MMP-939 are applied to gain knowledge of the specific
interactions of these molecules in the enzyme’s active site.
2.2. In vitro assays
Since matrix metalloproteinases—MMP-2 and -9 (gelatinase A
and B, respectively) in particular—have been given more and more
attention as possible drug and imaging targets, we aimed to
synthesize novel fluorinated compounds and the non-fluorinated
parents to study their inhibition potencies towards the afore-
mentioned proteases.
The synthetic fluorogenic substrate (7-methoxycoumarin-4-yl)
acetyl pro-Leu-Gly-Leu-[3-(2,4-dinitrophenyl)-L-2,3-diamino-pro-
pionyl]-Ala-Arg-NH2 was used in the affinity assays to study the
inhibition potency of the new compounds against MMP-2 and
MMP-9 as described previously42 (see Section 4). The resulting
IC50 values of the fluorimetric in vitro inhibition assays of
hydroxamates (R)- and (S)-1–4 and carboxylates (R)- and (S)-17–
20 are summarized in Table 1. The inhibition study with the
corresponding esters of the carboxylic and hydroxamic acids was
not performed, since it is known that esters generally show lower
activity than the corresponding acids.29
The respective MMP inhibition potencies of non-fluorinated
aminobutyric acid derivatives 17 are very low in the
with ca. 10-fold less potency for the (S)- compared to (R)-enan-
tiomer (Table 1). The corresponding hydroxamate derivatives (R)-
1 and (S)-1 were dramatically more potent showing IC50s of 2.0
and 4.6 nM for MMP-2 and MMP-9, respectively for the (R)-enan-
2. Results and discussion
2.1. Chemistry
Apart from the aliphatic
a-substituent, the target compounds
are analogues of CGS 27023A with regard to the N-substituents
as well as the zinc binding hydroxamate group. The compounds
of interest are depicted in Figure 1.
lM range
In order to prove our previous results33 and that of others19–21
with respect to the hydroxamic acid binding motif, the correspond-
ing carboxylic acids were also tested for their inhibition potencies.
The syntheses of
a-amino butyric acid and the a-amino pent-4-
enoic acid derivatives are shown in Scheme 1. The carboxylic acids
18 and 20 as well as the hydroxamic acids 2 and 4 were synthe-
sized in both enantiomeric forms carrying a fluorine substituent
tiomer, while the (S)-enantiomer is much less active (1.4 lM and
700 nM), which is in accordance with the observations for the
CGS compounds.24,25 The fluorinated carboxylic acid analogues
in the c-position. Additionally, their non-fluorinated versions (17,
(R)-18 and (S)-18 show inhibition potencies at only lM concentra-
19 and 1, 3, respectively) were prepared and tested in order to
get a deeper insight into the influence of fluorine on the CGS
27023A-analogues.
The synthesis can be divided into three major parts. The first
part is concerned with the synthesis of both enantiomers of
hydroxypinanone, which are then condensed to the corresponding
Schiff’s base.40 In the key step, the fluorinated building blocks are
tions and match with their non-fluorinated variations.
Surprisingly, in case of the fluorinated hydroxamic acids the differ-
ence between the two enantiomers is negligible. Moreover, the
selectivity is reversed in the case of MMP-9. While (R)-2 shows
IC50s of 6.4 nM and 12.3 nM for MMP-2 and -9, (S)-2 has a 10-fold
selectivity towards MMP-9 with IC50s of 32.8 nM (MMP-2) and
3.0 nM (MMP-9). Therefore, when comparing the non-fluorinated
hydroxamic acids (R)-1 and (S)-1 with their fluorinated analogues
(R)-2 and (S)-2, it can be pointed out that the terminal fluorine
substituent in fact does not have a major impact on the inhibition
potency in case of the (R)-enantiomer, but rather has a large effect
in case of the (S)-enantiomer. Thus, at least for the (S)-enantiomer
the fluorine does contribute to the binding at the enzyme’s active
site.
asymmetrically introduced in
a-position leading to enantiomeri-
cally enriched amino acid tert-butylesters after deprotection.41
Afterwards, these are substituted at their N-terminal position
using a modified literature procedure.29 The last step in the synthe-
sis is the hydrolysis of the esters followed by the introduction of
the hydroxamic acid functionality (Scheme 1).
The optically active
a-amino acid esters 5–8 were isolated in
32–58% yield and with 85–95% ee after purification by HPLC.
The first N-substitution reaction was performed with
p-methoxyphenylsulfonyl chloride in pyridine and led to the prod-
ucts in 36–70% yield after purification (9–12, Scheme 1d). The
picolyl substituent was introduced by treatment with an excess
of K2CO3 in DMF. After purification the products 13–16 were iso-
lated in 76–93% yields (e). The hydrolysis of the esters with TFA
The same trend of the less active carboxylic acids is visible in
the case of the unsaturated compounds. The non-fluorinated
pentenoic acid (R)-19 has an IC50 value in the
lM range for both
gelatinases, while (S)-19 was inactive as it was found for (R)-17
and (S)-17. The corresponding pentene hydroxamic acid derivative
(S)-3 shows high inhibition potencies of 6.3 nM and 6.4 nM for
MMP-2 and MMP-9, respectively. The (R)-3 is even more potent

M. Behrends et al. / Bioorg. Med. Chem. 23 (2015) 3809–3818
3811
O
O
O
H
O
S
a,b,c
d
e
H2N
H2N
N
OtBu
OtBu
OtBu
*
*
O
R
R
MeO
5
5
(R)- /(S)- (R = Et)
(R)-9/(S)-9 (R = Et)
6
6
10
10
11
(R)- /(S)- (R = CH2CH2F)
(R)- /(S)-
(R = CH2CH2F)
7/
7
11/
(R)- (S)-
(R)- (S)- (R = CH2CH=CH2)
(R = CH2CH=CH2)
(R)-8/(S)-8 (R = CH2CF=CH2)
(R)-12/(S)-12 (R = CH2CF=CH2)
N
O
N
O
O
O
N
g
N
OX
*
*
S
OX
S
N
H
O
O
R
R
MeO
X = tBu
MeO
f
h
X = H
X = tBu
X = H
---------------------------------------------------------
-----------------------------------------------------------
13/
(R)- (S)-
13 (R)-17/(S)-17 (R = Et)
21/
21 (R)-1/(S)-1 (R = Et)
22 (R)-2/(S)-2 (R = CH2CH2F)
(R)- (S)-
18
18
19
(R)-14/(S)-14 (R)- /(S)-
(R = CH2CH2F)
(R = CH2CH=CH2)
22/
(R)- (S)-
19/
3/ 3
(R)-23/(S)-23 (R)- (S)- (R = CH2CH=CH2)
(R)-15/(S)-15 (R)- (S)-
16/
16 (R)-20/(S)-20 (R = CH2CF=CH2)
24/
(R)- (S)-
24
4/
4
(R)- (S)-
(R)- (S)- (R = CH2CF=CH2)
Scheme 1. Synthetic strategy towards hydroxamate based MMP inhibitors. Reaction conditions: (a) hydroxypinanone, BF3ꢀEt2O, toluene,
D, 4 h; (b) n-BuLi, RBr or ROTs, THF,
ꢁ78 °C–rt; (c) 15% citric acid, THF, 0 °C–rt, 3 d; (d) p-MeOPhSO2Cl, pyridine, 0 °C–rt; (e) 3-(chloromethyl)pyridine hydrochloride, K2CO3, DMF, rt; (f) CF3CO2H, DCM, rt; (g)
HOBT, NMM, tBuONH3Cl, EDC, DCM, rt; (h) CF3CO2H, 40 °C, 3 h.
Table 1
the fluorinated pentene hydroxamic acids (R)-4 and (S)-4 show
very high potencies for MMP-2 and -9 with IC50 values of 9.3 nM
and 8.3 nM for (R)-4) and 7.2 nM and 4.9 nM for (S)-4, respectively.
Though the inhibition potencies of both enantiomers are very sim-
ilar, the (S)-enantiomer is slightly more potent, in contrast to pre-
vious findings where the respective (R)-enantiomer is significantly
more active. When comparing (R)-3 and (R)-4, though, the non-flu-
orinated compound has a higher inhibition potency for both MMP-
2 and MMP-9.
IC50 values of target amino acid and hydroxamic acid derivatives
X
O
O
N
S
Y
O
R
MeO
a
Compd
R
X
Y
IC50 (nM)
2.3. Molecular modeling
MMP-2 MMP-9
A crystal structure of MMP-939 was obtained from the protein
data bank (ID: 1GKC). The structure was built using the CHARMM
suite (Version c35b3),43 and the crystallographically observed
reverse hydroxamate inhibitor (N-2-[(2R)-2-{[formyl(hydroxy)
(R)-CGS 25966
(R)-CGS 27023Ac
(R)-17
(S)-17
(R)-1
(S)-1
(R)-18
(S)-18
(R)-2
(S)-2
(R)-19
(S)-19
(R)-3
(S)-3
(R)-20
(S)-20
(R)-4
CH(CH3)2
CH(CH3)2
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH2F
CH2CH2F
CH2CH2F
CH
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
NHOH
NHOH
OH
11b
27b
20d
8d
10,300
8800
OH
>100,000
2.0 0.2
1400
>100,000
34,700
6.4 1.8
32.8 16.4
2100
>100,000
3.9 0.3
6.3 1.5
35,700
>100,000
4.6 0.1
700 300
>100,000
>100,000
12.3 1.0
3.0 0.7
1700
>100,000
0.54 0.13
6.4 0.4
>100,000
>100,000
8.3 0.1
4.9 0.4
NHOH
NHOH
OH
amino]methyl}-4-methylpentanoyl]-N-3-dimethyl-L-valinamide)
was replaced by various MMPIs (compounds CGS 27023A, 1, 2, 3 and
4) as described in the Section 4. The hydroxamic acid of the MMPIs
was bound to the Zn2+ ion, and either of the two large aromatic
moieties (4-methoxyphenyl or 3-picolyl)can be placed into the deep
OH
NHOH
NHOH
OH
CH2CH2F
CH2CH@CH2
CH2CH@CH2
CH2CH@CH2
CH2CH@CH2
CH2CF@CH2
CH2CF@CH2
CH2CF@CH2
CH2CF@CH2
0
S1 binding pocket. A previous docking study suggested that the
OH
0
NHOH
NHOH
OH
OH
NHOH
NHOH
4-methoxyphenyl group is actually bound inside the S1 pocket
and 3-picolyl group located in the S2 solvent exposed pocket.44 In
0
this present study we investigated both possibilities, that is, inser-
>100,000
9.3 2.0
7.2 0.1
0
tion of the 3-picolyl group into the S1 binding pocket, and a second
0
set of calculations with the 4-methoxyphenyl group inserted into S1
(S)-4
binding pocket.
a
Standard deviations based on three independent experiments are given for IC50
in nM range.
s
From the first set of optimized structures we indeed observed
that the 4-methoxyphenyl group can bind also into the solvent
b
Ki values taken from Ref. 25.
(R)-configuration.
Ki values taken from Ref. 24.
c
0
0
exposed S2 pocket and the 3-picolyl group does fit into the S1
d
pocket too. However, a substantial empty cavity remained in the
distal end of the latter pocket (see Figs. S1–S4 in the SI). This find-
ing combined with the conclusions from the previous docking
study44 lead us to discontinue the investigation of this
arrangement.
The second set of cluster models was created, where the 4-
methoxyphenyl group was inserted in the S1 pocket and the 3-pi-
colyl group was inserted into the S2 pocket. From the calculations
it is observed that the 4-methoxyphenyl group fits well into the S1
with inhibition concentrations of 3.9 nM for MMP-2 and 0.54 nM for
MMP-9. It may be concluded that, in case of the unsaturated hydrox-
amic acids, the stereochemistry is not essential, but rather the allylic
double bond influences the binding to the MMPs’ active site.
The fluorinated pentenoic acids (R)-20 and (S)-20, in both enan-
tiomeric forms, are practically inactive towards both target
enzymes as their fluorine free parent compounds 19. In contrast,
0
0
0
pocket, and no large cavity remained as the 4-methoxyphenyl

3812
M. Behrends et al. / Bioorg. Med. Chem. 23 (2015) 3809–3818
group is deeply bound inside this pocket, and 3-picolyl group was
bound to the S20 pocket, see Figures S5–S8 in the SI. Interestingly all
models (MMPIs CGS 27023A and 1–4, in both enantiomeric forms)
showed very little deviation in the Znꢀ ꢀ ꢀO distances and Oꢀ ꢀ ꢀZnꢀ ꢀ ꢀO
bond angles (Table S2). Thus, the R substituents do not appear to
strongly influence the relative potencies via the binding of the
hydroxamate ligand to the zinc.
Therefore we turned our attention to the fluorine in the MMPIs.
Fluorine substituents are known to modulate the steric, electronic
and lipophilic parameters of a given drug molecule.38,39,45,46 Upon
inspection of the cluster model of compound (R)-2, short contacts
were found between the sulfonyl oxygen and the methyl group of
Ala189 (2.12 Å, angle 176°), which is present also in (S)-2 (2.13 Å,
angle 162°). Both enantiomers have short contacts between the
hydroxamate group to Glu402. For (R)-2 the N–Hꢀ ꢀ ꢀO@C (Glu402)
is 2.08 Å, angle 161° and for (S)-2 the N–Hꢀ ꢀ ꢀO@C (Glu402) is
2.09 Å, angle 170°. Most importantly, the fluorine in the R group
was noted to be facing towards a hydrogen of one of the methyl
groups of Leu187 (C–Fꢀ ꢀ ꢀH–C distance 1.65 Å, angle 161°) for the
(R)-2-enantiomer. Moreover, fluorine could also have a close con-
noted slight conformational changes of the R group as a result of
the change in the absolute configuration. In the (S)-compound,
the fluorinated R group is facing towards the Ala189 and His190,
while the fluorine itself is pointing towards the alpha-proton of
the His190 with a C–Fꢀ ꢀ ꢀH–C distance of 2.15 Å and the C–Fꢀ ꢀ ꢀH
angle is 126°. This fluorine is also in close contact to one of the
beta-protons of the His190, with a somewhat larger distance of
2.31 Å and a similar C–Fꢀ ꢀ ꢀH angle of 119°. Therefore, the fluorine
from the R group is interacting with the active site of the MMP-
9. In contrast, in the (R)-compounds the fluorinated R group seems
0
to be facing towards the 3-picolyl group located in the S2 pocket,
and it is therefore not strongly interacting directly with MMP-9.
This might be one reason for the fact that (R)-3 is more potent than
(R)-4.
In the optimized cluster model of (R)-CGS 27023A there are
close contacts to the crystallographically observed water molecule
(number 56) and with Ala189 and Glu402, see Figure S5a. The
strong hydrogen bonding interaction to the water is absent in
the (S)-CGS 27023A optimized model, instead the isopropyl R
group has a direct short contact with the Leu187 and Ala189.
Clearly, the Leu187 is a significantly weaker hydrogen bond accep-
tor than the water in the latter case, which might at least partially
explain why the (R)-CGS 27023A is more active. Furthermore, there
is more space for rotation of the isopropyl group in the (R)-CGS
27023A model and the ethyl group of (R)-1 than in case of (S)-
CGS 27023A and (S)-1, respectively, which may have further con-
tributed to the large difference in potency between the
enantiomers.
tact with
108°). In the (S)-2-enantiomer the fluorine has a similarly close
contact (C–Fꢀ ꢀ ꢀH–C distance 1.68 Å, angle 120°) to the -hydrogen
a-hydrogen of Leu187 (C–Fꢀ ꢀ ꢀH–C distance 2.01 Å, angle
a
of Leu187 and a hydrogen of the methyl group of Leu187 (C–Fꢀ ꢀ ꢀH–
C distance 1.99 Å, angle 129°) (Figs. 2 and S6a). A hydrogen bond
interaction requires a linear arrangement of the C–Fꢀ ꢀ ꢀH–X moi-
ety,48–50 and the sum of the van der Waals radii is 2.67 Å,47 there-
fore we attribute these short distances to be due to a steric clash.
However, this steric clash is only observed for a single rotamer of
the fluoromethyl group, and when the other two rotamers
(ꢁ120° or ꢁ240°) are optimized, they do not have such short con-
tacts, see Figures S6b and c. This might be one of the reasons for
similar potency of (R)-2 and (S)-2.
3. Conclusion
The herein presented fluorinated N,N-disubstituted amino acid
and hydroxamic acid derivatives present new examples of MMP
inhibitors based on CGS 27023A. The fluorine substituent in the
In contrast, the interactions of the enantiomers of compound 1
(these are the non-fluorinated parents of 2) within the S10 pocket
are rather different (Fig. S5b). Most prominently, (R)-1 shows a
short contact (2.15 Å, angle 178°) of one of the sulfonyl oxygens
to the methyl group of Ala189, which is almost identical to the val-
ues of (R)-2. Such an interaction is not present in (S)-1 (3.11 Å),
while very short contacts were found in (S)-2 (see above).
Moreover, there are different interactions of the enantiomers of 1
with Glu402. While the N–Hꢀ ꢀ ꢀO@C contact is 2.07 Å, angle 163°
for (R)-1, the N–Hꢀ ꢀ ꢀO@C distance is 1.86 Å, angle 168°, in (S)-1.
The same is true for the enantiomers of CGS 27023A (S@Oꢀ ꢀ ꢀH–C
Ala189, 2.16 Å, angle 153°. For the Glu402 interaction, N–
Hꢀ ꢀ ꢀO@C distance is 2.12 Å, angle 167° for (R)-CGS 27023A, and
N–Hꢀ ꢀ ꢀO@C contact is 2.13 Å, angle 165° for (S)-CGS 27023A.
c-position of the amino acid core of these compounds seems to
provide an additional possibility of interaction with the active site
of MMP-2 and MMP-9, thereby reducing the importance of the
stereocenter. Therefore both enantiomers of the fluorinated butyric
hydroxamates are potent inhibitors of MMP-2 and -9 (gelatinases
A and B), while only the (R)-enantiomer of CGS 27023A is active.
Also both enantiomers of the fluorinated and non-fluorinated
unsaturated hydroxamate derivatives show high inhibition poten-
cies against these enzymes. All of the corresponding carboxylic
acid derivatives are significantly less potent, which is already
known for similar compounds.51,52 Modelling studies revealed that
our MMPIs can bind inside the binding pockets of MMP-9 in a sim-
ilar fashion to CGS 27023A. The cluster models show that either of
the large aromatic substituents (4-methoxyphenyl or the 3-picolyl)
Interestingly, the distances of the methyl proton to the
a-proton
0
can fit into the S1 cavity. However, the 4-methoxyphenyl group
of Leu187 are quite similar in (R)-1 (1.72 Å and 1.73 Å) compared
to the corresponding distances in (R)-2 (1.65 Å and 2.01 Å), while
in the corresponding (S)-enantiomers the distances are signifi-
cantly different (1.99 Å and 1.68 Å in (S)-2 and 3.19 Å and 2.31 Å
in (S)-1). These differences might be a part of the explanation for
the different behavior of the enantiomers of fluorinated and non-
fluorinated compounds.
In the case of the allylic compounds 3, the (R)-enantiomer is 10
times more potent than the (S)-isomer. From Figure S7, it is
observed that (R)-3 shows many short contacts (with Gly186,
Ala189, Glu402 and Pro421), while (S)-3 has relatively fewer inter-
actions (with Ala189 and Glu402). When the allyl group of 3 is
replaced by a fluorinated allyl group in compounds 4, the selectiv-
ity for the enantiomers is reversed, but the differences are small
(see Table 1). For both enantiomers of compound 3, each of the R
groups are located in similar positions when compared to the
respective enantiomers of compound 4, see Figure S8. We only
0
can more deeply bind into the S1 pocket, while a substantial part
of the cavity remained empty in the distal end when the 3-picolyl
group is placed there. This might lead to different entropy effects
with respect to water replacement in the pocket. In contrast the
0
3-picolyl group fits perfectly into the solvent exposed S2 pocket.
Compared to the isopropyl group of CGS 27023A and the ethyl
0
group of (R)-1 reaching into the S1 pocket, the R group of the enan-
tiomeric MMPIs 2-4 shows different interactions with various resi-
dues in the binding site, while very little deviation is seen in the
Znꢀ ꢀ ꢀO distances and Oꢀ ꢀ ꢀZnꢀ ꢀ ꢀO bond angles. Close contacts of flu-
orine substituents of both enantiomers of hydroxamates 2 and 4
within the enzyme’s active site seem to cause practically equal
potencies for both enantiomers. The inversion of selectivity for
the (R)- versus (S)-enantiomers of 2 and 4 compared to the fluorine
free parents 1 and 3 in the MMP-9 assay is presumably also due to
a fluorine effect.

M. Behrends et al. / Bioorg. Med. Chem. 23 (2015) 3809–3818
3813
Figure 2. (R)-2 (left) and (S)-2 (right) MMPI optimized in the cluster model, reinserted into the MMP-9 protein environment (see also Fig. S6a in the Supporting information).
4.2. Syntheses
4. Experimental section
4.2.1. Synthesis of N-(4-methoxyphenylsulfonyl)aminoacid
esters 9–12—General procedure A
4.1. Materials and methods
A solution of the amino acid ester (1 equiv) in pyridine (2.5 mL/
mmol) was cooled to 0 °C. After stirring for 10 min, p-methoxy
phenylsulfonylchloride (1.05 equiv) was slowly added. The solu-
tion was warmed to room temperature and stirred for 48 h. DCM
was added to the reaction mixture and the organic phase was
washed with 1 M HCl, H2O and brine. After drying over magnesium
sulfate the solvent was removed under reduced pressure and the
residue purified by column chromatography [Cyclohexane/EtOAc,
2:1, (2% TEA was added to the eluent for fluorinated compounds)].
4.1.1. Analytical methods
Merck silica gel 60 (230–400 mesh) was used for column chro-
matography. Melting points (uncorrected) were determined on a
Stuart Scientific SMP 10 capillary melting point apparatus. The
optical rotations were determined with a Perkin–Elmer polarime-
ter 341 at different wavelengths and are given in degrees ꢂ cm3/
dm ꢂ g. 1H, 13C and 19F NMR spectra were recorded on Bruker
DPX300, ARX300, AV400 and Varian INOVA (500 MHz) and
Varian unity plus (600 MHz) spectrometers in
a solution of
CDCl3, CD3CN or DMSO-d6 using TMS as an internal standard.
CFCl3 was used as the internal standard for 19F NMR spectra.
Chemical shifts are expressed as d [ppm] values relative to the
internal standard. DEPT and two dimensional NMR techniques
(COSY, HMQC and HMBC) were employed to assign 1H and 13C
NMR signals. Mass spectrometry was performed on Waters-
Micromass GCT or Quattro LCZ, Bruker Daltronics MicroTof and
Themo-Fisher Scientific LTQ Orbitrap LTQ XL mass spectrometers.
Elemental analysis was obtained on Foss Heraeus CHN-O-Rapid
and Elementar-Analysesysteme VarioEL III analysers, respectively.
Analytical and preparative HPLC was performed on a Knauer RP-
HPLC with K-1800 pumps and a S-2500 UV-detector. Eluent A:
H2O/TFA (1%), eluent B: CH3CN/TFA (1%). The conditions used
were: analytical (Eurosphere 100-10C, 4.6 mm ꢂ 250 mm):
1.5 mL/min, gradient 90–30% A (0–30 min), 30% A (30–35 min),
gradient 30–90% A (35–40 min); preparative (Eurosphere 100-
10C, 20 mm ꢂ 250 mm): 18.5 mL/min, gradient 90–30% A (0–
12 min), 30% A (12–13 min) or 18.5 mL/min, gradient 90–25% A
(0–10 min), 25% A (10–13 min). All tested compounds were puri-
fied to >95% purity by HPLC. Purity was also confirmed by 1H and
13C NMR spectroscopy.
4.2.1.1.
tert-Butyl
N-(4-methoxyphenylsulfonyl)aminobu-
tanoate. (S)-Enantiomer, ((S)-9): Yield: 467 mg (36%), white solid.
Mp 82 °C. 1H NMR (300 MHz, CDCl3): d 0.92 (t, JH,H = 7.4 Hz, 3H),
3
1.27 (s, 9H, 6-CH3), 1.61 (m, 1H), 1.77 (m, 1H), 3.72 (ddd,
3JH,H = 9.1 Hz, JH,H = 6.9 Hz, JH,H = 5.2 Hz, 1H), 3.85 (s, 3H), 5.19
3
3
3
3
(d, JH,H = 9.1 Hz, 1H), 6.95 (dm, JH,H = 9.0 Hz, 2H), 7.78 (dm,
3JH,H = 9.0 Hz, 2 H) ppm. 13C NMR (75 MHz, CDCl3): d 9.2 (q), 26.8
(q), 27.7 (t), 55.6 (q), 57.1 (d), 82.2 (s), 114.1 (d), 129.4 (d), 131.4
(s), 162.9 (s), 170.8 (s) ppm. HRMS (ESI+): m/z 352.1192, calcd for
C
15H23NO5S + Na+, 352.1195, and m/z 681.2483, calcd for
(C15H23NO5S)2 + Na+, 681.2492. MS (GC/MS, 70 eV): m/z (%) = 329
(1) [M+], 228 (88) [M+ꢁC5H9O2], 171 (100) [M+ꢁC7H7O3S], 155
(5) [C6H5NO2S+], 123 (18), 107 (21) [C7H7O+], 77 (13) [C6H+5], 57
(15) [C4H+9]. Optical rotation (c 1.009, CHCl3):
[a]589 = +21.3,
20
20
20
20
20
[a
]
578 = +22.3, [
a]546 = +26.2, [a]436 = +54.4, [a]365 = +110.5.
(R)-Enantiomer, ((R)-9): Yield: 446 mg (39%), white solid. Mp
20
81 °C. Optical rotation (c 1.008, CHCl3):
[
a
]
589 = ꢁ20.6,
20
20
20
20
[a
]
578 = ꢁ22.0, [
a]
546 = ꢁ26.8, [
a]
436 = ꢁ54.1, [
a]
365 = ꢁ109.9.
4.2.1.2. tert-Butyl N-(4-methoxyphenylsulfonyl)-
c
-fluoro-
(S)-Enantiomer ((S)-10): Yield: 2200 mg
(70%), white solid. Mp 89–90 °C. 1H NMR (300 MHz, CDCl3): d
a-
aminobutanoate.
4.1.2. Materials
3
3
1.28 (s, 9H), 1.87–2.25 (m, JH,F = 26.2 Hz, JH,H = 5.5 Hz, 2H), 3.85
Unless otherwise specified, starting materials were purchased
from commercial suppliers and used without further purification.
The starting enantiomers of amino acid tert-butylesters 5–8
have been prepared according to known procedures.39,40,53
Spectroscopic data of these compounds are given in the
Supporting information. Solvents were purified by distillation and,
if necessary, dried according to standard procedures.
3
2
(s, 3H), 3.89 (m, JH,H = 8.0 Hz, 1H), 4.58 (dt, JH,F = 46.8 Hz,
3JH,H = 5.5 Hz, 2H), 5.34 (d, JH,H = 8.0 Hz, 1H), 6.96 (dm,
3
3JH,H = 8.8 Hz, 2H), 7.79 (dm, JH,H = 8.8 Hz, 2H). 13C NMR
3
2
(75 MHz, CDCl3): d 27.6 (q), 34.1 (dt, JC,F = 20.3 Hz), 52.7 (dd,
3JC,F = 3.7 Hz), 55.6 (q), 79.7 (dt, JC,F = 165.9 Hz), 82.9 (s), 114.2
1
(d), 129.5 (d), 130.9 (s), 163.1 (s), 170.2 (s). 19F NMR (282 MHz,

3814
M. Behrends et al. / Bioorg. Med. Chem. 23 (2015) 3809–3818
3
3
3
CDCl3): d ꢁ221.6 (tt, 2JH,F = 46.8 Hz, 3JH,F = 26.0 Hz). Elemental anal-
ysis: C15H22FNO5S (M = 347.402 g/mol), calcd C, 51.86; H, 6.38; N,
4.03; found: C, 51.92; H, 6.24; N, 3.96. HRMS (ESI+):
(dd, JH,H = 7.8 Hz, JH,H = 4.8 Hz, 1H), 7.72 (dm, JH,H = 8.9 Hz, 2H),
3
4
3
7.88 (dd, JH,H = 7.9 Hz, JH,H = 1.7 Hz, 1H), 8.49 (dd, JH,H = 4.8 Hz,
4JH,H = 1.5 Hz, 1H), 8.56 (d, JH,H = 1.9 Hz, 1 H) ppm. 13C NMR
4
C
15H22FNO5S + Na+,
calcd
370.1100,
found:
370.1094.
(100 MHz, CDCl3): d 10.7 (q), 24.2 (t), 27.6 (q), 46.2 (t), 55.4 (q),
61.9 (d), 81.7 (s), 113.9 (d), 123.0 (d), 129.3 (d), 131.4 (s), 133.7
(s), 136.0 (d), 148.5 (d), 149.2 (d), 162.7 (s), 169.7 (s) ppm.
(C15H22FNO5S)2 + Na+, calcd 717.2303, found: 717.2298. Optical
20
20
rotation (c 1.021, CHCl3):
[
a
]
589 = ꢁ23.5,
[
a
]
578 = ꢁ25.1,
20
20
20
20
20
[a
]
546 = ꢁ29.3, [
a
]
436 = ꢁ58.4, [
a
]
365 = ꢁ114.1.
Optical rotation (c 1.013, CHCl3): [
a]
589 = ꢁ43.3, [
a
]
578 = ꢁ45.5,
20
20
20
(R)-Enantiomer, ((R)-10): Yield: 1190 mg (41%), white solid. Mp
[a]
546 = ꢁ52.5, [
a]
436 = ꢁ96.2, [ 365 = ꢁ169.7.
a]
20
89 °C. Optical rotation (c 1.015, CHCl3):
[
a
]
589 = +24.7,
(R)-Enantiomer, ((R)-13): Yield: 394 mg (91%), white solid. Mp
20
20
20
20
20
[a
]
578 = +26.1, [
a]
546 = +30.4, [
a]
436 = +61.2, [
a
]
365 = +118.4.
171 °C. Optical rotation (c 1.011, CHCl3):
[
a]
589 = +44.8,
20
20
20
20
[a]578 = +47.0, [a]546 = +54.0, [a]436 = +98.7, [a]365 = +173.6.
4.2.1.3. tert-Butyl N-(4-methoxyphenylsulfonyl)aminopent-4-
enoate.
(S)-Enantiomer, ((S)-11): Yield: 1150 mg (48%), white
4.2.2.2. tert-Butyl N-(4-methoxyphenylsulfonyl)-N-(3-pyridyl-
methyl)-2-amino-4-fluorobutanoate. (S)-Enantiomer ((S)-14):
Yield: 1512 mg (85%), greenish, viscous oil. 1H NMR (300 MHz,
solid. Mp 58–59 °C. 1H NMR (300 MHz, CDCl3): d 1.29 (s, 9H),
3
2.40–2.48 (m, 2H), 3.85 (s, 3H), 3.88 (dt, JH,H = 9.0 Hz,
3JH,H = 5.8 Hz, 1H), 5.05–5.13 (m, 2H), 5.20 (d, JH,H = 8.9 Hz, 1H),
3
2
CDCl3): d 1.35 (s, 9H), 1.86 (m, 1H), 2.22 (ddt, JH,H = 13.6 Hz,
3
3
3
3
3JH,H = 9.7 Hz, JH,H = 6.0 Hz, 1H), 3.86 (s, 3H), 4.19–4.45 (m, 2H),
5.66 (ddt, JH,H = 15.9 Hz, JH,H = 11.2 Hz, JH,H = 7.1 Hz, 1H), 6.95
3
3
(dm, JH,H = 9.0 Hz, 2H), 7.78 (dm, JH,H = 9.0 Hz, 2 H). 13C NMR
(75 MHz, CDCl3): d 27.8 (q), 37.9 (t), 55.5 (d), 55.6 (q), 82.6 (s),
114.2 (d), 119.3 (t), 129.4 (d), 131.6 (2d), 163.0 (s), 170.0 (s).
2
3
3
4.36 (d, JH,H = 16.1 Hz, 1H), 4.49 (dd, JH,H = 7.7 Hz, JH,H = 6.6 Hz,
2
3
1H), 4.69 (d, JH,H = 16.2 Hz, 1H), 6.96 (dm, JH,H = 8.9 Hz, 2H), 7.25
3
3
3
(dd, JH,H = 7.9 Hz, JH,H = 4.8 Hz, 1H), 7.77 (dm, JH,H = 8.9 Hz, 2H),
7.82 (dm, 3JH,H = 8.4 Hz, 1H), 8.51 (s, 1H), 8.53 (s, 1H) ppm. 13C NMR
(R)-Enantiomer, ((R)-11): Yield: 2375 mg (56%), white solid. Mp
20
2
59 °C. Optical rotation (c 1.010, CHCl3):
[
a
]
589 = ꢁ17.1,
(75 MHz, CDCl3): d 27.7 (q), 31.8 (dt, JC,F = 20.5 Hz), 47.3 (t), 55.6
20
20
20
20
(q), 57.0 (dd, 3JC,F = 4.3 Hz), 80.1 (dt, JC,F = 166.3 Hz), 82.5 (s), 114.1
1
[a
]
578 = ꢁ18.4, [
a
]
546 = ꢁ20.4, [
a]
436 = ꢁ43.8, [
a]
365 = ꢁ88.5.
(d), 123.4 (d), 129.6 (d), 131.3 (s), 132.9 (s), 136.3 (d), 149.1 (d),
4.2.1.4. tert-Butyl N-(4-methoxyphenylsulfonyl)-
c
-fluoro-a-
149.4 (d), 163.0 (s), 169.0 (s) ppm. 19F NMR (282 MHz, CDCl3): d
aminopent-4-enoate. (S)-Enantiomer, ((S)-12): Yield: 1.27 g (49%),
2
3
3
ꢁ221.4 (ddt, JH,F = 46.9 Hz, JH,F = 28.3 Hz, JH,F = 22.8 Hz) ppm.
viscous oil. 1H NMR (300 MHz, CDCl3): d 1.30 (s, 9H), 2.58 (dd,
HRMS
439.1701;
(ESI+):
C21H27N2O5S+H+,
calcd
calcd
439.1703,
461.1522,
found:
found:
3
3
3JH,H = 5.7 Hz, JH,H = 3.6 Hz, 1H), 2.64 (d, JH,H = 5.9 Hz, 1H), 3.85
C
21H27N2O5S + Na+,
3
3
(s, 3H), 3.99 (t, JH,H = 5.6 Hz, 1H), 4.34 (dd, JH,F = 49.3 Hz,
461.1526; (C21H27N2O5S)2+H+, calcd 877.3328, found: 877.3320;
(C21H27N2O5S)2 + Na+, calcd 899.3147, found: 899.3146. MS (ESI+,
daughter ion experiment): m/z (%) 439 (15) [M++H+], 383 (100)
[439ꢁC4H8], 171 (5) [C7H7O3S+], 167 (10) [383ꢁC7H7O3SꢁCO2H], 92
2JH,H = 3.0 Hz, 1H), 4.63 (dd, JH,F = 17.1 Hz, JH,H = 3.0 Hz, 1H),
3
2
3
3
5.41 (s, 1H), 6.96 (dm, JH,H = 9.0 Hz, 2H), 7.79 (dm, JH,H = 9.0 Hz,
2H) ppm. 13C NMR (75 MHz, CDCl3):
d 27.6 (q), 36.4 (dt,
2JC,F = 27.5 Hz), 53.3 (d), 55.5 (q), 83.0 (s), 93.9 (dt, JC,F = 18.7 Hz),
2
(3) [C6H6N+]. Optical rotation (c 1.001, CHCl3): a 20
[ ]589 = +34.1,
1
114.1 (d), 129.4 (d), 131.3 (s), 161.0 (ds, JC,F = 257.1 Hz), 163.0
[ ] ] ] ]365 = n.d.
a 25078 = +35.5, [a 52406 = +41.1, [a 24036 = +74.5, [a 20
(s), 169.2 (s) ppm. 19F NMR (282 MHz, CDCl3): d ꢁ96.0 (ddt,
(R)-Enantiomer, ((R)-14): Yield: 587 mg (93%), greenish, viscous
3JH,F = 49.5 Hz, JH,F = 19.5 Hz, JH,F = 17.5 Hz, 1F) ppm. MS (ESI+,
daughter ion experiment): m/z (%) 360 (0) [M++H], 304 (8)
[M+ꢁC4H8], 284 (20) [304ꢁF], 244 (10) [304ꢁC3H4F], 214 (10)
[M+ꢁC5H9O2ꢁC2H2F], 188 (15) [M+ꢁC7H7O3S], 171 (100)
[C7H7O3S+], 107 (1) [C7H7O+], 88 (60), 77 (2) [C6H+5]. HRMS (ESI+):
3
3
20
20
oil. Optical rotation (c 1.001, CHCl3): [
a]
589 = ꢁ33.2, [
a
]
578 = ꢁ34.5,
20
20
20
[a]
546 = ꢁ39.9, [
a]
436 = ꢁ72.6, [ 365 = ꢁ124.9.
a]
4.2.2.3. tert-Butyl N-(4-methoxyphenylsulfonyl)-N-(3-pyridyl-
methyl)-2-amino-pent-4-enoate. (S)-Enantiomer, ((S)-15): Yield:
750 mg (55%), white solid. Mp 176–177 °C. 1H NMR (300 MHz,
C
16H22FNO5S + Na+, calcd 382.1100, found: 382.1096. Optical
20
20
20
rotation (c 1.005, CHCl3): [a]589 = +3.0, [a]578 = +3.1, [a]546 = +4.1,
[a]436 = +13.2, [a]365 = +36.8.
2
3
CDCl3): d 1.31 (s, 9H), 2.27 (ddd, JH,H = 14.7 Hz, JH,H = 8.0 Hz,
20
20
3JH,H = 6.9 Hz, 1H), 2.49 (dt, JH,H = 13.8 Hz, JH,H = 6.9 Hz, 1H), 3.86
(s, 3H), 4.49 (d, JH,H = 16.1 Hz, 1H), 4.51 (t, JH,H = 7.5 Hz, 1H), 4.69
3
3
(R)-Enantiomer, ((R)-12): Yield: 1.01 g (39%), viscous oil. Optical
2
3
20
20
20
rotation (c 0.998, CHCl3): [
a
]
589 = ꢁ0.8, [
a
]
578 = ꢁ1.0, [
a
]
546 = ꢁ1.2,
2
3
2
(d, JH,H = 16.6 Hz, 1H), 4.89 (dd, JH,H = 17.1 Hz, JH,H = 1.5 Hz, 1H),
20
20
[a
]
436 = ꢁ3.9, [ 365 = ꢁ10.2.
a
]
4.99 (dd, 3JH,H = 10.2 Hz, 2JH,H = 1.5 Hz, 1H), 5.60 (ddt, 3JH,H = 17.0 Hz,
3JH,H = 10.3 Hz, 3JH,H = 6.7 Hz, 1H), 6.94 (dm, JH,H = 9.0 Hz, 2H), 7.25
3
4.2.2. Synthesis of N-(4-methoxyphenylsulfonyl)-N-(3-pyridyl-
methyl)-aminoacid esters 13–16—General procedure B
3
3
3
(dd, JH,H = 7.9 Hz, JH,H = 4.9 Hz, 1H), 7.73 (dm, JH,H = 9.0 Hz, 2H),
3
3
7.86 (d, JH,H = 7.9 Hz, 1H), 8.50 (d, JH,H = 4.0 Hz, 1H), 8.54 (d,
4JH,H = 1.7 Hz, 1H) ppm. 13C NMR (75 MHz, CDCl3): d 27.8 (q), 35.5
(t), 46.6 (t), 55.6 (q), 60.2 (d), 82.3 (s), 114.1 (d), 118.4 (t), 123.3 (d),
129.6 (d), 131.5 (s), 133.0 (d), 133.7 (s), 136.6 (d), 148.5 (d), 149.0
(d), 163.0 (s), 169.3 (s) ppm. HRMS (ESI+): C22H28N2O5S+H+, calcd
433.1797, found: 433.1812; C22H28N2O5S + Na+, calcd 455.1617,
found: 455.1630, (C22H28N2O5S)2+H+, calcd 865.3516, found:
865.3532; (C22H28N2O5S)2 + Na+, calcd 887.3336, found: 887.3345.
The appropriate aminoacid ester 9–12 (1 equiv) was dissolved
in DMF (5–10 mL/mmol). K2CO3 (10 equiv) was added and the sus-
pension stirred for 15 min. Then, 3-(chloromethyl) pyridinium
chloride (1.1 equiv) was added and the reaction mixture was stir-
red for 48 h at room temperature. The suspension was diluted with
H2O and extracted with EtOAc. The combined organic phases were
washed with H2O and brine, dried over magnesium sulfate and the
solvent was removed in vacuo. The crude products were purified
by column chromatography [Cyclohexane/EtOAc, 3:1, (2% TEA
was added to the eluent for fluorinated compounds)].
Optical rotation (c 1.001, CHCl3): [a 20
]
589 = ꢁ26.4, [a 20
578 = ꢁ27.3,
]
[
a 20
]
546 = ꢁ31.2, [a 20
]
436 = ꢁ56.6, [a 20
365 = ꢁ97.7.
]
(R)-Enantiomer ((R)-15): Yield: 2010 mg (67%), white solid. Mp
177 °C. Optical rotation (c 1.009, CHCl3):
[a]578 = +27.8, [a]546 = +31.8, [a]436 = +57.2, [a]365 = +99.1.
20
4.2.2.1. tert-Butyl N-(4-methoxyphenylsulfonyl)-N-(3-pyridyl-
methyl)-aminobutanoate. (S)-Enantiomer ((S)-13): Yield: 505 mg
(88%), white solid. Mp 170 °C. 1H NMR (400 MHz, CDCl3): d 0.80
[
a]
589 = +26.7,
20
20
20
20
3
2
(t, JH,H = 7.4 Hz, 3H), 1.31 (s, 9H), 1.49 (ddd, JH,H = 14.1 Hz,
4.2.2.4. tert-Butyl N-(4-methoxyphenylsulfonyl)-N-(3-pyridyl-
methyl)-2-amino-4-fluoropent-4-enoate. (S)-Enantiomer, ((S)-
3JH,H = 11.5 Hz, JH,H = 6.1 Hz, 1H), 1.77 (m, 1H), 3.84 (s, 3H), 4.31
(dd, JH,H = 8.4 Hz, JH,H = 6.7 Hz, 1H), 4.50 (d, JH,H = 16.5 Hz, 1H),
4.71 (d, JH,H = 16.5 Hz, 1H), 6.94 (dm, JH,H = 9.0 Hz, 2H), 7.24
3
3
3
2
16): Yield: 1428 mg (76%), yellow, viscous oil. 1H NMR (300 MHz,
2
3
3
CDCl3):
d
1.35 (s, 9H), 2.48 (m, JH,F = 19.2 Hz, 1H), 2.73

M. Behrends et al. / Bioorg. Med. Chem. 23 (2015) 3809–3818
3815
3
3
1
(td, JH,F = 15.0 Hz, JH,H = 6.8 Hz, 1H), 3.86 (s, 3H), 4.14 (dd,
(dt, JC,F = 163.1 Hz), 114.4 (d), 123.5 (d), 129.6 (d), 130.8 (s), 133.9
3JH,F = 49.7 Hz, JH,H = 3.1 Hz, 1H), 4.39 (d, JH,H = 16.3 Hz, 1H),
4.55 (dd, JH,F = 17.2 Hz, JH,H = 3.0 Hz, 1H), 4.58 (t, JH,H = 7.3 Hz,
1H), 4.65 (d, JH,H = 16.3 Hz, 1H), 6.95 (d, JH,H = 9.0 Hz, 2H), 7.23
(ddd, JH,H = 7.9 Hz, JH,H = 4.8 Hz, JH,H = 0.7 Hz, 1H), 7.78 (m, 1H),
(s), 136.4 (d), 148.0 (d), 148.8 (d), 162.7 (s), 171.2 (s) ppm. 19F NMR
2
2
3
2
3
2
3
(282 MHz, DMSO-d6): d ꢁ219.3 (ddt, JH,F = 47.1 Hz, JH,F = 27.8 Hz,
3JH,F = 22.3 Hz) ppm. HRMS (ESI+): C17H19FN2O5S+H+, calcd 383.1077,
2
3
3
3
4
found: 383.1073;
405.0892; (C17H19FN2O5S)2+H+, calcd 765.2076, found: 765.2071;
C
17H19FN2O5S + Na+, found: 405.0896, found:
3
3
4
7.78 (d, JH,H = 9.0 Hz, 2H), 8.51 (dd, JH,H = 4.6 Hz, JH,H = 1.7 Hz, 2
H) ppm. 13C NMR (75 MHz, CDCl3):
d
27.8 (s), 34.0 (d,
(C17H19FN2O5S)2 + Na+,
calcd
787.1895,
found:
787.1887.
2JC,F = 28.1 Hz), 47.4 (s), 55.6 (s), 57.6 (s), 82.8 (s), 93.4 (d,
HRMS (ESIꢁ): C17H19FN2O5SꢁH+, calcd 381.0926, found: 381.0930;
(C17H19FN2O5SꢁH+)2+H+, calcd 763.1925, found: 763.1930;
(C17H19FN2O5SꢁH+)2 + Na+, calcd 785.1744, found: 785.1758.
(R)-Enantiomer, ((R)-18): Yield: 352 mg (90%), white solid. Mp
155–156 °C.
2JC,F = 19.1 Hz), 114.2 (s), 123.3 (s), 129.8 (s), 131.4 (s), 132.8 (s),
1
136.4 (s), 149.1 (s), 149.5 (s), 161.5 (d, JC,F = 256.5 Hz), 163.1 (s),
168.4 (s) ppm. 19F NMR (282 MHz, CDCl3):
d
ꢁ97.6 (m,
3JH,F = 50.0 Hz, JH,F = 19.2 Hz, JH,F = 17.2 Hz, JH,F = 15.0 Hz) ppm.
HRMS (ESI+): C22H27FN2O5S+H+, calcd 451.1703, found: 451.1701;
C22H27FN2O5S + Na+, calcd 473.1522, found: 473.1521; C22H27
FN2O5S)2+H+, calcd 901.3328, found: 901.3320; (C22H27FN2O5S)2 +
Na+, calcd 923.3147, found: 923.3144. Optical rotation (c 1.010,
3
3
3
4.2.3.3. N-(4-Methoxyphenylsulfonyl)-N-(3-pyridylmethyl)-ami-
nopent-4-enoic acid. (S)-Enantiomer, ((S)-19): Yield: 312 mg (89%),
colorless, viscous oil. 1H NMR (400 MHz, DMSO-d6): d 2.16 (ddd,
2JH,H = 15.0 Hz, 3JH,H = 8.4 Hz, 3JH,H = 7.4 Hz, 1H), 2.42 (m, 1H), 3.76 (s,
20
20
20
20
CHCl3): [
a
]
589 = ꢁ6.3, [
a]
578 = ꢁ6.8, [
a
]
546 = ꢁ7.9, [
a]
436 = ꢁ14.2,
20
3
2
2
[
a]
365 = ꢁ22.9.
3H), 4.35 (dd, JH,H = 8.7 Hz, JH,H = 6.2 Hz, 1H), 4.36 (d, JH,H =
2
3
(R)-Enantiomer, ((R)-16): Yield: 523 g (58%), yellow, viscous oil.
17.3 Hz, 1H), 4.51 (d, JH,H = 16.9 Hz, 1H), 4.80 (dd, JH,H = 17.2 Hz,
2JH,H = 1.6 Hz, 1H), 4.84 (dd, JH,H = 10.3 Hz, JH,H = 1.5 Hz, 1H), 5.49
20
20
3
2
Optical rotation (c 1.004, CHCl3):
[a
]
589 = +7.7,
[a
]
578 = +8.1,
20
20
20
3
3
3
[
a]
546 = +9.3, [
a]
436 = +16.3, [
a
]
365 = +28.4.
(ddt, JH,H = 17.0 Hz, JH,H = 10.3 Hz, JH,H = 6.7 Hz, 1H), 6.99 (dm,
3JH,H = 9.0 Hz, 2H), 7.25 (dd, JH,H = 7.8 Hz, JH,H = 4.8 Hz, 1H), 7.66
(dm, 3JH,H = 8.9 Hz, 2H), 7.70 (m, 1H), 8.36 (d, 3JH,H = 4.1 Hz, 1H), 8.46
(s, 1 H) ppm. 13C NMR (100 MHz, DMSO-d6): d 34.1 (t), 46.5 (t), 55.7
(q), 59.7 (d), 114.3 (d), 117.8 (t), 123.1 (d), 129.5 (d), 131.0 (s), 133.8
(d), 134.0 (s), 135.9 (d), 148.1 (d), 149.2 (d), 162.6 (s), 171.2 (s) ppm.
HRMS (ESI+): C18H20N2O5S+H+, calcd 377.1171, found: 377.1186;
3
3
4.2.3. Hydrolysis of the tert-butyl esters—General procedure C
The according amino acid ester 13–16 (1 equiv) was dissolved
in dry DCM (20 mL/mmol) in a dry Young tube. TFA (20 mL/mmol)
was added to the solution and the tube was sealed. The reaction
mixture was stirred at room temperature for 4 h. Afterwards the
solvent was removed under reduced pressure. The residue was dis-
solved in chloroform and washed with aqueous citric acid (pH ꢃ 4).
The aqueous layer was extracted with chloroform and the com-
bined organic phases were dried over magnesium sulfate. After
removal of the solvent the products were obtained in high purities
as viscous oils and, if necessary, purified by HPLC.
C
18H20N2O5S + Na+, calcd 399.0991, found: 399.1004; (C18H20N2
O5S)2+H+, calcd 753.2264, found: 753.2259; (C18H20N2O5S)2 + Na+,
calcd 775.2084, found: 775.2094. HRMS (ESI–): C18H20N2O5SꢁH+, calcd
375.1015, found: 375.1035; (C18H20N2O5S)2ꢁH+, calcd 751.2108,
found: 751.2141. MS (ESI+, daughter ion experiment): m/z (%) 377
(75) [M++H+], 331 (75) [377ꢁH+ꢁCO2H], 280 (42) [C13H16N2O3S+],
240 (8) [331ꢁC6H6N], 200 (20) [C8H10NO3S+], 171 (50) [C7H7O3S+],
161 (100) [331ꢁC7H7O3S], 123 (8) [C7H7O+2].
4.2.3.1.
N-(4-Methoxyphenylsulfonyl)-N-(3-pyridylmethyl)-
aminobutanoic acid. (S)-Enantiomer, ((S)-17): Yield: 194 mg (91%),
(R)-Enantiomer, ((R)-19): Yield: 196 mg (92%), colorless, viscous
oil.
colorless, viscous oil. 1H NMR (300 MHz, DMSO-d6): d 0.70 (t,
3JH,H = 7.3 Hz, 3H), 1.45 (ddd, JH,H = 14.2 Hz, JH,H = 8.7 Hz,
2
3
3JH,H = 7.3 Hz, 1H), 1.75 (dt, JH,H = 14.0 Hz, JH,H = 6.8 Hz, 1H), 3.83
2
3
4.2.3.4. N-(4-Methoxyphenylsulfonyl)-N-(3-pyridylmethyl)-2-
amino-4-fluoropent-4-enoic acid. (S)-Enantiomer, ((S)-20): Yield:
200 mg (85%), yellowish solid. Mp 172 °C. 1H NMR (300 MHz,
3
3
(s, 3H), 4.23 (dd, JH,H = 8.9 Hz, JH,H = 6.0 Hz, 1H), 4.43 (d,
2JH,H = 16.9 Hz, 1H), 4.61 (d, JH,H = 16.9 Hz, 1H), 7.07 (dm,
2
3JH,H = 9.0 Hz, 2H), 7.38 (dd, JH,H = 7.8 Hz, JH,H = 4.9 Hz, 1H), 7.74
3
3
DMSO-d6):
d
2.59 (m, 1H), 2.84 (ddd, 3JH,F = 15.7 Hz,
3
3
(dm, JH,H = 9.0 Hz, 2H), 7.84 (d, JH,H = 7.9 Hz, 1H), 8.47 (d,
3JH,H = 4.1 Hz, 1H), 8.57 (s, 1 H) ppm. 13C NMR (75 MHz, DMSO-d6):
d 11.0 (q), 23.2 (t), 46.4 (t), 55.7 (q), 61.5 (d), 114.3 (d), 123.4 (d),
129.5 (d), 131.0 (s), 134.3 (s), 136.3 (d), 148.0 (d), 148.9 (d), 162.6
(s), 171.7 (s) ppm. HRMS (ESI+): C17H20N2O5S+H+, calcd 365.1171,
2JH,H = 12.6 Hz, JH,H = 5.5 Hz, 1H), 3.91 (s, 3H), 4.30 (dd,
3
3JH,F = 51.6 Hz, JH,H = 3.1 Hz, 1H), 4.53 (d, JH,H = 16.9 Hz, 1H), 4.62
2
2
3
2
2
(dd, JH,F = 17.8 Hz, JH,H = 3.0 Hz, 1H), 4.66 (d, JH,H = 16.8 Hz, 1H),
3
3
3
4.69 (dd, JH,H = 5.7 Hz, JH,H = 3.0 Hz, 1H), 7.14 (d, JH,H = 9.1 Hz,
3
2H), 7.45 (m, 1H), 7.80 (d, JH,H = 9.0 Hz, 2H), 7.88 (m, 1H), 8.55 (s,
found: 365.1169;
C
17H20N2O5S + Na+, calcd 387.0991, found:
1H), 8.63 (s, 1H), 12.94 (s, 1H) ppm. 13C NMR (75 MHz, DMSO-d6):
387.0988; (C17H20N2O5S)2+H+, calcd 729.2264, found: 729.2267;
(C17H20N2O5S)2 + Na+, calcd 751.2084, found: 751.2085. MS (ESI+,
daughter ion experiment): m/z (%) 365 (90) [M++H+], 319 (100)
[365ꢁH+ꢁCO2H], 280 (65) [C13H16N2O3S+], 228 (15) [319ꢁC6H6N],
200 (30) [228ꢁC2H5], 171 (55) [C7H7O3S+].
2
d 32.6 (dt, JC,F = 28.0 Hz), 46.7 (t), 55.7 (q), 57.0 (d), 93.2 (dt,
2JC,F = 18.4 Hz), 114.3 (d), 123.4 (d), 129.4 (d), 129.6 (s), 130.7 (s),
1
136.6 (d), 147.7 (d), 148.7 (d), 161.7 (ds, JC,F = 255.3 Hz), 162.7
(s), 169.9 (s) ppm. 19F NMR (282 MHz, DMSO-d6): d ꢁ96.2 (m,
3JH,F = 51.5 Hz, JH,F = 17.8 Hz, JH,F = 15.7 Hz) ppm. HRMS (ESI+):
3
3
(R)-Enantiomer, ((R)-17): Yield: 112 mg (96%), colorless, viscous
oil.
C
18H19FN2O5S+H+, calcd 395.1077, found: 395.1088; C18H19FN2
O5S + Na+, calcd 417.0896, found: 417.0905; (C18H19FN2O5S)2+H+,
calcd 789.2076, found: 789.2096; (C18H19FN2O5S)2 + Na+, calcd
811.1895, found: 811.1897. HRMS (ESI–): C18H19FN2O5SꢁH+, calcd
4.2.3.2. N-(4-Methoxyphenylsulfonyl)-N-(3-pyridylmethyl)-2-
amino-4-fluorobutanoic acid. (S)-Enantiomer, ((S)-18): Yield:
472 mg (96%), white solid. Mp 156 °C. 1H NMR (300 MHz, DMSO-d6):
393.0920,
found:
393.0940;
(C18H19FN2O5Sꢁ
H+)2+H+,
calcd 87.1919, found: 787.1955; (C18H19FN2O5SꢁH+)2 + Na+, calcd
809.1739, found: 809.1766. MS (ESI+, daughter ion experiment):
m/z (%) 395 (12) [M++H+], 179 (22) [M+ꢁC2H2FꢁC7H7O3S], 171
(31) [C7H7O3S+], 165 (100) [M+ꢁC3H4FꢁC7H7O3S], 147 (10), 123
(8), 107 (18) [C7H7O+], 92 (20) [C6H7N+]. MS (ESIꢁ, daughter ion
experiment): m/z (%) 393 (2) [MꢁꢁH+], 277 (27) [393ꢁC5H5FO2ꢁ],
171 (100) [C7H7O3Sꢁ], 157 (4) [C6H5O3Sꢁ], 115 (18) [C5H4FOꢁ2 ].
3
3
d 1.85 (m, JH,F = 22.3 Hz, 1H), 2.19 (m, JH,F = 27.9 Hz, 1H), 3.84 (s,
3H), 4.32 (dt, 2JH,F = 47.1 Hz, 3JH,H = 5.7 Hz, 2H), 4.43 (d,
2JH,H = 17.2 Hz, 1H), 4.49 (m, 1H), 4.62 (d, JH,H = 16.8 Hz, 1H), 7.09
2
3
3
3
(dm, JH,H = 8.9 Hz, 2H), 7.39 (dd, JH,H = 7.8 Hz, JH,H = 4.8 Hz, 1H),
7.75 (dm, 3JH,H = 8.9 Hz, 2H), 7.82 (m, 1H), 8.49 (d, 3JH,H = 3.7 Hz, 1H),
8.55 (s, 1H), 13.02 (s br, 1H) ppm. 13C NMR (75 MHz, DMSO-d6): d
30.9 (dt, 2JC,F = 20.2 Hz), 46.8 (t), 55.7 (q), 56.3 (dd, 3JC,F = 5.0 Hz), 80.4

3816
M. Behrends et al. / Bioorg. Med. Chem. 23 (2015) 3809–3818
(R)-Enantiomer, ((R)-20): Yield: 204 mg (91%), yellowish solid.
Mp 172–173 °C.
(R)-Enantiomer, ((R)-22): Yield: 353 mg (91%), white solid. Mp
20
60 °C. Optical rotation (c 1.004, CHCl3):
[a
]589 = ꢁ14.8,
20
20
20
20
[a]
578 = ꢁ15.7, [
a]
546 = ꢁ18.1, [
a]
436 = ꢁ34.3, [ 365 = ꢁ62.8.
a
]
4.2.4. Synthesis of the hydroxamates 21–24—General procedure
D
4.2.4.3. tert-Butyl N-(4-methoxyphenylsulfonyl)-N-(3-pyridyl-
methyl)-2-aminopent-4-en-hydroxamate. (S)-Enantiomer, ((S)-23):
Yield: 306 mg (92%), white solid. Mp 65–66 °C. 1H NMR (300 MHz,
The N,N-disubstituted amino acid (1 equiv) was suspended in
DCM (15 mL/mmol). HOBT (1 equiv) was added and the resulting
mixture stirred for five minutes. After addition of NMM (5 equiv)
the acid was dissolved. Subsequently, O-tert-butylhydroxylamine
(3 equiv) was added and stirred for 5 min. EDC (1.3 equiv) was
added and the reaction mixture was stirred overnight at room
temperature. The solution was washed with water and the
aqueous phase was extracted with DCM. The combined organic
layers were washed with brine, dried over magnesium
sulfate and the solvent was removed under reduced pressure.
The crude products were purified by column chromatography
[Cyclohexane/EtOAc, 1:3, (2% TEA was added to the eluent for flu-
orinated compounds)].
2
3
CDCl3): d 1.21 (s, 9H), 2.16 (dt, JH,H = 13.8 Hz, JH,H = 6.8 Hz, 1H),
2
3
3
2.60 (ddd, JH,H = 14.0 Hz, JH,H = 8.1 Hz, JH,H = 7.0 Hz, 1H), 3.86 (s,
3H), 4.29 (dd, 3JH,H = 8.3 Hz, 3JH,H = 6.9 Hz, 1H), 4.58 (s, 2H), 4.94 (d,
2JH,H = 2.9 Hz, 1H), 4.97 (d, JH,H = 3.1 Hz, 1H), 5.39 (ddt,
2
3JH,H = 14.1 Hz, 3JH,H = 9.6 Hz, 3JH,H = 6.9 Hz, 1H), 6.93 (dm,
3JH,H = 8.9 Hz, 2H), 7.21 (dd, JH,H = 7.7 Hz, JH,H = 4.9 Hz, 1H), 7.64
3
3
3
3
(dm, JH,H = 9.0 Hz, 2H), 7.72 (d, JH,H = 7.9 Hz, 1H), 8.49 (d,
3JH,H = 3.7 Hz, 1H), 8.56 (d, 4JH,H = 1.5 Hz, 1H), 8.70 (s, 1H) ppm. 13
C
NMR (75 MHz, CDCl3): d 26.2 (q), 33.9 (t), 45.8 (t), 55.6 (q), 57.6
(d), 82.2 (s), 114.4 (d), 119.0 (t), 123.3 (d), 129.3 (d), 131.0 (s),
132.5 (d), 132.8 (s), 136.4 (d), 148.8 (d), 149.8 (d), 163.2 (s), 167.7
(s) ppm. Elemental analysis: C22H29N3O5S (M = 447.18 g/mol), calcd
C, 59.04; H, 6.53; N, 9.39; found: C, 59.11; H, 6.57; N, 9.10. HRMS
(ESI+): C22H29N3O5S+H+, calcd 448.1906, found: 448.1907;
4.2.4.1. tert-Butyl N-(4-methoxyphenylsulfonyl)-N-(3-pyridyl-
methyl)-2-aminobutan-hydroxamate. (S)-Enantiomer, ((S)-21):
Yield: 173 mg (78%), white solid. Mp 66 °C. 1H NMR (300 MHz,
C
22H29N3O5S + Na+,
calcd
895.3734,
found:
895.3730;
(C22H29N3O5S)2+H+, calcd 470.1726, found: 470.1722; (C22H29N3
O5S)2 + Na+, calcd 917.3554, found: 917.3552. MS (ESI+, daughter
ion experiment): m/z (%) 448 (10) [M++H+], 392 (100) [448ꢁC4H8],
331 (32) [448ꢁC5H11NO2], 279 (8) [331ꢁC4H6], 240 (7), 171 (30)
3
DMSO-d6): d 0.69 (t, JH,H = 7.2 Hz, 3H), 1.23 (s, 9H), 1.44 (tq,
3
2JH,H = 14.8 Hz, JH,H = 7.4 Hz, 1H), 1.90 (m, 1H), 3.85 (s, 3H), 4.11
3
(m, 1H), 4.56 (s, 2H), 6.91 (d, JH,H = 8.9 Hz, 2H), 7.20 (dd,
3
3
3JH,H = 7.8 Hz, JH,H = 4.9 Hz, 1H), 7.61 (d, JH,H = 8.9 Hz, 2H), 7.71 (d,
[C7H7O3S+], 161 (20) [331ꢁC7H7O3S], 125 (11), 107 (2) [C7H7O+],
3JH,H = 7.8 Hz, 1H), 8.48 (d, JH,H = 3.7 Hz, 1H), 8.54 (d, JH,H = 1.5 Hz,
1H), 8.59 (s, 1H) ppm. 13C NMR (75 MHz, DMSO-d6): d 10.6 (q), 22.9
(t), 26.2 (q), 45.7 (t), 55.6 (q), 59.5 (d), 82.2 (s), 114.4 (d), 123.2 (d),
129.2 (d), 131.3 (s), 132.9 (s), 136.5 (d), 148.8 (d), 149.9 (d), 163.2
(s), 168.2 (s) ppm. HRMS (ESI+): C21H29N3O5S+H+, calcd 436.1906,
3
4
92 (8) [C6H6N+]. Optical rotation (c 0.998, CHCl3): [
a]
589 = ꢁ30.4,
20
20
20
20
20
[a
]
578 = ꢁ31.9, [
a]
546 = ꢁ37.0, [
a
]
436 = ꢁ69.1, [ 365 = ꢁ126.5.
a]
(R)-Enantiomer, ((R)-23): Yield: 130 mg (73%), white solid. Mp
20
65–66 °C. Optical rotation (c 1.010, CHCl3):
[
a]
589 = +29.6,
20
20
20
20
[a
]
578 = +31.2, [
a]546 = +36.0, [a]436 = +66.9, [a]365 = +121.9.
found: 436.1898;
C
21H29N3O5S + Na+, calcd 458.1726, found:
458.1716; (C21H29N3O5S)2+H+, calcd 871.3734, found: 871.3717;
(C21H29N3O5S)2 + Na+, calcd 893.3554, found: 893.3534. MS (ESI+,
daughter ion experiment): m/z (%) = 436 (50) [M++H+], 380 (100)
[436ꢁC4H8], 319 (18) [436ꢁH+ꢁCONHOC(CH3)3], 264 (6)
[436ꢁH+ꢁC7H7O3S], 228 (4) [319ꢁC6H6N], 171 (12) [C7H7O3S+].
4.2.4.4.
tert-Butyl N-(4-methoxyphenylsulfonyl)-N-(3-pyridyl-
methyl)-2-amino-4-fluoropent-4-en-hydroxamate. (S)-Enantiomer,
((S)-24): Yield: 107 mg (61%), white solid. Mp 60–61 °C. 1H NMR
3
(300 MHz, CDCl3):
d
1.23 (s, 9H), 2.26 (ddd, JH,F = 17.5 Hz,
2JH,H = 14.7 Hz, JH,H = 6.2 Hz, 1H), 2.80 (ddd, JH,F = 19.6 Hz, JH,H
=
3
3
2
Optical rotation (c 1.007, CHCl3): [a 20
]
589 = ꢁ37.2, [a 20
578 = ꢁ39.3,
]
3
3
14.7 Hz, JH,H = 8.2 Hz, 1H), 3.86 (s, 3H), 4.15 (dd, JH,F = 49.7 Hz,
a 20
]
546 = ꢁ45.2, [a 20
]
436 = ꢁ84.7, [a 20
365 = ꢁ156.2.
]
2JH,H = 3.1 Hz, 1H), 4.48 (dd, JH,F = 17.1 Hz, JH,H = 3.1 Hz, 1H), 4.50
3
2
[
(d, 2JH,H = 16.2 Hz, 1H), 4.59 (m, 1H), 4.62 (d, JH,H = 16.0 Hz, 1H), 6.93
2
(R)-Enantiomer, ((R)-21): Yield: 101 mg (83%), white solid. Mp 65–
66 °C. Optical rotation (c 1.004, CHCl3): [a 20
]
589 = +37.2, [a 20
]578 = +39.1,
3
3
3
(d, JH,H = 8.9 Hz, 2H), 7.20 (dd, JH,H = 7.8 Hz, JH,H = 4.8 Hz, 1H), 7.67
(t, 3JH,H = 8.9 Hz, 3H), 8.49 (d, 3JH,H = 3.8 Hz, 1H), 8.53 (d, 4JH,H = 1.7 Hz,
1H), 8.69 (s, 1 H) ppm. 13C NMR (75 MHz, CDCl3): d 26.2 (q), 32.8 (dt,
2JC,F = 27.9 Hz), 45.9 (t), 54.9 (d), 55.7 (q), 82.5 (s), 93.9 (dt,
2JC,F = 18.5 Hz), 114.4 (d), 123.3 (d), 129.5 (d), 130.9 (s), 132.5 (s), 136.4
(d), 149.1 (d), 145.0 (d), 161.1 (ds, 1JC,F = 256.4 Hz), 163.4 (s), 166.9 (s)
[
a 20
]
546 = +44.9, [a 24306 = +84.3, [a 20
]
]365 = +155.4.
4.2.4.2. tert-Butyl N-(4-methoxyphenylsulfonyl)-N-(3-pyridyl-
methyl)-2-amino-4-fluorobutan-hydroxamate. (S)-Enantiomer,
((S)-22): Yield: 355 mg (75%), white solid. Mp 59–60 °C. 1H NMR
3
ppm. 19F NMR (282 MHz, CDCl3): d ꢁ98.4 (ddd, JH,F = 49.6 Hz,
3
(300 MHz, CDCl3): d 1.24 (s, 9H), 1.72 (m, JH,F = 22.5 Hz, 1H), 2.22
3
2
3JH,F = 17.9 Hz, JH,F = 17.6 Hz) ppm. HRMS (ESI+): C22H28FN3O5S+H+,
3
(m, JH,F = 31.6 Hz, 1H), 3.86 (s, 3H), 4.25 (m, JH,F = 47.6 Hz, 2H),
3
3
calcd 466.1812, found: 466.1819; C22H28FN3O5S + Na+, calcd 488.1631,
found: 488.1634; (C22H28FN3O5S)2 + H+, calcd 931.3546, found:
931.3565; (C22H28FN3O5S)2 + Na+, calcd 953.3384, found: 953.3384.
MS (ESI+, daughter ion experiment): m/z (%) 466 (18) [M++H+], 410
(100) [466ꢁC4H8], 349 (13) [466ꢁH+ꢁCONHOC(CH3)3], 179 (11)
[410ꢁC7H7O3S+ꢁC3H4F], 171 (30) [C7H7O3S+], 92 (8) [C6H6N+]. Optical
4.49 (d, JH,H = 16.2 Hz, 1H), 4.55 (m, 1H), 4.61 (d, JH,H = 16.0 Hz,
1H), 6.94 (d, 3JH,H = 8.8 Hz, 2H), 7.22 (dd, 3JH,H = 7.8 Hz,
3JH,H = 4.8 Hz, 1H), 7.62–7.71 (m, JH,H = 8.9 Hz, 3H), 8.49–8.53 (m,
3
2H), 8.81 (m, 1 H) ppm. 13C NMR (75 MHz, CDCl3): d 26.9 (s), 30.6
2
3
(d, JC,F = 19.5 Hz), 45.8 (s), 53.9 (d, JC,F = 3.0 Hz), 55.6 (s), 80.0 (d,
1JC,F = 165.6 Hz), 82.4 (s), 114.5 (s), 123.4 (s), 129.2 (s), 130.8 (s),
132.6 (s), 136.4 (s), 149.1 (s), 149.8 (s), 163.3 (s), 167.4 (s) ppm. 19
F
rotation (c 0.996, CHCl3): [a 20
]
589 = ꢁ6.5, [a 20
]
578 = ꢁ6.8, [a 20
546 = ꢁ7.9,
]
ꢁ222.2 (ddt, 2JH,F = 47.5 Hz,
[
a 20
]
436 = ꢁ14.3, [a 20
365 = ꢁ24.9.
]
NMR (282 MHz, CDCl3):
d
3JH,F = 31.5 Hz, JH,F = 22.6 Hz) ppm. HRMS (ESI+): C21H28FN3O5S+H+,
3
(R)-Enantiomer ((R)-24): Yield: 112 mg (63%), white solid. Mp
C
21H28FN3O5S + Na+, calcd
20
calcd 454.1812, found: 454.1804;
59–60 °C. Optical rotation (c 1.023, CHCl3):
[a]589 = +8.5,
476.1631, found: 476.1623; (C21H28FN3O5S)2+H+, calcd 907.3546,
found: 907.3544; (C21H28FN3O5S)2 + Na+, calcd 929.3365, found:
929.3370. MS (GC/MS, 70 eV): m/z (%) 454 (100) [M++H+], 398 (97)
[M+ꢁC4H8], 337 (15) [M+ꢁCONHOC(CH3)3], 317 (4) [337ꢁHF], 246
(3) [337ꢁC6H6N], 171 (15) [C7H7O3S+]. Optical rotation (c 1.005,
20
20
20
20
[a]578 = +8.7, [a]546 = +10.1, [a]436 = +18.2, [a]365 = +31.8.
4.2.5. Synthesis of the hydroxamic acids—General procedure E
In a dried Young tube, the appropriate hydroxamate 21–24 was
dissolved in fresh TFA (50 mL/mmol) under argon. The tube was
sealed and the solution stirred at 40 °C for 12 h. Then the reaction
mixture was evaporated to dryness in vacuo. The residue was
CHCl3): [a 20
] ] ] ]436 = +40.7,
589 = +17.9, [a 25708 = +18.7, [a 25406 = +21.7, [a 20
[ ]
a 20
365 = +75.1.

M. Behrends et al. / Bioorg. Med. Chem. 23 (2015) 3809–3818
3817
dissolved in chloroform (100 mL/mmol) and washed with aqueous
citric acid (pH ꢃ 4). The aqueous layer was extracted with chloro-
form and the combined organic phases were dried over magne-
sium sulfate. After evaporation of the solvent the products were
obtained as viscous oils and, if necessary, purified by HPLC.
[M++H+], 331 (15) [M+ꢁCONHOH], 279 (4) [C13H15N2O3S+], 240 (8)
[331ꢁC5H4N], 171 (100) [C7H7O3S+], 161 (30) [C10H13N2+], 92 (45)
[C6H6N+].
(R)-Enantiomer, ((R)-3): Yield: 33 mg (38%), colorless, viscous
oil.
4.2.5.1. N-(4-Methoxyphenylsulfonyl)-N-(3-pyridylmethyl)-2-
4.2.5.4. N-(4-Methoxyphenylsulfonyl)-N-(3-pyridylmethyl)-2-
amino-butanhydroxamic
acid.
(S)-Enantiomer,
((S)-1):
amino-4-fluoropent-4-enhydroxamic acid.
(S)-Enantiomer,
Yield: 60 mg (40%), cloudy, highly viscous liquid. 1H NMR
((S)-4): Yield: 62 mg (66%), colorless, viscous oil. 1H NMR
3
3
2
(300 MHz, CD3CN): d 0.71 (t, JH,H = 7.4 Hz, 3H), 1.40 (m, 1H), 1.66
(400 MHz, CD3CN): d 2.35 (ddd, JH,F = 18.1 Hz, JH,H = 14.9 Hz,
3
3
3
2
3
(m, 1H), 3.86 (s, 3H), 4.18 (dd, JH,H = 8.7 Hz, JH,H = 6.6 Hz, 1H),
3JH,H = 6.6 Hz, 1H), 2.74 (ddd, JH,F = 18.9 Hz, JH,H = 14.8 Hz, JH,H
=
2
2
4.74 (d, JH,H = 17.4 Hz, 1H), 4.86 (d, JH,H = 17.4 Hz, 1H), 7.03 (d,
8.4 Hz, 1H), 3.85 (s, 3H), 4.34 (dd, 3JH,F = 50.3 Hz, 2JH,H = 3.3 Hz, 1H),
3JH,H = 9.0 Hz, 2H), 7.71 (d, 3JH,H = 9.1 Hz, 2H), 7.91 (dd,
4.56 (dd, JH,F = 17.3 Hz, JH,H = 3.3 Hz, 1H), 4.57 (dd, JH,H = 8.2 Hz,
3
2
3
3
3
3
3JH,H = 8.1 Hz, JH,H = 5.8 Hz, 1H), 8.50 (d, JH,H = 8.1 Hz, 1H), 8.58
3JH,H = 6.5 Hz, 1H), 4.81 (s, 2H), 7.01 (d, JH,H = 9.0 Hz, 2H), 7.66 (d,
3
3
3
(d, JH,H = 5.7 Hz, 1H), 8.77 (s, 1 H) ppm. 13C NMR (75 MHz,
3JH,H = 9.0 Hz, 2H), 7.86 (dd, JH,H = 8.0 Hz, JH,H = 5.8 Hz, 1H), 8.41
CD3CN): d 10.7 (q), 23.8 (t), 46.0 (t), 56.7 (q), 59.7 (d), 115.6 (d),
127.7 (d), 130.5 (d), 131.6 (s), 141.0 (d), 141.1 (s), 142.1 (d), 146.4
(d), 164.6 (s), 168.3 (s) ppm. HRMS (ESI+): C17H21N3O5S + H+, calcd
380.1280, found: 380.1263; C17H21N3O5S + Na+, calcd 402.1100,
found: 402.1082. MS (ESI+, daughter ion experiment): m/z (%) 380
(100) [M+H+], 319 (25) [M+ꢁCONHOH], 228 (5) [319ꢁC6H6N], 171
(30) [C7H7O3S+], 92 (10) [C6H6N+].
(d, 3JH,H = 8.2 Hz, 1H), 8.59 (d, 3JH,H = 5.5 Hz, 1H), 8.73 (s, 1 H) ppm.
13C NMR (100 MHz, CD3CN): d 33.2 (dt, JC,F = 28.0 Hz), 46.1 (t),
2
2
55.5 (d), 56.7 (q), 94.5 (dt, JC,F = 18.7 Hz), 115.7 (d), 127.5 (d),
130.5 (d), 131.5 (s), 140.1 (s), 141.4 (d), 142.6 (d), 145.7 (d), 162.5
1
(ds, JC,F = 255.6 Hz), 164.7 (s), 166.8 (s) ppm. 19F NMR (282 MHz,
3
3
3
CD3CN):
d
ꢁ97.3 (ddd, JH,F = 50.2 Hz, JH,F = 18.2 Hz, JH,F
=
17.7 Hz) ppm. HRMS (ESI+): C18H20FN3O5S+ + H+, calcd 410.1186,
found: 410.1174; (C18H20 FN3O5S+)2 + H+, calcd 819.2294, found:
819.2278; (C18H20FN3O5S+)2 + Na+, calcd 841.2113, found:
841.2096. MS (ESI+, daughter ion experiment): m/z (%) 410 (5)
[M++H+], 349 (11) [M+ꢁCONHOH], 179 (23) [349ꢁC7H7O3S+], 171
(100) [C7H7O3S+], 135 (18) [C8H11N2+], 123 (20) [C7H7O+2], 92 (50)
[C6H6N+].
(R)-Enantiomer, ((R)-1): Yield: 38 mg (43%), colorless, viscous
oil.
4.2.5.2. N-(4-Methoxyphenylsulfonyl)-N-(3-pyridylmethyl)-2-
amino-4-fluorobutanhydroxamic acid. (S)-Enantiomer, ((S)-2):
Yield: 49 mg (42%), colorless, viscous oil. 1H NMR (600 MHz,
3
3
CD3CN): d 1.76 (m, JH,F = 23.7 Hz, 1H), 2.14 (ddd, JH,F = 29.1 Hz,
(R)-Enantiomer, ((R)-4): Yield: 57 mg (58%), colorless, viscous
oil.
3
2JH,H = 11.8 Hz, JH,H = 6.1 Hz, 1H), 3.85 (s, 3H), 4.20–4.42 (m, 2H),
3
3
2
4.48 (dd, JH,H = 8.9 Hz, JH,H = 6.1 Hz, 1H), 4.76 (d, JH,H = 17.1 Hz,
2
3
1H), 4.81 (d, JH,H = 17.1 Hz, 1H), 7.01 (d, JH,H = 8.9 Hz, 2H), 7.68
(d, JH,H = 8.9 Hz, 2H), 7.88 (dd, JH,H = 8.1 Hz, JH,H = 5.7 Hz, 1H),
4.3. In vitro assays
3
3
3
3
3
8.43 (d, JH,H = 7.7 Hz, 1H), 8.58 (d, JH,H = 5.3 Hz, 1H), 8.74 (s, 1 H)
The synthetic fluorogenic substrate (7-methoxycoumarin-4-yl)
acetyl pro-Leu-Gly-Leu-[3-(2,4-dinitrophenyl)-L-2,3-diamino-pro-
ppm. 13C NMR (150 MHz, CD3CN): d 31.3 (dt, JC,F = 20.0 Hz), 46.1
2
3
1
(t), 55.0 (dd, JC,F = 3.6 Hz), 56.78 (q), 81.4 (dt, JC,F = 164.2 Hz),
115.7 (d), 127.6 (d), 130.2 (d), 131.4 (s), 140.4 (s), 141.2 (d), 142.3
(d), 146.1 (d), 164.7 (s), 167.3 (s) ppm. 19F NMR (564 MHz,
pionyl]-Ala-Arg-NH2 (R&D Systems, Minneapolis, USA) was used
as described previously.42 All compounds shown in Table 1 were
assayed with respect to the inhibition of human active MMP-2
and MMP-9 by pre-incubating MMP-2 (1 nM) or MMP-9 (2 nM)
and inhibitor compounds at varying concentrations (10 pM–
1 mM) in 50 mM Tris HCl (pH 7.5), containing 0.2 M NaCl, 5 mM
CD3CN):
d
ꢁ221.4
(tdd,
2JH,F = 47.1 Hz,
3JH,F = 29.0 Hz,
3JH,F = 23.7 Hz) ppm. HRMS (ESI+): C17H20FN3O5S+ + H+, calcd
398.1186, found: 398.1181. C17H20FN3O5S+ + Na+, calcd 420.1105,
found: 420.0999; (C17H20FN3O5S+)2 + H+, calcd 795.2294, found:
795.2300; (C17H20FN3O5S+)2 + Na+, calcd 817.2113, found:
817.2116. MS (ESI+, daughter ion experiment): m/z (%) 398 (10)
[M++H+], 337 (16) [M+ꢁCONHOH], 171 (100) [C7H7O3S+], 167 (30)
[337ꢁC7H7O3S], 135 (16) [C8H11N+2], 123 (22) [C7H7O2+], 107 (5)
[C7H7O+], 92 (52) [C6H6N+].
CaCl2, 20
quot of substrate (10
90 L of the pre-incubated MMP/inhibitor mixture, and the activity
lM ZnSO4 and 0.05% Brij 35 at 37 °C for 30 min. An ali-
l
L of a 5 M solution) was then added to
l
l
was determined at 37 °C by following fluorescent product release
with time. The fluorescence changes were monitored using a
Fusion Universal Microplate Analyzer (Packard Bioscience,
Massachusetts, USA) with excitation and emission wavelengths
set to 330 and 390 nm, respectively. Reaction rates were measured
from the initial 10 min of the reaction profile where product
release was linear with time and plotted as a function of inhibitor
dose. From the resulting inhibition curves, the IC50 value for each
inhibitor was calculated by nonlinear regression analysis, per-
formed using Grace 5.1.8 software (Linux).
(R)-Enantiomer, ((R)-2): Yield: 45 mg (49%), colorless, viscous
oil.
4.2.5.3. N-(4-Methoxyphenylsulfonyl)-N-(3-pyridylmethyl)-2-
aminopent-4-enhydroxamic acid. (S)-Enantiomer, ((S)-3): Yield:
77 mg (30%), colorless, highly viscous liquid. 1H NMR (400 MHz,
2
3
CD3CN): d 2.14 (dt, JH,H = 14.3 Hz, JH,H = 6.8 Hz, 1H), 2.45 (dt,
2JH,H = 14.2 Hz, 3JH,H = 7.7 Hz, 1H), 3.84 (s, 3H), 4.31 (t,
3JH,H = 7.6 Hz, 1H), 4.61 (d, 2JH,H = 16.8 Hz, 1H), 4.73 (d,
4.4. Molecular modeling
2JH,H = 16.8 Hz, 1H), 4.92 (s, 1H), 4.95 (d, JH,H = 5.6 Hz, 1H), 5.47
3
3
3
3
(ddt, JH,H = 17.3 Hz, JH,H = 10.4 Hz, JH,H = 6.9 Hz, 1H), 7.00 (d,
The crystal structure of an MMP-9 was obtained from the protein
data bank 1GKC.39 The structure was built using the CHARMM suite
(Version c35b3),43 and the crystallographically observed reverse
hydroxamate inhibitor (N-2-[(2R)-2-{[formyl(hydroxy)amino]
3JH,H = 8.8 Hz, 2H), 7.30 (dd, JH,H = 7.6 Hz, JH,H = 4.9 Hz, 1H), 7.70
3
3
3
3
(d, JH,H = 8.9 Hz, 2H), 7.81 (d, JH,H = 7.8 Hz, 1H), 8.41 (s, 1H), 8.53
(s, 1H) ppm. 13C NMR (100 MHz, CD3CN): d 35.2 (t), 46.8 (t), 56.6
(q), 58.0 (d), 115.4 (d), 119.1 (t), 124.5 (d), 130.5 (d), 132.1 (d),
133.9 (s), 135.9 (s), 137.7 (d), 148.5 (d), 149.5 (d), 164.4 (s), 167.4
(s) ppm. Exact mass (ESI+): C18H21N3O5S + H+, calcd 392.1280,
methyl}-4-methylpentanoyl]-N3-dimethyl-L-valinamide) was removed.
The various MMPIs (compounds 2, 3, 4 and CGS 27023A) were man-
ually inserted into the binding pocket using GAUSSVIEW (Version
4.1.2).54 An initial relaxation procedure of the entire protein struc-
ture was performed using the CHARMM27 force field.55,56 A cluster
found: 392.1271;
C
18H21N3O5S + Na+, calcd 414.1100, found:
414.1094. MS (ESI+, daughter ion experiment): m/z (%) 392 (10)

3818
M. Behrends et al. / Bioorg. Med. Chem. 23 (2015) 3809–3818
model57 was created by including the residues surrounding the
MMPIs, and the remaining protein environment was not included.
The residues in the cluster were: Trp338, Asp185, Gly186, Leu187,
His190, Tyr393, Val398, His401, Glu402, His 405, Pro421, Typ423,
Leu188, His411, Ala189, Zn498, for a total of 274 atoms (excluding
the atoms from the MMPIs). The geometry of the MMPIs was subse-
quently optimized using the PM6-DH258–60-semi-empirical
Hamiltonian inside the frozen protein cluster model using the
MOPAC2012 program.61
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Financial support by the Deutsche Forschungsgemeinschaft
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